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Graph-based open-ended survey on concerns related to COVID-19

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Graph-based open-ended survey on concerns related to COVID-19 Tatsuro KawamotoID1*, Takaaki Aoki2, Michiko Ueda3 1 Artificial Intelligence Research Center, National Institute of Advanced Industrial Science and Technology, Tokyo, Japan, 2 Faculty of Education, Kagawa University, Takamatsu, Japan, 3 Faculty of Political Science and Economics, Waseda University, Tokyo, Japan * [email protected] * [email protected] a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 Data Availability Statement: All raw data are available from https://github.com/tatsuro- kawamoto/opinion_graphs. Data Availability Statement: All raw data are available from https://github.com/tatsuro- kawamoto/opinion_graphs. PLOS ONE RESEARCH ARTICLE a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 OPEN ACCESS OPEN ACCESS Citation: Kawamoto T, Aoki T, Ueda M (2021) Graph-based open-ended survey on concerns related to COVID-19. PLoS ONE 16(8): e0256212. https://doi.org/10.1371/journal.pone.0256212 OPEN ACCESS Citation: Kawamoto T, Aoki T, Ueda M (2021) Graph-based open-ended survey on concerns related to COVID-19. PLoS ONE 16(8): e0256212. https://doi.org/10.1371/journal.pone.0256212 Citation: Kawamoto T, Aoki T, Ueda M (2021) Graph-based open-ended survey on concerns related to COVID-19. PLoS ONE 16(8): e0256212. https://doi.org/10.1371/journal.pone.0256212 Editor: Paolo Barucca, University College London, UNITED KINGDOM Received: December 15, 2020 Accepted: August 2, 2021 Published: August 13, 2021 Copyright: © 2021 Kawamoto 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. Received: December 15, 2020 Accepted: August 2, 2021 Published: August 13, 2021 Copyright: © 2021 Kawamoto et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Abstract The COVID-19 pandemic is an unprecedented public health crisis with broad social and economic consequences. We conducted four surveys between April and August 2020 using the graph-based open-ended survey (GOS) framework, and investigated the most pressing concerns and issues for the general public in Japan. The GOS framework is a hybrid of the two traditional survey frameworks that allows respondents to post their opinions in a free-for- mat style, which can subsequently serve as one of the choice items for other respondents, just as in a multiple-choice survey. As a result, this framework generates an opinion graph that relates opinions and respondents. We can also construct annotated opinion graphs to achieve a higher resolution. By clustering the annotated opinion graphs, we revealed the characteristic evolution of the response patterns as well as the interconnectedness and multi-faceted nature of opinions. Substantively, our notable finding is that “social pressure,” not “infection risk,” was one of the major concerns of our respondents. Social pressure refers to criticism and discrimination that they anticipate receiving from others should they contract COVID-19. It is possible that the collectivist nature of Japanese culture coupled with the government’s policy of relying on personal responsibility to combat COVID-19 explains some of the above findings, as the latter has led to the emergence of vigilantes. The presence of mutual surveillance can contribute to growing skepticism toward others as well as fear of ostracism, which may have negative consequences at both the societal and individual levels. * [email protected] PLOS ONE PLOS ONE Introduction Funding: T.A. and T.K.: JSPS Grants-in-Aid for Scientific Research Grant No. 18K18604 https:// www.jsps.go.jp/english/e-grants/index.html M.U.: JSPS Grants-in-Aid for Scientific Research Grant No. 20H01584 https://www.jsps.go.jp/english/e- grants/index.html T.A.: Research and Regional Cooperation for Crisis Management Shikoku, Grant number N/A https://www.kagawa-u.ac.jp/iecms/ The funders had no role in study design, data The COVID-19 pandemic is an unprecedented event with a myriad of consequences. Without a doubt, it is one of the most serious public health crises in recent history. However, it is more than a public health challenge. Various measures that were introduced to prevent the spread of the virus have caused major disruptions in economic activities and social life. Reduced busi- ness activities and associated job losses, school closures, movement restrictions, and social dis- tancing have affected multiple aspects of our lives, presenting new challenges that every single member of society has to face in addition to the disease itself. Thus, the COVID-19 pandemic 1 / 11 PLOS ONE | https://doi.org/10.1371/journal.pone.0256212 August 13, 2021 PLOS ONE Graph-based open-ended survey on concerns related to COVID-19 has given rise to multi-faceted issues and concerns for many individuals, ranging from the consequences of the infection itself to the economic and social ramifications of the pandemic. Throughout this paper, we refer to these concerns and issues as opinions. collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist. Using the graph-based open-ended survey (GOS) framework [1], this paper seeks to under- stand such multi-faceted opinions expressed by the general population in Japan between April and August 2020. The GOS framework is a hybrid of the two traditional survey frameworks: multiple choice and free format. In a multiple-choice survey, respondents are presented with a list of items from which they could choose one or more that were most applicable to them. While this is a widely used framework owing to its ease of implementation, the potential answers must be anticipated and compiled as a list by those conducting the survey. Thus, the multiple-choice survey may not be the most appropriate framework to use if the purpose is to gauge complex and multi-faceted opinions, such as people’s concerns during an unprece- dented event like the COVID-19 pandemic. Their concerns and opinions may not be clear a priori because of the complex and rapidly developing nature of the event. Introduction The second frame- work is the free-format survey in which the respondents post their opinions as texts. However, analysing such texts can be arbitrary to a certain extent and challenging in a large-scale survey, including this study. In the GOS framework, respondents can post their opinions in a free-format style, which can subsequently serve as one of the choice items for other respondents, just as in the multiple- choice survey. Because survey respondents can select others’ opinions that they agree with, the GOS framework constitutes a graph (or network) of responses at the end. This graph makes statistical evaluation of the free-format responses feasible, while preserving the diversity of respondents’ opinions that increases spontaneously as respondents express their own opinions. A major contribution of this paper is to reveal the most pressing issues and concerns of the general public through the opinions expressed by the respondents. In addition, by conducting the same survey using the GOS framework several times, we were able to capture the evolution of those opinions. Our analysis shows that the variety of major opinions rapidly changed during the study period, and that several opinions were strongly related. Traditional survey methods are less suitable to capture such characteristic behaviours. These results indicate that studying opin- ions under a rapidly changing situation, such as the COVID-19 pandemic, is indeed an ideal application of the GOS framework. Our study period corresponded to the middle of the first wave of cases in Japan (April 2020), the period in which the number of cases temporarily diminished (May 2020), and the beginning and the middle of the second wave (July and August 2020, respectively), which was larger than the first one in terms of the number of cases, but resulted in fewer deaths (see Fig 1 for the evolution of the confirmed cases). The first case of COVID-19 in Japan was reported on January 16. The Japanese government declared a state of emergency in major metropolitan areas on April 7, and expanded the coverage to the entire country on April 16. The Japanese government implemented neither a lockdown nor strict movement restrictions. Non-essential businesses were urged to either temporarily close or operate for reduced hours, but non-com- pliance was not penalised. The state of emergency was lifted on May 25. Introduction As of August 31, the total number of positive cases was 67,077, and the number of COVID-19-related deaths was 1,278, which amounted to 10.15 deaths per one million people [2]. Materials and methods We conducted four rounds of an online survey of the Japanese adult population aged 20–59 between April and August 2020. The respondents were asked about their most pressing 2 / 11 PLOS ONE | https://doi.org/10.1371/journal.pone.0256212 August 13, 2021 PLOS ONE Graph-based open-ended survey on concerns related to COVID-19 Fig 1. Survey periods and the daily new confirmed cases of COVID-19 in Japan. https://doi.org/10.1371/journal.pone.0256212.g001 Fig 1. Survey periods and the daily new confirmed cases of COVID-19 in Japan. https://doi.org/10.1371/journal.pone.0256212.g001 concerns. Specifically, the question asked (in Japanese) was, “What is the concern and the diffi- culty that you face in your daily life and economic activities? Please provide concrete and suc- cinct answers.” The first survey was conducted between April 13 and 19, 2020. The subsequent three surveys were conducted on the following dates: May 21–26, July 3–7, and August 5–9, 2020. In each round, a set of screening questions was sent to approximately 10,000 individuals who are members of commercial web panels. We then selected a sample of approximately 6,000 respondents each time, based on their demographic characteristics, to represent the Jap- anese general population in terms of their residency area, sex, and age groups. An invitation to our survey was sent to these selected individuals through a survey company. We used all the responses in our analysis and no further criteria has been applied to screen the respondents. The number of respondents who participated in the four rounds of the survey were 2103, 1516, 1729, and 1659, respectively. Their demographic attributes are reported in S1 Table. The survey participants were informed of the purpose of the study prior to their participa- tion, and had the option to quit at any time. The respondents provided explicit digital consent that the information they provided could be used for the purpose of this study. The data were completely anonymous. This study, including the use of digital consent, was approved by the Ethics Review Committee on Human Research of Waseda University (approval number: 2020–050). Graph-based open-ended survey The results of the GOS were used to generate a bipartite graph consisting of two sets of vertices representing the opinions and respondents, respectively. Each edge connects a pair of opinions and respondent vertices, and indicates that a respondent supports the opinion. We refer to this graph as an opinion graph (see S1 File for a comment on the definition of the opinion graph). The generation process of the opinion graph is illustrated in Fig 2. At the beginning of the survey, we prepared a set of opinion vertices (which can be an empty set) as the initial opinions (choices). The description of the initial opinions is provided in S1 File. When a respondent responds to a question, several opinions are sampled uniformly and randomly, and presented to the respondent; the minimum number of samples is eight, while respondents can opt to refer to up to 24 opinions. Whenever a respondent supports a presented opinion(s), the GOS generates a respondent vertex connected to the selected opinion vertices. When a respondent expresses new opinions, the GOS generates corresponding opinion vertices connected to the newly added respondent vertex. The numbers of opinion vertices and respondent vertices The results of the GOS were used to generate a bipartite graph consisting of two sets of vertices representing the opinions and respondents, respectively. Each edge connects a pair of opinions and respondent vertices, and indicates that a respondent supports the opinion. We refer to this graph as an opinion graph (see S1 File for a comment on the definition of the opinion graph). The generation process of the opinion graph is illustrated in Fig 2. At the beginning of the survey, we prepared a set of opinion vertices (which can be an empty set) as the initial opinions The results of the GOS were used to generate a bipartite graph consisting of two sets of vertices representing the opinions and respondents, respectively. Each edge connects a pair of opinions and respondent vertices, and indicates that a respondent supports the opinion. We refer to this graph as an opinion graph (see S1 File for a comment on the definition of the opinion graph). The generation process of the opinion graph is illustrated in Fig 2. At the beginning of the survey, we prepared a set of opinion vertices (which can be an empty set) as the initial opinions (choices). PLOS ONE | https://doi.org/10.1371/journal.pone.0256212 August 13, 2021 Graph-based open-ended survey The description of the initial opinions is provided in S1 File. When a respondent responds to a question, several opinions are sampled uniformly and randomly, and presented to the respondent; the minimum number of samples is eight, while respondents can opt to refer to up to 24 opinions. Whenever a respondent supports a presented opinion(s), the GOS generates a respondent vertex connected to the selected opinion vertices. When a respondent expresses new opinions, the GOS generates corresponding opinion vertices connected to the newly added respondent vertex. The numbers of opinion vertices and respondent vertices PLOS ONE | https://doi.org/10.1371/journal.pone.0256212 August 13, 2021 3 / 11 PLOS ONE Graph-based open-ended survey on concerns related to COVID-19 Fig 2. Example of the generation process of an opinion graph. https://doi.org/10.1371/journal.pone.0256212.g002 g 2. Example of the generation process of an opinion graph. Fig 2. Example of the generation process of an opinion graph. https://doi.org/10.1371/journal.pone.0256212.g002 https://doi.org/10.1371/journal.pone.0256212.g002 increase simultaneously as the GOS is carried out. The above procedure constitutes an opinion graph. We expect a set of similar opinion vertices to be connected via respondent vertices. Simi- larly, we expect the respondent vertices to be classified into groups, in which the vertices have a similar response pattern. The classification of opinions and respondents is realised through clustering of an opinion graph, which we refer to as the opinion groups and respondent groups, respectively. Clustering of the opinion graphs A number of clustering algorithms have been proposed for graphs and bipartite graphs in the literature [3–7]. Here, we used the Markov chain Monte Carlo method, which was imple- mented in a software called graph-tool [8] that performs a Bayesian inference of groups under the assumption of a stochastic block model [9]. The algorithm identifies a statistically signifi- cant group structure and estimates the number of groups in a non-parametric manner. All the clustering results in this study are inferences of unnested (i.e. non-hierarchical) stochastic block models. We confirmed that, while the nested variant does not raise the resolution con- siderably, it often subdivides a large opinion group into smaller sets, which is apparently an overfit. PLOS ONE | https://doi.org/10.1371/journal.pone.0256212 August 13, 2021 Annotated opinion graphs In addition to the clustering of raw opinion graphs, we also considered annotated opinion graphs to achieve a higher resolution by adding more information externally through annotation. Based on the collected opinions, we asked three annotators to classify the opinions into 10 groups: (1) infection risk, (2) social pressure & future prospect, (3) financial issues, (4) (restric- tion of) travel, (5) government policies, (6) mask (shortage), (7) mask (discomfort), (8) other issues, (9) no concerns, and (10) invalid responses. The opinions that may not exclusively belong to one of these groups are left unannotated. The last item refers to opinions that are not directly related to the question that was asked. More detailed descriptions of these groups are provided in S1 File. The annotators classified opinions independently, and we distinguished annotations according to different annotators. The maximum number of annotation labels was 30 for each survey. We denoted the total number of annotation labels as K, which can be less than 30 if some labels are not used. As shown in Fig 3, based on annotators’ decisions, we constructed a K-dimensional vector that represents a prior probability distribution of the group assignment for each opinion vertex. The kth element is η (0) if the vertex has the kth annotation label; otherwise, 4 / 11 PLOS ONE | https://doi.org/10.1371/journal.pone.0256212 August 13, 2021 PLOS ONE Graph-based open-ended survey on concerns related to COVID-19 Fig 3. Schematic of an annotated opinion graph. A raw opinion graph is shown at the top, and the prior distribution for each vertex is shown as a bar plot at the bottom. Fig 3. Schematic of an annotated opinion graph. A raw opinion graph is shown at the top, and the prior distribution for each vertex is shown as a bar plot at the bottom Fig 3. Schematic of an annotated opinion graph. A raw opinion graph is shown at the top, and the prior distribution for each vertex is shown as a bar plot at the bottom. https://doi.org/10.1371/journal.pone.0256212.g003 https://doi.org/10.1371/journal.pone.0256212.g003  (0 <   η), where  and η are determined to normalise the vector to unity. Therefore, the annotated vertices have biased prior distributions, whereas all unannotated vertices have a uni- form prior distribution.  (0 <   η), where  and η are determined to normalise the vector to unity. Annotated opinion graphs Therefore, the annotated vertices have biased prior distributions, whereas all unannotated vertices have a uni- form prior distribution. These prior distributions were incorporated into the Bayesian inference using the bfield parameter in graph-tool. The set of opinion vertices with similar prior distributions tends to be classified into the same group. Because we used a non-parametric inference method, the algo- rithm eventually identifies the parsimonious number of opinion groups. Alternatively, one could use an approach discussed in [10] to incorporate annotations in the inference. PLOS ONE | https://doi.org/10.1371/journal.pone.0256212 August 13, 2021 Response patterns In each round, 191 (9.1%), 127 (8.4%), 119 (6.9%), and 117 (7.1%) respondents expressed their own opinions, while the remaining respondents selected opinion(s) that were applicable to them from a list presented to them. Fig 4 shows the clustering results of the raw opinion graphs. For the first and fourth surveys, no group structure was identified. In contrast, for the second and third surveys, opinion Fig 4. Clustering results of the opinion graphs. These graphs only show randomly sampled connected subgraphs instead of the entire dataset, for illustration purposes. The opinion vertices are aligned at the bottom, while the respondent vertices are aligned at the top. Both types of vertices are sorted based on the inferred group assignments. The colour of each vertex represents the group assignment. The pink vertices are the set of opinions that can be coded as no concerns. htt //d i /10 1371/j l 0256212 004 Fig 4. Clustering results of the opinion graphs. These graphs only show randomly sampled connected subgraphs instead of the entire dataset, for illustration purposes. The opinion vertices are aligned at the bottom, while the respondent vertices are aligned at the top. Both types of vertices are sorted based on the inferred group assignments. The colour of each vertex represents the group assignment. The pink vertices are the set of opinions that can be coded as no concerns. https://doi.org/10.1371/journal.pone.0256212.g004 https://doi.org/10.1371/journal.pone.0256212.g004 5 / 11 PLOS ONE | https://doi.org/10.1371/journal.pone.0256212 August 13, 2021 PLOS ONE Graph-based open-ended survey on concerns related to COVID-19 Fig 5. Response patterns of the four surveys. The rows indicate opinion groups and the columns indicate respondent groups. In each table, the two respondent groups are indicated as groups A and B. https://doi.org/10.1371/journal.pone.0256212.g005 Fig 5. Response patterns of the four surveys. The rows indicate opinion groups and the columns indicate respondent groups. In each table, the two respondent groups are indicated as groups A and B. https://doi.org/10.1371/journal.pone.0256212.g005 of the four surveys. The rows indicate opinion groups and the columns indicate respondent groups. In each table, the two icated as groups A and B. Fig 5. Response patterns of the four surveys. The rows indicate opinion groups and the columns indicate respondent groups. In each table, the two respondent groups are indicated as groups A and B. https://doi.org/10.1371/journal.pone.0256212.g005 graphs were partitioned into two groups. PLOS ONE | https://doi.org/10.1371/journal.pone.0256212 August 13, 2021 Response patterns In both cases, the vertices in the smaller respondent group are highly connected to the opinion vertices that can be coded as no concerns, while the respondents in the other group supported the opinions on various issues, such as infection risk and financial concerns. The clustering in Fig 4 is the most agnostic result based purely on the collected responses. However, their resolution is insufficient, as the raw opinion graph is only classified into a few groups or not classified at all. Fig 5 shows the response patterns of respondents that were obtained through the clustering of annotated opinion graphs. The (i, j) element is the group- wise propensity (normalised in each column) towards opinion group i for respondent group j. More precisely, this is the fraction of the number of edges between groups i and j. Although the number of identified opinion groups in each survey was nine (first survey), six (second sur- vey), and seven (third and fourth surveys), we added empty opinion groups in Fig 5 to obtain the same number of rows in all surveys. In contrast to the results reported in Fig 4, where we observed only one group of respondents for the first and fourth surveys, we can now observe two groups analogous to the results of the second and third surveys: respondents who had spe- cific concerns (group A) and those who did not (group B). 6 / 11 PLOS ONE | https://doi.org/10.1371/journal.pone.0256212 August 13, 2021 PLOS ONE Graph-based open-ended survey on concerns related to COVID-19 Fig 6. Palette diagrams of the four surveys. https://doi.org/10.1371/journal.pone.0256212.g006 Fig 6. Palette diagrams of the four surveys. https://doi.org/10.1371/journal.pone.0256212.g006 https://doi.org/10.1371/journal.pone.0256212.g006 To further reveal the details about response patterns, we show the palette diagrams [11, 12] in Fig 6. The palette diagram is essentially a streamplot, which is a stack plot with varying ori- gin axes. The streamplot is a common visualization for time-series data, for example, the evo- lution of car companies’ market share. In the palette diagram, the vertical axis represents the normalized propensity (i.e., vertical thickness represents unity) for each opinion group that a respondent supports. The set of normalised propensity patterns for all respondents are opti- mally aligned in a horizontal fashion, so the global distribution of the response pattern is better understood. In other words, the respondent vertex indices correspond to the timestamps in the time-series data. PLOS ONE | https://doi.org/10.1371/journal.pone.0256212 August 13, 2021 Response patterns The palette diagrams in Fig 6 show the microscopic response patterns of respondents. The tables in Fig 5 show how these patterns can be summarised at a macroscopic level. 7 / 11 PLOS ONE | https://doi.org/10.1371/journal.pone.0256212 August 13, 2021 PLOS ONE Graph-based open-ended survey on concerns related to COVID-19 The results presented in these two figures suggest that there was a sizable block of respon- dents who did not express any specific concerns throughout the study period. These respon- dents were identified as a distinct group, relatively isolated from others, through graph clustering. The evolution of the number of respondents in this group is negatively correlated with the number of daily cases, which peaked during our fourth survey, as expected. As indi- cated in Fig 6, these respondents were less likely to support other opinion groups. The results presented in these two figures suggest that there was a sizable block of respon- dents who did not express any specific concerns throughout the study period. These respon- dents were identified as a distinct group, relatively isolated from others, through graph For the other opinion groups, it should be noted that many of these groups, including gov- ernment policies and travel, emerged spontaneously from the opinions expressed by respon- dents themselves, as they were not included in the initial opinions displayed at the beginning of the survey. On the other hand, the opinions belonging to infection risk, social pressure & future prospect, and financial issues were included in the initial opinions. It should also be noted, however, that the initial opinions become less likely to be displayed to the respondents as more respondents post their own opinions. Therefore, the initial opinions are unlikely to have a significant influence on the result. In all four surveys, the opinions of respondent group A (shown in Fig 5) tended to focus on the following opinion groups: infection risk, social pressure & future prospect, financial issues, and travel. The size of these opinion groups varied considerably across surveys. For example, travel, which covers issues associated with difficulties with traveling and stay-at-home “requests,” was not a major issue in April, as there were no formal movement restrictions in Japan. It became more prevalent in the second and third surveys (May and July) when the number of cases diminished temporarily. PLOS ONE | https://doi.org/10.1371/journal.pone.0256212 August 13, 2021 Conclusion and discussion In this paper, we surveyed the general adult population of Japan four times using the GOS framework to investigate the issues and concerns of people in the midst of the COVID-19 pan- demic. We classified the opinions of respondents by aggregating the response patterns and annotations through graph clustering. As a result, we revealed the characteristic evolution of the response patterns, particularly on infection risk. The survey results indicated that the most pressing concerns of the general public changed through the various phases of the pandemic. Many of our respondents also had multiple issues and concerns. Such a fine evolution of people’s opinions as well as the multi-faceted nature of these opinions may not be adequately captured by the traditional multiple-choice survey method, which required us to predict the opinions to be included in a list of potential choices. Presenting survey respondents with a pre-determined set of responses can be particularly chal- lenging when the situation changes on a daily basis, as in the case of the current pandemic. Therefore, this is an ideal situation for an open-ended survey, in which the group labels are determined a posteriori. As a hybrid of the two traditional survey methods, the GOS frame- work takes advantage of both methods and successfully reveals the multi-faceted and rapidly changing features of people’s opinions. Substantively, our notable finding is that “social pressure,” not “infection risk,” was one of the major concerns of our respondents. Social pressure refers to criticism and discrimination that they anticipate receiving from others should they contract COVID-19. Although stigmati- zation and social rejection of those with an infectious disease have been reported both in past pandemics and the current one [13–15], it is noteworthy that individuals in the general popu- lation, who were neither actual COVID patients nor high-risk people (e.g., healthcare work- ers), widely anticipated and worried about stigmatization and ostracism even when their chance of contracting COVID was infinitesimally small, as in our study period. While a full explanation of the factors underlying such anticipated social stigma is beyond the scope of the current paper, it is possible that the collectivist nature of Japanese culture cou- pled with the government’s policy of relying on personal responsibility to combat COVID-19 explains some of the above findings, as the latter has led to the emergence of vigilantes. Response patterns By August, however, it no longer constituted a major opinion as the infection risk again became the dominant concern with the rising number of cases. In the first survey (April 2020), our respondents also tended to complain about the govern- ment’s handling of the pandemic (government policies) as well as express concerns regarding the status of hospitals that were overwhelmed by the rapidly rising number of patients; this was captured in the other issues group. However, as indicated in Fig 5, the fraction of such con- cerns quickly decreased in subsequent surveys as the focus of respondents shifted to other issues, such as financial concerns. It is noteworthy that social pressure & future prospect always constituted one of the major opinion groups throughout our study period. This group mainly refers to the negative social consequences of contracting the disease, rather than the physical consequences. Many respon- dents expressed fears that they might be ostracised by others if they contract the disease; thus, this opinion group largely represents the social pressure that respondents felt even before test- ing positive for COVID-19. Similarly, financial issues was another group that appeared as a siz- able block in all of the surveys, indicating that the economic consequences of the pandemic were among the major concerns of respondents. Fig 6 reveals that many respondents expressed multiple concerns and opinions, instead of selecting a single issue as their most pressing concern. While financial issues, social pressure & future prospect, and no concerns tended to appear on their own, other types of issues were more likely to appear in combination with other opinion groups, suggesting that many of our respondents faced multiple issues and concerns during the pandemic. Finally, our results show that infection risk became the dominant concern by the time we conducted the fourth survey, which coincided with the peak period of the second wave of cases (August). As shown in Fig 6, although the infection risk group was one of many other opinions for most respondents until the third survey, it became a prominent group in the fourth survey. The number of respondents who specified infection risks as their only concern was highest in the fourth survey, which suggests that infection risk became the dominant issue for a large number of respondents. 8 / 11 PLOS ONE | https://doi.org/10.1371/journal.pone.0256212 August 13, 2021 PLOS ONE Graph-based open-ended survey on concerns related to COVID-19 PLOS ONE | https://doi.org/10.1371/journal.pone.0256212 August 13, 2021 Conclusion and discussion In col- lectivism, people tend to place greater value on group objectives than on personal ones [16]; thus, those who do not prioritize group goals (in this particular context, public health) are more likely to be ostracized and excluded. In addition, the Japanese government’s emphasis on self-restraint, instead of introducing strict official rules, created an environment in which vigi- lantes, or self-appointed police, emerged and were actively engaged in reporting and attacking people not complying with the government “guidelines.” The activities of vigilantes, for whom the special term “jishuku keisatsu” (self-restraint police) was coined, were widely reported during the pandemic in Japan. They included instances of harsh criticism, harassment, and vandalism toward those who were considered “rule-breakers.” For instance, vehicles from non-local areas were vandalized when travel restraint requests were in place; stores that did not follow the government’s request to operate at reduced hours were anonymously threatened, and COVID-19 patients who did not comply with the government guidelines were severely criticized [17, 18]. In addition, the Japanese tend to blame COVID patients for contracting the disease, and one study reported that people in Japan were much more likely to think that it was a COVID patient’s fault that they contracted the disease, citing their own responsibility, compared to those in other developed countries [19]. Thus, our respondents might also have expected that they would be blamed for their action, because people would be likely to perceive it as a conse- quence of their action and non-compliance. PLOS ONE | https://doi.org/10.1371/journal.pone.0256212 August 13, 2021 9 / 11 PLOS ONE Graph-based open-ended survey on concerns related to COVID-19 Such an attitude and the presence of mutual surveillance can contribute to growing skepti- cism toward others as well as fear of ostracism, which may have negative consequences at both the societal and individual levels. This is especially true as rejection sensitivity is known to be relatively higher in Japan [20]. There was even one instance of suicide by a COVID patient in January 2021, who was supposedly motivated by self-blame and the fear of ostracization by others [21]. Our findings suggest that many members of Japanese society felt pressure associated with COVID-related social rejection during the early phase of the pandemic. S1 Table. Demographic characteristics of the survey respondents. (PDF) S1 Table. Demographic characteristics of the survey respondents. (PDF) Conclusion and discussion Stigmatization and blaming of non-compliers may have detrimental effects on the general population, especially in a collectivist culture, but we have a very limited understanding of their effects, let alone their existence, during the current pandemic. Thus, highlighting the significance of social pressure for members of a collectivist society during a public health crisis is an important substantive contribution of the present study. Understanding the potential psychological effects of social pressure and conducting cross-cultural comparisons of such effects constitute an important future research agenda. Finally, let us discuss the role of annotation in the clustering of opinion graphs. If every respondent supports only one issue or concern, then the resulting opinion graph would have a relatively simple group structure, in which each respondent group would be densely connected to the corresponding opinion group. However, as observed in Figs 5 and 6, the opinion graphs in the present survey have more complex group structures because a significant number of respondents support multiple opinions with different meanings. The annotations were needed to achieve a higher resolution because a graph with a complex group structure is difficult to distinguish from a uniformly random graph, that is, pure noise. Although the annotations made the group structures clear, it should also be noted that clus- tering annotated opinion graphs is far from trivial. First, because the annotators’ classifications were performed independently, just as the responses of the surveys, the decisions of the anno- tators are subjective and do not coincide precisely (see S1 File for the consistency among the annotators). In addition, not all opinion vertices were annotated, and there were no annota- tions for respondent vertices. Considering the fact that the responses to open-ended questions are often classified (i.e. annotated) in a completely manual manner, our statistical inference approach that brings together all the information from respondents and annotators is a con- siderable improvement. PLOS ONE | https://doi.org/10.1371/journal.pone.0256212 August 13, 2021 References 1. Kawamoto T, Aoki T. Democratic classification of free-format survey responses with a network-based framework. Nature Machine Intelligence. 2019; 1(7):322–327. https://doi.org/10.1038/s42256-019- 0071-y 2. Ministry of Health Labour and Welfare. On the number of COVID-19 cases.; 2020. https://www.mhlw. go.jp/stf/covid-19/kokunainohasseijoukyou.html#h2_1. 3. Dhillon IS. Co-Clustering Documents and Words Using Bipartite Spectral Graph Partitioning. In: Pro- ceedings of the Seventh ACM SIGKDD International Conference on Knowledge Discovery and Data Mining. KDD’01. New York, NY, USA: Association for Computing Machinery; 2001. p. 269aˆ??274. Available from: https://doi.org/10.1145/502512.502550. 4. Lee DD, Seung HS. Learning the parts of objects by non-negative matrix factorization. Nature. 1999; 401(6755):788–791. https://doi.org/10.1038/44565 5. Kluger Y, Basri R, Chang JT, Gerstein M. Spectral biclustering of microarray data: coclustering genes and conditions. Genome research. 2003; 13(4):703–716. https://doi.org/10.1101/gr.648603 6. Larremore DB, Clauset A, Jacobs AZ. Efficiently inferring community structure in bipartite networks. Phys Rev E. 2014; 90:012805. https://doi.org/10.1103/PhysRevE.90.012805 7. Gerlach M, Peixoto TP, Altmann EG. A network approach to topic models. Science Advances. 2018; 4(7). https://doi.org/10.1126/sciadv.aaq1360 PMID: 30035215 8. Peixoto TP. The graph-tool python library; 2014. Available from: http://figshare.com/articles/graph_tool/ 1164194. 9. Peixoto TP. Bayesian stochastic blockmodeling. “Advances in Network Clustering and Blockmodeling”, edited by Doreian P, Batagelj V, Ferligoj A, ( Wiley, New York, 2019). 2017;. 10. Hric D, Peixoto TP, Fortunato S. Network structure, metadata, and the prediction of missing nodes and annotations. Phys Rev X. 2016; 6:031038. 11. Noguchi C, Kawamoto T. Evaluating network partitions through visualization. arXiv. 2019;1906.00699. 12. Noguchi C, Kawamoto T. Palette diagram: A Python package for visualization of collective categorical data. arXiv. 2020;2011.01934. 13. Siu JYm. The SARS-Associated Stigma of SARS Victims in the Post-SARS Era of Hong Kong. Qual Health Res. 2008; 18(6):729–738. https://doi.org/10.1177/1049732308318372 14. Lee S, Chan LYY, Chau AMY, Kwok KPS, Kleinman A. The experience of SARS-related stigma at Amoy Gardens. Social Science & Medicine. 2005; 61(9):2038–2046. https://doi.org/10.1016/j. socscimed.2005.04.010 15. Bagcchi S. Stigma during the COVID-19 pandemic. Lancet Infect Dis. 2020; 20(7):782. https://doi.org/ 10.1016/S1473-3099(20)30498-9 16. Markus HR, Kitayama S. Culture and the self: Implications for cognition, emotion, and motivation. Psy- chological Review. 1991; 98(2):224–253. https://doi.org/10.1037/0033-295X.98.2.224 17. Osaki T. Japan’s ‘virus vigilantes’ take on rule-breakers and invaders.; 2020. The Japan Times, May 13. 18. Michel P. Understanding the need to shame someone on social media for not exercising self-restraint during a pandemic.; 2020. The Japan Times, May 16. 19. Miura A, Hiraishi K, Nakanishi D. Do they get what they deserved?: Exploring “situational power” with social psychology. Kagaku. Writing – original draft: Tatsuro Kawamoto, Michiko Ueda. Writing – original draft: Tatsuro Kawamoto, Michiko Ueda. PLOS ONE | https://doi.org/10.1371/journal.pone.0256212 August 13, 2021 Author Contributions Conceptualization: Tatsuro Kawamoto, Takaaki Aoki, Michiko Ueda. Conceptualization: Tatsuro Kawamoto, Takaaki Aoki, Michiko Ueda. Data curation: Tatsuro Kawamoto, Takaaki Aoki, Michiko Ueda. Funding acquisition: Takaaki Aoki, Michiko Ueda. Methodology: Tatsuro Kawamoto. Data curation: Tatsuro Kawamoto, Takaaki Aoki, Michiko Ueda. Project administration: Tatsuro Kawamoto. Project administration: Tatsuro Kawamoto. 10 / 11 PLOS ONE | https://doi.org/10.1371/journal.pone.0256212 August 13, 2021 PLOS ONE Graph-based open-ended survey on concerns related to COVID-19 Software: Tatsuro Kawamoto. Visualization: Tatsuro Kawamoto, Takaaki Aoki. References 2020; 90(10):906–908. 20. Garris CP, Ohbuchi Ki, Oikawa H, Harris MJ. Consequences of interpersonal rejection: A cross-cultural experimental study. Journal of Cross-Cultural Psychology. 2011; 42(6):1066–1083. https://doi.org/10. 1177/0022022110381428 21. Rich M, Hida H. Pandemic despair fuels rates of suicide for Japanese women; 2021. New York Times, Feb 23. 11 / 11 PLOS ONE | https://doi.org/10.1371/journal.pone.0256212 August 13, 2021

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Beyond the Disease: Contextualized Implications of the COVID-19 Pandemic for Children and Young People Living in Eastern and Southern Africa

Frontiers in public health

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PERSPECTIVE published: 19 October 2020 doi: 10.3389/fpubh.2020.00504 1Children and young people is an inclusive term that refers to any person aged 24 years or younger (1, 2). Beyond the Disease: Contextualized Implications of the COVID-19 Pandemic for Children and Young People Living in Eastern and Southern Africa Kaymarlin Govender 1*, Richard Gregory Cowden 2, Patrick Nyamaruze 1, Russell Murray Armstrong 1 and Luann Hatane 3 1 Health Economics and HIV/AIDS Research Division, University of KwaZulu-Natal, Durban, South Africa, 2 Human Flourishing Program, Institute for Quantitative Social Science, Harvard University, Cambridge, MA, United States, 3 Paediatric-Adolescent Treatment Africa, Cape Town, South Africa Edited by: Edited by: Marie Leiner, Texas Tech University Health Sciences Center, United States Reviewed by: Luis Alvaro Moreno Espinoza, The College of Chihuahua, Mexico Satinder Aneja, Sh d U i it I di Reviewed by: Luis Alvaro Moreno Espinoza, The College of Chihuahua, Mexico Satinder Aneja, Sharda University, India *Correspondence: Kaymarlin Govender [email protected] *Correspondence: Kaymarlin Govender [email protected] Specialty section: This article was submitted to Children and Health, a section of the journal Frontiers in Public Health INTRODUCTION The outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is likely to create unprecedented challenges in Eastern and Southern Africa (ESA), a region where health systems are fragile, socioeconomic inequalities exist, and public health crises of HIV and tuberculosis are rampant. Many countries in this region instituted nationwide public health control measures (e.g., social distancing requirements, stay-at-home orders) to minimize the spread of SARS-CoV-2 and reduce the burden of the coronavirus disease 2019 (COVID-19) pandemic on health systems. Although such measures are designed to flatten the curve of SARS-CoV-2 transmission, they often present unique direct and indirect consequences for specific subpopulations. This paper provides an analysis of the implications of COVID-19 and related public health interventions for the well-being of children and young people (CYP)1 living in ESA. We discuss responses that should be implemented to Received: 02 June 2020 Accepted: 06 August 2020 Published: 19 October 2020 Keywords: COVID-19, Eastern and Southern Africa, health, well-being, children, young people The coronavirus disease 2019 (COVID-19) pandemic has created extraordinary challenges and prompted remarkable social changes around the world. The effects of COVID-19 and the public health control measures that have been implemented to mitigate its impact are likely to be accompanied by a unique set of consequences for specific subpopulations living in low-income countries that have fragile health systems and pervasive social-structural vulnerabilities. This paper discusses the implications of COVID-19 and related public health interventions for children and young people living in Eastern and Southern Africa. Actionable prevention, care, and health promotion initiatives are proposed to attenuate the negative effects of the pandemic and government- enforced movement restrictions on children and young people. Edited by: Marie Leiner, Texas Tech University Health Sciences Center, United States Reviewed by: Luis Alvaro Moreno Espinoza, The College of Chihuahua, Mexico Satinder Aneja, Sharda University, India *Correspondence: Kaymarlin Govender [email protected] Citation: Govender K, Cowden RG, Nyamaruze P, Armstrong RM and Hatane L (2020) Beyond the Disease: Contextualized Implications of the COVID-19 Pandemic for Children and Young People Living in Eastern and Southern Africa. Front. Public Health 8:504. doi: 10.3389/fpubh.2020.00504 October 2020 | Volume 8 | Article 504 Frontiers in Public Health | www.frontiersin.org 1 COVID-19 and CYP in Eastern and Southern Africa Govender et al. out on immunization activities during the pandemic (8). Many countries in ESA already had sub-optimal rates of immunization for vaccinable diseases (e.g., measles, polio) before the COVID- 19 pandemic. For example, 2018 estimates indicate that Angola and Ethiopia accounted for 45% of all infants in ESA who were un- or under-vaccinated for diphtheria, tetanus, and pertussis (9). Immunization activities in this region are likely to be disrupted by social responses to COVID-19 (e.g., reluctance to attend vaccination sessions for fear of exposure) and the effects of public health control measures (e.g., border closures and travel disruptions can impact vaccine accessibility). These conditions raise the risk of sudden outbreaks of vaccine-preventable diseases occurring when social distancing restrictions are eased. For children who already have a compromised immune system (e.g., those living with HIV), likelihood of mortality from vaccine- preventable conditions (e.g., measles) is higher if immunizations are not received (10). mitigate the negative effects of the COVID-19 pandemic on CYP in the region (for a summary, see Table 1). mitigate the negative effects of the COVID-19 pandemic on CYP in the region (for a summary, see Table 1). MEDICAL CARE NEEDS OF CHILDREN LIVING WITH UNSUPPRESSED VIRAL LOADS, LOW CD4 COUNTS, AND TUBERCULOSIS INFECTIONS Previous disease outbreaks have demonstrated that when health systems are overwhelmed, deaths from vaccine-preventable (e.g., tuberculosis) and treatable conditions (e.g., HIV) tend to increase. COVID-19 is likely to adversely affect the many CYP living with HIV in the region, especially those who are not aware that they are HIV positive and those with unsuppressed viral loads and low CD4 counts. Estimates from countries in ESA (e.g., Kenya, South Africa) indicate that up to 37% of HIV positive CYP are living with unsuppressed viral loads (3, 4). Unsuppressed viral loads (and low CD4 counts) increase vulnerability to opportunistic infections, including respiratory-related conditions (5). HIV testing must be paired with SARS-CoV-2 testing to detect the concurrent presence of these viruses in CYP. Those who test positive for SARS-CoV-2 should be monitored closely (if asymptomatic) or treated for COVID-19 symptoms, whereas those who test positive for HIV should immediately be placed on antiretroviral treatment (ART). The latter is particularly important for young people who are at increased risk of being severely affected by COVID-19, including those who have not disclosed their HIV status and those who defer seeking HIV treatment during the pandemic. For CYP on ART, continuation of comprehensive ART is crucial to achieving optimal adherence and viral suppression. While acknowledging the importance of initiating measures to minimize the spread of SARS-CoV-2, delivery of immunization services is essential to maintaining the health of children through vaccinations for preventable diseases. Particularly in ESA where health care systems are under-resourced, finding a balance between containing transmission of SARS-CoV-2 and continuing immunization programs is critical. Planning is needed to ensure that unvaccinated children are prioritized by immunization initiatives (e.g., large-scale, home-based immunization campaigns) and developing contingency plans to circumvent immunization campaign disruptions caused by homebound orders related to COVID-19. PHYSICAL, PSYCHOLOGICAL, AND SOCIAL CONSEQUENCES OF HUMAN MOBILITY RESTRICTIONS Recently modeled projections indicate that a 6 months disruption of ART could lead to an additional 465,000 AIDS- related deaths in ESA in the next 12 months (6). As countries in the region implement COVID-19-related public health control measures, there is a need to allocate multi-month prescriptions and refills to reduce the frequency of visits to clinical settings and maintain access to HIV prevention services, including condoms and pre-exposure prophylaxis (7). This will ensure that patients have enough treatment during stay-at-home orders and limit unnecessary visits to health care facilities, thereby reducing the risk of exposure to SARS-CoV-2. Children with unsuppressed viral loads who contract SARS-CoV-2 will need to be placed immediately in high care facilities to manage complications from co-infections. As SARS-CoV-2 rapidly spreads across the world, it is inducing a considerable degree of fear and anxiety among people. Measures that have been implemented to contain the virus, including restrictions on freedom of mobility, limits to physical social contact, and imposed isolation and quarantine, can negatively impact the health and well-being of CYP (11). The consequences of stay-at-home orders are likely to be exacerbated in low- resource countries where financial capacity to support CYP is limited (12). Stress that is triggered by homebound orders can weaken immune systems of growing children and increase their susceptibility to infections (13). CYP who are forced into sedentary lifestyles are at higher risk of developing non- communicable chronic illnesses (e.g., diabetes, hypertension), which is already a growing concern in low-resource contexts such as ESA (14). Because young people living with HIV are more vulnerable to mental illness, especially depression (15), coping with a public health emergency like COVID-19 might compound pre-existing psychological distress. with access to health work psychosocial support School-based information detection and treatment of Community outreach/door immunization for children d th vo W ns • U in an DISRUPTIONS TO IMMUNIZATION PROGRAMS on interventions are targeted ystems level strategies Legislation/polic uninterrupted primary health h intensified SARS-CoV-2 ance creening for SARS-CoV-2 and 19 symptoms e to allocate multi-month HIV prescription refills, while ing for stock-outs CoV-2 testing to include ment of medication availability herence to medication among pediatric high care facilities are e for severe COVID-19 patients discharged and recovered 19 patients • Increase budge and SARS-CoV programs are st during lockdow • SARS-CoV-2 su integrated into e • Increase legislat to human rights during lockdow llance systems to monitor food ages and health and nutrition of CYP • Accommodate CYP into stay-a homebound po those without a equipment or w the home to ex e possible, use innovative ways to e mental health support to people lockdown periods (e.g., edicine, web-based counseling ellness services SMS information • Mental health s classified as an service • Public health in child-friendly an r food ion • A C h th e th ways to o people seling formation • M c se • P c d e s a p d • S in • In to d V-2 V-2 and th HIV e ability mong ties are patient red m messaging otocols [e.g., n should be OVID-19 resp sential servic monito d nutrit ovative pport t (e.g., d couns MS, inf RS-CoV S-CoV ti-mont s, while s nclude on avail ation a re facilit ID-19 p recove d SAR or SAR ms te mult n refills ck-outs ng to in dicatio medica gh car e COVI d and systems to m nd health and YP ible, use inno ntal health su down periods e, web-based s services S platform rnet pro nizatio the CO d an ess h intensified nce creening fo 9 sympto e to allocat prescriptio ng for stoc oV-2 testin ment of med erence to m pediatric hi for severe discharged cross-p er inter pp]) l immu d into t sidered eillance ages an s of CY e poss de men g lockd edicine ll ern an omot alth s rovid are w urveil riage COVID Contin nd TB monito ARS ssess nd ad PLHI DISRUPTIONS TO IMMUNIZATION PROGRAMS Levels at which prevention, care, and health promotion interventions are targeted ral strategies onitoring and School-based and community level strategies Health systems level strategies Legislation/policy strategies cy to adopt ion behaviors (e.g., ment measures, hand stancing) cy for HIV viral load , and SARS-CoV-2 D-19 symptoms and ools to promote nce monitor healthy levels tion refills early and h stocks of required • Support integrated community prevention, surveillance, and early detection of HIV/TB and SARS-CoV-2 by linking CYP to community social and health services • Increase family- and community-based support for YPLHIV through alternative avenues (e.g., social media groups, online ART adherence clubs and peer support) that comply with social distancing requirements • Telephonic counseling for those with high viral loads • Capacitate YPLHIV to fulfill curriculum-based learning away from school (e.g., online platforms, community library, home schooling) • Provide uninterrupted primary health care with intensified SARS-CoV-2 surveillance • Triage screening for SARS-CoV-2 and COVID-19 symptoms • Continue to allocate multi-month HIV and TB prescription refills, while monitoring for stock-outs • SARS-CoV-2 testing to include assessment of medication availability and adherence to medication among YPLHIV • Ensure pediatric high care facilities are available for severe COVID-19 patients • Monitor discharged and recovered • Increase budgets to ensure HIV, TB, and SARS-CoV-2 testing and treatment programs are stepped up and continued during lockdowns • SARS-CoV-2 surveillance should be integrated into existing HIV/TB programs • Increase legislative capacity to respond to human rights abuses against YPLHIV during lockdowns ategies ensure HIV, TB, sting and treatment d up and continued ance should be g HIV/TB programs apacity to respond ses against YPLHIV cal activity needs of me orders or particularly for s to special ave limited space in s should be ntial primary health ntions must be sitive to the abilities of children on the e and effective e (Continued) eeds for pace healt be hildre ve tinue vity needs rs or larly for cial ed space d be mary heal ust be o the of childre ffective (Continue l activity needs orders or articularly for o special e limited space should be al primary heal ons must be tive to the bilities of childre the and effective (Continue cross-platform messaging and ver internet protocols [e.g., App]) capacities and v al immunization should be ed into the COVID-19 response nsidered an essential service • Accelerate rese development of SARS-CoV-2 va d Southern Africa. en are hedules DISRUPTIONS TO IMMUNIZATION PROGRAMS Long-term stay-at-home orders that have been implemented to contain the spread of SARS-CoV-2 have disrupted vaccination campaigns and immunization activities, which increases the risk of children contracting other infectious diseases. Measles immunization campaigns have been delayed in 24 countries and will be canceled in 13 others, with millions of children missing Restrictions to mobility imposed by lockdowns will make it difficult for CYP living in ESA to access health services. As funding and health care services are scaled up to Frontiers in Public Health | www.frontiersin.org October 2020 | Volume 8 | Article 504 2 d strategies for promoting well-being among children and young people in Eastern and Southern Africa. ion, care, an unity level education on d immunizati on campaign adolescents, s of making le e to students er support television ) to be trained d learning an ntact with lea essential ( di omotion interv ealth systems le During lockdown services and wor designated as es resourced to acc children with age safe e-education cost-free child he report the occurr violence ntegrate human nto the COVID-1 response Strengthen case multi-sectoral ref support at-risk a Health workers t Os a ene nd O, no preve d com loca aigns an rig NGO aigns and s grate dren a io to ed ble g to rep ontac seekin loca shed relief ams milies t l and Os a ttlene and GO, no igns and s grate ren a o to ed ble gr o rep ontac eekin loca hed f elief f ms lies t and Os an lene and d O, no h preve nd com nd loca mpaigns man righ d NGO mpaigns s, and s ntegrate hildren a adio ps to ed erable g re to rep e contac d seekin nd loca erished al relief f grams amilies t cial and NGOs a bottlene ss and d NGO, no h preve nd com nd loca mpaigns man rig d NGO paigns s, and tegrate ildren a adio ps to e rable g e to rep conta seekin nd loca rished al relief grams amilies ial and GOs a ottlene ss and NGO, no ern a omot alth s Provid are w urvei riage COVID Contin nd T monito SARS sses nd a YPLH manage COVID-19 and its psychosocial effects, it is important that essential counseling and social support services remain accessible. Innovative approaches need to be developed and implemented to provide mental health support to CYP during the COVID-19 pandemic (e.g., telemedicine, virtual peer support, online counseling and wellness services). Where such services are not feasible, community health workers and families need to be supported to care for CYP. Relaxing lockdown restrictions by creating opportunities for CYP to engage in physical activity will improve physical and psychological health. age-appropriate services, safe e-education platforms, and cost- free child helplines for children to report incidences of abuse or violence. Caregivers also need to be offered guidance on communicating in clear and sensitive language to children about risks, concerns, and protective measures related to SARS-CoV-2 transmission and infection. Children from many impoverished households in ESA are also likely to fare poorly at homeschooling or distance learning due to challenges accessing electricity, electronic devices (e.g., computers), and the internet. Government and private sector partnerships with schools are needed to provide learners and caregivers with resources to facilitate meaningful remote learning opportunities. Basic Education Ministries should identify ways of supplying learners with printed reading materials through community health workers and community centers that are applying COVID-19 safety measures. Caregivers of children must be given support to implement simple routines that maintain typical eating windows, incorporate time for educational activities (e.g., reading), and include recreational activities that adhere to public health control measures. Teachers should be encouraged to stay in regular contact with learners and caregivers during school closures to ensure that learners understand and can engage with educational material. Teachers also need to be trained to remotely teach children living with disabilities, and caregivers should be given assistance with making distance learning accessible to children with disabilities. In low-income countries, radio and television education broadcasts may be more effective than e-learning (22). Rapidly creating age- and grade-appropriate educational radio and television programs in different languages can support learning during school closures. In ESA, unemployment rates remain relatively high, with young people being disproportionately affected (16). Most of the employable young people in the region rely on the informal sector for income. In countries that were confronted with food insecurity before the pandemic (e.g., Zimbabwe), stringent public health control measures that are now linked to COVID-19 are exacerbating hunger and poverty among young people. IMPACT OF SCHOOL CLOSURES ON HEALTH, SAFETY, AND LEARNING As a result of COVID-19 stay-at-home orders, many children have experienced a disruption in formal education. Nationwide school closures are likely to have negative implications for the educational experiences of many children, especially those living in ESA where schools lack sufficient infrastructure to support the educational needs of children while stay- at-home orders are enforced. Government-sponsored school nutritional programs (e.g., feeding schemes) are prevalent in many countries in the region (17). Closing schools immediately restricts access to these programs, which many children depend on. Poor nutrition has been associated with worse educational outcomes in children, weakened immune systems, susceptibility to opportunistic infections, and premature mortality (18). During periods of prolonged school closures, there is a need to ensure children continue to have access to food. South Africa recently increased household funding through a child support grant that provides an additional R300 per child and R500 per caregiver each month (19). Similar initiatives are required in other countries in the ESA region. There is evidence to suggest that most children who are infected with SARS-CoV-2 have mild symptoms or are low transmitters of the virus (23). As countries consider whether or not to re-open schools, the best interests of children and overall public health should be considered. Decisions need to be based on localized prevalence rates of SARS-CoV-2, the ability of schools to adhere to COVID-19 safety regulations, and an assessment of the benefits of classroom-based instruction vis-à- vis remote learning (24). ern a omot alth s Provid are w urvei riage COVID Contin nd T monito SARS sses nd a YPLH It is crucial that governments institute social protection measures to cushion the informal economy and provide food subsidies for those living in poverty. ern a omot alth s Provid are w urvei riage COVID Contin nd T monito SARS sses nd a YPLH strategies for promoting well-being among children and young people in Eastern Levels at which prevention, care, and health promo al strategies onitoring and School-based and community level strategies Health y to adopt on behaviors (e.g., ment measures, hand ancing) y for HIV viral load and SARS-CoV-2 -19 symptoms and ols to promote nce onitor healthy levels on refills early and stocks of required • Support integrated community prevention, surveillance, and early detection of HIV/TB and SARS-CoV-2 by linking CYP to community social and health services • Increase family- and community-based support for YPLHIV through alternative avenues (e.g., social media groups, online ART adherence clubs and peer support) that comply with social distancing requirements • Telephonic counseling for those with high viral loads • Capacitate YPLHIV to fulfill curriculum-based learning away from school (e.g., online platforms, community library, home schooling) • Monitor stigma discrimination and human • Prov care surve • Triag COV • Cont and mon • SAR asse and YPL • Ensu avail • Mon COV wns t r ercise e and • S sh ne orkers d to • W p d te le in Easte health pro Hea B hat s sed • P c s • T C • C a m • S a a Y • E a • M es sed tive online pport) th rements h high um-bas ine ockdow suppor s that regula and exe ased phasiz alth wo ues and rvices vide C during lo ies to s outines ty and health a s are ea to emp ach hea lth issu alth se to prov s of YPLHIV d nd communit developing ro physical activi s to support h en restrictions d information kly routines mmunity outre g mental hea sic mental he communities hts abuses regivers a ldren with orporate p als mmunities ivities whe hool-base pport wee pport com h detectin ivering ba milies ili d righ • Car chi inco me • Co act • Sch sup • Sup with del fam en are hedules en are hedules n the g a and ces s aders) f care Levels at which prevention, care, and health promotion interventions are targeted School-based and community level strategies Health systems level strategies Legislation/policy strategies a • Provide community-wide education on vaccination guidelines and immunization schedules • School-based immunization campaigns to reach older children and adolescents, even when schools are closed • Ensure equitable access to COVID-19 vaccine once available • Schools to consider ways of making learning materials easily accessible to students • Launch multi-media learner support initiatives (e.g., radio and television educational programming) • Where possible, teachers to be trained on conducting remote-based learning and encouraged to stay in contact with learners (e.g., radio, television) • Caregivers to assist with essential home based learning skills (e g reading NA • Implement a national policy to mitigate the immediate impact of school closures and facilitate continuity of education for all students through remote learning • Provide household grants to support out-of-pocket costs to access learning material prevention, care, and health promotion interventions are targeted d community level Health systems level strategies Legislation/policy strategies ty-wide education on lines and immunization munization campaigns to en and adolescents, even closed • Ensure equitable access to COVID-19 vaccine once available der ways of making learning ccessible to students dia learner support dio and television amming) eachers to be trained on e-based learning and ay in contact with learners sion) st with essential i kill ( di NA • Implement a national policy to mitigate the immediate impact of school closures and facilitate continuity of education for all students through remote learning • Provide household grants to support out-of-pocket costs to access learning material ues mic and and ity sk s ued) n re-open sider conte ion: SARS mong CYP n rates, pr COVID-19 tes among i ecisions on hould cons combinat ajectory am ansmission everity of C mortality rat egative imp e • ems to be tbreaks l health syste OVID-19 out citate loca nsive to C ools • Capac respo in sch ning where centers collect ere to mote learn mation on w ommunity n place) to e and adhe te learn on on w munity ace) to d adhe mo mati om n p e an assistance ks) in both open and s during d civil society “watch dogs” stribution essage service; ssistance s) in both pen and during civil society watch dogs” stribution essage service; ssistance s) in both open and during d civil society watch dogs” stribution essage service; ished families with relief funds and food rams milies to be linked with al and development GOs and communities ottlenecks and breakdowns s and delivery y of hunger/malnutrition and support them through rapid linkage to community food distribution and feeding programs g social grants to improv security • Roll-out large-scale fo programs (e.g., food b urban and rural areas • Keep food supply cha accessible to commun lockdowns • Parliamentary process organizations to serve to monitor corruption i of resources GO, non-governmental organization; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2; SMS, sho od with es kdowns through rapid linkage to community food distribution and feeding programs security • Roll-out large-scale fo programs (e.g., food b urban and rural areas • Keep food supply cha accessible to commun lockdowns • Parliamentary process organizations to serve to monitor corruption of resources tal organization; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2; SMS, sho large-scale fo s (e.g., food b nd rural areas od supply cha ble to commun ns ntary process tions to serve or corruption rces us 2; SMS, sho large-scale fo s (e.g., food b nd rural areas od supply cha ble to commu ns ntary process tions to serve or corruption rces us 2; SMS, sho security Roll-out program urban an Keep foo accessib lockdow Parliame organizat to monito of resour e coronaviru y ams • • • y syndrome o community eeding progra ute respiratory nkage t n and fe vere acu ough rapid lin od distribution RS-CoV-2, se d th es downs thr foo l organization; SA preve com loca aigns n rig NGO aigns and s grate ren a o to ed ble g o rep ontac eekin loca hed elief ams ilies t and Os a tlene and O, no COVID-19 and CYP in Eastern and Southern Africa Govender et al. Frontiers in Public Health | www.frontiersin.org FOOD INSECURITY IN FAMILIES AND COMMUNITIES In ESA countries that have been affected by COVID-19, public health measures designed to control the spread of SARS-CoV- 2 has stalled economies and severely impacted the livelihood of people. Many people in ESA are employed informally, have low-paid contract positions, or receive hourly wages. The abrupt closure of many businesses (formal and informal) has resulted in a sudden loss of income for numerous people, with household food and health security being threatened. The World Food Programme (38) has warned that more than 200 million people could be pushed into acute food insecurity by the COVID-19 pandemic, many of which will be residents of ESA countries. ESA also has an immense number of children orphaned by AIDS Double orphans, in particular, are likely to end up living on the street, in youth- and child-headed households, or with extended family members who are likely to experience further financial strain because of the increased dependency ratio (39). As COVID-19 stay-at-home orders confine people to their homes, some young women may not have the opportunity to distance themselves from perpetrators of abuse or seek in-person support and health services for experienced abuse. Countries in ESA (e.g., Kenya, South Africa) have reported increases in the incidence of gender-based violence since COVID-19 homebound orders began (34, 35). Periods of confinement or lockdown accentuate the need to reach the most vulnerable groups with social safety nets. While it may be difficult to reach vulnerable populations when country-level COVID-19 public health control measures are in place, civil society organizations and NGOs need to actively monitor incidents of human rights violations by law enforcement and military personnel who enforce stay-at-home orders and social distancing measures. More broadly, civil society organizations ought to be involved in mitigating unintended consequences of the pandemic, including gender-based violence, discrimination, and food insecurity. p y ( ) Food insecurity will limit the availability of nutritional food choices, which could detract from optimal immune system functioning and reduce the effectiveness of ART among those who are living with HIV. Addressing the impact of income loss in lower-income households through allocation of cash transfers can ease the burden of food insecurity. For example, South Africa has implemented the COVID-19 Social Relief of Distress grant that is paid to individuals who are currently unemployed and do not receive any other form of social grant. DISPROPORTIONATE IMPLICATIONS OF VIOLENCE, HUMAN RIGHTS ABUSES, AND LIMITS ON ACCESS TO SERVICES FOR MARGINALIZED GROUPS School closures during times of crisis can heighten children’s risk of exploitation, abuse, and violence (20). During homebound restrictions, families and communities need to be vigilant and protect children from harm. Countries may benefit from adopting the seven strategies outlined in the INSPIRE package (21). INSPIRE is designed to assist countries and communities to focus on prevention programs and services that have the greatest potential to reduce violence against children. This package has been successfully used in low- and middle-income countries, including those in ESA. During stay-at-home periods, social and child protection services must be designated as essential services and sufficiently resourced to support children with The COVID-19 pandemic is accentuating social-structural inequalities, which tend to disproportionately affect marginalized people and those living in financially precarious situations (25). As countries implement public health policies to minimize transmission of SARS-CoV-2, young girls and women, people who identify as LGBTI, people who engage in sex work, informal traders, and street children are more likely to be targets of police brutality (26, 27). There have also been reports of misuse of emergency powers by governments to target marginalized and vulnerable populations (27). For example, a number of LGBTI shelter residents in Uganda were falsely arrested and incarcerated October 2020 | Volume 8 | Article 504 6 COVID-19 and CYP in Eastern and Southern Africa Govender et al. for approximately 50 days on the pretext of violating COVID-19 lockdown regulations (28). Young sex workers may have fewer avenues to protect themselves and are more prone to being victims of violence from police and other sex workers (29). Some younger sex workers may have children, which might increase their risk-taking propensity as they search for income and food to support their families. and health scientists to support the dissemination of accurate information about individual and group vulnerability to COVID- 19, safe prevention and health promotion measures, and effective treatment approaches. With so many sources of information available to CYP, government-supported initiatives are needed to ensure that the public is informed about where to acquire credible information about COVID-19. Caregivers must be empowered to provide accurate, age-appropriate information to children about stigma and supervise exposure of CYP to information about COVID-19. Innovative, ongoing support services are also needed to assist CYP who are infected with SARS-CoV- 2 or recovering from COVID-19 to cope with stigma and its psychosocial consequences. DISPROPORTIONATE IMPLICATIONS OF VIOLENCE, HUMAN RIGHTS ABUSES, AND LIMITS ON ACCESS TO SERVICES FOR MARGINALIZED GROUPS Access to contraceptives is also a challenge with the imposition of COVID-19 stay-at-home requirements. Restricted mobility, reduced availability of public transportation, and closures of non-essential retail outlets and youth centers limits the capacity of young people to access contraceptives. Shortages of these commodities may precipitate risky sexual practices and unintended pregnancies (30), both of which were already long-standing issues in ESA before lockdowns were imposed in response to COVID-19 (31, 32). For young people living with HIV, condom shortages may increase the likelihood of onward HIV transmission. Further, fear associated with contracting SARS-CoV-2 is preventing individuals from attending public clinics (33). Closure of non-governmental organizations (NGOs) and community centers also places additional strain on the homeless and street children who ordinarily rely on those sources for food, clothing, and basic hygiene products. Frontiers in Public Health | www.frontiersin.org REFERENCES a systematic review and meta-analysis. EClinicalMedicine. (2018) 1:28–42. doi: 10.1016/j.eclinm.2018.06.002 1. World Health Organization. Guidance on Ethical Considerations in Planning and Reviewing Research Studies on Sexual and Reproductive Health in Adolescents. (2018). Available online at: https://apps.who.int/iris/bitstream/ handle/10665/273792/9789241508414-eng.pdf?ua=1 (accessed May 3, 2020). 11. World Vision. Children’s Voices in Times of COVID-19: Continued Child Activism in the Face of Personal Challenges. (2020). Available online at: https://www.wvi.org/publications/report/child-participation/childrens- voices-times-covid-19-continued-child-activism (accessed April 13, 2020). 2. United Nations. Convention on the Rights of the Child. (1989). Available online at: https://treaties.un.org/doc/Treaties/1990/09/19900902%2003-14%20AM/ Ch_IV_11p.pdf (accessed May 16, 2020). 2. United Nations. Convention on the Rights of the Child. (1989). Available online at: https://treaties.un.org/doc/Treaties/1990/09/19900902%2003-14%20AM/ Ch_IV_11p.pdf (accessed May 16, 2020). 12. Madhi SA, Gray GE, Ismail N, Izu A, Mendelson M, Cassim N, et al. COVID-19 lockdowns in low- and middle-income countries: success against COVID-19 at the price of greater costs. S Afr Med J. (2020) 110:724–6. doi: 10.7196/SAMJ.2020.v110i8.15055 3. Boerma RS, Boender TS, Bussink AP, Calis JC, Bertagnolio S, Rinke de Wit TF, et al. Suboptimal viral suppression rates among HIV-infected children in low-and middle-income countries: a meta-analysis. Clin Infect Dis. (2016) 63:1645–54. doi: 10.1093/cid/ciw645 3. Boerma RS, Boender TS, Bussink AP, Calis JC, Bertagnolio S, Rinke de Wit TF, et al. Suboptimal viral suppression rates among HIV-infected children in low-and middle-income countries: a meta-analysis. Clin Infect Dis. (2016) 63:1645–54. doi: 10.1093/cid/ciw645 13. Kołodziej J. Effects of stress on HIV infection progression. HIV AIDS Rev. (2016) 15:13–6. doi: 10.1016/j.hivar.2015. 07.003 4. Njuguna I, Neary J, Mburu C, Black D, Beima-Sofie K, Wagner AD, et al. Clinic-level and individual-level factors that influence HIV viral suppression in adolescents and young adults: a national survey in Kenya. AIDS. (2020) 34:1065–74. doi: 10.1097/QAD.0000000000002538 4. Njuguna I, Neary J, Mburu C, Black D, Beima-Sofie K, Wagner AD, et al. Clinic-level and individual-level factors that influence HIV viral suppression in adolescents and young adults: a national survey in Kenya. AIDS. (2020) 34:1065–74. doi: 10.1097/QAD.0000000000002538 14. Vancampfort D, Mugisha J, Richards J, De Hert M, Lazzarotto AR, Schuch FB, et al. Dropout from physical activity interventions in people living with HIV: a systematic review and meta-analysis. AIDS Care. (2017) 29:636– 43. doi: 10.1080/09540121.2016.1248347 15. Woollett N, Cluver L, Bandeira M, Brahmbhatt H. Identifying risks for mental health problems in HIV positive adolescents accessing HIV treatment in Johannesburg. Child Adol Psych Men. (2017) 29:11–26. doi: 10.2989/17280583.2017.12 83320 5. Anígilájé EA, Aderibigbe SA, Adeoti AO, Nweke NO. FOOD INSECURITY IN FAMILIES AND COMMUNITIES While cash transfers can assist many low-income households, this may not be sufficient to avert food insecurity. Large-scale roll out of food assistance programs (e.g., food banks) in both urban and rural areas is needed to supplement cash transfers, ensure access to life-sustaining food, and prevent social unrest and hunger riots. Food security at home could be improved through home-delivered meals facilitated by local organizations (e.g., NGOs, municipalities). School feeding programs need to be reintroduced, with the option of community sites becoming key distribution points that are accompanied by screening for COVID-19 symptoms, follow-up, and monitoring of children from affected households. Therapeutic nutrition ought to be provided to children who are malnourished or receiving ART. In the long-term, providing lower-income households with direct livelihood support through financed projects to develop small NGOs with established networks are more likely to have access to marginalized populations and should act as conduits between recipients and donors that offer shelter, access to food, and other essential services. Retail shops and youth centers that provide sexual and reproductive health services should be classified as essential services to ensure continued provision of contraceptives and medical treatment to young people. Upholding the rights of all citizens, including marginalized groups, should be a cornerstone of the COVID-19 response. Legal and psychosocial support services should also be accessible to CYP who may require “crisis response” interventions. COVID-19 has provoked social stigma and discriminatory behaviors (36). People who are already living with a stigmatized health condition (e.g., HIV) could face dual stigmas if they contract SARS-CoV-2. Stigmatization can deter health-seeking behaviors and contribute to more severe health problems (37). Local broadcasters ought to regularly feature medical experts October 2020 | Volume 8 | Article 504 Frontiers in Public Health | www.frontiersin.org 7 COVID-19 and CYP in Eastern and Southern Africa Govender et al. and other institutional capacities in ESA, such as education and national security. scale livestock and agricultural activities will both support child nutrition and mitigate the strain of food shortages and increases in food prices. ACKNOWLEDGMENTS The authors acknowledge the Swedish International Development Agency (Sida) and the South African National Research Foundation (NRF) for supporting this work. The authors acknowledge the Swedish International Development Agency (Sida) and the South African National Research Foundation (NRF) for supporting this work. AUTHOR CONTRIBUTIONS KG, RC, and PN conceptualized the manuscript. RC and PN led the writing of the manuscript. KG, RC, PN, RA, and LH provided critical revisions, edited, and finalized the manuscript. CONCLUSION The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author. The COVID-19 pandemic has prompted extraordinary measures around the world to slow the pace of SARS-CoV-2 transmission and minimize the public health consequences of the disease. Though necessary, some of these measures may have direct and indirect implications for specific subpopulations. Public health control orders should be cognizant of the unique needs of CYP, particularly those with underlying health conditions and who live in impoverished conditions. Countries in ESA will need to balance responding directly to the COVID-19 pandemic with upholding human rights and supporting CYP, particularly more vulnerable groups (e.g., children living with HIV, young women), to ensure food, education, and counseling services are available during government-enforced movement restrictions. More generally, the COVID-19 public health crisis highlights the importance of providing fiscal support to improve health systems REFERENCES Available online at: https://www.unicef.org/ media/media_96644.html (accessed May 7, 2020). 23. Ludvigsson JF. Systematic review of COVID-19 in children shows milder cases and a better prognosis than adults. Acta Paediatrica. (2020) 109:1088– 95. doi: 10.1111/apa.15270 34. John N, Casey SE, Carino G, McGovern T. Lessons never learned: crisis and gender-based violence. Dev World Bioeth. (2020) 20:65–8. doi: 10.1111/dewb. 12261 24. United Nations Children’s Fund. Framework for Reopening Schools. (2020). Available online at: https://www.unicef.org/media/68366/file/Framework- for-reopening-schools-2020.pdf (accessed May 8, 2020). 35. Mutavati A, Zaman M. Fighting the ‘Shadow Pandemic’ of Violence Against Women & Children During COVID-19. (2020). Available online at: https:// www.un.org/africarenewal/web-features/coronavirus/fighting-%E2%80 %98shadow-pandemic%E2%80%99-violence-against-women-children- during-covid-19 (accessed April 27, 2020). 25. Joint United Nations Programme on HIV/AIDS. Sex Workers Must Not be Left Behind in the Response to COVID-19. (2020). Available online at: https:// www.unaids.org/en/resources/presscentre/pressreleaseandstatementarchive/ 2020/april/20200408_sex-workers-covid-19 (accessed April 23, 2020). 36. United Nations Children’s Fund. Social Stigma Associated With COVID-19. (2020). Available online at: https://www.unicef.org/media/65931/file/Social%20stigma%20associated%20 with%20the%20coronavirus%20disease%202019%20(COVID-19).pdf (accessed May 14, 2020). 26. Platt L, Elmes J, Stevenson L, Hol V, Rolles S, Stuart R. Sex workers must not be forgotten in the COVID-19 response. Lancet. (2020) 396: 9– 11. doi: 10.1016/S0140-6736(20)31033-3 27. Joint United Nations Programme on HIV/AIDS. UNAIDS Condemns Misuse and Abuse of Emergency Powers to Target Marginalized and Vulnerable Populations. (2020). Available online at: https://www.unaids. org/en/resources/presscentre/pressreleaseandstatementarchive/2020/april/ 20200409_laws-covid19 (accessed April 22, 2020). 37. Stangl AL, Earnshaw VA, Logie CH, van Brakel W, Simbayi LC, Barré I, et al. The Health Stigma and Discrimination Framework: a global, crosscutting framework to inform research, intervention development, and policy on health-related stigmas. BMC Med. (2019) 17:31. doi: 10.1186/s12916-019-1271-3 28. McCool A. Court Orders Release of LGBT+ Ugandans Arrested for ’Risking Spreading Coronavirus’. (2020). Available online at: https://www.reuters.com/ article/us-health-coronavirus-uganda-lgbt/court-orders-release-of-jailed- lgbt-ugandans-after-coronavirus-charges-dropped-idUSKBN22U2DO (accessed May 6, 2020). 38. World Food Programme. COVID-19 Will Double Number of People Facing Food Crises Unless Swift Action is Taken. (2020). Available online at: https://www.wfp.org/news/covid-19-will-double- number-people-facing-food-crises-unless-swift-action-taken (accessed May 15, 2020). 29. Global Network of Sex Work Projects. Policy Brief: Young Sex Workers. (2016). Available online at: https://www.nswp.org/sites/nswp.org/files/ Policy%20Brief%20Young%20Sex%20Workers%20-%20NSWP,%202016.pdf (accessed April 24, 2020). 39. United Nations Children’s Fund. Africa’s Orphaned Generation. (2003). Available online at: https://www.unicef.org/sowc06/pdfs/africas_orphans.pdf (accessed May 19, 2020). 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. 30. United Nations Population Fund. REFERENCES Tuberculosis, before and after antiretroviral therapy among HIV-infected children in Nigeria: what are the risk factors? PLoS ONE. (2016) 11:e0156177. doi: 10.1371/journal.pone.0156177 6. Jewell BL, Mudimu E, Stover J, ten Brink D, Phillips AN, Smith JA, et al. Potential effects of disruption to HIV programs in sub-Saharan Africa caused by COVID-19: results from multiple mathematical models. Lancet. (2020). doi: 10.1016/S2352-3018(20)30211-3. [Epub ahead of print]. 16. Chigunta F, Schnurr J, James-Wilson D, Torres V. Being “Real” About Youth Entrepreneurship in Eastern and Southern Africa: Implications for Adults, Institutions and Sector Structures. Geneva: International Labour Organization (2005). 7. Joint United Nations Programme on HIV/AIDS. Thai Hospitals to Provide Three- to Six-Month Supplies of Antiretroviral Therapy. (2020). Available online at: https://www.unaids.org/en/resources/presscentre/featurestories/ 2020/march/20200325_thailand (accessed March 12, 2020). 17. Lesley D, Alice W, Carmen B, Donald B. Global School Feeding Sourcebook: Lessons From 14 Countries. London: Imperial College Press (2016). 18. United Nations Development Programme. Africa Human Development Report 2012: Towards a Food Secure Future. (2012). Available online at: http://hdr. undp.org/sites/default/files/reports/240/ahdr_2012.pdf (accessed April 14, 2020). 8. United Nations. COVID-19 Isolation Threatens Life-Saving Vaccinations for Millions of Children Globally. (2020). Available online at: https://news.un.org/ en/story/2020/04/1061612 (accessed April 21, 2020). 19. South African Government. President Cyril Ramaphosa: Additional Coronavirus COVID-19 economic and social relief measures. (2020). 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Available online at: https://www.youngpeopletoday.org/wp-content/ uploads/2019/04/Unesco_EUP_Report_2018_LOW_RES.pdf (accessed April 25, 2020). org/press-releases/dont-let-children-be-hidden-victims-covid-19-pandemic (accessed April 15, 2020). org/press-releases/dont-let-children-be-hidden-victims-covid-19-pandemic (accessed April 15, 2020). 21. World Health Organization. INSPIRE Handbook: Action for Implementing the Seven Strategies for Ending Violence Against Children. Geneva: World Health Organization (2018). 33. African News Agency. High Percentage of HIV-Positive People Skipping Treatment Over Covid-19 Fears. (2020). Available online at: https:// www.africannewsagency.com/news-politics/HIV-positive-people-in-SA- skipping-treatment-over-Covid-19-fears-24770911 (accessed April 19, 2020). 22. United Nations Children’s Fund. Radio Programs Help Keep Children Learning in Lake Chad Crisis. (2017). Frontiers in Public Health | www.frontiersin.org REFERENCES Impact of the COVID-19 Pandemic on Family Planning and Ending Gender-Based Violence, Female Genital Mutilation and Child Marriage. (2020). Available online at: https://www. unfpa.org/sites/default/files/resource-pdf/COVID-19_impact_brief_for_ UNFPA_24_April_2020_1.pdf (accessed April 26, 2020). Copyright © 2020 Govender, Cowden, Nyamaruze, Armstrong and Hatane. 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. Schaefer R, Gregson S, Benedikt C. Widespread changes in sexual behaviour in eastern and southern Africa: challenges to achieving global HIV targets? Longitudinal analyses of nationally representative surveys. J Int Aids Soc. (2019) 22:e25329. doi: 10.1002/jia2.25329 32. United Nations Educational, Scientific and Cultural Organization. Situational Analysis on Early and Unintended Pregnancy in Eastern and Southern Africa. October 2020 | Volume 8 | Article 504 Frontiers in Public Health | www.frontiersin.org 9

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Insulin Resistance and Oxidative Stress: In Relation to Cognitive Function and Psychopathology in Drug-Naïve, First-Episode Drug-Free Schizophrenia

Frontiers in psychiatry

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1 Department of Psychiatry, The First Affiliated Hospital, Zhengzhou University, Zhengzhou, China, 2 Biological Psychiatry International Joint Laboratory of Henan/Zhengzhou University, Zhengzhou, China, 3 Henan Psychiatric Transformation Research Key Laboratory/Zhengzhou University, Zhengzhou, China, 4 Academy of Medical Sciences/Zhengzhou University, Zhengzhou, China, 5 Illawarra Health and Medical Research Institute and University of Wollongong, Wollongong, NSW, Australia, 6 Lifespan Changes in Brain and Cognition (LCBC), Department of Psychology, University of Oslo, Oslo, Norway, 7 Norwegian Center for Mental Disorders Research (NORMENT), Institute of Clinical Medicine, University of Oslo, Oslo, Norway, 8 Psychotic Disorders Program, UMass Memorial Medical Center, University of Massachusetts Medical School, Worcester, MA, United States, 9 Henan Mental Hospital, The Second Affiliated Hospital of Xinxiang Medical University, Xinxiang, China, 10 Henan Key Lab of Biological Psychiatry of Xinxiang Medical University, Xinxiang, China, 11 International Joint Research Laboratory for Psychiatry and Neuroscience of Henan, Xinxiang, China Edited by: Xiancang Ma, First Affiliated Hospital of Xi’an Jiaotong University, China Reviewed by: Manli Huang, Zhejiang University, China Philip D. Harvey, University of Miami, United States *Correspondence: Yongfeng Yang [email protected] Xueqin Song [email protected] †These authors have contributed equally to this work Reviewed by: Manli Huang, Zhejiang University, China Philip D. Harvey, University of Miami, United States *Correspondence: Yongfeng Yang [email protected] Xueqin Song [email protected] Objective: The present study aimed to examine whether insulin resistance and oxidative stress are associated with cognitive impairment in first-episode drug-free schizophrenia (SZ) patients. Methods: Ninety first-episode SZ patients and 70 healthy controls were enrolled. Fasting insulin (FINS) and markers of oxidative stress [oxidized glutathione (GSSG), superoxide dismutase (SOD), nitric oxide (NO) and uric acid (UA) levels] were measured in serum before pharmacological treatment was initiated. Psychiatric symptoms and cognitive function were assessed with the Positive and Negative Syndrome Scale (PANSS) and MATRICS Consensus Cognitive Battery (MCCB), respectively. In addition, the homeostatic model assessment of insulin resistance (HOMA-IR) was also studied. †These authors have contributed equally to this work Specialty section: This article was submitted to Schizophrenia, a section of the journal Frontiers in Psychiatry Received: 23 February 2020 Accepted: 26 October 2020 Published: 19 November 2020 Specialty section: This article was submitted to Schizophrenia, a section of the journal Frontiers in Psychiatry Results: HOMA-IR and serum levels of GSSG and NO were significantly higher in SZ patients than in healthy controls (P < 0.001), while the serum levels of SOD were significantly lower than in healthy controls (P < 0.001). ORIGINAL RESEARCH published: 19 November 2020 doi: 10.3389/fpsyt.2020.537280 HOMA-IR, GSSG and NO levels were significantly correlated to the total cognitive function scores of the patient group (r = −0.345,−0.369,−0.444, respectively, P < 0.05). But these factors were not co-related to the cognitive functions in the healthy control group. And, levels of SOD, UA were not associated with the total cognitive function scores in both the patient and the healthy control groups. NO was positively correlated with general pathological and the total score in the PANSS, and was negatively correlated with six cognitive domains (r = −0.316 to −0.553, P < 0.05). Insulin Resistance and Oxidative Stress: In Relation to Cognitive Function and Psychopathology in Drug-Naïve, First-Episode Drug-Free Schizophrenia Qi Tao 1,2,3,4†, Yu Miao 1,2,3†, Huihui Li 1,2,3, Xiuxia Yuan 1,2,3, Xufeng Huang 5, Yunpeng Wang 1,6, Ole A. Andreassen 1,7, Xiaoduo Fan 8, Yongfeng Yang 9,10,11* and Xueqin Song 1,2,3* Qi Tao 1,2,3,4†, Yu Miao 1,2,3†, Huihui Li 1,2,3, Xiuxia Yuan 1,2,3, Xufeng Huang 5, Yunpeng Wang 1,6, Ole A. Andreassen 1,7, Xiaoduo Fan 8, Yongfeng Yang 9,10,11* and Xueqin Song 1,2,3* INTRODUCTION Studies have found that increased IR may occur earlier in first-episode SZ patients than in healthy controls (9). Ringen (10) also proposed that IR has been associated with SZ. Citation: Tao Q, Miao Y, Li H, Yuan X, Huang X, Wang Y, Andreassen OA, Fan X, Yang Y and Song X (2020) Insulin Resistance and Oxidative Stress: In Relation to Cognitive Function and Psychopathology in Drug-Naïve, First-Episode Drug-Free Schizophrenia. Front. Psychiatry 11:537280. doi: 10.3389/fpsyt.2020.537280 November 2020 | Volume 11 | Article 537280 Frontiers in Psychiatry | www.frontiersin.org Tao et al. Schizophrenia, Insulin Resistance, Oxidative Stress Conclusions: The levels of insulin resistance and oxidative stress are elevated, and correlated with the severity of cognitive impairment in drug-naïve, first-episode SZ patients. Treatment approaches targeting on reducing insulin resistance and oxidative stress may improve cognitive function in SZ patients. Conclusions: The levels of insulin resistance and oxidative stress are elevated, and correlated with the severity of cognitive impairment in drug-naïve, first-episode SZ patients. Treatment approaches targeting on reducing insulin resistance and oxidative stress may improve cognitive function in SZ patients. Keywords: schizophrenia, insulin resistance, oxidative stress, cognitive impairment, psychopathology Participants p All subjects in this study were approved by the Ethics Committee of the First Affiliated Hospital of Zhengzhou University and provided written informed consent. Inpatients 18 and 45 years old diagnosed with first-episode SZ (disease duration <2 years) were recruited. The inclusion criteria for patients were: (1) diagnosis of first episode SZ based on the Diagnostic and Statistical Manual of Mental Disorders fourth version (DSM-IV) criteria and confirmed using the Structured Clinical Interview for DSM-IV (SCID) (25); (2) never treated with antipsychotic medications or other psychotropics; (3) the PANSS total score >60 points. Exclusion criteria included: (1) diagnose neurological or other mental illnesses, autoimmune diseases, diabetes and other organic diseases; (2) alcohol or substance abuse history; (3) pregnant or lactating women; (4) have taken any antibiotics, anti-inflammatory agents or probiotics in the past month; (5) major changes in living environment or diet in the past month. Recruitment of normal-weight healthy control subjects through advertisement, they had the same exclusion criteria as the patient group; Moreover, none of them had a history of any psychiatric diseases. Studies have found that the levels of oxidized products such as GSSG and NO are increased, and the levels of antioxidant products such as SOD are decreased in patients with schizophrenia (11), and such changes are associated with cognitive function and psychopathology (12). Studies have shown that that oxidative stress causes cognitive impairment by damaging neurons (13). The excitatory amino acid Glu and NMDA receptors are closely related to oxidative stress, which may cause cognitive impairment through the Glu-NMDAR- NO pathway (14). Glu can activate NMDA receptors (15), and produce excessive oxide and nitric oxide (NO), leading to neuronal excitotoxicity involved in the development of various neuropsychiatric diseases (16). Oxidative stress may be an intermediary mechanism of NMDA receptor dysfunction involved in the occurrence of schizophrenia. Boskovic suggested that oxidative stress may be involved in the occurrence and development of SZ (17). UA scavenges singlet oxygen and free radicals, and it can also trap peroxynitrite anions (free radicals in the ONOO –), thereby reduce the damage mediated by ONOO–. INTRODUCTION Thus, UA is an effective neuroprotective compounds (18). In addition, serum levels of UA can indicate the oxidative stress state of the body. Houlihan et al. (19) studied that UA at a high level may improve memory-related behaviors in cognitive function. However, there is no definitive conclusion on the relationship between UA and cognitive function. Schizophrenia (SZ) is a chronic severe mental illness with mainly unknown etiology, which incurs heavy burden on the persons affected, their families and the society (1). Cognitive impairment has been increasingly recognized as a core feature of SZ, and is associated with reduced social functioning, which is important for the prognosis of patients. N-methyl-D- aspartate (NMDA) receptor hypofunction has been implicated in pathophysiology of SZ (2). Previous studies have suggested that cognitive impairment is related to the hypofunction of the NMDA receptor, a type of ionic glutamate (Glu) receptor (3). Long-term potentiation (LTP) is induced through the NMDA receptor pathway to enhance learning and memory (4). When the NMDA receptor is over-activated, it causes excitatory toxicity to neural cells, leading to cell damage and death. Moreover, the proper function of the NMDA channel in the central nervous systems has been reported to be regulated by many other factors (5, 6). Previous studies have reported an associations between IR, oxidative stress and the risk of type 2 diabetes (20, 21), but there has been little research on the associations between IR, oxidative stress and SZ. In addition, several lines of evidence showed that the antioxidant defense system may be disrupted in SZ patients, and excessive free radical production and oxidative stress damage response may be involved in the pathogenesis of SZ (22–24). Few studies have focused on the association between IR, oxidative stress and cognitive function in SZ patients. The present study was to investigate if serum levels of biomarkers reflecting IR and oxidative stress are elevated in patients with drug-naïve, first episode SZ, and whether such biomarkers are associated with cognitive function in these patients. Insulin resistance (IR) refers to the decreased efficiency of insulin in the promotion of glucose uptake and use. Insulin have effect on brain function, as it regulates the activity of NMDA and improves synaptic plasticity (7). When the biological efficacy of insulin decreases, the learning and memory function are reduced. Chen et al. believed that IR seems to play a role in cognitive impairment in SZ (8). Frontiers in Psychiatry | www.frontiersin.org Assessments In a multiple regressing model including HOMA-IR, GSSG, NO as predictors, and the total MCCB score as the dependent variable, we found that only HOMA-IR had an effect on the MCCB composite score (t = −2.321, P < 0.05). Biochemical Measurements Table 4 shows that within the patient groups, the NO levels were positively correlated with the general pathological score and total score in the PANSS assessment (r = 0.323,0.375, respectively, P < 0.05, Table 4). After controlling for age, gender, disease duration and smoking status, we found that these biological indicators (FINS, HOMA-IR, GSSG, NO, SOD, UA) showed no correlation with the scores of PANSS (positive symptom score, negative symptom score, general pathology score, and PANSS total score) (P > 0.001). Table 4 shows that within the patient groups, the NO levels were positively correlated with the general pathological score and total score in the PANSS assessment (r = 0.323,0.375, respectively, P < 0.05, Table 4). After controlling for age, gender, disease duration and smoking status, we found that these biological indicators (FINS, HOMA-IR, GSSG, NO, SOD, UA) showed no correlation with the scores of PANSS (positive symptom score, negative symptom score, general pathology score, and PANSS total score) (P > 0.001). After all subjects were enrolled, 5 ml of venous blood was collected from the elbow under fasting condition (12 h fasting) by full-time laboratory personnel in the morning of the next day from 6:30 a.m. to 7:30 a.m. to avoid the influence of biological rhythm changes of the measured factors. Venous blood samples were collected from the elbows into EDTA anticoagulant tubes at 4◦C for 10 min at 3,000 r/rain to separate the upper serum. FPG levels were measured by the glucose oxidase method and an automated analyzer (Roche Diagnostics, C8000, Germany), serum FINS levels were measured by radioimmunoassay (Elecsys 2010, Roche, Basel, Switzerland); GSSG was measured by a microenzyme method (A061-1,China); Serum SOD levels was measured using the kits and Roche automatic biochemical analyzer (Roche Diagnostics, C8000, Germany); NO levels was detected by one-step method (A013- 2,China); The serum uric acid (UA)levels was measured by uricase-peroxidase method (Roche, C720, Switzerland). The current group of non-smokers had never smoked before. The homeostasis model of assessment of insulin resistance (HOMA- IR) was calculated using the following formula: HOMA-IR = FPG (mmol/L) × FINS (mu/L)/22.5 (28). Body mass index (BMI) = height/body mass ² (kg/m ²). Assessments The psychiatric symptoms of SZ were assessed in all enrolled patients using the Positive and Negative Syndrome Scale November 2020 | Volume 11 | Article 537280 Frontiers in Psychiatry | www.frontiersin.org 2 Schizophrenia, Insulin Resistance, Oxidative Stress Tao et al. (PANSS) (26). The PANSS was administered by a professionally trained and experienced psychiatrist. All subjects received a baseline cognitive evaluation using the MATRICS Consensus Cognitive Battery (MCCB) (27). It involves seven cognitive areas: (1) Speed of Processing Information; (2) Attention and Vigilance Awareness; (3) Working Memory; (4): Verbal Learning; (5) Visual Learning; (6) Reasoning and Problem Solving; (7) Social Cognition. The MCCB scoring program generates T-scores that are standardized and corrected for age and sex (27). The “cognitive composite” is the standardized sum of the seven domains. Training, data collection and data quality assurance were implemented or supervised by experienced psychologists in accordance with the guidelines outlined in the MCCB manual (27). (PANSS) (26). The PANSS was administered by a professionally trained and experienced psychiatrist. All subjects received a baseline cognitive evaluation using the MATRICS Consensus Cognitive Battery (MCCB) (27). It involves seven cognitive areas: (1) Speed of Processing Information; (2) Attention and Vigilance Awareness; (3) Working Memory; (4): Verbal Learning; (5) Visual Learning; (6) Reasoning and Problem Solving; (7) Social Cognition. The MCCB scoring program generates T-scores that are standardized and corrected for age and sex (27). The “cognitive composite” is the standardized sum of the seven domains. Training, data collection and data quality assurance were implemented or supervised by experienced psychologists in accordance with the guidelines outlined in the MCCB manual (27). were significantly lower than those of the healthy control group (P < 0.05, Table 1). Table 2 shows that FPG levels in SZ patients showed an increased trend but fell short of statistical significance (t = 1.448, P = 0.150); However, FINS, HOMA-IR, GSSG, and NO levels were higher in the SZ patients than in the healthy controls (P < 0.001). On the other hand, the serum SOD levels were lower in the SZ patients than in the healthy controls (t = −3.703, P < 0.001). Table 3 shows that HOMA-IR, GSSG and NO were significantly correlated to the total cognitive function scores of the patient group (r = −0.345, −0.369, −0.444, respectively, P < 0.05, Table 3). DISCUSSION Previous studies have reported associations between IR, oxidative stress and the risk of type 2 diabetes (20), but there has been little research on the associations between IR, oxidative stress and SZ. In addition, few studies have focused on the association between IR, oxidative stress and cognitive function in SZ patients. Our study found that serum marker levels for insulin resistance and oxidative stress were increased compared to healthy controls in first-episode untreated SZ patients, and that the levels were associated with cognitive function impairment. We also found correlations between several indicators of oxidative stress and clinical symptoms in the SZ patient group. To our knowledge, the present study is the first to show associations between cognitive performance and markers of IR and oxidative stress in first- episode untreated SZ patients. Statistical Analysis SPSS 21.0 statistical software was used for all data analysis. All numeric variables data were expressed as mean ±SD. All categorical variables data were expressed as ratios and frequencies. Group comparison was performed using an unpaired t-test, and Chi-square test was used for testing independence among categorical variables. The correlations between IR, oxidative stress and cognitive function was computed by Pearson’s correlation. two-sided P < 0.05 indicated that the difference was statistically significant. Serum FINS and HOMA-IR were increased in first- episode SZ patients, consistent with the previous study by Spelman (9). Other published studies have shown that the development of IR may be caused by decreased insulin tyrosine kinase receptor activity, abnormal insulin signaling, decreased glucose transportation, decreased glucose phosphorylation and decreased glycogen synthase activity (29). As a result of IR, the activity of the cholinergic system in the hippocampus and other brain regions is significantly decreased, which can lead to neuronal degeneration and aggravated cognitive impairment. Our results showed that FINS levels and HOMA-IR were positively correlated with the severity of cognitive impairment in SZ patients. This effect may be related to the following aspects: Frontiers in Psychiatry | www.frontiersin.org RESULTS Table 1 shows that there were no significant differences in age, education, gender, smoking status, and BMI between the SZ patients and healthy control groups (P > 0.05). In the seven domains of cognitive function, the scores of the SZ patient group November 2020 | Volume 11 | Article 537280 Frontiers in Psychiatry | www.frontiersin.org 3 Tao et al. Schizophrenia, Insulin Resistance, Oxidative Stress TABLE 1 | Demographic and clinical characteristics of the study sample. Patients Healthy controls Group comparison Characteristics N = 90 N = 70 Mean (SD) Mean (SD) t/χ2 p Age (years) 21.5 ± 7.7 23.4 ± 5.4 −1.592 0.114 Education (years) 10.4 ± 2.6 11.1 ± 2.4 −1.63 0.106 BMI (kg/m2) 21.5 ± 2.2 21.1 ± 2.4 1.052 0.295 Disease duration (months) 5.9 ± 6.3 N (%) N (%) Gender −0.397 0.692 Male 44 (48.9) 32 (45.7) Female 46 (51.1) 38 (54.3) Smoking status 1.056 0.293 Yes 5 (0.06) 7 (0.10) No 85 (0.94) 63 (0.90) PANSS-total 84.2 ± 12.7 PANSS-positive 23.2 ± 4.8 PANSS-negative 22.4 ± 5.9 PANSS-general 38.5 ± 7.8 MCCB composite score 28.6 ± 16.5 45.1 ± 19.4 −5.790 0.000 SOP 30.6 ± 9.4 51.7 ± 10.0 −12,334 0.000 CPT-IP 32.5 ± 10.9 50.7 ± 9.2 −10.201 0.000 WMS-III 38.8 ± 9.4 55.6 ± 10.6 −9.632 0.000 HVLT 37.6 ± 7.6 50.7 ± 10.0 −8.465 0.000 BVMT 39.8 ± 10.1 57.2 ± 12.1 −8.925 0.000 NAB 40.1 ± 9.8 46.1 ± 10.5 −3.351 0.001 MSCEIT 39.0 ± 11.6 56.3 ± 15.1 −7.375 0.000 PANSS-positive, Positive Symptom; PANSS-negative, Negative Symptom; PANSS-general, General Pathological Score; PANSS-total, PANSS Total Score. TABLE 2 | Comparison of insulin resistance and oxidative stress measures between the two groups. Patient group Healthy control group t P Items (N = 90) (N = 70) BIOCHEMICAL MEASURES FPG (mmol/L) 4.4 ± 0.5 4.3 ± 0.7 1.448 0.150 FINS (mmol/L) 11.1 ± 3.3 6.1 ± 1.1 10.338 0.000 HOMA-IR (mU/L) 2.2 ± 0.7 1.2 ± 0.3 10.790 0.000 GSSG 30.9 ± 7.6 16.8 ± 5.8 11.667 0.000 NO 103.9 ± 54.5 32.2 ± 33.6 8.839 0.000 SOD 198.6 ± 43.1 231.7 ± 29.1 −3.703 0.000 UA 292.2 ± 87.5 273.6 ± 60.1 1.303 0.195 FPG, fasting plasma glucose; FINS, fasting insulin; HOMA-IR, insulin resistance index; GSSG, oxidized glutathione; NO, nitric oxide; SOD, superoxide dismutase; UA, uric acid. Schizophrenia, Insulin Resistance, Oxidative Stress Tao et al. Tao et al. RESULTS TABLE 1 | Demographic and clinical characteristics of the study sample. Patients Healthy controls Group comparison Characteristics N = 90 N = 70 Mean (SD) Mean (SD) t/χ2 p Age (years) 21.5 ± 7.7 23.4 ± 5.4 −1.592 0.114 Education (years) 10.4 ± 2.6 11.1 ± 2.4 −1.63 0.106 BMI (kg/m2) 21.5 ± 2.2 21.1 ± 2.4 1.052 0.295 Disease duration (months) 5.9 ± 6.3 N (%) N (%) Gender −0.397 0.692 Male 44 (48.9) 32 (45.7) Female 46 (51.1) 38 (54.3) Smoking status 1.056 0.293 Yes 5 (0.06) 7 (0.10) No 85 (0.94) 63 (0.90) PANSS-total 84.2 ± 12.7 PANSS-positive 23.2 ± 4.8 PANSS-negative 22.4 ± 5.9 PANSS-general 38.5 ± 7.8 MCCB composite score 28.6 ± 16.5 45.1 ± 19.4 −5.790 0.000 SOP 30.6 ± 9.4 51.7 ± 10.0 −12,334 0.000 CPT-IP 32.5 ± 10.9 50.7 ± 9.2 −10.201 0.000 WMS-III 38.8 ± 9.4 55.6 ± 10.6 −9.632 0.000 HVLT 37.6 ± 7.6 50.7 ± 10.0 −8.465 0.000 BVMT 39.8 ± 10.1 57.2 ± 12.1 −8.925 0.000 NAB 40.1 ± 9.8 46.1 ± 10.5 −3.351 0.001 MSCEIT 39.0 ± 11.6 56.3 ± 15.1 −7.375 0.000 PANSS positive Positive Symptom; PANSS negative Negative Symptom; PANSS general General Pathological Score; PANSS total PANSS Total Score TABLE 1 | Demographic and clinical characteristics of the study sample. TABLE 2 | Comparison of insulin resistance and oxidative stress measures between the two groups. Patient group Healthy control group t P Items (N = 90) (N = 70) BIOCHEMICAL MEASURES FPG (mmol/L) 4.4 ± 0.5 4.3 ± 0.7 1.448 0.150 FINS (mmol/L) 11.1 ± 3.3 6.1 ± 1.1 10.338 0.000 HOMA-IR (mU/L) 2.2 ± 0.7 1.2 ± 0.3 10.790 0.000 GSSG 30.9 ± 7.6 16.8 ± 5.8 11.667 0.000 NO 103.9 ± 54.5 32.2 ± 33.6 8.839 0.000 SOD 198.6 ± 43.1 231.7 ± 29.1 −3.703 0.000 UA 292.2 ± 87.5 273.6 ± 60.1 1.303 0.195 FPG, fasting plasma glucose; FINS, fasting insulin; HOMA-IR, insulin resistance index; GSSG, oxidized glutathione; NO, nitric oxide; SOD, superoxide dismutase; UA, uric acid. TABLE 2 | Comparison of insulin resistance and oxidative stress measures between the two groups. FPG, fasting plasma glucose; FINS, fasting insulin; HOMA-IR, insulin resistance index; GSSG, oxidized glutathione; NO, nitric oxide; SOD, superoxide dismutase; UA, uric acid. ma glucose; FINS, fasting insulin; HOMA-IR, insulin resistance index; G IL-1α, IL-1β, IL-6, and TNF-α. RESULTS Thus, oxidative stress reduces the recruitment of P13K by interfering with the phosphorylation of insulin receptor substrates, thereby inducing IR. On the other hand, High levels of free fatty acids lead to an increase in ROS and reactive nitrogen species (RNS), thereby activating the oxidative stress mechanisms that damage DNA, proteins and lipids. Additionally, high levels of free fatty acids can activate a range of intracellular signaling pathways that are closely related to IR and other cellular functions. The dysregulation of free fatty acids leads to neurodegenerative disease by promoting the phosphorylation of tau in the hippocampus (44). Insulin inhibits the activity of glycogen synthesis kinase-3β (GSK- 3β), which phosphorylates tau, pyruvate dehydrogenase, and glycogen synthase. GSK-3β activation under insulin deficiency or IR promotes the phosphorylation of tau and the inactivation of pyruvate dehydrogenase and glycogen, affecting energy metabolism and acetylcholine synthesis. Thus, the interplay between IR and oxidative stress factors can synergistically impair cognitive functions in the SZ patients (45). by increased GSSG and NO levels accompanied by decreased SOD levels (34), and such an imbalance may correlate with impaired cognitive functions (12). NO, as a signaling molecule, may be involved in the impairment of cognitive function in SZ patients through the following mechanisms: (1) activation of the hypothalamus- pituitary-adrenal axis, secretion of prolactin, and corticosteroids, and elevation of hormones levels and thus activating the dopaminergic neurons in the hypothalamus, causing positive symptoms and cognitive impairment (35); (2) NMDA receptor activation, which is involved in the release of Glu and dopamine, and causes cognitive deficits and mental disorders (36); and (3) reduction in nitrite ions and superoxide anions, which react to the formation of peroxynitrite anions, leading to neurotoxic effects. Previous studies have suggested that the antioxidant defense system is disrupted in SZ patients, and excessive free radical production and oxidative stress damage response may be involved in the pathogenesis of SZ (22, 23). SOD and UA are both effective indicators of antioxidant capacity, and SOD as an enzyme can effectively scavenge oxygen free radicals in the body, representing an important component of the free radical defense system. Our results showed that SOD levels were significantly decreased in SZ patients compared with healthy control subjects, which was consistent with the study by Reyazuddin (37), indicating that there may be a high level of oxidative stress in SZ patients. RESULTS Additionally, high levels of free fatty acids can activate a range of intracellular signaling pathways that are closely related to IR and other cellular functions. The dysregulation of free fatty acids leads to neurodegenerative disease by promoting the phosphorylation of tau in the hippocampus (44). Insulin inhibits the activity of glycogen synthesis kinase-3β (GSK- 3β), which phosphorylates tau, pyruvate dehydrogenase, and glycogen synthase. GSK-3β activation under insulin deficiency or IR promotes the phosphorylation of tau and the inactivation of pyruvate dehydrogenase and glycogen, affecting energy metabolism and acetylcholine synthesis. Thus, the interplay between IR and oxidative stress factors can synergistically impair cognitive functions in the SZ patients (45). This study has several advantages. Our results were based on a relatively large number of drug-naïve SZ patients. The status of being drug-naïve and first episodes are important in removing the impact of medications on the associations between IR and oxidative stress and cognitive impairment in SZ TABLE 3 | Insulin resistance, oxidative stress, and cognitive function. Indicators HOMA-IR GSSG NO SOD UA r P r P r P r P r P MCCB composite −0.345 0.001 −0.369 0.003 −0.444 0.000 0.200 0.143 −0.113 0.350 score (Patient group) MCCB composite score 0.020 0.891 −0.153 0.230 0.001 0.997 0.002 0.992 0.118 0.412 (Healthy control group) TABLE 4 | Correlation of FINS, HOMA-IR, GSSG, NO, SOD, and UA values with PANSS in patient group (N = 90). Indicators FINS HOMA-IR GSSG NO SOD UA r P r P r P r P r P r P PANSS-positive 0.041 0.763 0.146 0.283 0.119 0.401 0.159 0.260 −0.081 0.591 −0.191 0.176 PANSS-negative 0.012 0.930 0.020 0.886 −0.082 0.562 0.213 0.130 −0.125 0.409 0.012 0.931 PANSS-general 0.078 0.568 0.013 0.924 0.011 0.936 0.323 0.020 0.024 0.874 −0.142 0.317 PANSS-total 0.065 0.636 0.059 0.663 0.016 0.908 0.375 0.006 −0.083 0.585 −0.138 0.331 E 3 | Insulin resistance, oxidative stress, and cognitive function. that the PKC-D subtype is a potential activator of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, and the activation of NADPH oxidase can increase the production of reactive oxygen species(ROS) (42). Normally, insulin receptor substrates (IRS-1 and IRS-2) are mainly distributed in low density microsomes (LDMs). However, but upon activating these receptors, phosphatidylinositol 3-kinase (P13K) is recruited to LDMs (43). Oxidative stress can interfere with P13K migration from the cytoplasm to LDMs, thereby inducing IR. Frontiers in Psychiatry | www.frontiersin.org RESULTS (4) Long-term IR can increase the content of highly active oxidative groups and decrease the activity of antioxidant enzymes, causing neuronal cell apoptosis (32). (1) IR may be accompanied by insulin-like growth factor-1 (IGF- 1) resistance, which competitively inhibits the binding of insulin to the insulin receptor (30). Other studies have reported elevated plasma FINS levels and decreased IGF-1 in SZ patients (31). (2) High FINS levels can disturb the insulin signaling pathway and decrease metalloproteinase (IDE) levels, leading to intracellular and extracellular Amyloid- β deposition, and causing neuronal degeneration. (3) IR can promote neuronal inflammation by enhancing the release of pro-inflammatory cytokines such as In the present study, we found that the levels of oxidized products (GSSG, and NO) were significantly increased in SZ patients, while SOD levels were significantly decreased, and that these levels were correlated to some cognitive function domains. Numerous studies have revealed an imbalance between the oxidative and antioxidant systems in SZ patients (33). Evidenced November 2020 | Volume 11 | Article 537280 Frontiers in Psychiatry | www.frontiersin.org 4 Schizophrenia, Insulin Resistance, Oxidative Stress Tao et al. Tao et al. Schizophrenia, Insulin Resistance, Oxidative Stress TABLE 3 | Insulin resistance, oxidative stress, and cognitive function. Indicators HOMA-IR GSSG NO SOD UA r P r P r P r P r P MCCB composite −0.345 0.001 −0.369 0.003 −0.444 0.000 0.200 0.143 −0.113 0.350 score (Patient group) MCCB composite score 0.020 0.891 −0.153 0.230 0.001 0.997 0.002 0.992 0.118 0.412 (Healthy control group) TABLE 4 | Correlation of FINS, HOMA-IR, GSSG, NO, SOD, and UA values with PANSS in patient group (N = 90). Indicators FINS HOMA-IR GSSG NO SOD UA r P r P r P r P r P r P PANSS-positive 0.041 0.763 0.146 0.283 0.119 0.401 0.159 0.260 −0.081 0.591 −0.191 0.176 PANSS-negative 0.012 0.930 0.020 0.886 −0.082 0.562 0.213 0.130 −0.125 0.409 0.012 0.931 PANSS-general 0.078 0.568 0.013 0.924 0.011 0.936 0.323 0.020 0.024 0.874 −0.142 0.317 PANSS-total 0.065 0.636 0.059 0.663 0.016 0.908 0.375 0.006 −0.083 0.585 −0.138 0.331 by increased GSSG and NO levels accompanied by decreased SOD levels (34), and such an imbalance may correlate with impaired cognitive functions (12). RESULTS NO, as a signaling molecule, may be involved in the impairment of cognitive function in SZ patients through the following mechanisms: (1) activation of the hypothalamus- pituitary-adrenal axis, secretion of prolactin, and corticosteroids, and elevation of hormones levels and thus activating the dopaminergic neurons in the hypothalamus, causing positive symptoms and cognitive impairment (35); (2) NMDA receptor activation, which is involved in the release of Glu and dopamine, and causes cognitive deficits and mental disorders (36); and (3) reduction in nitrite ions and superoxide anions, which react to the formation of peroxynitrite anions, leading to neurotoxic effects. Previous studies have suggested that the antioxidant defense system is disrupted in SZ patients, and excessive free radical production and oxidative stress damage response may be involved in the pathogenesis of SZ (22, 23). SOD and UA are both effective indicators of antioxidant capacity, and SOD as an enzyme can effectively scavenge oxygen free radicals in the body, representing an important component of the free radical defense system. Our results showed that SOD levels were significantly decreased in SZ patients compared with healthy control subjects, which was consistent with the study by Reyazuddin (37), indicating that there may be a high level of oxidative stress in SZ patients. Other studies noted that the presence of the oxidative stress indicators SOD and NO in first-episode SZ patients is associated with cognitive impairment, consistent with the results of the present study. Our study did not find a correlation between UA levels and cognitive function impairment in SZ patients. whether SZ patients have a lower level of UA remains controversial (38–40). Hyperglycemia which mediates oxidative stress by directly that the PKC-D subtype is a potential activator of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, and the activation of NADPH oxidase can increase the production of reactive oxygen species(ROS) (42). Normally, insulin receptor substrates (IRS-1 and IRS-2) are mainly distributed in low density microsomes (LDMs). However, but upon activating these receptors, phosphatidylinositol 3-kinase (P13K) is recruited to LDMs (43). Oxidative stress can interfere with P13K migration from the cytoplasm to LDMs, thereby inducing IR. Thus, oxidative stress reduces the recruitment of P13K by interfering with the phosphorylation of insulin receptor substrates, thereby inducing IR. On the other hand, High levels of free fatty acids lead to an increase in ROS and reactive nitrogen species (RNS), thereby activating the oxidative stress mechanisms that damage DNA, proteins and lipids. REFERENCES 11. Bitanihirwe BK, Woo TU. 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ETHICS STATEMENT The studies involving human participants were reviewed and approved by Ethics Committee of the First Affiliated Hospital of Zhengzhou University. The patients/participants provided their RESULTS Other studies noted that the presence of the oxidative stress indicators SOD and NO in first-episode SZ patients is associated with cognitive impairment, consistent with the results of the present study. Our study did not find a correlation between UA levels and cognitive function impairment in SZ patients. whether SZ patients have a lower level of UA remains controversial (38–40). This study has several advantages. Our results were based on a relatively large number of drug-naïve SZ patients. The status of being drug-naïve and first episodes are important in removing the impact of medications on the associations between IR and oxidative stress and cognitive impairment in SZ patients. However, the present study also has limitations. Our results were based on analysis of cross-sectional samples. Future Hyperglycemia, which mediates oxidative stress by directly or indirectly activating the diacylglycerol (DAG) -PKC pathway, is the main source of oxidative stress (41). Studies have shown November 2020 | Volume 11 | Article 537280 5 Schizophrenia, Insulin Resistance, Oxidative Stress Tao et al. follow-up studies that have measurements at multiple time points may strengthen our conclusions. In addition, unmeasured confounders in the present study may have some impact on our results thus future replication studies are warranted. written informed consent to participate in this study. Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article. In summary, in the present work, we report abnormal IR and oxidative stress factors in first-episode untreated SZ patients, and show how they are correlated to on cognitive function. There was an association between IR and oxidative stress in the patients. The current findings need to be replicate, but suggest that IR may be a peripheral biological marker of cognitive dysfunction development in SZ patients, and could play a role in the pathological disease processes. AUTHOR CONTRIBUTIONS XS and YY contributed the conception and the design of the study. YM and HL were in charge of conducting clinical assessment and collecting fasting blood samples. QT, YM, and XY undertook the statistical analysis. QT, YM, and YY wrote the draft of the paper. YM revised the paper. XH, YW, OA, and XF were responsible for supervision and reviewing. All authors approved the submitted version. FUNDING The datasets generated for this study will not be made publicly available this data is confidential. Funding for this study was provided by grants from the National Natural Science Foundation of China (Nos. 81971253 and 81571318 to XS), Medical science and technology foundation of health and family planning commission of Henan Province (SBGJ2018028 to XS), School and Hospital Co-incubation Funds of Zhengzhou University (No. 2017-BSTDJJ-04 to XS), and Henan Clinical Research Center for Mental Disorders (2019-zxkfkt-004). REFERENCES Insulin and glucagon signaling in the central nervous system. Rev Endocr Metab Disord. (2013) 14:365– 75. doi: 10.1007/s11154-013-9258-4 19. Houlihan LM, Wyatt ND, Harris SE, Hayward C, Gow AJ, Marioni RE, et al. Variation in the uric acid transporter gene (SLC2A9) and memory performance. Hum Mol Genet. (2010) 19:2321–30. doi: 10.1093/hmg/ddq097 8. Zhao WQ, Chen H, Quon MJ, Alkon DL. Insulin and the insulin receptor in experimental models of learning and memory. Eur J Pharmacol. (2004) 490:71–81. doi: 10.1016/j.ejphar.2004.02.045 20. Hirsch GE, Heck TG. 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Insulin-like growth factor I and anthropometric parameters in a Danish population. Exp Clin Endocrinol Diabetes. (2012) 120:171– 4. doi: 10.1055/s-0031-1301289 44. Schubert M, Brazil DP, Burks DJ, Kushner JA, Ye J, Flint CL, et al. Insulin receptor substrate-2 deficiency impairs brain growth and promotes tau phosphorylation. J Neurosci. (2003) 23:7084–92. doi: 10.1523/JNEUROSCI.23-18-07084.2003 45. Gasparini L, Netzer WJ, Greengard P, Xu HX. Does insulin dysfunction play a role in Alzheimer’s disease? Frontiers in Psychiatry | www.frontiersin.org REFERENCES Trends Pharmacol Sci. (2002) 23:288– 93. doi: 10.1016/S0165-6147(02)02037-0 31. Venkatasubramanian G, Chittiprol S, Neelakantachar N, Naveen MN, Thirthall J, Gangadhar BN, et al. Insulin and insulin-like growth factor-1 abnormalities in anti psychotic-naive schizophrenia. Am J Psychiat. (2007) 164:1557–60. doi: 10.1176/appi.ajp.2007.070 20233 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. 32. Mota M, Banini BA, Cazanave SC, Sanyal AJ. Molecular mechanisms of lipotoxicity and glucotoxicity in nonalcoholic fatty liver disease. Metabolism. (2016) 65:1049–61. doi: 10.1016/j.metabol.2016.02.014 33. Gu F, Chauhan V, Chauhan A. Glutathione redox imbalance in brain disorders. Curr Opin Clin Nutr Metab Care. (2015) 18:89–95. doi: 10.1097/MCO.0000000000000134 Copyright © 2020 Tao, Miao, Li, Yuan, Huang, Wang, Andreassen, Fan, Yang and Song. 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. 34. Ballesteros A, Jiang P, Summerfelt A, Du XM, Chiappelli J, O’Donnell P, et al. No evidence of exogenous origin for the abnormal glutathione redox state in schizophrenia. Schizophr Res. (2013) 146:184–9. doi: 10.1016/j.schres.2013.02.001 Frontiers in Psychiatry | www.frontiersin.org November 2020 | Volume 11 | Article 537280

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Axe: A Novel Approach for Generic, Flexible, and Comprehensive Monitoring and Adaptation of Cross-Cloud Applications

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Axe: A Novel Approach for Generic, Flexible, and Comprehensive Monitoring and Adaptation of Cross-Cloud Applications J¨org Domaschka, Daniel Seybold, Frank Griesinger, and Daniel Baur University of Ulm, Institute of Information Resource Management, Albert-Einstein-Allee 43, 89081 Ulm, Germany {joerg.domaschka,daniel.seybold, frank.griesinger,daniel.baur}@uni-ulm.de http://www.uni-ulm.de/in/omi University of Ulm, Institute of Information Resource Management, Albert-Einstein-Allee 43, 89081 Ulm, Germany {joerg.domaschka,daniel.seybold, frank.griesinger,daniel.baur}@uni-ulm.de http://www.uni-ulm.de/in/omi Abstract. The vendor lock-in has been a major problem since cloud computing has evolved as on the one hand side hinders a quick transi- tion between cloud providers and at the other hand side hinders an ap- plication deployment over various clouds at the same time (cross-cloud deployment). While the rise of cross-cloud deployment tools has to some extend limited the impact of vendor lock-in and given more freedom to operators, the fact that applications now are spread out over more than one cloud platform tremendously complicates matters: Either the operator has to interact with the interfaces of various cloud providers or he has to apply custom management tools. This is particularly true when it comes to the task of auto-scaling an application and adapting it to load changes. This paper introduces a novel approach to monitoring and adaptation management that is able to flexibly gather various mon- itoring data from virtual machines distributed across cloud providers, to dynamically aggregate the data in the cheapest possible manner, and finally, to evaluate the processed data in order to adapt the application according to user-defined rules. 1 Introduction Since the beginning of cloud computing, vendor lock-in has been a major prob- lem. It is still around mainly due to the fact that cloud standards such as CIMI [6] and OCCI [16] have not been widely adopted by cloud providers. Tools abstracting the differences between cloud providers, and thus allowing multi- cloud deployment—the capability to deploy one application at different cloud platforms using the same application specification—have been a first step to overcome vendor lock-in. Yet, it is only cross-cloud deployment—the capability to spread a single application instance across different cloud providers—that en- ables users to take full advantage of different providers and their capabilities. In particular, it enables trading offthe properties of application requirements against the offerings on a per-component or even per-component instance basis. This for instance allows a hybrid-cloud deployment where a database containing J¨org Domaschka, Daniel Seybold, Frank Griesinger, and Daniel Baur 2 sensitive data is deployed in a private cloud, while the rest of the application resides in different public clouds. Both approaches, multi-cloud and cross-cloud, give the application opera- tor the chance to change its current application deployment and to adapt to changed conditions such as the workload, e.g., more load than originally antici- pated, and changed environmental conditions, e.g., the prices of other operators have changed. In order to benefit from these features, however, the application operators need to be able to actively judge the quality of the current deploy- ment. For pure multi-cloud systems the application operator may refer to the monitoring tools of the currently selected cloud operator. While basic monitoring data may come for free on some cloud providers, often the user needs advanced metrics that either cost (Amazon, Rackspace) or require him to set up own mon- itoring tools. In addition to that, he has to familiarise with the user interfaces of various cloud providers. For cross-cloud deployment using the providers’ monitoring infrastructure is technically feasible, but tremendously increases the effort as multiple tools have to be used in parallel. Moreover, it is difficult to access metrics that involve the crossing of provider domains (such as network traffic from provider A to provider B). Furthermore is hard to access application-specific or component- specific metrics. Also, a sophisticated and configurable aggregation on the met- rics is currently not easily possible. Finally, while most cloud providers support a simple approach to auto-scaling for application adaptation, e.g. 1 Introduction metrics-based scale-out, there is currently no built-in mechanism that supports a cross-cloud adaptation of applications. In this paper, we address these issues by introducing Axe, a generic, flexible, and extensible monitoring and adaptation engine for cross-cloud deployments. Besides the fact, that we introduce the tool, our contributions are as follows: (i) We present a powerful API that enables the specification of rules independent of the concrete deployment. (ii) We discuss a heuristic of how to reduce the cross- cloud provider network traffic and hence reduce costs. (iii) We introduce the first engine to deal with the Scalability Rule Language (SRL)[8, 12]. All of the features are embedded in Cloudiator, our cross-cloud, multi-tenancy deployment and application management tool [2, 7]. This document is structured as follows: Section 2 introduces background on Cloudiator and Scalability Rule Language (SRL) and further defines require- ments towards our approach. Section 3 introduces our approach by presenting the individual tools of our platform and their configuration. It also discusses ar- chitectural options and introduces our architecture as well as the API. Section 4 exhibits the current status and upcoming tasks. Section 5 discusses related work, before we conclude with a report on our current status and open issues. 2.1 Requirements and Constraints For supporting in-depth analyses of existing deployments, several requirements have to be considered: (a) The fact that on the one hand, the monitoring of large-scale applications does generate huge amounts of data and on the other hand cloud providers usually charge for network traffic that leaves their data centre gives motivation that as much of data processing shall happen within the domain of individual cloud providers. (b) In order to avoid single points of fail- ures, the architecture of a monitoring solution should not rely on a centralised approach, but rather favour distributed approaches with no central entity. As the amount of monitoring data usually increases with the number of allocated virtual machines (VMs), the resources assigned to monitoring shall increase with the size of the application. (c) The operators of a cloud application may discover that they have to monitor further high-level or even low-level metrics or need monitoring to happen at a higher resolution. Hence, it is necessary that monitor- ing properties can be changed also after an application has been deployed. (d) The same considerations that hold for monitoring, have to hold for scaling rules. In addition, it is necessary that rules can be defined in a generic way without having to know the exact number of instances per component in advance. (e) The monitoring platform has to be able to capture application-specific metrics. 2 Background The design of Axe has been heavily driven by constraints of cross-cloud environ- ments. In addition to that, Axe builds heavily on earlier work. In the following, 3 3 Axe we first introduce the constraints and derive requirements from them. In the next step, we present our Cloudiator tool that we use as the basis for the Axe im- plementation [7]. Finally, we roughly describe the Scalability Rule Language that constitutes the meta-model for our monitoring and scaling solution. 1 https://github.com/cloudiator 2.2 The Cloudiator Tool Cloudiator1 is a cross-cloud deployment tool that also supports adaptation and re-deployment. In this section, we present the Cloudiator architecture to the extend necessary to understand how it embeds Axe. Figure 1 summarises the architecture as of the original Cloudiator tool (green), but also the en- hancements of Axe (yellow). The Axe specific components Aggregation, Scaling Engine, and Visor are introduced in detail in Section 3. The figure shows that Cloudiator consists of a home domain for which Colosseum is the entry point offering a JSON-based REST interface. This is used by a graphical Web-based user interface, but can also be used by adapters and automatisation tools. It also comprises various registries that store the Cloudiator users, information about cloud providers, the cloud accounts of the users, and meta-information about cloud offerings such as the operating sys- tems of images. Moreover, the home domain contains a repository of application components together with their life-cycle handlers as well as applications com- posed of these components. In addition, the registries contain information about started VMs and the component instances deployed on them as well as about the J¨org Domaschka, Daniel Seybold, Frank Griesinger, and Daniel Baur 4 Remote Domain Home Domain Colosseum Scaling Engine VM Lifecycle-Agent Visor REST Docker Container Registries Sword Workers Application Repo. Aggregator EC2 Openstack VM VM VM TSDB Aggr. UI Fig. 1. The Cloudiator architecture Fig. 1. The Cloudiator architecture wiring between the component instances. Finally, they hold the workers syncing the registries with the cloud provider information, and executing the provision- ing of virtual machines or the installation of application components on virtual machines. The Sword abstraction layer realises the communication with the various cloud provider APIs based on Apache jclouds2. The remote domain comprises all VMs at various cloud providers as well as the component instances running on them. In addition to that it contains Cloudiator’s life cycle agent on each of the VMs that the home domain uses in order to distribute component instances over VMs and to poll the status of the component instances when it needs to be shown in the user interface. 2.3 Scalability Rule Language The SRL [8] is a provider-agnostic description language. It provides expressions to define the monitoring raw metric values from VMs and component instances and also mechanisms to compose higher-level metrics from raw metrics. More- over, it comprises mechanisms to express events and event patterns on metrics and metric values. Finally, SRL captures thresholds on the events and actions to be executed when thresholds are violated. A simple SRL rule in prose may be add a new instance of this distributed database if (i) all instances have a 5 minute average CPU load > 60%, (ii) at least one instance has a 1 minute average CPU load > 85%, and (iii) the total number of instances is < 6. 2 https://jclouds.apache.org/ 3 Approach This section sketches our approach in order to realise a flexible monitoring and adaptation tool that satisfies the requirements imposed on cross-cloud tool- ing (cf. Section 2). Basically, our auto-scaling process maps to the MAPE loop [15, 11] consisting of the following phases: monitoring, analysis, planning, and exe- cution of changes. With respect to our setting, this means that first, we have to 5 Axe retrieve monitoring data from the virtual machines and component instances. In a second step, the raw data gathered there has to be aggregated and processed. Third, the rule processing has to happen on the aggregated data and finally, the resulted rule has to be executed. 3.1 Visor: Gathering Monitoring Data In order to be able to gather the raw monitoring data from the VMs and compo- nent instances, we introduce Visor as a monitoring agent to the remote domain. Just as the life-cycle agent, Visor is deployed on every VM and provides a re- mote interface the home domain uses in order to configure a particular Visor instance. This allows Visor to adopt to the application and to only collect the required metrics, thus saving space and bandwidth. Visor supports the captur- ing of data on a per component instance basis as well as on a per-VM basis. The sooner is achieved by sensors monitoring basic system properties on virtual ma- chine level, e.g. by accessing system properties such as CPU load. The latter is done by exploiting the fact that all component instances are run inside a Docker3 container and the resource consumption can be retrieved on a per-container ba- sis. By default, Visor offers various sensors supporting system metrics such as CPU load, memory consumption, disk I/O, and network I/O. In order to support custom metrics, Visor supports the implementation of custom sensors, by providing an easy-to-implement Java interface. It exploits the dynamic class loading properties of Java in order to be able to add those implementations at runtime. For supporting application-specific metrics that can only be retrieved from within an application such as the length of queues or the degree to which buffers have been filled, Visor offers a telnet-based interface where applications can push their metrics data to. This interface is compatible with the carbon daemon of graphite4, thus allowing an easy migration to Visor. 4 http://graphite.readthedocs.org/en/latest/carbon-daemons.html 3 http://www.docker.io 4 3.2 Buffering Monitoring Data A key element when computing higher-level metrics especially over larger time- windows is the need to buffer raw monitoring data. Time-series databases (TS- DBs) have been designed to store timestamped data in an efficient way and also to provide quick access to the stored data. Many TSDB implementations sup- port applying functions on stored data right out of the box what makes them a perfect match not only for buffering, but also for aggregation (cf. Section 3.3). The following paragraphs first derive a strategy on how to implement buffering including the constraints and then compares TSDBs found in literature and the open source community with respect to the required properties. 6 J¨org Domaschka, Daniel Seybold, Frank Griesinger, and Daniel Baur Strategy With respect to our requirements (cf. Section 2) the buffering and therefore the TSDB approach needs to be able to work with limited resources, have no single point of failure, and increase available resources when more VMs are being used. In order to cope with these requirements, we use the following approach: from each VM acquired for an application, we reserve a configurable amount of memory and storage (e.g. 10%) that we further split between a local storage area and a shared storage area. Both storage areas are managed by a TSDB instance running on the VM. The Visor instance running on this VM will then feed all monitoring data to the TSDB. The TSDB will store data from its local Visor in the local storage area and further relay the data to other TSDBs where it is stored in the shared storage area. This feature avoids that a TSDB becomes a single point of failure, but still enables quick access to local data. In order to keep network traffic between cloud providers low, any TSDB will only select other TSDBs running in the same cloud to replicate its data. If not enough instances are available to reach the desired replication degree, the maximum possible degree is used. Hence, this concludes to a ring-like topology that has been introduced in peer-to-peer systems [3] and is also used by distributed databases [13]. Table 1. Details of considered times series databases Name KairosDB OpenTSDB InfluxDB Version 1.0.0 2.1.0 0.9.0 Datastore H2/Cassandra HBase BoltDB Distributed no/yes yes yes InMemory yes/no no yes Table 1. 5 https://influxdb.com/docs/v0.9/introduction/overview.html 6 http://www.datastax.com/ 3.2 Buffering Monitoring Data Details of considered times series databases Selection of TSDB Table 1 shows a comparison of established TSDB imple- mentations [10] and several of their properties. The results are intermediate as our evaluation is this ongoing (cf. Section 4.1). Selection of TSDB Table 1 shows a comparison of established TSDB imple- mentations [10] and several of their properties. The results are intermediate as our evaluation is this ongoing (cf. Section 4.1). The for us relevant details of the TSDBs are its maturity, available datastores, support of distribution and in memory storage. The TSDB should be in some mature state in order to provide a stable version, client libraries and an available documentation. Following the strategy exposed in Section 3.2 the datastores shall be lightweight and ideally support an in memory mode. Also they have to offer a distributed architecture to ensure horizontal scaling and replication. OpenTSDB offers the best maturity regarding the version number. The un- derlying datastore HBase supports distribution but regarding the architecture of HBase [9] an in-memory mode is missing. Also, it is not a lightweight data- store [10] and an automated set-up as required in our scenario is not a trivial task and hard to script. Consequently, OpenTSDB is not an applicable solution. From its capabilities InfluxDB seems suited for the outlined approach. Yet, the recently released version 0.9.0 comes with extensive changes in the stor- 7 Axe age architecture and API design compared to 0.8.05. Given these changes there currently are no client implementations for version 0.9.0 available. KairosDB also provides a mature version 1.0.0. It supports the single-site, in-memory datastore H2 and the distributed Cassandra datastore supporting scalable to a hundreds of instances [13]. While Cassandra’s resource usage can be limited, in-memory storage is only supported in the commercial version6. Following this comparison KairosDB is currently the most appropriate TSDB to use in Axe based on maturity, distribution and the possibility to limit the resource consumption of Cassandra. 3.3 Aggregation In order to make use of the time series produced by the various raw metrics, these have to be aggregated. Aggregation includes for instance the computation of average values, of maxima, minima, or simply the normalisation of values. In addition to that, aggregation may include merging of metrics, e.g. when comput- ing the average of averages. Hence, aggregation is always application-specific. Strategy The strategy followed by Axe is based on the metric and metric aggregation concepts provided by SRL (cf. Section 2). In particular, it supports the hierarchical aggregation of metrics with an unlimited depth. In addition, it supports the use of time-bound or element-bound windows specifying the interval of a time series to be used for computations. Finally, the user may specify a schedule for each metric that defines how often a value of a metric shall be computed. In order to satisfy the requirement for minimum network traffic and scale of the monitoring system, Axe performs aggregation as close to the data source as possible. Hence, all aggregations that require input data from a single VM will be performed on this VM. We refer to this computation to happen in the host scope. For this approach only the local storage is accessed and no communication is required which further reduces latency. Aggregations that need input only from VMs from a particular cloud are performed in cloud scope. Such computations exclusively access the shared space spanning a cloud. While it is desirable to dis- tribute all computations of a particular cloud scope amongst the affected VMs the definition of a suitable heuristic is currently work in progress. Finally, com- putations that require input from multiple clouds happen in cross-cloud scope (or global scope). These are performed in the home domain of Cloudiator. It is important to note that values for higher-level aggregated, metrics have to be buffered just as the values of any other metric as well. Here, we use the following strategy to write to our storage platform: Values from local scope computations are treated just like values from raw metrics. Values from compu- tations in cloud scope are written to the shared store of their cloud. The results J¨org Domaschka, Daniel Seybold, Frank Griesinger, and Daniel Baur 8 from cross-cloud scope computations are stored in a possibly distributed TSDB operated at the home domain. 3.3 Aggregation Using this set of hierarchical scopes, we expect to have effectively minimised latency and network traffic while at the same time having equally loaded all VMs with monitoring tasks and hence also equally spread the risk of failures. The deployment of the aggregation tasks onto the Aggregators residing in the system, and hence the decision which scope to use for it, is handled by the Scaling Engine component. Engine component. API The API provided by Colosseum in order to configure the monitoring and aggregation functionality of Axe as described above mainly supports the power of SRL. Yet, in order to ease the specification of sensors and aggregation functions independent from the number of deployed virtual machines and the cloud they are currently deployed on, we offer a richer interface. Monitor doMonitorVms(AppInst app, Component comp, SensorDescription sens); 3.5 Architecture Above descriptions and discussions lead to the architecture from Figure 1 and whose main components are (i) the Scaling Engine, (ii) the Aggregator, and ( iii) Visor. The latter has already been introduced in earlier work [2]. ( ) y [ ] The Scaling Engine is the central managing environment of Axe that con- trols the distribution and outsourcing of the computation-heavy work to highly scalable and loosely coupled components, the Aggregators. Nevertheless, it is possible to scale the Scaling Engine up to having one instance per scaling rule. The aggregations are managed and executed by the Aggregators in the sys- tem. Due to the design of the system, this can be done in parallel. Also, for their implementation, the focus has been set to minimise latency. The Scaling Engine is the central managing environment of Axe that con- trols the distribution and outsourcing of the computation-heavy work to highly scalable and loosely coupled components, the Aggregators. Nevertheless, it is possible to scale the Scaling Engine up to having one instance per scaling rule. p g g p g p g The aggregations are managed and executed by the Aggregators in the sys- tem. Due to the design of the system, this can be done in parallel. Also, for their implementation, the focus has been set to minimise latency. Fig. 2. API example. This method will trigger the monitoring of all VMs of this ap- plication instance where component comp has been installed using sensor sens. The methods (cf. Figure 2 for an example) for defining raw metrics consist of filters (e.g. by the component type) specifying all instances to be monitored, and a sensor description defining what to monitor. The sensor description consists of scheduling information and information which sensor type to be deployed on Visor. The return value of such an invocation can be used in further methods to define higher-level metrics (cf. Figure 3). Here, a map functionality is used to specify the high level metric: That is, for each ingoing (raw) metric a new metric is created (e.g. average CPU usage in the last 5 minutes). The API also supports reduce-like semantics where a single metric is generated from all input metrics (e.g. average of above averages). Monitor mapAggregatedMonitors(FormulaQuantifier quantifier, Schedule schedule, Window window, FormulaOperator op, List<Monitor> monitors); Fig. 3. API example. This method will install an aggregation triggered according to a schedule, based on an operator, and using a window of elements operating. 9 Axe 3.4 Auto-Scaling In general, auto-scalers can be categorised in five different classes [14]. For Axe we adopt SRL which mainly belongs to the threshold-based rules as well as time series analysis class. SRL links a set of threshold-based conditions with each other using binary operators. In addition, any set of thresholds may be linked to the values produced by the metrics. Furthermore, any of such constructs has attached a set of scaling actions to be executed whenever the condition has been satisfied. So far, Axe supports to trigger the scale out and scale in of components. Yet, the implementation of further actions is underway. The triggering of rules leads to an invocation of the Cloudiator functionality to bring up a new or shut down an existing VM. Strategy The auto-scaling functionality of Axe builds on top of the monitoring capabilities. In particular any of the conditions connected via Boolean operators is considered to be a metric on its own taking the values 0 or 1. When the metric turns to 1 the respective action will be triggered and forwarded as request to the other Cloudiator tools, in particular Colosseum. These tasks are executed by the Scaling Engine component. API The scaling API provides the capability to attach an action to a monitor. The action itself is described in terms of the component to deal with, the scaling type, and its parameters. For instance for horizontal scaling, the parameters are the amount of instances to add/remove, and the allowed maximum and minimum number of instances of that component. 4.3 Scaling Engine So far Axe supports horizontal scaling actions. Vertical scaling is currently being implemented. Furthermore, we work reducing the burden for the user when im- plementing scaling rules. Therefore, we plan to encapsulate SRL’s complexity in a simpler language possible inspired from complex-event-processing languages [17]. While SRL and with it Axe adopts concepts from auto-scaling concepts based on threshold-based and time series analysis, other concepts exist that include queuing theory, control theory, and reinforcement learning [14]. Accordingly, Axe borrows all its strengths from SRL, but also the weaknesses and could profit from the integration of other techniques. For instance, reinforcement learning might be handled in external processing tools, that constantly adjust the scaling rules. Future work includes the evaluation of such approaches. 4 Current Status and Future Work The following presents the current status and gives an outlook on our planned work. We distinguish these aspects for data collection in a TSDB, data aggrega- tion, and scaling. J¨org Domaschka, Daniel Seybold, Frank Griesinger, and Daniel Baur J¨org Domaschka, Daniel Seybold, Frank Griesinger, and Daniel Baur 10 4.2 Aggregators Currently, the aggregation functionality is implemented for KairosDB and sup- ports aggregation from and to arbitrary KairosDB instances. We plan to extend these capabilities to fit all predefined operators of SRL. We currently implement aggregators for other databases as well to support the TSDB evaluation. 4.1 Time-series Database The current version of Axe uses KairosDB with the Cassandra as a datastore. Cassandra is configured to use only a low portion of a VM’s resources to keep the impact on the components running on that VM small. Upcoming work comprises a performance-oriented evaluation of InfluxDB and other NoSQL databases fo- cusing on their capabilities for managing time-series data. Further, the Zipkin framework7 will be evaluated on its suitability for cross-cloud applications. 7 urlhttps://github.com/openzipkin/zipkin 8 7 urlhttps://github.com/openzipkin/zipkin 8 http://aws.amazon.com/en/cloudwatch/ 9 http://ganglia.sourceforge.net/ 10 https://www.nagios.org/ 8 http://aws.amazon.com/en/cloudwatch/ 9 9 http://ganglia.sourceforge.net/ 10 https://www.nagios.org/ 5 Related Work We compare related work with respect to monitoring and auto-scaling. Cloud monitoring Lifting monitoring to the cloud comes along with vari- ous requirements compared to traditional server monitoring [1]. Tools provided by cloud providers, such as Amazon’s CloudWatch8 suffer from vendor lock- in. Also, additional tools are required when data from different cloud providers shall be aggregated. Established open source monitoring tools such as Ganglia9 or Nagios10 are designed to monitor large distributed systems, but struggle with 10 https://www.nagios.org/ 11 Axe the dynamic of cloud environments. More cloud-aware monitoring systems such as Zipkin—which is based on Dapper [18]—can cope with the dynamic cloud environment and offer a rich functionality. Yet, in order to scale the monitor- ing system manual actions or additional tools are necessary. Compared to Axe none of the mentioned tools supports a reduction of communication overhead for cross-cloud applications. Auto-scaling techniques In contrast to similar scaling engines [4], Axe is not tied to a specific language, but targets to be open for various approaches. Cloud orchestration tools such as Apache Brooklyn11, the rules are simple threshold-based and any more complex rules have to be defined in an exter- nal monitoring tool. Axe in Cloudiator goes beyond this, as it provides an integrated and easy-to-use solution that even allows changes of the scalability configuration at runtime. Several projects deal with integrated auto-scaling mechanisms for cloud ser- vices. One of them is the EU project CELAR[5]. Auto-scaling in CELAR is based on a multi-level description of combined metrics. By that metrics are as- signed to a certain level and when violations occur, the scaling is based on the top level of the topology. While Axe also supports a multi-level description of metrics, it goes beyond the CELAR approach due to the fact that it realises metric aggregation and analysis in a distributed and hierarchical manner. 11 http://brooklyn.incubator.apache.org/ 12 https://github.com/cloudiator 11 http://brooklyn.incubator.apache.org/ p y p 12 https://github.com/cloudiator References 1. Aceto, G., Botta, A., De Donato, W., Pescap`e, A.: Cloud monitoring: A survey. Computer Networks 57(9), 2093–2115 (2013) ( ) ( ) 2. Baur, D., Wesner, S., Domaschka, J.: Towards a Model-based Execution Ware for Deploying Multi-Cloud Applications. In: Proceedings of the 2nd International Workshop on Cloud Service Brokerage September 2014 (2014) 3. Clarke, I., Sandberg, O., Wiley, B., Hong, T.: Freenet: A distributed anonymous information storage and retrieval system. In: Federrath, H. (ed.) Designing Privacy Enhancing Technologies, Lecture Notes in Computer Science, vol. 2009, pp. 46–66. Springer Berlin Heidelberg (2001) ( ) 4. Copil, G., Moldovan, D., Truong, H.L., Dustdar, S.: Sybl: An extensible language for controlling elasticity in cloud applications. In: Cluster, Cloud and Grid Com- puting (CCGrid), 2013 13th International Symposium on. pp. 112–119 (May 2013) 5. Copil, G., Moldovan, D., Truong, H.L., Dustdar, S.: Multi-level elasticity control of cloud services. In: Basu, S., Pautasso, C., Zhang, L., Fu, X. (eds.) Service-Oriented Computing, Lecture Notes in Computer Science, vol. 8274, pp. 429–436. Springer Berlin Heidelberg (2013), http://dx.doi.org/10.1007/978-3-642-45005-1_31 ( ) p g 6. DMTF: Cloud Infrastructure Management Interface (CIMI) Model and RESTful HTTP-based Protocol (2013) ( ) 7. Domaschka, J., Baur, D., Seybold, D., Griesinger, F.: Cloudiator: A Cross-Cloud, Multi-Tenant Deployment and Runtime Engine. In: 9th Symposium and Summer School On Service-Oriented Computing (2015) ( ) 8. Domaschka, J., Kritikos, K., Rossini, A.: Towards a Generic Language for Scala- bility Rules. In: Proceedings of CSB 2014: 2nd International Workshop on Cloud Service Brokerage (2014 (To Appear)) g ( ( )) 9. George, L.: HBase: The Definitive Guide. O’Reilly Media, 1 edn. (2011) 10. Goldschmidt, T., Jansen, A., Koziolek, H., Doppelhamer, J., Breivold, H.P.: Scal- ability and Robustness of Time-Series Databases for Cloud-Native Monitoring of Industrial Processes. In: 2014 IEEE 7th International Conference on Cloud Com- puting, Anchorage, AK, USA, June 27 - July 2, 2014. pp. 602–609 (2014) ( ) 11. Jacob, B., Lanyon-Hogg, R., Nadgir, D., Yassin, A.: A Practical Guide to the IBM Autonomic Computing Toolkit. IBM redbooks, IBM Corporation, International Technical Support Organization (2004) 12. Kritikos, K., Domaschka, J., Rossini, A.: SRL: A Scalability Rule Language for Multi-cloud Environments. In: CloudCom, 2014 IEEE 6th International Conference on. pp. 1–9 (Dec 2014) pp ( ) 13. Lakshman, A., Malik, P.: Cassandra: A Decentralized Structured Storage System. SIGOPS Oper. Syst. Rev. 44(2), 35–40 (Apr 2010) ( ) ( ) 14. 6 Conclusions The integrated scaling solutions of current cloud orchestration tools lack an sup- port for sophisticated implementations of auto-scaling techniques. Only such a solution can achieve highly dynamic applications, with the ability to adjust their configuration at runtime in order to cope with unexpected changes of work- load. In this paper, we introduced Axe, a novel, cloud provider-independent approach of cloud application monitoring and application adaptation manage- ment. Axe supports distributed monitoring of cross-cloud applications and also comes with a distributed, hierarchical aggregation of monitored metrics reduc- ing the network traffic across cloud providers. The adaptation of the Scalability Rules Language (SRL) enables the expression of powerful scaling rules based on hierarchical metrics, complex events and threshold. The platform is scalable in itself and hence also supports large-scale applications. It has been integrated in our Cloudiator deployment tool12. Acknowledgements The research leading to these results has received funding from the European Community’s Seventh Framework Programme (FP7/2007- 2013) under grant agreement number 317715 (PaaSage) and from the European Community’s Framework Programme for Research and Innovation HORIZON 2020 (ICT-07-2014) under grant agreement number 644690 (CloudSocket). J¨org Domaschka, Daniel Seybold, Frank Griesinger, and Daniel Baur 12 References Lorido-Botran, T., Miguel-Alonso, J., Lozano, J.: A review of auto-scaling tech- niques for elastic applications in cloud environments. Journal of Grid Computing 12(4), 559–592 (2014) ( ) ( ) 15. Maurer, M., Breskovic, I., Emeakaroha, V., Brandic, I.: Revealing the mape loop for the autonomic management of cloud infrastructures. In: ISCC 2011. pp. 147– 152 (June 2011) ( ) 16. Open Grid Forum: Open Cloud Computing Interface - Core (2011) 17. Paschke, A., Kozlenkov, A., Boley, H.: A homogeneous reaction rule language for complex event processing. In: 33rd VLDB 2007 (2007) 17. Paschke, A., Kozlenkov, A., Boley, H.: A homogeneous complex event processing. In: 33rd VLDB 2007 (2007) ( ) 18. Sigelman, B.H., Barroso, L.A., Burrows, M., Stephenson, P., Plakal, M., Beaver, D., Jaspan, S., Shanbhag, C.: Dapper, a large-scale distributed systems tracing infrastructure. Tech. rep., Google, Inc. (2010)

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English

Volatile Compound Screening Using HS-SPME-GC/MS on Saccharomyces eubayanus Strains under Low-Temperature Pilsner Wort Fermentation

Microorganisms

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Received: 21 April 2020; Accepted: 15 May 2020; Published: 18 May 2020 Abstract: The recent isolation of the yeast Saccharomyces eubayanus has opened new avenues in the brewing industry. Recent studies characterized the production of volatile compounds in a handful set of isolates, utilizing a limited set of internal standards, representing insufficient evidence into the ability of the species to produce new and diverse aromas in beer. Using Headspace solid-phase microextraction followed by gas chromatography-mass spectrometry (HS-SPME-GC-MS), we characterized for the first time the production of volatile compounds in 10 wild strains under fermentative brewing conditions and compared them to a commercial lager yeast. S. eubayanus produces a higher number of volatile compounds compared to lager yeast, including acetate and ethyl esters, together with higher alcohols and phenols. Many of the compounds identified in S. eubayanus are related to fruit and floral flavors, which were absent in the commercial lager yeast ferment. Interestingly, we found a significant strain × temperature interaction, in terms of the profiles of volatile compounds, where some strains produced significantly greater levels of esters and higher alcohols. In contrast, other isolates preferentially yielded phenols, depending on the fermentation temperature. This work demonstrates the profound fermentation product differences between different S. eubayanus strains, highlighting the enormous potential of this yeast to produce new styles of lager beers. Keywords: yeast; volatile compounds; S. eubayanus; beer; lager Kamila Urbina 1,2,†, Pablo Villarreal 1,2,†, Roberto F. Nespolo 2,3,4, Ricardo Salazar 5, Rocio Santander 6 and Francisco A. Cubillos 1,2,* 1 Departamento de Biología, Facultad de Química y Biología, Universidad de Santiago de Chile, 9160000 Santiago, Chile; [email protected] (K.U.); [email protected] (P.V.) 1 Departamento de Biología, Facultad de Química y Biología, Universidad de Santiago de Chile, 9160000 Santiago, Chile; [email protected] (K.U.); [email protected] (P.V.) 2 Millennium Institute for Integrative Biology (iBio), 7500565 Santiago, Chile; [email protected] 3 Instituto de Ciencias Ambientales y Evolutivas, Universidad Austral de Chile, 5090000 Valdivia, Chile 4 Center of Applied Ecology and Sustainability (CAPES), 8331150 Santiago, Chile 5 Laboratorio de Electroquímica del Medio Ambiente, LEQMA, Departamento de Química de los Materiales, 9160000 Santiago, Chile; [email protected] (K.U.); [email protected] (P.V.) 2 Millennium Institute for Integrative Biology (iBio), 7500565 Santiago, Chile; [email protected] 3 Instituto de Ciencias Ambientales y Evolutivas, Universidad Austral de Chile, 5090000 Valdivia, Chile 4 Center of Applied Ecology and Sustainability (CAPES), 8331150 Santiago, Chile 5 4 Center of Applied Ecology and Sustainability (CAPES), 8331150 Santiago, Chile pp gy y ( ), g , 5 Laboratorio de Electroquímica del Medio Ambiente, LEQMA, Departamento de Química de los Materiales Facultad de Química y Biología, Universidad de Santiago de Chile, 9160000 Santiago, Chile; [email protected] pp gy y ( ) g 5 Laboratorio de Electroquímica del Medio Ambiente, LEQMA, Departamento de Química de los Materiales, Facultad de Química y Biología, Universidad de Santiago de Chile, 9160000 Santiago, Chile; [email protected] 6 Laboratorio de Cinética y Fotoquímica, Departamento de Ciencias del Ambiente, Facultad de Química y Biología, Universidad de Santiago de Chile, 9160000 Santiago, Chile; [email protected] * Correspondence: [email protected] † These authors contributed equally to this work. † These authors contributed equally to this work. microorganisms Microorganisms 2020, 8, 755; doi:10.3390/microorganisms8050755 Article Volatile Compound Screening Using HS-SPME-GC/MS on Saccharomyces eubayanus Strains under Low-Temperature Pilsner Wort Fermentation Kamila Urbina 1,2,†, Pablo Villarreal 1,2,†, Roberto F. Nespolo 2,3,4, Ricardo Salazar 5, Rocio Santander 6 and Francisco A. Cubillos 1,2,* Kamila Urbina 1,2,†, Pablo Villarreal 1,2,†, Roberto F. Nespolo 2,3,4, Ricardo Salazar 5, Rocio Santander 6 and Francisco A. Cubillos 1,2,* www.mdpi.com/journal/microorganisms 1. Introduction Beer is the most popular and widespread alcoholic drink in the world, offering a wide variety of styles and flavors around the globe [1]. The production of this beverage is determined by well-defined processes, such as malting, grinding and macerating the grains, cooking the wort, and finally fermenting [2]. The final fermented product contains unique organoleptic properties, as a result of www.mdpi.com/journal/microorganisms Microorganisms 2020, 8, 755; doi:10.3390/microorganisms8050755 www.mdpi.com/journal/microorganisms 2 of 19 Microorganisms 2020, 8, 755 a delicate balance between aromas and flavors provided by the main ingredients used during the process: malt, water, hops, and yeast [3,4]. Industrial beer production is dominated by yeasts belonging to the Saccharomyces genus, mainly Saccharomyces cerevisiae responsible for ale beer fermentation, while S. pastorianus (a hybrid between S. cerevisiae × S. eubayanus) is used for lager beers. Unlike ales, which are fermented by S. cerevisiae in a range of 16–25 ◦C, lagers are fermented by S. pastorianus at lower temperatures (8–15 ◦C), mostly because of the cryotolerant contribution of S. eubayanus to the lager hybrid [5]. Lager beer represents more than 90% of the beer produced worldwide and is the most popular style consumed [6]. During fermentation, yeast cells produce a wide range of secondary metabolites, including volatile compounds (VCs) responsible for the complex flavors and aromas found in beer. VCs are molecules with low molecular weight and rapid dispersion in the environment and determine the different organoleptic qualities of beer [7,8]. Some of the VCs found in beer are synthesized by yeast cells through several metabolic pathways, emphasizing that a significant contribution to the organoleptic properties of beer is provided by the particular strain used for fermentation [2]. Hence, the strains’ genotype, together with environmental factors during fermentation, determines to a large extent the presence of these compounds in the final product [9]. For example, S. pastorianus (employed in the production of Pilsner-style lager) [10,11] produces at low temperatures a subtle organoleptic profile mainly characterized by the presence of ester compounds such as ethyl acetate, ethyl hexanoate, and isoamyl acetate. On the other hand, S. cerevisiae (employed to produce ale craft beer fermented at high temperatures compared to lager) [10,11] produces a wider range of VCs like β-myrcene, ethyl octanoate, β-linalool, ethyl decanoate, octanoic acid, decanoic acid, ethyl acetate, and ethyl propionate, among others, providing a more complex organoleptic profile in beer [11,12]. 1. Introduction The environmental stresses faced during the fermentation process, such as the presence of spoilage microorganisms, the fermentation temperature, nitrogen availability, and carbon sources in the wort, also impact the VC profile of the beer [2,7]. Therefore, depending on these variables, yeast cells produce different concentrations and types of VCs, allowing them to regulate their metabolism in terms of nitrogen uptake, membrane fluidity, and biomass generation [1,6]. The most relevant VCs produced by yeast are esters, phenols, and alcohols derived from the Ehrlich route [2,10]. However, most of the strains used in beer fermentation produce very similar and smooth organoleptic profiles, as found in many lager beers. While ale beers are marked by a broad array of esters and higher alcohols [1], lagers are generally characterized by a limited set of flavors and aromas [13]. For example, two commercial lager strains (Frisinga-TUM 3470 and Securitas-TUM 193) produced fewer esters and higher alcohols compared to a set of ale strains [14]. Interestingly, this set of ale strains showed different VC production profiles, demonstrating a pervasive intra-specific phenotypic variability among ale strains and a considerable impact on the final fermented product [14]. The lack of a wide range of VCs in lager styles is mostly explained by the low genetic diversity among S. pastorianus strains, strongly limiting the variety of lager styles. Consequently, the identification of new strains able to provide unique organoleptic profiles is an interesting approximation to expand the repertoire of lager beers and address the demands for new beer styles with different alcohol content and new sensory profiles [15,16]. A novel wild yeast with the capacity to be used in the brewing industry is S. eubayanus. The isolation of this cryotolerant yeast was initially reported by Libkind et al., 2011 [17] from Nothofagus forests in Argentina, and since then, it has been found in North America [18,19], East Asia [20], New Zealand [21], and, most recently, in Chile [22]. Interestingly, recent reports have shown considerable diversity of S. eubayanus in Patagonia [19,22]. Moreover, Patagonian strains exhibited significantly different fermentative profiles depending on the latitude from where the strains were obtained, in that isolates from northern Patagonian populations showed a better fermentative performance compared to isolates obtained from southern Patagonia and North American populations. However, in general, S. eubayanus attenuation levels were lower compared to the S. pastorianus W34/70 commercial strain, yet some strains still showed commercially accepted attenuation levels [22]. 1. Introduction In this context, S. eubayanus has caught the 3 of 19 Microorganisms 2020, 8, 755 interest of researchers and beer producers given the novel fermentative properties, such as fermentation at low temperatures (12 ◦C), efficient maltose utilization, and production of VCs (e.g., volatile-esters) that are pleasant in the final product [6]. Despite these attractive traits, the phenotypic diversity associated with the production of VCs among S. eubayanus isolates in wort fermentation has not been thoroughly characterized, constraining the potential utilization and knowledge of these yeasts. Furthermore, VC production in the S. eubayanus type strain has only been performed using HS-GC-FID (headspace gas chromatography with flame-ionization detector), where few VCs were quantified using internal standards [6,23]. Alternative approaches, such as HS-SPME-GC-MS (headspace solid-phase microextraction followed by gas chromatography-mass spectrometry), allows identifying a wider set of VCs whose identity is unknown before analysis. Furthermore, using this approach, relative ratios between samples can be compared, representing an alternative for brewing to identify potential novel aromas and flavors in beer. In this study, we sought to investigate and characterize for the first time the fermentative and VC profile of different S. eubayanus strains from different Patagonian localities under fermentative conditions at low temperature (12 ◦C). To accomplish this, micro-fermentations were screened using the headspace solid-phase microextraction followed by gas chromatography-mass spectrometry method (HS-SPME-GC-MS) as a sensitive analytical means to identify VCs. The data gathered here demonstrate that a broad array of VCs is produced by wild S. eubayanus yeasts and highlights the potential of new strains for lager beer fermentation. 2.1. Yeast Strains Used in this Study The S. eubayanus strains used in this study were obtained from bark samples from Nothofagus pumilio trees (lenga) collected in 10 different localities in Chile. All the strains were previously identified and described by Nespolo et al., 2020 [22] (Table S1). Briefly, strains were isolated from approximately 1 g of bark samples and immediately incubated in a 15 mL tubes containing 10 mL of enrichment media. This media contained: 2% yeast nitrogen base, 1% raffinose, 2% peptone, and 8% ethanol [24]. Samples were incubated for two weeks at 20 ◦C without agitation and were subsequently vortexed and plated (5 µL) onto YPD agar (1% yeast extract, 2% peptone, 2% glucose, and 2% agar). Isolated colonies were stored in glycerol 20% v/v and stored at −80 ◦C in the Molecular Genetics Laboratory yeast collection at Universidad de Santiago de Chile. The strains are available upon request. 2.2. Micro-Fermentation Assay Micro-fermentation assays were performed on two genetically different S. eubayanus isolates from each location (Table S1). The different fermentations were made in beer wort “Pilsner Connoisseur” (Muntons), which was sterilized at 100 ◦C for 25 min. An initial inoculum of 5 mL was prepared in 6 ◦P beer wort, where a single colony from each strain was used. The sample was incubated for 24 h at 20 ◦C in constant agitation at 150 rpm. Subsequently, the inoculum was transferred to 50 mL of 12 ◦P beer wort and kept at 20 ◦C with shaking at 150 rpm for another 24 h. The cell cultures obtained were centrifuged at 5000 rpm for 5 min and used to calculate the final cell concentration for use in each micro-fermentation, according to the formula described by White and Zainasheff[25]. The micro-fermentation assay was performed in triplicate in 50 mL of previously oxygenated (15 mg/L) 12 ◦P beer wort and supplemented with 0.3 ppm of ZnCl2. Airlocks with 30% of glycerol were used. The weight of the bottles was measured throughout the progress of the fermentation process, registering on an analytical balance the CO2 loss over time (g/L). The commercial strain S. pastorianus W34/70 (Sp.W34/70) was used as a fermentation positive control. Similarly, micro-fermentations at 12 ◦C followed the same procedure. 4 of 19 Microorganisms 2020, 8, 755 2.4. HS-SPME-GC-MS Analysis Analysis of the production of all VCs was carried out by the combination of four analytical procedures: headspace, solid-phase microextraction, gas chromatography, and mass spectrometry (HS-SPME-GC-MS, Thermo Scientifics, Whaltam, MA, USA). A headspace vial was loaded with 2 mL of sample and equilibrated at extraction temperature (60 ◦C) for 30 min. Then, a 50/30 µm divinylbenzene/carboxenpolydimethylsiloxane fiber (DVB/Car/PDMS; Supelco, Bellefonte, PA, USA) was introduced within the headspace vial in order to extract the volatiles, through a silicon septum for 30 min. Targeted volatiles loaded in the fiber were analyzed by using a GC-MS (Thermo Scientifics Trace GC Ultra, equipped with Thermo Scientifics ISQ quadrupole mass spectrometer and autosampler Thermo Scientifics Triplus) and GC-MS/MS (Thermo Scientifics Trace 1300 GC, equipped with Thermo Scientifics TSQ Triple quadrupole mass spectrometer and autosampler Thermo Scientifics Triplus RHS) in full-scan mode. The desorption of the fiber was carried out (splitless mode) at 250 ◦C for 5 min and cleaning at 270 ◦C for 15 min. Helium was passed at a constant flow (1.2 mL/min) for serving as a carrier gas. Mass spectrometry detection was performed under electron impact (EI) ionization at 70 eV by operating in the full-scan acquisition mode in the 40–400 m/z range. Ion source and transfer line temperature were maintained at 250 ◦C, respectively. Potential emanations were analyzed using Xcalibur Software (Thermo Electron Corporation) matching mass spectra with those saved in the National Institute of Standards and Technology (NIST) MS Spectral Library 2014. Chromatographic peaks were considered “unknown” when their similarity index (SI) and reverse similarity index (RSI) were less than 850 and discarded in this identification process. These parameters refer to the degree at which the target spectrum matches the standard spectrum in the NIST Library (a value of 1000 indicates a perfect fit). Selected chromatographic peaks were checked with their respective chemical standards and Kovats indexes [26]. 2.3. Ammonium and Amino Acid Analysis Ammonium and Amino acid consumption was evaluated at different time-points during the fermentation process. For this, a 100 mm cannula was inserted into the rubber stopper to perform periodical sampling. Samples were obtained at 24, 48, and 168 h by extracting 0.5 to 1 mL of fermented beer wort. Each sample was centrifuged at 13,000 rpm for 5 min, and the supernatant was recovered and stored at −20 ◦C. Fermented wort was processed following the protocol described by Gomez-Alonzo et al. 2007. Briefly, 120 µL of the sample was incubated with 20 µL of diethylethoxymethylen-emalonate (DEEMM, SIGMA) for 24 h at room temperature in a solution containing 580 µL of a borate buffer (pH = 9) and 250 µL of absolute methanol (Fisher Chemical). The sample was heated at 70 ◦C for 2 h to allow the complete degradation of excess DEEMM and reagent byproducts. After the derivatization reaction, 20 µL of the processed samples were analyzed by High-Performance Liquid Chromatography (HPLC, Shimadzu) with a C18-HL column (250 mm × 4.6 mm), a binary gradient (phase A: phase B:), and a flow rate of 0.9 mL/min. For detection, a photodiode array detector (Shimadzu, Kokyo, Japan) set at 270, and 280 nm was used. The different compounds were identified according to the retention time and UV-vis spectral characteristics of the derivatives of the corresponding standards and were quantified using the internal standard’s method. 3.1. Differences in Fermentation Capacity Across S. eubayanus Isolates To evaluate the fermentative capacity and differences in kinetic profiles of S. eubayanus strains when brewing wort, we performed a micro-fermentation assay using 20 strains obtained from different sites in central and Chilean Patagonia [22] (Figure 1a, Table S1). For this, strains were inoculated in beer wort, and CO2 release was measured for 14 days (see Methods). The strains exhibiting the greatest levels of CO2 release at the end of the fermentation were CL1107.1 (Nahuelbuta National Park, p-value = 0.422, ANOVA) and CL600.1 (Antillanca National Park, p-value = 0.294, ANOVA), neither of them exhibited significant differences when compared to the commercial strain (Sp.W34/70) (Figure 1b). Interestingly, these two strains were obtained from different sampling sites (central Chile and northern Chilean Patagonia, Figure 1a, and Figure S1). On the other hand, strains with the lowest fermentative capacity were CL1002.1 (Torres del Paine National Park, p-value < 0.0001, ANOVA), CL814.1 (Magallanes National Reserve, p-value < 0.0001, ANOVA), CL815.1 (Magallanes National Park, p-value < 0.0001), CL606.1 (Vicente Pérez Rosales National Park, p-value < 0.0001, ANOVA), and CL801.1 (Karukinka Natural Park, p-value < 0.0001, ANOVA), all of them from the central and southern Chilean Patagonia sampling sites (Figure 1a,b and Figure S1). To determine whether fermentation performance correlated with climate and/or geographic conditions, we performed a correlation test between CO2 release and: i) latitude and ii) average yearly temperature. We found a significant correlation for both parameters: latitude (Pearson r = −0.58, p-value < 0.001, t-Test) and average yearly temperature per isolation locality (Pearson r = 0.59, p-value < 0.001, t-Test) vs. CO2 loss (Figure 1c,d). The observed differences could correlate with fermentation capacity and CO2 release, indicating different fermentative potentials between strains depending on their geographic origin. 2.5. Statistical Analyses All statistical analyses were performed using biological triplicates. One-way ANOVAs (Analysis of Variance) and Pearson non-parametric correlations (t-Test) were performed using GraphPad Prism 8.01 for Windows, GraphPad Software, La Jolla, CA, USA, www.graphpad.com. The differences were considered statistically significant at p-values < 0.05. A principal component analysis (PCA) was performed using R software [27], and the “prcomp” package stats 3.6.0 and plotted using the “ggbiplot” package. Temperature data for the correlation analyses was obtained from the Center of Climate 5 of 19 Microorganisms 2020, 8, 755 and Resilience Research (http://www.cr2.cl/datos-de-temperatura/). The data obtained correspond to measurements of daily average temperature recorded in 33 stations of the Chilean Meteorological Center and in 196 stations of the General Direction for Water during 2018. and Resilience Research (http://www.cr2.cl/datos-de-temperatura/). The data obtained correspond to measurements of daily average temperature recorded in 33 stations of the Chilean Meteorological Center and in 196 stations of the General Direction for Water during 2018. 3.2. Volatile Compound Production in S. eubayanus Isolates To characterize the VC production profiles of the S. eubayanus strains, we analyzed the fermented wort by HS-SPME-GC-MS VCs and identified VCs whose identity is unknown, rather than using internal standards for commonly known compounds. To attain this, we selected 10 strains based on the two following criteria: (i) One representative isolate from each sampling site and (ii) significantly different fermentative capacities (CO2 release levels) between strains to maximize the genetic and phenotypic diversity. Therefore, we selected strains with high fermentative capacity (CL1107.1 and CL450.1), mid-fermentation capacity (CL711.2, CL216.1, CL905.1, CL602.1, and CL812.1), and low fermentative capacity (CL606.1, CL1002.1, and CL814.1) (Figure 1b). HS-SPME-GC-MS analyses using the 10 selected strains, together with the Sp.W34/70 lager control, allowed the identification of 55 different compounds in all fermented wort (Figure 2a, Table S2). Out of these compounds, 52 were found in S. eubayanus strains and 22 were found in the lager strain (Sp.W34/70) (Figure 2a). Interestingly, 32 compounds were exclusive to S. eubayanus and were not detected in Sp.W34/70. In comparison, only three compounds (ethyl hexanoate, ethyl heptanoate, and ethyl dihydrocinnamate) were found solely in the commercial strain Sp.W34/70, resulting in 19 compounds common to both species (Figure 2a, Table S2). These results demonstrate that all compounds (except ethyl hexanoate, ethyl heptanoate, and ethyl dihydrocinnamate) generated by the lager strain are found in at least one wild isolate. Among all the analyzed compounds, esters represent the main fraction of the VCs identified (26 in wild S. eubayanus and 15 in Sp.W34/70) (Figure 2b,c), where 14 compounds were found in common across both species (Figure 2b). Interestingly, of the whole set of VCs identified, only a single higher alcohol, phenethyl alcohol, was detected by HS-SPME-GC-MS. In contrast, no phenolic compounds 6 of 19 p-value f them Microorganisms 2020, 8, 755 (Magallanes National 0 0001 ANOVA) were identified in the lager strain. However, in wild S. eubayanus, we discovered two different phenolic compounds: 4-vinylguaiacol (4-VG), which was found in all strains and 2,4-Di-tert-butylphenol, which was only identified in CL1002.1 and CL602.1. A negative and significant correlation was found between the number of VCs produced at the end of the fermentation process and fermentation capacity (Pearson r = −0.35, p-value < 0.05, t-Test, Pearson Correlation). 3.2. Volatile Compound Production in S. eubayanus Isolates In this context, strains with a higher fermentative capacity produced a lower number of (identified) compounds, suggesting that the production of a wider number of VCs is not directly related to high fermentation capacity (Figure 2d). from the central and southern Chilean Patagonia sampling sites (Figure 1a,b and Figure S1). To determine whether fermentation performance correlated with climate and/or geographic conditions, we performed a correlation test between CO2 release and: i) latitude and ii) average yearly temperature. We found a significant correlation for both parameters: latitude (Pearson r = −0.58, p- value < 0.001, t-Test) and average yearly temperature per isolation locality (Pearson r = 0.59, p-value < 0.001, t-Test) vs. CO2 loss (Figure 1c,d). The observed differences could correlate with fermentation capacity and CO2 release, indicating different fermentative potentials between strains depending on their geographic origin. Figure 1. Saccharomyces eubayanus fermentation performance in lager wort. (a) The geographical location of the sampling sites and the strain codes from each locality. (b) Total CO2 loss [g/L] in Figure 1. Saccharomyces eubayanus fermentation performance in lager wort. (a) The geographical location of the sampling sites and the strain codes from each locality. (b) Total CO2 loss [g/L] in fermentations carried out by S. eubayanus strains. Lager strain S. pastorianus W34/70 was used as a fermentation control. Different letters reflect statistically significant differences between strains with a p-value < 0.05, one-way ANOVA. (c) Latitude and (d) annual average temperature at the isolation site of each strain vs. CO2 loss (Pearson Correlation). Each dot represents a fermentation replicate. Three biological replicates were analyzed per strain. Figure 1. Saccharomyces eubayanus fermentation performance in lager wort. (a) The geographical location of the sampling sites and the strain codes from each locality. (b) Total CO2 loss [g/L] in Figure 1. Saccharomyces eubayanus fermentation performance in lager wort. (a) The geographical location of the sampling sites and the strain codes from each locality. (b) Total CO2 loss [g/L] in fermentations carried out by S. eubayanus strains. Lager strain S. pastorianus W34/70 was used as a fermentation control. Different letters reflect statistically significant differences between strains with a p-value < 0.05, one-way ANOVA. (c) Latitude and (d) annual average temperature at the isolation site of each strain vs. CO2 loss (Pearson Correlation). Each dot represents a fermentation replicate. Three biological replicates were analyzed per strain. 7 of 19 Microorganisms 2020, 8, 755 Figure 2. 3.3. Main Volatile Compounds Identified in Fermented Wort 3.3. Main Volatile Compounds Identified in Fermented Wort To improve our comparative analysis of VC production profiles across strains, we selected compounds comprising the highest relative amounts, representing at least 45% of the total VC area detected and that were identified in at least five S. eubayanus strains (Table 1, Figure 3). In this way, we selected 12 compounds, most of them aliphatic esters. Of these, ethyl octanoate (aliphatic ester), ethyl hexadecanoate (aliphatic ester), phenethyl alcohol (aromatic alcohol), and n-decanoic acid (carboxylic acid) were found in all strains in similar relative amounts (Figure 3). On the other hand, among the aliphatic esters detected by HS-SPME-GC-MS, ethyl decanoate was the most abundant VC, especially in the CL1002.1 isolate from Torres del Paine National Park (3300 ± 879.8, p-value < 0.05, ANOVA) (Table 1). Other compounds, such as isoamyl decanoate, phenethyl acetate (aromatic ester), phenethyl decanoate (aromatic ester), and octanoic acid (carboxylic acid) (Figure 3), were exclusively detected in a subset of strains (Table 1). Within the detected off-flavors, 4-VG (phenols) exhibited one of the highest area contributions to the profiles of the strains, being identified at similar levels in all strains (p-value > 0.05, ANOVA, Figure 3), except for CL602.1 (p-value < 0.05, ANOVA), exhibiting a lower detected relative amount compared to the other strains (98.8 ± 11.4). To improve our comparative analysis of VC production profiles across strains, we selected compounds comprising the highest relative amounts, representing at least 45% of the total VC area detected and that were identified in at least five S. eubayanus strains (Table 1, Figure 3). In this way, we selected 12 compounds, most of them aliphatic esters. Of these, ethyl octanoate (aliphatic ester), ethyl hexadecanoate (aliphatic ester), phenethyl alcohol (aromatic alcohol), and n-decanoic acid (carboxylic acid) were found in all strains in similar relative amounts (Figure 3). On the other hand, among the aliphatic esters detected by HS-SPME-GC-MS, ethyl decanoate was the most abundant VC, especially in the CL1002.1 isolate from Torres del Paine National Park (3300 ± 879.8, p-value < 0.05, ANOVA) (Table 1). Other compounds, such as isoamyl decanoate, phenethyl acetate (aromatic ester), phenethyl decanoate (aromatic ester), and octanoic acid (carboxylic acid) (Figure 3), were exclusively detected in a subset of strains (Table 1). 3.2. Volatile Compound Production in S. eubayanus Isolates (b) Total esters identified in beer wort fermented with wild Chilean S. eubayanus (Blue) strains and S. pastorianus W34/70 (Red). (c). Main chemical groups detected in fermented beer wort. (d) Correlation between the number of VCs produced and fermentative capacity. Each point depicts a biological replicate. the Sp.W34/70 strain. (a) Total VCs identified in beer wort fermented by wild Chilean S. eubayanus (Blue) strains and S. pastorianus W34/70 (Red). (b) Total esters identified in beer wort fermented with wild Chilean S. eubayanus (Blue) strains and S. pastorianus W34/70 (Red). (c). Main chemical groups detected in fermented beer wort. (d) Correlation between the number of VCs produced and fermentative capacity. Each point depicts a biological replicate. 3.2. Volatile Compound Production in S. eubayanus Isolates Volatile compounds identified in lager beer micro-fermentations using wild S. eubayanus and the Sp.W34/70 strain. (a) Total VCs identified in beer wort fermented by wild Chilean S. eubayanus (Blue) strains and S. pastorianus W34/70 (Red). (b) Total esters identified in beer wort fermented with wild Chilean S. eubayanus (Blue) strains and S. pastorianus W34/70 (Red). (c). Main chemical groups detected in fermented beer wort. (d) Correlation between the number of VCs produced and fermentative capacity. Each point depicts a biological replicate. Figure 2. Volatile compounds identified in lager beer micro-fermentations using wild S. eubayanus and the Sp.W34/70 strain. (a) Total VCs identified in beer wort fermented by wild Chilean S. eubayanus (Blue) strains and S. pastorianus W34/70 (Red). (b) Total esters identified in beer wort fermented with wild Chilean S. eubayanus (Blue) strains and S. pastorianus W34/70 (Red). (c). Main chemical groups detected in fermented beer wort. (d) Correlation between the number of VCs produced and fermentative capacity. Each point depicts a biological replicate. Figure 2. Volatile compounds identified in lager beer micro-fermentations using wild S. eubayanus d h / l d f d b f d b ld h l b Figure 2. Volatile compounds identified in lager beer micro-fermentations using wild S. eubayanus and Figure 2. Volatile compounds identified in lager beer micro-fermentations using wild S. eubayanus and the Sp.W34/70 strain. (a) Total VCs identified in beer wort fermented by wild Chilean S. eubayanus (Blue) strains and S. pastorianus W34/70 (Red). (b) Total esters identified in beer wort fermented with wild Chilean S. eubayanus (Blue) strains and S. pastorianus W34/70 (Red). (c). Main chemical groups detected in fermented beer wort. (d) Correlation between the number of VCs produced and fermentative capacity. Each point depicts a biological replicate. Figure 2. Volatile compounds identified in lager beer micro-fermentations using wild S. eubayanus and the Sp.W34/70 strain. (a) Total VCs identified in beer wort fermented by wild Chilean S. eubayanus (Blue) strains and S. pastorianus W34/70 (Red). (b) Total esters identified in beer wort fermented with wild Chilean S. eubayanus (Blue) strains and S. pastorianus W34/70 (Red). (c). Main chemical groups detected in fermented beer wort. (d) Correlation between the number of VCs produced and fermentative capacity. Each point depicts a biological replicate. and the Sp.W34/70 strain. (a) Total VCs identified in beer wort fermented by wild Chilean S. eubayanus (Blue) strains and S. pastorianus W34/70 (Red). 3.3. Main Volatile Compounds Identified in Fermented Wort 3.3. Main Volatile Compounds Identified in Fermented Wort Within the detected off-flavors, 4-VG (phenols) exhibited one of the highest area contributions to the profiles of the strains, being identified at similar levels in all strains (p-value > 0.05, ANOVA, Figure 3), except for CL602.1 (p-value < 0.05, ANOVA), exhibiting a lower detected relative amount compared to the other strains (98.8 ± 11.4). 8 of 19 Microorganisms 2020, 8, 755 Table 1. Main Volatile Compounds detected in S. eubayanus. Table 1. Main Volatile Compounds detected in S. eubayanus. 3.3. Main Volatile Compounds Identified in Fermented Wort 3.3. Main Volatile Compounds Identified in Fermented Wort Relative Amount (×106) and SEM Strains Chemical Group Compound Name RI CL1107.1 CL1002.1 CL905.1 CL814.1 CL812.1 CL711.2 CL606.1 CL602.1 CL450.1 CL216.1 Aliphatic esters Ethyl octanoate 1192.64 506.1 ± 91.8 1400 ± 226.4 483.1 ± 250.2 1300 ± 1100 446.4 ± 144 276.7 ± 31.7 812.9 ± 123.5 228.8 ± 76 230.2 ± 73.7 819.5 ± 151.7 Ethyl decanoate 1391.77 1400 ± 522.4 3300 ± 879.8 1700 ± 734.6 1500 ± 1200 1100 ± 118.7 870.3 ± 219.4 2400 ± 635.3 141.7 ± 38.3 362 ± 152.8 802.2 ± 253.5 Ethyl dodecanoate 1443.83 793.6 ± 226 1000 ± 289.6 1600 ± 357.4 164.5 ± 96.9 325.8 ± 11.7 868.5 ± 260.4 872.2 ± 73.8 123.7 ± 17 335.3 ± 125.3 186 ± 30.2 Isoamyl decanoate 1590.63 99.7 ± 25.7 180.5 ± 43.5 102.1 ± 28.9 50.8 ± 30 42.4 ± 2.1 74.9 ± 8.1 129.4 ± 29.3 N/D N/D 33.5 ± 10.5 Ethyl tetradecanoate 1790.87 160.3 ± 50.9 89.8 ± 28.2 228.2 ± 38.9 23.7 ± 8 54.3 ± 10.5 166 ± 50 115.2 ± 21.6 49.7 ± 6.8 104.5 ± 38.3 N/D Ethyl hexadecanoate 1991.92 552 ± 241.4 169 ± 53.7 666 ± 179.7 53.4 ± 7.4 161.3 ± 34.4 363 ± 85.2 242.5 ± 37.5 229.4 ± 65.1 274.3 ± 115.2 136.9 ± 20 Aromatic esters Phenethyl acetate 1250.12 490.8 ± 79.1 189.8 ± 17 522.4 ± 233.1 212.9 ± 172.2 232.1 ± 78.9 501.7 ± 95.5 360.2 ± 84.7 N/D N/D 312.6 ± 79.2 Phenethyl decanoate 2062.65 149.5 ± 32.8 369.7 ± 62.8 N/D N/D 27.7 ± 5.3 123.7 ± 40.7 170.8 ± 25.3 N/D N/D N/D Aromatic alcohol Phenethyl Alcohol 1116.19 2900 ± 1600 1500 ± 409.3 3800 ± 971.5 1800 ± 1400 2500 ± 992.4 3700 ± 1100 2000 ± 627 2900 ± 805 3200 ± 565.2 3200 ± 1000 Carboxylic acid Octanoic acid 1171.39 483 ± 91.8 N/D 633.5 ± 101.3 N/D 22.9 ± 17.4 N/D N/D 12.5 ± 2.4 N/D 39 ± 13.1 n-Decanoic acid 1360.8 200 ± 59 198 ± 8.8 127.5 ± 11 93.3 ± 43.8 93.4 ± 7.8 163.3 ± 25.6 158.3 ± 16.1 N/D 120.4 ± 28.9 81.9 ± 25.6 Phenols 4-vinylguaiacol 1317.45 281.4 ± 11.2 310.9 ± 22.3 238.7 ± 39.4 250.1 ± 167.4 258.7 ± 53.9 287.5 ± 21.8 292.7 ± 17.6 98.8 ± 11.4 118.1 ± 15 284.1 ± 14.5 Compounds detected in S. 3.3. Main Volatile Compounds Identified in Fermented Wort 3.3. Main Volatile Compounds Identified in Fermented Wort eubayanus fermentation by headspace solid-phase microextraction followed by gas chromatography-mass spectrometry (HS-SPME-GC-MS). SEM: standard error of the mean; CAS: Chemical Abstracts Service; N/D: Not Detected; RI: Kovats retention indices. 9 of 19 Microorganisms 2020, 8, 755 Microorganisms 2020, 8 organisms 2020, 8, 755 9 of g , , Figure 3. Relative amounts of the main volatile compounds in fermented wort at 12 °C. The strains are distributed according to their fermentative capacity from high to low levels (left to right). Different letters reflect statistically significant differences between strains with a p-value < 0.001, one-way ANOVA. No-letters strains do not show statistically significant differences. Figure 3. Relative amounts of the main volatile compounds in fermented wort at 12 ◦C. The strains are distributed according to their fermentative capacity from high to low levels (left to right). Different letters reflect statistically significant differences between strains with a p-value < 0.001, one-way ANOVA. No-letters strains do not show statistically significant differences. Figure 3. Relative amounts of the main volatile compounds in fermented wort at 12 °C. The strains are distributed according to their fermentative capacity from high to low levels (left to right). Different letters reflect statistically significant differences between strains with a p-value < 0.001, one-way ANOVA. No-letters strains do not show statistically significant differences. Figure 3. Relative amounts of the main volatile compounds in fermented wort at 12 ◦C. The strains are distributed according to their fermentative capacity from high to low levels (left to right). Different letters reflect statistically significant differences between strains with a p-value < 0.001, one-way ANOVA. No-letters strains do not show statistically significant differences. Figure 3. Relative amounts of the main volatile compounds in fermented wort at 12 °C. The strains are distributed according to their fermentative capacity from high to low levels (left to right). Different letters reflect statistically significant differences between strains with a p-value < 0.001, one-way ANOVA. No-letters strains do not show statistically significant differences. Figure 3. Relative amounts of the main volatile compounds in fermented wort at 12 ◦C. The strains are distributed according to their fermentative capacity from high to low levels (left to right). Different letters reflect statistically significant differences between strains with a p-value < 0.001, one-way ANOVA. No-letters strains do not show statistically significant differences. 3.3. Main Volatile Compounds Identified in Fermented Wort 3.3. Main Volatile Compounds Identified in Fermented Wort To reduce the dimensionality of our dataset and interpret the profile of VC production for each strain, we performed a global principal component analysis (PCA) using the 12 selected VCs (Figure 4). The two first component explained 46.6% and 37% of the observed variation, respectively, allowing us to classify the S. eubayanus strain profiles in four groups: (Q1) CL450.1 and CL602.1, (Q2) CL812.1, CL216.1 and CL814.1, (Q3) CL905.1, CL1107.1 and CL711.2, and (Q4) CL606.1 and CL1002.1. To reduce the dimensionality of our dataset and interpret the profile of VC production for each strain, we performed a global principal component analysis (PCA) using the 12 selected VCs (Figure 4). The two first component explained 46.6% and 37% of the observed variation, respectively, allowing us to classify the S. eubayanus strain profiles in four groups: (Q1) CL450.1 and CL602.1, (Q2) CL812.1, CL216.1 and CL814.1, (Q3) CL905.1, CL1107.1 and CL711.2, and (Q4) CL606.1 and CL1002.1. Interestingly, strains in Q2 produced higher amounts of VCs, such as phenethyl alcohol and ethyl tetradecanoate, while those in Q3 showed higher areas for isoamyl decanoate and ethyl decanoate. This analysis suggests that strains with similar aromatic profiles do not have a common geographical or genetic origin, with isolates from central and southern Chile being found in all groups. Similarly, except for phenethyl alcohol (r = −0.75, p-value < 0.05, t-Test, Pearson Correlation), no significant correlation was found (p-value> 0.05, t-Test, Pearson Correlation) when relating latitude (origin of the strains) with 10 of 19 yde and ) These Microorganisms 2020, 8, 755 (linalool, 1-octanol an b ld h d 4 h the relative amount of the primary VC produced (Figure S2). Altogether, our results show the broad spectrum of VC profiles in S. eubayanus strains from different sites, and highlight the organoleptic potential for innovation in the brewing industry of this newly discovered yeast. results demonstrate that fermentation temperature differentially modulates the total number of VCs depending on the strain and genetic background, likely impacting the aromatic profiles of the beers produced (Table 2, Table S3). Figure 4. Principal Component Analysis of VC production across S. eubayanus strains. Distribution of the selected strains based on the leading VCs produced. a: Phenethyl Alcohol, b: Ethyl hexadecanoate, c: Octanoic acid, d: Ethyl tetradecanoate, e: Phenethyl acetate, f: Ethyl dodecanoate, g: n-Decanoic acid, h: Isoamyl decanoate, i: 4-vinyl guaiacol, j: Ethyl decanoate, k: Phenethyl decanoate, l: Ethyl Figure 4. 3.3. Main Volatile Compounds Identified in Fermented Wort 3.3. Main Volatile Compounds Identified in Fermented Wort Principal Component Analysis of VC production across S. eubayanus strains. Distribution of the selected strains based on the leading VCs produced. a: Phenethyl Alcohol, b: Ethyl hexadecanoate, c: Octanoic acid, d: Ethyl tetradecanoate, e: Phenethyl acetate, f: Ethyl dodecanoate, g: n-Decanoic acid, h: Isoamyl decanoate, i: 4-vinyl guaiacol, j: Ethyl decanoate, k: Phenethyl decanoate, l: Ethyl octanoate. The strains were distributed in four quadrants, Q1: First group. Q2: Second group. Q3: Third group. Q4: Fourth group. Figure 4. Principal Component Analysis of VC production across S. eubayanus strains. Distribution of Figure 4. Principal Component Analysis of VC production across S. eubayanus strains. Distribution of Figure 4. Principal Component Analysis of VC production across S. eubayanus strains. Distribution of the selected strains based on the leading VCs produced. a: Phenethyl Alcohol, b: Ethyl hexadecanoate, c: Octanoic acid, d: Ethyl tetradecanoate, e: Phenethyl acetate, f: Ethyl dodecanoate, g: n-Decanoic acid, h: Isoamyl decanoate, i: 4-vinyl guaiacol, j: Ethyl decanoate, k: Phenethyl decanoate, l: Ethyl Figure 4. Principal Component Analysis of VC production across S. eubayanus strains. Distribution of the selected strains based on the leading VCs produced. a: Phenethyl Alcohol, b: Ethyl hexadecanoate, c: Octanoic acid, d: Ethyl tetradecanoate, e: Phenethyl acetate, f: Ethyl dodecanoate, g: n-Decanoic acid, h: Isoamyl decanoate, i: 4-vinyl guaiacol, j: Ethyl decanoate, k: Phenethyl decanoate, l: Ethyl octanoate. The strains were distributed in four quadrants, Q1: First group. Q2: Second group. Q3: Third group. Q4: Fourth group. Figure 4. Principal Component Analysis of VC production across S. eubayanus strains. Distribution of the selected strains based on the leading VCs produced. a: Phenethyl Alcohol, b: Ethyl hexadecanoate, c: Octanoic acid, d: Ethyl tetradecanoate, e: Phenethyl acetate, f: Ethyl dodecanoate, g: n-Decanoic acid, h: Isoamyl decanoate, i: 4-vinyl guaiacol, j: Ethyl decanoate, k: Phenethyl decanoate, l: Ethyl Figure 4. Principal Component Analysis of VC production across S. eubayanus strains. Distribution of the selected strains based on the leading VCs produced. a: Phenethyl Alcohol, b: Ethyl hexadecanoate, c: Octanoic acid, d: Ethyl tetradecanoate, e: Phenethyl acetate, f: Ethyl dodecanoate, g: n-Decanoic acid, h: Isoamyl decanoate, i: 4-vinyl guaiacol, j: Ethyl decanoate, k: Phenethyl decanoate, l: Ethyl octanoate. The strains were distributed in four quadrants, Q1: First group. Q2: Second group. Q3: Third group. Q4: Fourth group. 3.4. Fermentation Temperature Impacts the Volatile Compound Profile in S. eubayanus Strains 3.4. Fermentation Temperature Impacts the Volatile Compound Profile in S. eubayanus Strains Depending on the strain and fermentation temperature, producers can fine-tune the aromatic profiles of beverages. We compared the temperature dependence of S. eubayanus strains and their impact on the production of VCs in brewing wort at the end of fermentation. For this, we selected two strains (CL905.1 and CL602.1) with (i) similar fermentative capacity (Figure 1b) and (ii) different aromatic profiles and located in different quadrants in the PCA (Figure 4). In this way, we performed new fermentation batches at 12 ◦C and 20 ◦C and identify the VCs produced at both temperatures using HS-SPME-GC-MS. Fermentative profiles were similar between strains at 20 ◦C and did not show statistically significant differences for fermentation CO2 release. At 12 ◦C, the strains show statistically significant differences for fermentation CO2 release with a p-value < 0.001, ANOVA (Figure S3). We found that VC profiles differed in a strain- and temperature-dependent manner. Notably, at 12 ◦C, the two isolates produced 16 compounds in common, rising to 20 when fermentation was run at 20 ◦C (Table S3). In strain CL905.1, we identified 23 and 32 compounds at 12 ◦C and 20 ◦C, respectively, while the opposite was observed in CL602.1, where we identified 24 and 21 VCs at 12 ◦C and 20 ◦C, respectively. In the case of CL602.1, the main chemical group affected by temperature was the aliphatic esters, while in CL905.1, the main chemical groups with more compounds detected at higher temperature were aliphatic esters (1-hexanol, 2-ethyl, propanoic acid, 2-methyl,3-hydroxy-2,2,4-trimethylpentyl ester and ethyl 9-hexadecanoate), aliphatic alcohols (linalool, 1-octanol and 1-decanol), aromatic aldehydes (benzaldehyde, benzeneacetaldehyde and benzaldehyde, 4-methyl), and other compounds (1,1,3-trimethyl-3-phenylindane) (Table S3). These results demonstrate that fermentation temperature differentially modulates the total number of VCs depending on the strain and genetic background, likely impacting the aromatic profiles of the beers produced (Table 2, Table S3). 11 of 19 Microorganisms 2020, 8, 755 Table 2. Main Volatile Compounds detected in CL905.1 and CL602.1. 3.4. Fermentation Temperature Impacts the Volatile Compound Profile in S. eubayanus Strains Relative Abundance (×106) and SEM Strain CL905.1 CL602.1 Chemical Group Compound RI 12 ◦C 20 ◦C 12 ◦C 20 ◦C Aliphatic esters Ethyl octanoate 1192.63881 483.1 ± 250.2 1566.67 ± 251.66 228.8 ± 76 1566.67 ± 120.19 Ethyl 9-decenoate 1384.56268 448.5 ± 165.1 622.93 ± 232.23 137.5 ± 63.5 622.93 ± 116.55 Ethyl decanoate 1391.77014 1700 ± 734.6 3666.67 ± 1011.60 141.7 ± 38.3 3666.67 ± 409.61 Isoamyl decanoate 1644.71354 102.1 ± 28.9 162.40 ± 44.51 N/D 162.40 ± 21.72 Ethyl tetradecanoate 1786.82537 228.2 ± 38.9 267.33 ± 52.60 49.7 ± 6.8 267.33 ± 23.47 Ethyl hexadecanoate 1991.92208 666 ± 179.7 553.43 ± 59.25 N/D 553.43 ± 48.51 Aromatic esters Phenethyl acetate 1250.12471 522.4 ± 233.1 687.80 ± 328.51 N/D 687.80 ± 189.00 Aromatic alcohols Phenethyl Alcohol 1116.18753 3800 ± 971.5 8600.00 ± 346.41 2900 ± 805 8600.00 ± 851.14 Aromatic aldehydes Benzaldehyde, 4-methyl 1083.5138 N/D 170.50 ± 58.41 42.1 ± 6 170.50 ± 21.10 Carboxylic acids Octanoic acid 1171.38687 633.5 ± 101.3 N/D 12.5 ± 2.4 131.80 ± 13.44 Dodecanoic acid 1556.73878 38.3 ± 3.4 1866.67 ± 404.15 N/D 1866.67 ± 135.36 Phenols 4-vinylguaiacol 1317.44761 238.7 ± 39.4 308.03 ± 76.01 98.8 ± 11.4 290.93 ± 38.64 Compounds detected in S. eubayanus fermentation by HS-SPME-GC-MS. SEM: standard error of the mean; CAS: Chemical Abstracts Service; N/D: Not detected; RI: Kovats retention indices. 12 of 19 Microorganisms 2020, 8, 755 We next selected the compounds with the highest area percentage (representing approximately 65% of the area of the whole peak) in CL905.1 and CL602.1 strains (Figure 5, Table 2). In this way, we identified 12 main compounds at 12 ◦C and 20 ◦C, for which relative areas were compared between both strains. As previously mentioned, not only the number of VCs changed with temperature, but we also found differences in the relative amount produced depending on the fermentation temperature. For example, ethyl octanoate (aliphatic ester) and phenethyl alcohol (aromatic alcohol, higher alcohol) (Figure 5, Table 2) significantly increased at 20 ◦C (p-value > 0.001, ANOVA), independently of the strain. On the other hand, higher levels in the relative amount of 4-VG at 20 ◦C compared to 12 ◦C was only observed in CL602.1 (p-value > 0.001, ANOVA) and not in CL905.1 (Figure 5). Similarly, other ester compounds significantly increased with temperature solely in CL602.1, such as: ethyl 9-decanoate, ethyl decanoate, isoamyl decanoate, and phenethyl acetate (p-value > 0.001, ANOVA). 3.4. Fermentation Temperature Impacts the Volatile Compound Profile in S. eubayanus Strains Depending on the strain, a higher fermentation temperature allowed us to find novel compounds undetected at 12 ◦C. For example, benzaldehyde 4-methyl (aromatic aldehyde) was exclusively detected in CL905.1 at 20 ◦C, but not at 12 ◦C. In contrast, in CL602.1, this compound was detected at both temperatures (p-value < 0.001, ANOVA). These results demonstrate that fermentation temperature results in different profiles in both strains in terms of the number of compounds, but also in their relative amounts, representing a strain × temperature specific interaction. Microorganisms 2020, 8, x FOR PEER REVIEW 1 of 21 Figure 5. Relative amounts of main volatile compounds identified in fermented wort at 12 and 20 °C. CL905.1 (Choshuenco National Park) and CL602.1 (Antillanca National Park) were fermented at 12 °C and 20 °C, respectively. Different letters reflect statistically significant differences between strains with a p-value < 0.001, one-way ANOVA. No-letters strains do not show statistically significant differences. Figure 5. Relative amounts of main volatile compounds identified in fermented wort at 12 and 20 ◦C. CL905.1 (Choshuenco National Park) and CL602.1 (Antillanca National Park) were fermented at 12 ◦C and 20 ◦C, respectively. Different letters reflect statistically significant differences between strains with a p-value < 0.001, one-way ANOVA. No-letters strains do not show statistically significant differences. Figure 5. Relative amounts of main volatile compounds identified in fermented wort at 12 and 20 °C. CL905.1 (Choshuenco National Park) and CL602.1 (Antillanca National Park) were fermented at 12 °C and 20 °C, respectively. Different letters reflect statistically significant differences between strains with a p-value < 0.001, one-way ANOVA. No-letters strains do not show statistically significant differences. Figure 5. Relative amounts of main volatile compounds identified in fermented wort at 12 and 20 ◦C. CL905.1 (Choshuenco National Park) and CL602.1 (Antillanca National Park) were fermented at 12 ◦C and 20 ◦C, respectively. Different letters reflect statistically significant differences between strains with a p-value < 0.001, one-way ANOVA. No-letters strains do not show statistically significant differences. 13 of 19 Microorganisms 2020, 8, 755 13 of 19 3.5. Nitrogen Consumption Differentially Impacts the Prodcution of Volatile Compounds 3.5. Nitrogen Consumption Differentially Impacts the Prodcution of Volatile Compounds The ability to assimilate different nitrogen sources in the brewing wort directly impacts the physiology of yeasts during the fermentation process. Simultaneously, the differential consumption of nitrogen sources could affect the aroma and flavor profiles of fermented wort [1,28–30]. 3.4. Fermentation Temperature Impacts the Volatile Compound Profile in S. eubayanus Strains Different letters depict significant differences between strains with a p-value < 0.05, one-way ANOVA. Figure 6. Differential yeast assimilable nitrogen (YAN) consumption in CL905.1 and CL602.1 strains at 12 ◦C and 20 ◦C. (a) Ammonium and amino acid consumption kinetics at 24, 48, and 168 h. Blue dots: 12 ◦C fermentation; Red dots: 20 ◦C fermentation. (*) depict significant differences between temperatures with a p-value < 0.001, one-way ANOVA. (b) Main differences in amino Acid consumption between temperatures. Different letters depict significant differences between strains with a p-value < 0.05, one-way ANOVA. Figure 6. Differential yeast assimilable nitrogen (YAN) consumption in CL905.1 and CL602.1 strains at 12 °C and 20 °C. (a) Ammonium and amino acid consumption kinetics at 24, 48, and 168 h. Blue dots: 12 °C fermentation; Red dots: 20 °C fermentation. (*) depict significant differences between temperatures with a p-value < 0.001, one-way ANOVA. (b) Main differences in amino Acid consumption between temperatures. Different letters depict significant differences between strains with a p-value < 0.05, one-way ANOVA. Figure 6. Differential yeast assimilable nitrogen (YAN) consumption in CL905.1 and CL602.1 strains at 12 ◦C and 20 ◦C. (a) Ammonium and amino acid consumption kinetics at 24, 48, and 168 h. Blue dots: 12 ◦C fermentation; Red dots: 20 ◦C fermentation. (*) depict significant differences between temperatures with a p-value < 0.001, one-way ANOVA. (b) Main differences in amino Acid consumption between temperatures. Different letters depict significant differences between strains with a p-value < 0.05, one-way ANOVA. Subsequently, we analyzed the consumption of amino acids in wort. In this way, we found that their consumption rate was slower compared to ammonium, where both strains exhibited significant differences at 24 h between 12 ◦C and 20 ◦C (Figure 6a, Table S4). Strain CL905.1 consumed 82.44 ± 1.95 mg N/L at 12 ◦C, while a higher value was reached (125.77 ± 2.13 mg N/L, p-value < 0.001, ANOVA) when the fermentation temperature was set to 20 ◦C. Similarly, CL602.1 showed an increase in amino acid consumption between 12 ◦C (91.56 ± 8.41 mg N/L) and 20 ◦C (130.36 ± 3.28 mg N/L, Subsequently, we analyzed the consumption of amino acids in wort. In this way, we found that their consumption rate was slower compared to ammonium, where both strains exhibited significant differences at 24 h between 12 ◦C and 20 ◦C (Figure 6a, Table S4). 3.4. Fermentation Temperature Impacts the Volatile Compound Profile in S. eubayanus Strains In this context, to determine the nitrogen consumption profiles of the two strains (CL905.1 and CL602.1) at 12 ◦C and 20 ◦C during fermentation, yeast assimilable nitrogen (YAN) consumption was measured by HPLC after 24, 48, and 168 h of fermentation (Figure 6). In the case of ammonium, that represents 29.02% (Table S4) of the overall YAN in the beer wort, 21.98% ± 1.31% of all the available ammonium was consumed by 24 h at 20 ◦C, while at 12 ◦C, a similar fraction (14.82% ± 10.50%, p-value < 0.001, one-way ANOVA) was reached at 48 h (Figure 6a, Table S4). In this way, when we compared the effect of temperature over the consumption of ammonium in both strains, significant differences were observed only at 24 h (p-value < 0.05, ANOVA) (Figure 6a), while at 168 h, most of the ammonium was already consumed in both strains, suggesting a temperature dependent (and not strain dependent) nitrogen consumption rate. Microorganisms 2020 8 x FOR PEER REVIEW 3 of 21 Figure 6. Differential yeast assimilable nitrogen (YAN) consumption in CL905.1 and CL602.1 strains at 12 °C and 20 °C. (a) Ammonium and amino acid consumption kinetics at 24, 48, and 168 h. Blue dots: 12 °C fermentation; Red dots: 20 °C fermentation. (*) depict significant differences between temperatures with a p-value < 0.001, one-way ANOVA. (b) Main differences in amino Acid consumption between temperatures. Different letters depict significant differences between strains with a p-value < 0.05, one-way ANOVA. Figure 6. Differential yeast assimilable nitrogen (YAN) consumption in CL905.1 and CL602.1 strains at 12 ◦C and 20 ◦C. (a) Ammonium and amino acid consumption kinetics at 24, 48, and 168 h. Blue dots: 12 ◦C fermentation; Red dots: 20 ◦C fermentation. (*) depict significant differences between temperatures with a p-value < 0.001, one-way ANOVA. (b) Main differences in amino Acid consumption between temperatures. Different letters depict significant differences between strains with a p-value < 0.05, one-way ANOVA. Figure 6. Differential yeast assimilable nitrogen (YAN) consumption in CL905.1 and CL602.1 strains at 12 °C and 20 °C. (a) Ammonium and amino acid consumption kinetics at 24, 48, and 168 h. Blue dots: 12 °C fermentation; Red dots: 20 °C fermentation. (*) depict significant differences between temperatures with a p-value < 0.001, one-way ANOVA. (b) Main differences in amino Acid consumption between temperatures. 3.4. Fermentation Temperature Impacts the Volatile Compound Profile in S. eubayanus Strains Strain CL905.1 consumed 82.44 ± 1.95 mg N/L at 12 ◦C, while a higher value was reached (125.77 ± 2.13 mg N/L, p-value < 0.001, ANOVA) when the fermentation temperature was set to 20 ◦C. Similarly, CL602.1 showed an increase in amino acid consumption between 12 ◦C (91.56 ± 8.41 mg N/L) and 20 ◦C (130.36 ± 3.28 mg N/L, 14 of 19 Microorganisms 2020, 8, 755 p-value < 0.001, ANOVA) (Table S4), demonstrating differential amino acid consumption depending solely on the fermentation temperature, but not on these genetic backgrounds. Of all the amino acids analyzed in this study (Table S5), solely glutamic acid and aspartic acid significantly differed at 12 ◦C (Figure 6b) (p-value <0.05, ANOVA) between strains, while for the rest of the amino acids, the differences were due to an increase in fermentation temperature, independently of the strain (Figure 6b). Additionally, we determined that, for both strains, several amino acids, such as aspartic acid, glutamic acid, serine, and threonine, were rapidly consumed in the wort (>60%) at 24 h, demonstrating the preference of S. eubayanus strains for these nitrogen sources (Figure 7, Table S5). On the other hand, amino acids such as histidine, glycine, tyrosine, and cysteine had lower consumption levels (<30%) at 24 h (low preference) and were efficiently consumed (>80%) only at 168 h (Table S7). Figure 6. Differential yeast assimilable nitrogen (YAN) consumption in CL905.1 and CL602.1 strains at 12 °C and 20 °C. (a) Ammonium and amino acid consumption kinetics at 24, 48, and 168 h. Blue dots: 12 °C fermentation; Red dots: 20 °C fermentation. (*) depict significant differences between temperatures with a p-value < 0.001, one-way ANOVA. (b) Main differences in amino Acid consumption between temperatures. Different letters depict significant differences between strains with a p-value < 0.05, one-way ANOVA. Figure 7. Preferred amino acid consumption in S. eubayanus strains at 24 h. Gray: CL905.1; Orange: CL602.1. (****) depict significant differences between amino acids with a p-value < 0.001, one-way ANOVA. Figure 7. Preferred amino acid consumption in S. eubayanus strains at 24 h. Gray: CL905.1; Orange: CL602.1. (****) depict significant differences between amino acids with a p-value < 0.001, one-way ANOVA. Figure 7. Preferred amino acid consumption in S. eubayanus strains at 24 h. Gray: CL905.1; Orange: CL602.1. (****) depict significant differences between amino acids with a p-value < 0.001, one-way ANOVA Figure 7. Preferred amino acid consumption in S. 3.4. Fermentation Temperature Impacts the Volatile Compound Profile in S. eubayanus Strains eubayanus strains at 24 h. Gray: CL905.1; Orange: CL602.1. (****) depict significant differences between amino acids with a p-value < 0.001, one-way ANOVA. 4. Discussion Beer is a complex mixture of ingredients, brewed from raw materials including water, malt, hops, and yeast, that contains a broad range of different components that react and interact at all stages of the brewing process [2]. Yeasts employed in industrial fermentation processes can convert relatively high concentrations of sugars into ethanol, carbon dioxide, and a wide range of secondary metabolites [31], which impact in several ways the aromatic profile of beer. While these compounds are only produced at low concentrations, they are responsible for the complex aromas of fermented beverages. Currently, alternative yeast strains are being considered to provide new and innovative beers [32]. Under this scenario, and considering the recent market trends, bioprospecting research on pioneering wild yeast strains with the capacity to produce new beers is becoming an attractive field in food research. In this way, the recent finding of S. eubayanus in Patagonia is a crucial element for beer microbial innovation [6,9,17,22]. In this study, we analyzed the fermentation capacity and VC production of a genetically diverse set of Patagonian S. eubayanus strains from Chile, previously described by Nespolo et al. [22]. To our knowledge, this represents the first study that explores the landscape of VCs produced during beer fermentation in S. eubayanus using HS-SPME-GC-MS. The differences observed between these strains relate to those previously reported, where strains with higher stress tolerance developed a greater fermentative capacity than other strains [22,33]. Accordingly, the worldwide increase in beer consumption and the continuous growth of microbrewers highlight the need for the characterization of the aromatic profile of novel beers, requiring tools to evaluate beer quality and authenticity. In this context, the HS-SPME-GC-MS method, used in this 15 of 19 Microorganisms 2020, 8, 755 analysis, has been reported as a fast and sensitive technique for the identification of VCs in both industrial and craft beers [10,34]. This technique develops reproducible data, with the improvement that avoids the injection of the standards, favoring several advantages, such as the time and cost of the analysis [10]. In HS-SPME (headspace solid-phase microextraction), analytes are extracted into a thin fused-silica fiber coated with the extracting phase by immersing it in the headspace of solid or liquid samples. 4. Discussion Then, by GC-MS, compound identification is based on mass spectra matching with the standard NIST 08 MS library and on the comparison of retention indices (RI) sourced from the NIST Standard Reference Database MS Spectral Library 2014. The identification of the VCs by comparing the experimental mass spectra based on spectral similarity with those stored in the NIST MS library database, without chemical reference standards, should be regarded as putative compounds identifications (Level 2). Moreover, the determination of Kovats retention indices (RI) and their match with those available in previous literature [10,34] represents a useful tool for identification purposes, being independent of the operating conditions, except for the polarity of the used stationary phase. Furthermore, considering complex matrices as wort fermentation, which can contain hundreds of VCs to be analyzed, this analytical approach described above has the advantage that avoids the injection of the pure standards, favoring the time and cost of the analysis and the limitation that several standards are not commercially available. y In this study, we compared the VC production profile of two different yeast species, S. pastorianus (Sp.W34/70, commercial lager strain) and S. eubayanus (wild isolates from Chilean Patagonia) using HS-SPME-GC-MS. The main differences between S. eubayanus and S. pastorianus lie in the number (51 vs. 22) and the relative amounts of esters and phenols identified. These findings reflect the aromatic profile of the commercial strain, which likely lost the ability to produce a broad array of VCs due to a complex brewing domestication process. During this process, the fermentative capacity of the strain was prioritized over the production of complex aromatic profiles [35]. Esters were found in a higher proportion in the two yeast species analyzed in our study. These compounds are produced during the fermentation process and are usually characteristic of young beers [10]. Moreover, other compounds were identified, such as higher alcohols and phenols, of which the production is influenced by several factors, such as wort composition, fermentation parameters, and beer maturation state [34]. The presence of these compounds, together with volatile-esters, is responsible for contributing to the final beer flavor [1,8]. One of the most distinctive VCs produced by wild yeast is 4-VG [8]. The aroma and flavor descriptors used for this phenolic compound (identified in all S. eubayanus stains) are wide-ranging, and include stable, barnyard, horsey, leathery, smoky, spicy, clove, medicinal, and others (Dzialo M. et al. 2017; Lentz M. 4. Discussion 2018), contributing significantly to the organoleptic profile. The role of phenols in the aromatic profile of the beer is questionable; in individual beer styles, such as Bavarian wheat beers and Belgian white beers (Lentz M. 2018), the phenolic flavors are desired and contribute to the style of the beer. However, the same VCs are perceived as undesirable in other fermented beverages and are commonly referred to as “phenolic off-flavors” (POF). Similar studies using other S. eubayanus strains [36], indicated that this species produces higher levels of esters, phenols, and higher alcohols in fermentations at low temperatures (15 ◦C), compared to Sp.W34/70. Indeed, our results followed the same trend, as we detected an additional set of VCs previously uncharacterized in lager beers. In this context, considering the great and novel diversity of VCs produced, the S. eubayanus strains considered in our study show an innovative potential for alternative beer production. The volatile fraction of beer includes over 800 different compounds, but only several dozen of these are considered flavor active [37]. In this context, we selected compounds that could be relevant to discriminate the set of S. eubayanus strains considered in our study and performed a PCA. When analyzing the distribution of the isolates, according to their aromatic profile, we observed a random distribution of the S. eubayanus strains. There are no strains grouped by fermentation capacity or geographic isolation zone. By dividing the PCA into four quadrants (Q1–Q4), it is remarkable that strains with similar fermentative profiles are distributed in opposite quadrants. Given this, the quantity and variety of the VCs produced by S. eubayanus isolates seem to be strain-specific traits, and reflect the 16 of 19 16 of 19 Microorganisms 2020, 8, 755 great genetic diversity present in this species, as reported previously [19,22]. In this context, the genetic diversity and the different VCs identified in S. eubayanus resemble those described for S. cerevisiae, rather than S. pastorianus, likely determined by the genetic diversity of the species. S. eubayanus is not the sole species to produce large amounts of volatile esters. For example, S. cerevisiae strain Safale S-04 (Belgium) produces a wide range of these compounds, such as: ethyl acetate, isoamyl acetate, phenethyl acetate, ethyl decanoate, ethyl hexanoate, and ethyl octanoate [38,39], responsible for giving the fermented wort a fruity profile and in some cases a mixture with solvent aromas [7]. 4. Discussion These profiles are attractive in the worldwide beer market, and the development of lager beers with fruity aromatic profiles fermented at low temperatures could likely be of interest in the global beer industry. p p y g y The parameters that affect the response of yeasts during the biosynthesis of aromatic compounds are diverse [7]. The production of active aromatic compounds is directly related to the yeast strain used in fermentation [1], as the genome of each strain is unique and will ultimately impact the final aroma profile of the beverage [40]. This is very well reported in S. cerevisiae [12,41–43], while in S. eubayanus, only a handful of studies have evaluated the aromatic profile in beer [6,23,36]. Other relevant parameters reported are wort composition, oxygen [44], yeast assimilable nitrogen (YAN) [11,45,46], and temperature of fermentation [7,47,48]. In the present study, it was possible to observe that temperature exerted an effect on the total production of VCs depending on the genetic background of the S. eubayanus strains. These changes have been previously reported in yeast of the genus Saccharomyces, where fermentation temperatures above 20 ◦C modified the production of VCs in beer and wine [47]. A special case was the POF 4-VG, where no changes were observed in strain CL905.1, while in CL602.1, its content significantly increased when the fermentation temperature was increased (12◦to 20 ◦C). Most reports focused on the study of 4-VG have set out to find ways to reduce their production within the fermentation process by modulating precursors in the wort [8]; however, our results also demonstrate the impact of strain × temperature on the production of 4-VG. From a physiological perspective, none of the parameters analyzed in this study may solely explain the changes in the VCs produced in S. eubayanus strains, suggesting a complex temperature × strain response under fermentative conditions. An example is the YAN content. During alcoholic fermentation, the cells import and metabolize the YAN in the wort together with other nutrients to produce biomass as well as VCs [1,30,49,50]. Therefore, depending on the amount and type of assimilated YAN, different VCs will be generated, thus determining the aromatic profiles of the fermented wort [7,50]. The YAN content can be separated into two groups, ammonium and amino acids. Ammonium is the primary nitrogen source in beer and wine yeasts [30]. 4. Discussion Nitrogen consumption has been widely studied in wine yeast, establishing hierarchies in nitrogen source utilization [29]. For example, ammonium, glutamine, glutamate, and asparagine are considered good or preferred nitrogen sources. In S. eubayanus, we found that ammonium, aspartic acid, glutamic acid, serine, and threonine nitrogen sources were rapidly consumed and may represent the preferred nitrogen sources for wort fermentation. Interestingly, no differences were observed between strains in terms of such preferences, although temperature significantly impacted the nitrogen consumption rate at early timepoints. 5. Conclusions Sensory properties of beer are influenced by the wide variety of VCs produced by yeast. The Patagonian populations of S. eubayanus comprise phenotypically distinct individuals, representing a niche of innovation for a wide range of volatile compounds in beer. Overall, S. eubayanus strains produce a more significant number of volatile compounds compared to a commercially available lager yeast. In this context, fermentation temperature plays an essential role, together with the genetic background of the S. eubayanus strain, in determining the VC profile of the resulting beer. The variation of the fermentation temperature can increase (CL905.1) or decrease (CL602.1) the amount and type of VCs depending on the strain, generating a fine-tuning mechanism on the total production of compounds that contribute to the complete and complex aromatic profile of the beer. HS-SPME-GC-MS is a handy tool to analyze the main VCs present in the fermented wort, allowing the reliable identification of the 17 of 19 17 of 19 Microorganisms 2020, 8, 755 large variety of compounds produced during the fermentation process. In this way, the procedures that were followed in this report lay the foundation to study a wider set of strains, deepening our understanding of the catalog of VCs produced by S. eubayanus natural isolates, which have an enormous innovation potential. large variety of compounds produced during the fermentation process. In this way, the procedures that were followed in this report lay the foundation to study a wider set of strains, deepening our understanding of the catalog of VCs produced by S. eubayanus natural isolates, which have an enormous innovation potential. Supplementary Materials: The following are available online at http://www.mdpi.com/2076-2607/8/5/755/s1. Figure S1 Fermentation profiles of S. eubayanus strains from central and southern Chile in beer wort. The fermentative kinetics of geographically-grouped wild yeast strains represent their fermentative capacity, as measured by CO2 loss (g/L). (a) Central strains: Region of Maule, Bío-Bío and La Araucanía. (b) Southern strains: Region of Los Ríos, Los Lagos, and Aysén. (c) Far southern strains: Region of Magallanes. As a negative control, we used beer wort without yeast cells. The commercial lager strain S. pastorianus W34/70 was used as a fermentation control. Figure S2. Pearson Correlation between the main volatile compounds identified and latitude. Each point depicts an average of the relative amount produced. Figure S3. Fermentation profiles of CL905.1 and CL602.1 strains in beer wort. References 1. Pires, E.J.; Teixeira, J.A.; Branyik, T.; Vicente, A.A. Yeast: The soul of beer’s aroma—A review of flavour-active esters and higher alcohols produced by the brewing yeast. Appl. Microbiol. Biotechnol. 2014, 98, 1937–1949. [CrossRef] [PubMed] 1. Pires, E.J.; Teixeira, J.A.; Branyik, T.; Vicente, A.A. Yeast: The soul of beer’s aroma—A review of flavour-active esters and higher alcohols produced by the brewing yeast. Appl. Microbiol. Biotechnol. 2014, 98, 1937–1949. [CrossRef] [PubMed] 2. Olaniran, A.O.; Hiralal, L.; Mokoena, M.P.; Pillay, B. Flavour-active volatile compounds in beer: Production, regulation and control. J. Inst. Brew. 2017, 123, 13–23. [CrossRef] 2. Olaniran, A.O.; Hiralal, L.; Mokoena, M.P.; Pillay, B. 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The molecular biology of fruity and floral aromas in beer and other alcoholic beverages. FEMS Microbiol. Rev. 2019, 43, 193–222. [CrossRef] [PubMed] 5. Baker, E.P.; Hittinger, C.T. Evolution of a novel chimeric maltotriose transporter in Saccharomyces eubayanus from parent proteins unable to perform this function. PLoS Genet. 2019, 15, e1007786. [CrossRef] 5. Baker, E.P.; Hittinger, C.T. Evolution of a novel chimeric maltotriose transporter in Saccharomyces eubayanus from parent proteins unable to perform this function. PLoS Genet. 2019, 15, e1007786. [CrossRef] 6. Gibson, B.; Geertman, J.A.; Hittinger, C.T.; Krogerus, K.; Libkind, D.; Louis, E.J.; Magalhaes, F.; Sampaio, J.P. New yeasts-new brews: Modern approaches to brewing yeast design and development. FEMS Yeast Res. 2017, 17. [CrossRef] 6. Gibson, B.; Geertman, J.A.; Hittinger, C.T.; Krogerus, K.; Libkind, D.; Louis, E.J.; Magalhaes, F.; Sampaio, J.P. New yeasts-new brews: Modern approaches to brewing yeast design and development. FEMS Yeast Res. 2017, 17. [CrossRef] 7. Dzialo, M.C.; Park, R.; Steensels, J.; Lievens, B.; Verstrepen, K.J. 5. Conclusions The fermentative kinetics of both wild yeast strains represent their fermentative capacity, as measured by CO2 loss (g/L). (a) Fermentation at 12 ◦C. (b) Fermentation at 20 ◦C. Table S1. Saccharomyces eubayanus isolates used in this work. Table S2. Presence/Absence table of Volatile compounds detected in S. eubayanus. Table S3. Presence/Absence of Volatile compounds detected in CL905.1 and CL602.1 strain at two fermentation temperatures. Table S4. Total YAN consumption. Table S5. YAN consumption at 24 h of Fermentation. Table S6. YAN consumption at 48 h of Fermentation. Table S7 YAN consumption at 168 h of Fermentation. Author Contributions: K.U.: Investigation, Data curation, and Formal analysis; P.V.: Writing—Original Draft, Formal analysis and Methodology; R.S. (Rocio Santander): Data curation and Methodology; R.F.N.: Conceptualization, resources, and supervision, R.S. (Ricardo Salazar): Conceptualization, resources, and supervision, F.A.C.: Conceptualization, Methodology, Writing—Reviewing and Editing, and Funding acquisition. All authors have read and agreed to the published version of the manuscript. Acknowledgments: F.A.C. was supported by the Comisión Nacional de Investigación Científica y Tecnológica CONICYT FONDECYT 1180161 and Millennium Institute for Integrative Biology (iBio). P.V. was funded by FONDECYT POSTDOCTORAL 3200575 and Universidad de Santiago de Chile 021843CR_POSTDOC. R.S. (Ricardo Salazar) was supported by CONICYT FONDECYT 1170352. R.F.N. was supported by FIC ‘Transferencia Levaduras Nativas para Cerveza Artesanal’ and CONICYT FONDECYT 1180917. K.U. was funded by USA1899–Vridei 021943CR-PAP Universidad de Santiago de Chile. R.S. 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https://bmcneurol.biomedcentral.com/track/pdf/10.1186/s12883-020-01876-0

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Association between white matter impairment and cognitive dysfunction in patients with ischemic Moyamoya disease

BMC neurology

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Liu et al. BMC Neurology (2020) 20:302 https://doi.org/10.1186/s12883-020-01876-0 Liu et al. BMC Neurology (2020) 20:302 https://doi.org/10.1186/s12883-020-01876-0 Open Access Abstract Background: Ischemic Moyamoya disease is one of the important causes of stroke, which leads to severe impairment in cognitive functions. This cognitive impairment occurs prior to stroke. However, the cognitive functions that are impaired and the mechanisms of these impairments have not been determined. Methods: We analyzed 12 patients with Moyamoya disease and 12 controls. All participants underwent cognitive tests and magnetic resonance imaging (MRI) scans. The diffusion tensor imaging (DTI) data was processed using Tract-Based Spatial Statistics (TBSS). Significantly different white matter areas were correlated with different cognitive functions. Results: There were significant differences in intelligence and subtraction between the patients and controls (p < 0.05). The parameters of DTI such as fractional anisotropy (FA), mean diffusivity (MD), axial diffusivity (AD), and radial diffusivity (RD) have different changes in anterior thalamic radiation, inferior fronto-occipital fasciculus (IFO), superior longitudinal fasciculus (SLF), uncinate fasciculus (UF), inferior longitudinal fasciculus, forceps minor, and other regions between the two groups. Conclusion: Left UF and IFO may be the key brain regions affecting arithmetic function, while bilateral IFO has an effect on intelligence. RD and AD may be better indicators for early prediction of chronic white matter damage than FA, while MD tends to have a comprehensive indirect change. There is cognitive impairment in ischemic MMD, which is closely related to white matter impairment. Trial registration: Clinical Trial Registration, Unique identifier: ChiCTR1900023610. Registered 4 June 2019 – Prospective study registered. Keywords: Moyamoya disease, Cognitive dysfunction, Diffusion tensor imaging, Tract-based spatial statistics, Ischemic cerebrovascular disease * Correspondence: [email protected] g †Ziqi Liu and Shihao He contributed equally to this work. 1Department of Neurosurgery, Beijing Tiantan Hospital, Capital Medical University, 119 South Fourth Ring West Road, Fengtai District, Beijing 100070, China †Ziqi Liu and Shihao He contributed equally to this work. 1Department of Neurosurgery, Beijing Tiantan Hospital, Capital Medical University, 119 South Fourth Ring West Road, Fengtai District, Beijing 100070 China 2Center of Stroke, Beijing Institute for Brain Disorders, Beijing 10069, China Full list of author information is available at the end of the article Background participants. The study included 12 patients with MMD (5 men, 7 women; mean age 42.83 ± 8.80 years old; mean education: 9.83 ± 4.09 years) from the Neurosurgery Department of Beijing Tiantan Hospital which is affiliated to the Capital Medical University and Peking University International Hospital between June 2019 and December 2019. Moreover, the control group included 12 volunteers (7 men, 5 women; mean age 39.33 ± 10.82 years old, mean education: 12.42 ± 3.42 years). There was no significant difference in sex composition, age, education level, and risk factors be- tween the two groups (P > 0.05). Details of Suzuki Stage could be found in Tables 1 and 2. g Moyamoya disease (MMD) is a congenital cerebrovas- cular malformation that leads to progressive stenosis of the main arteries of the brain and compensatory proliferation of small puffy vessels in the base of brain [1], which eventually leads to serious cerebro- vascular accidents, particularly stroke. Previously, the risk of dementia after stroke was reported to be as high as 30% for all kinds of diseases [2], while the risk of dementia before stroke was approximately 9– 14% [3]. In a small sample study, about two-thirds of patients with MMD have cognitive impairment [4], however, not all patients with cognitive dysfunction suffer from stroke. MMD has a significant effect on the cognitive abilities of adults without stroke, and in about a quarter of patients [5], the degree of cogni- tive impairment may affect daily function. It is con- cerning that MMD patients without stroke have a high rate of dementia. The impairment of cognitive function in MMD has been widely studied. Previous studies have found that MMD patients have signifi- cantly lower executive function, attention, and short- term memory than controls [5–7], but the difference in the structure of the brain between patients and controls is not known. Neuroimaging is a uniquely noninvasive way to study brain structure. Therefore, we used neuroimaging to find a link between different parts of the brain and cognitive functions. © The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Liu et al. BMC Neurology (2020) 20:302 Liu et al. BMC Neurology (2020) 20:302 Page 2 of 11 Background The inclusion criteria of patients were as follows:(1) All patients should meet the Guidelines for Diagnosis and Treatment of Moyamoya Disease (Spontaneous Occlusion of the Circle of Willis), the research com- mittee on the pathology and treatment of spontan- eous occlusion of the circle of willis; health labour sciences research grant for research on measures for Table 1 Basic information of patients and controls Patients (n = 12) Controls (n = 12) Statistics Variables Mean ± SD Mean ± SD P values Sex (M:F) 05:07 07:05 0.414 Age (years) 42.83 ± 8.80 39.33 ± 10.82 0.394 Education 9.83 ± 4.09 12.42 ± 3.42 0.107 Medical history, n (%) Hypertension 2 (16.7) 2 (16.7) 1 Dyslipidemia 3 (25.0) 1 (8.3) 0.273 Smoking history 3 (25.0) 2 (16.7) 0.615 Alcohol taking 1 (8.3) 2 (16.7) 0.537 Suzuki Stage Left 1 2 (16.7) 2 2 (16.7) 3 5 (41.7) 4 2 (16.7) 5 1 (8.3) 6 0 Right 1 1 (8.3) 2 1 (8.3) 3 8 (66.7) 4 1 (8.3) 5 1 (8.3) 6 0 Table 1 Basic information of patients and controls Neuroimaging, especially diffusion tensor imaging (DTI), is important for studying brain structure and function. It has been reported that DTI is highly sen- sitive to changes in the microstructure of white mat- ter diffusion characteristics, which is mainly used for nervous system disease with white matter damage but rarely in MMD. Previously, Kazumata et al. [8] found that the mean fractional anisotropy (FA) of white matter tracts in the lateral prefrontal lobe, cingulate region, and inferior parietal region was significantly correlated with processing speed, executive function, and working memory. However, the number of such studies in MMD is limited. We aimed to study white matter fiber bundle damage in MMD patients without stroke by DTI to investigate whether the site of dam- age in white matter fiber bundles is associated with certain cognitive impairment and thus, determine the cause of cognitive impairment in patients without stroke. Values are numbers of cases (%) unless otherwise indicated. Mean values are presented with SDs Values are numbers of cases (%) unless otherwise indicated. Mean values are presented with SDs Abbreviations: F female, M male, SD standard deviation Methods Patients This prospective study was approved by the research ethics committee of Beijing Tiantan Hospital affiliated to Capital Medical University (KYSQ2019–058-01). Written informed consent was obtained from all Liu et al. BMC Neurology (2020) 20:302 Page 3 of 11 Liu et al. BMC Neurology Table 2 Patients characteristics Sex Education (year) Clinical Presentation Suzuki Grade Medical History Vessel Stenosis or Occlusion Pattern Left Right Left Right F 15 TIA 3 4 None ACA* MCA* ICAO* MCA* F 9 Asymptomatic 2 3 None MCA MCA M 12 Asymptomatic 1 3 Hypertension ICAS ICAS MCA F 4 Asymptomatic 3 1 None ACA* MCA* ICAS F 8 TIA 3 3 None ACA* MCA* F 3 TIA 1 3 Dyslipidemia ICAS ACA MCA M 15 TIA 3 3 None ACA MCA ACA MCA F 9 TIA 5 3 None ICAO* ICAO* M 9 TIA 3 3 Dyslipidemia ICAS ACA MCA MCA* Hypertension F 16 TIA 4 5 None ICAO* ACA MCA ICAO* ACA MCA M 9 Asymptomatic 4 3 None ICAO* ICAS ACA MCA M 9 Asymptomatic 2 2 Dyslipidemia MCA ICAS Abbreviations: F female, M male, TIA transient ischemic attack, ACA anterior cerebral artery, MCA middle cerebral artery, ICAS internal carotid artery stenosis, ICAO internal carotid artery occlusion; *The asterisk stands for occluded vessels. (for example: ACA* means anterior cerebral artery occlusion) The black word stands for narrow vessels. (for example: MCA means middle cerebral artery stenosis) Abbreviations: F female, M male, TIA transient ischemic attack, ACA anterior cerebral artery, MCA middle cerebral artery, ICAS internal carotid artery stenosis, ICAO internal carotid artery occlusion; *The asterisk stands for occluded vessels. (for example: ACA* means anterior cerebral artery occlusion) The black word stands for narrow vessels. (for example: MCA means middle cerebral artery stenosis) internal carotid artery occlusion; *The asterisk stands for occluded vessels. (for example: ACA* means anterior cerebral artery occlusion) The black word stands for narrow vessels. (for example: MCA means middle cerebral artery stenosis) internal carotid artery occlusion; *The asterisk stands for occluded vessels. (for example: ACA* means anterior cerebral artery occlusion) The black word stands for narrow vessels. (for example: MCA means middle cerebral artery stenosis) no history of drugs usage that could affect cognitive function. Methods Patients intractable diseases [9]; (2) In the MMD patients group, there was no previous ischemic or hemorrhagic attack, and in the case of intracranial la- cunar cerebral infarction, the lesion area should be less than 1.5 cm; (3) righthand dominance; (4) being free of dementia, or depression; and (5) no major psy- chiatric disease or other medical conditions. MRI acquisition MRI data were obtained using a 3.0-Tesla MR system (Verio A Tim + Dot System, Siemens, Germany). Volu- metric T1 (three-dimension, 3D) gradient echo was ac- quired in the sagittal plane with a thickness of 1 mm (flip angle = 8, time of repetition (TR) = 2300 ms, time of echo (TE) = 3.25 ms, matrix =256 × 256, FOV = 250 × 250 mm). DTI following echo planar imaging (EPI) was acquired in 30 directions (flip angle = 180°, voxel size = 1.8 × 1.8 × 4.0 mm3, TR =3600 ms, TE = 95 ms, matrix = 128 × 128, FOV = 230 × 230 mm, 25 cuts with 4 mm thickness, b-value = 1000). The exclusion criteria of patients were as follows: (1) Acute stage of cerebral infarction and other neuropsychiatric diseases, severe systemic diseases, and severe systemic diseases (e.g., AD, Parkinson’s disease); (2) any contraindications for MR scans (e.g., metal implants); (3) Manifestation of any medications that could affect cognitive abilities; (4) fatigue or hunger; or (5) an inability to complete the tasks independently. Tract-based spatial statistics analysis By means of social recruitment, we released recruit- ment advertisements for the control group. A total of 20 people was included in the control group. Through asking medical history, we recorded clinical variables such as age, sex and past medical history, and con- ducted cognitive test after MRI examination. 8 people were excluded through exclusion criteria because of brain cysts (1), incomplete cognitive test (3), large head movement or low signal-to-noise ratio (SNR) during MRI scanning (4). Twelve people were finally included in the control group and matched with the MMD group. Tract-based spatial statistics analysis DTI preprocessing used PANDA [10] pipeline which was conducted through FMRIB Software Library (FSL 5.0.9, University of Oxford, UK, http://www.fmrib.ox.ac. uk/fsl). Firstly, in the raw DTI images, using the FSL Eddy Correction Tool [11], eddy current distortions and motion artifacts were corrected. Then, the cor- rected DTI images were stripped to remove non-brain tissues like the skull and muscle by the FSL Brain Ex- traction Tool (BET) [12]. Secondly, each individual images, including FA, mean diffusivity (MD), and three eigenvalues λ1, λ2 and λ3, were calculated using the FSL diffusion tensor analysis toolkit (FDT) [13]. Axial diffusivity (AD) was generally accepted as the largest eigenvalue (λ1), while radial diffusivity (RD) Inclusion criteria for the control group were as fol- lows: no clinical evidence of psychiatric or neuro- logical disease, no brain damage on routine MRI, and Liu et al. BMC Neurology (2020) 20:302 Page 4 of 11 Page 4 of 11 Liu et al. BMC Neurology (2020) 20:302 was defined as the mean of the two smaller eigen- values (λ2 and λ3). Then MD was calculated as the mean of the three eigenvalues (λ1, λ2, and λ3). Next, we analyzed the FA images of all the patients and controls in the Tract-Based Spatial Statistics (TBSS) analysis [14] within FSL following the standard pipe- line (www.fsl.fmrib.ox.ac.uk/fsl/fslwiki/TBSS). All FA images were nonlinear-registered to Montreal Neuro- logical Institute 152 (MNI 152) space through the FSL registration tool FNIRT, and the mean FA images and skeleton (FA threshold 0.2) were created then. Fi- nally, all participants’ FA images were projected onto this skeleton to create normalized skeletonized FA images. Then, similarly, through the nonlinear trans- formation of FA images, AD, MD, and RD images were all registered to the Montreal Neurological Insti- tute (MNI) standard space and individual skeletonized images were generated for next analysis. Statistical analysis Voxel-wise statistics across participants were put into ef- fect for each voxel of FA images. We used 5000 permu- tations and Threshold-Free Cluster Enhancement (TFCE) to correct multiple comparisons. Considering re- sults of the voxel-wise analyses, we reported the signifi- cant clusters ≥15 voxels, labeled them according to the Johns Hopkins University JHU-ICBM-tracts atlas. Then we binarized the TFCE corrected statistical maps into masks (uncorrected p < 0.05). Finally, Pearson correl- ation analysis was performed between the significant cognitive scores and clusters of white matter fibers for patients and healthy controls, (p < 0.05) [24] in SPSS 19.0 (IBM Corp. Released 2010. IBM SPSS Statistics for Windows, Version 19.0. Armonk, NY: IBM Corp.). Simi- larly, we repeated the same analyses for the AD, MD, and RD values, but we binarized the TFCE corrected statistical maps into masks with corrected p < 0.05. Tract-based spatial statistics analysis determine the degree of cognitive decline in daily life [22]. Additional details on the cognitive assessment questions can be found elsewhere [23]. The partici- pants were tested using computer workstations by neuropsychologists who were blinded to the clinical data. The interval between neuropsychological testing and MRI examination was < 5 days. Cognition acquisition All cognitive assessment programs were tested using the Online Psychological Experimental System. Choice reaction time (RT) was used as the baseline condition and the index of movement ability. The objective of using the basic RT task was to determine manual re- sponse effect and main processing speed. The choice RT task was adapted from the simple RT task from Butterworth’s Dyscalculia Screener [15]. In all 30 tri- als of this task, a white fixation cross and a white dot were presented on a black screen. The former was presented in the center of the screen, and the latter was presented on the left or right side of the fixation cross (It was present on the left side in half the cases). Participants should press “Q” or “P” with their left or right fingers if the dot appeared on the left or right side of the fixation cross, respectively. The inter- stimulus interval was randomized between 1500 and 3000 ms. Nonverbal matrix reasoning was used to as- sess general intelligence and reasoning ability. The task was adapted from the abstract reasoning ability part of Raven’s Progressive Matrices (Raven, 2000). The mental rotation was used to evaluate the visual- spatial ability [16]. Verbal working memory was used to measure working memory capacity [17]. A multi- step, multilocation search task was used to evaluate executive function [18]. Simple and complex subtrac- tion problems were used to assess simple calculation ability and magnitude representation [19]. Word- memory ability and visual short-term memory were measured with the short-term memory (STM) span for Chinese words and phrases and the picture STM test, respectively [20]. The Edinburgh Handedness Inventory was used to investigate left and right- handedness [21]. The AD8 questionnaire was used to Cognition result BMC Neurology (2020) 20:302 Table 3 Summary of neuropsychologic assessments in each group Variables Patients (n = 12) Controls (n = 12) Statistics Mean ± SD Mean ± SD P values AD-8 1.08 ± 1.311 0.42 ± 0.67 0.131 CRT_RT 999.25 ± 809.84 483.42 ± 249.89 0.055 CRT_ACC 93.00 ± 17.29 99 ± 1.48 0.256 SPM 13.92 ± 4.91 25.17 ± 8.56 0.001** ROT 12.08 ± 5.65 18.33 ± 7.40 0.030* VWM1 7.33 ± 1.88 8.67 ± 1.23 0.053 VWM2 5.42 ± 1.51 7.83 ± 1.64 0.001** SUB 29.92 ± 14.50 43.83 ± 9.18 0.011* COMSUB 13.50 ± 6.65 24.33 ± 6.07 0.000** WORDM 55.67 ± 14.27 69.67 ± 9.22 0.009** PICTM 68.33 ± 17.97 76.33 ± 4.33 0.148 EXCUT1 0.58 ± 2.39 −0.83 ± 2.89 0.204 EXCUT2 −1.67 ± 4.05 −3.00 ± 2.26 0.333 ANXIETY 1.92 ± 2.28 0.50 ± 0.80 0.062 DEPRESS 0.50 ± 0.91 0.17 ± 0.39 0.259 Abbreviation: SD standard deviation, CRT_RT/ACC Choice reaction time_ reaction time/ accuracy, SPM Raven’s Standard Progressive Matrices, ROT Mental rotation, VWM verbal working memory, digit span, 1, Recite in order, 2, Recite in reverse order, SUB Simple subtraction, COMSUB Complex subtraction, WORDM word-memory, PICTM picture-memory, EXCUT Executive function,1, same direction, 2, Opposite direction, ANXIETY, Hamilton Anxiety Scale, DEPR ESS Hamilton Depression Scale *p < 0.05, **p<0.01 left ATR (r = −0.656, p = 0.021), left IFO (r = −0.748, p = 0.005), and left UF (r = −0.622, p = 0.031) were negatively correlated as well. The complex subtraction and MD of left IFO (r = −0.651, p = 0.022) and left UF (r = −0.623, p = 0.031) and RD of left IFO (r = − 0.697, p = 0.012) were negatively correlated. left ATR (r = −0.656, p = 0.021), left IFO (r = −0.748, p = 0.005), and left UF (r = −0.622, p = 0.031) were negatively correlated as well. The complex subtraction and MD of left IFO (r = −0.651, p = 0.022) and left UF (r = −0.623, p = 0.031) and RD of left IFO (r = − 0.697, p = 0.012) were negatively correlated. Discussion Diffusion tensor imaging (DTI) has been proven to be an effective method for detecting white matter integ- rity and exploring the relationship between brain microstructure and cognitive function [25–28]. This is one of the few studies on white matter and cognitive function in asymptomatic ischemic MMD patients. Moreover, our analysis method and the patient selec- tion criteria are more reasonable than that of previ- ous studies, and we found asymptomatic ischemic MMD white matter lesions and cognitive changes. We have performed and explained each of the correl- ation analysis. Abbreviation: SD standard deviation, CRT_RT/ACC Choice reaction time_ reaction time/ accuracy, SPM Raven’s Standard Progressive Matrices, ROT Mental rotation, VWM verbal working memory, digit span, 1, Recite in order, 2, Recite in reverse order, SUB Simple subtraction, COMSUB Complex subtraction, WORDM word-memory, PICTM picture-memory, EXCUT Executive function,1, same direction, 2, Opposite direction, ANXIETY, Hamilton Anxiety Scale, DEPR ESS Hamilton Depression Scale *p < 0.05, **p<0.01 Abbreviation: SD standard deviation, CRT_RT/ACC Choice reaction time_ reaction time/ accuracy, SPM Raven’s Standard Progressive Matrices, ROT Mental rotation, VWM verbal working memory, digit span, 1, Recite in order, 2, Recite in reverse order, SUB Simple subtraction, COMSUB Complex subtraction, WORDM word-memory, PICTM picture-memory, EXCUT Executive function,1, same direction, 2, Opposite direction, ANXIETY, Hamilton Anxiety Scale, DEPR ESS Hamilton Depression Scale *p < 0.05, **p<0.01 values. MD and AD values of the patient group in forceps minor, bilateral superior longitudinal fascic- ulus (SLF), bilateral ATR, bilateral inferior frontal- occipital fasciculus (IFO), and left uncinate fasciculus (UF) were higher than those in the control group (TFCE corrected p < 0.05); RD values of the patient group were higher in the forceps minor, left IFO, left ATR, and left UF than those in the control group (TFCE corrected p < 0.05). The details are shown in Fig. 1 and Table 4. y In the cognitive results, we found that the main cognitive impairments in the MMD group were mainly in the areas of logical reasoning, short-term memory, executive function, and calculation, which was similar to that reported in previous studies; how- ever, our analysis was detailed. Previous studies have found that MMD patients have significantly lower ex- ecutive function, attention, and short-term memory than normal control groups. Long-term ischemia in MMD patients leads to cognitive impairment that in- cludes selective impairment of these cognitive func- tions. Cognition result Cognitive tests were performed on 12 MMD patients and 12 controls, and it was found that the function of Raven’s Standard Progressive Matrices (SPM), Mental rotation (ROT), verbal working memory 2(VWM2), Sim- ple subtraction (SUB), Complex subtraction (COMSUB), and word-memory (WORDM) in the patient group were all significantly lower than that in the control group (p < 0.05). Moreover, there were very significant differences in RAVEN, VWM2, COMSUB, and WORDM functions between the patients and the control group (p < 0.01). Moreover, it is noteworthy that, although differences in VWM1 (p = 0.053) and ANXIETY (p = 0.062) were insig- nificant, there was a decline in the scores in the patient group. More cognitive details could be found in Table 3. Microstructural changes in white matter fibers Compared with the control group, we found changes in FA, MD, AD, and RD in the patient group. With 5000 permutations and TFCE, we found that the FA value differences between patient group and control group were found in the forceps minor, right anterior thalamic radiation (ATR), and right frontal occipital fasciculus (MMD<healthy controls (HC), TFCE uncor- rected p < 0.05). The differences in MD, AD, and RD values were more extensive than the differences in FA Page 5 of 11 Liu et al. Discussion MMD mostly causes narrowing or occlusion of the internal carotid artery, middle cerebral artery, and anterior cerebral artery, and the regions supported by these vessels are mostly located in the anterior half of the brain, including the frontal lobe, temporal lobe, and parietal lobe. The main aim of this study was to analyze whether there was substantial damage to the white matter in these areas. Cognition result g y In the control group, we found that the simple sub- traction and AD of left IFO (r = 0.612, p = 0.034) and forceps minor (r = 0.701, p = 0.011) was positively corre- lated; additionally, simple subtraction and MD of forceps minor (r = 0.582, p = 0.047) was positively correlated. Verbal working memory 2 and MD value of left IFO (r = 0.710, p = 0.010) and forceps minor (r = 0.695, p = 0.012) was positively correlated. Moreover, verbal work- ing memory 2 and AD value of left IFO (r = 0.679, p = 0.015) and forceps minor (r = 0.697, p = 0.012) was posi- tively correlated. Other brain regions were not found to be correlated with each index of DTI. The result is shown in Fig. 2 and Fig. 3. Correlation of DTI index and cognition We analyzed the correlation between DTI indicators, in- cluding FA, MD, AD, and RD values, and cognitive func- tion with significant differences between the patient and control groups. In the patient group, there was a negative correl- ation between Raven reasoning test and MD in bilat- eral IFO (left r = −0.645, p = 0.023; right r = −0.73, p = 0.007), right SLF (r = −0.585, p = 0.046), and left UF (r = −0.576, p = 0.050). Moreover, Raven reasoning test and AD of right SLF (r = −0.673, p = 0.016) were negatively correlated. Simple subtraction and MD of left ATR (r = −0.642, p = 0.024), left IFO (r = −0.686, p = 0.014), and left UF (r = −0.669, p = 0.017) were negatively correlated. Simple subtraction and RD of While analyzing the differences in white matter fiber microstructure between the MMD group and Page 6 of 11 Liu et al. BMC Neurology (2020) 20:302 Liu et al. BMC Neurology Fig. 1 Differences in FA, MD, RD, and AD values in different regions of the white matter between the patient group and control group after 5000 permutations and TFCE corrected p<0.05 (MD, AD, RD) or TFCE uncorrected p<0.05 (FA) Fig. 1 Differences in FA, MD, RD, and AD values in different regions of the white matter between the patient group and control group after 5000 permutations and TFCE corrected p<0.05 (MD, AD, RD) or TFCE uncorrected p<0.05 (FA) g. 1 Differences in FA, MD, RD, and AD values in different regions of the white matter between the patient group and control group after 5000 ermutations and TFCE corrected p<0.05 (MD, AD, RD) or TFCE uncorrected p<0.05 (FA) Fig. 1 Differences in FA, MD, RD, and AD values in different regions of the white matter between the patient gr permutations and TFCE corrected p<0.05 (MD, AD, RD) or TFCE uncorrected p<0.05 (FA) Fig. 1 Differences in FA, MD, RD, and AD values in different regions of the white matter between the patient group and control group after 5000 permutations and TFCE corrected p<0.05 (MD, AD, RD) or TFCE uncorrected p<0.05 (FA) values observed in MMD patients and the increase in MD, RD, and AD values over a wide area are due to a change caused by the increase of cellulose water composition caused by ischemia and the damage of myelin sheath. Correlation of DTI index and cognition BMC Neurology (2020) 20:302 Table 4 Significantly different white matter regions Index Number of voxels Peak coordinate (MNI) Label X Y Z FA MMD < HC 39 −17 50 12 forceps minor 23 38 33 27 ATR R 23 15 57 5 IFO R 22 −9 −17 67 SLF L 19 28 17 4 SLF R 18 27 21 0 UF R MD MMD > HC a forceps minor SLF R ATR L ATR R IFO L IFO R UF L AD MMD > HC a forceps minor ATR L ATR R SLF L SLF R IFO L IFO R ILF L UF L RD MMD > HC a IFO L ATR L forceps minor UF L Table 4: DTI index and significant different cluster in JHU-ICBM-tracts atlas and peak coordinate in MNI. Abbreviation: ATR anterior thalamic radiation, IFO inferior frontal-occipital fasciculus, SLF superior longitudinal fasciculus, UF uncinate fasciculus, ILF inferior longitudinal fasciculus, L left, R right. aThe voxels of the cluster which is combined with several regions are too large to locate the peak coordinate disease risk between the enrolled patients and the control group, and the DTI analysis adopted the method of self-selecting ROI in the SPM8. However, there were no significant differences in age, sex, edu- cation level, and risk between the patient group and the control group, excluding diseases such as diabetes that may cause damage to white matter. Furthermore, we used TBSS to analyze DTI data, which is more technically advanced than SPM8. Although there are several differences, many of the results are similar. For example, Kazumata et al. found that the DTI index changes in the knee and bilateral longitudinal bundle of the corpus callosum, which coincides with our results. The correlation analysis of cognition and DTI parame- ters showed that the cognitive function reduction of MMD patients mainly focuses on Raven’s reasoning test, subtraction, and complex subtraction. Therefore, we mainly discussed the arithmetic ability and logical rea- soning ability of patients. Math calculation is a complex skill and not a sim- ple process [37–39]. It requires many cognitive pro- cesses, including attention, working memory, and processing speed, in addition to specific mathematical skills. Previous studies have reported that math calcu- lation was associated with atypical brain function as well as atypical brain structure and connectivity [40]. The study of Navas-Sanchez et al. Correlation of DTI index and cognition [41] on 13 math- ematically gifted children and 23 controls found that in the mathematically gifted children, the FA value increased in the bilateral SLF, IFO, ATR, and left UF. Li et al. [42] found that the FA value reduction of left ILF and bilateral IFO was negatively correlated with the decline of calculation ability in the study of math- ematical subtraction ability of 47 children. In the study of 30 adults with dyscalculia and 17 controls, Kucian et al. [43] found that the FA value of bilateral SLF in dyscalculia adults was reduced, and this corre- lated with mathematical ability. In our study, the FA value of these regions in MMD patients decreased, but was not correlated with mathematical subtraction. On the contrary, in the left ATR, IFO, and UF, there was a significant negative correlation between RD and MD values and mathematical subtraction ability. Add- itionally, this indicates that RD value may be a more sensitive indicator than FA value. In the previous meta-analysis [13], it was found that the higher the MD value in the UF region, the worse the attention, processing speed, and working memory. Although this does not explicitly point to mathematical power, it will undoubtedly have an impact on math calculation. Many studies [44–47] have reported a correlation be- tween the left hemisphere, but not the right hemi- sphere, and computational power, which we speculate Table 4: DTI index and significant different cluster in JHU-ICBM-tracts atlas and peak coordinate in MNI. Abbreviation: ATR anterior thalamic radiation, IFO inferior frontal-occipital fasciculus, SLF superior longitudinal fasciculus, UF uncinate fasciculus, ILF inferior longitudinal fasciculus, L left, R right. aThe voxels of the cluster which is combined with several regions are too large to locate the peak coordinate and RD values change, and the increase in MD may be due to indirect reasons, while the FA value does not change as most of the cognitive functions are re- served. In addition, it is worth mentioning that both FA and MD change with age [33–35], therefore, age factors were considered in the inclusion of patients and controls to exclude the influence of age. Previous studies by Kazumata et al. [36] found that the changes in FA, MD, and RD parameters in MMD patients were relatively extensive, while there was al- most no change in AD parameters. This is not en- tirely consistent with our results. Correlation of DTI index and cognition First, the pathological process of MMD is considered to be a kind of ischemic change, and long-term brain main blood supply artery ische- mia is bound to lead to overall brain ischemia and hypoxia. Although the damage at the cellular level is unknown, but this kind of chronic ischemia may cause structural changes in the cells, and this is con- sistent with an increase in the MD, AD and RD values. Therefore, we have reason to suspect that MMD ischemic injury can damage the myelin sheath and affect the transmission function of white matter fiber tracts, and thus, affect its areas of cognitive function. In the early days of the change, mainly AD the control group, we observed changes in FA, MD, AD, RD, and other indicators. In general, compared with the control group, the MMD group showed a decrease in FA and an increase in MD, AD, and RD values, but brain area with reduced FA was relatively small (TFCE uncorrected p < 0.05), while the MD, AD, and RD showed differences over a wide range of brain area in the patient group (TFCE corrected p < 0.05). This is a very interesting phenomenon. Accord- ing to previous studies, RD and AD are thought to be more sensitive measures of neurodegeneration changes than FA [29]. AD value is highly sensitive to the maturation of white matter and the increase in water components, while RD value is closely related to demyelination changes and myelin sheath diameter. MD is a reflection of cellularity, edema, and necrosis [30–32]. We suspect that the local decrease in FA Page 7 of 11 Page 7 of 11 Liu et al. Correlation of DTI index and cognition Although all patients were selected as asymptomatic ischemic MMD patients, there was a significant difference in Liu et al. BMC Neurology (2020) 20:302 Page 8 of 11 Liu et al. BMC Neurology Fig. 2 Correlation of cognition and WM regions in MMD group Fi C l f d WM MMD f iti d WM i i MMD Fig. 2 Correlation of cognition and WM regions in MMD group and hemorrhage in MMD, inclusion and exclusion criteria limit the number of patients. Although there was no statistical difference between patients and the controls, it is not so well matched with controls (con- trol group has more males, younger patients and more schooling). Larger numbers of matched controls were needed for better homogeneity. Second, for some cognitive test with several significant differ- ences, such as the rotation, we did not find associated brain regions to explain the differences. Third, al- though we included patients with bilateral MMD, the occlusive degree and location of bilateral vessels in these patients was not uniform, and the small cohort limited further stratification. Moreover, previous stud- ies have proved that DKI parameters, such as MK, are more sensitive than DTI parameters. Therefore, may be related to right-handedness and the dominant hemisphere. The Raven’s test is a purely nonverbal intelligence test. In previous studies for 16 adolescents [48], there was a significant positive correlation between FA value of the right suboccipital tract and IQ. Although we did not ob- serve the intelligence related to the FA values in these areas, a decrease in FA, MD, and AD values was ob- served in the left ILF and right IFO. Furthermore, in the correlation analysis, a negative correlation between MD values of bilateral IFO, left UF, and right SLF and raven score was observed. Funding This study was supported by the Beijing Municipal Science & Technology Commission (Z151100004015077-DR) and Beijing municipal health system high-level health technical personnel training program (2015–3-041-DR). The funding helped our researchers learn and analysis MRI techniques and helped test cognitive function in patient and control groups. Authors’ contributions Ischemic MMD has a unique effect on the cognitive function of the brain, especially in arithmetic and intelligence. Left UF and IFO may be the key brain regions affecting arithmetic function, while bilateral IFO has an effect on intelligence. Long-term chronic ischemia has damage to the white matter of both sides of the brain, but the left hemisphere is more serious than the right hemisphere. RD and AD may be better indicators for early prediction of chronic white matter damage than FA, while MD tends to be a represent a comprehensive indirect change. All the above evidences indicate that there is cognitive im- pairment in ischemic MMD, which is closely related to white matter impairment. RW, ZL, SH had full access to all of the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis. Study concept and design was performed by RW, ZL and SH. Acquisition of MRI and clinical data performed by ZL, ZX, RD, ZY and CX. The neuropsychological cognitive test was carried out by ZL, SH and LY. Analysis and interpretation of data performed by ZL, SH, RD. Drafting of the manuscript performed SH, RD, ZL, ZX. Critical revision of the manuscript for important intellectual content performed by RW, ZL, SH, and RD. Statistical analysis performed by ZL, SH, ZY and CX. Study supervision performed by RD, and RW. All authors have read and approved the manuscript. Limitations Our research has some limitations. First, the sample size of this study is not enough. Due to infarction Liu et al. BMC Neurology (2020) 20:302 Page 9 of 11 Liu et al. BMC Neurology Fig. 3 Correlation of cognition and WM regions in control group Fig. 3 Correlation of cognition and WM regions in control group we considered adding DKI scanning to improve our project. Finally, we would like to have a comparison before and after surgery to see if there is a possibility of improvement in these different brain regions. These studies are already under way. imaging; TBSS: Tract-Based Spatial Statistics; ATR: anterior thalamic radiation; IFO: inferior fronto-occipital fasciculus; SLF: superior longitudinal fasciculus; UF: uncinate fasciculus; ILF: inferior longitudinal fasciculus Acknowledgments Th k S S h f Thank Star South for her help and supports every day. Thank my roommates, especially Peng Wang, for being quiet when I study in dormitory. 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Homozygous MTAP deletion in primary human glioblastoma is not associated with elevation of methylthioadenosine

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1 Department of Cancer Systems Imaging, The University of Texas MD Anderson Cancer Center, Houston, TX, USA. 2 Department of Cancer Biology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA. 3 MD Anderson UT Health Graduate School of Biomedical Sciences, Houston, TX, USA. 4 Department of Chemistry and Lewis-Sigler Institute for Integrative Genomics, Princeton University, Princeton, NJ, USA. 5 Department of Neurosurgery, Henry Ford Hospital, Detroit, MI, USA. 6 The Jackson Laboratory for Genomic Medicine, Farmington, CT, USA. 7 Department of Neuro-Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA. 8 Department of Pathology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA. 9 Department of Medicine, Harvard Medical School, and Division of Signal Transduction, Beth Israel Deaconess Medical Center, Boston, MA, USA. 10 SPOROS Bioventures, Houston, TX, USA. ✉email: [email protected]; [email protected] NATURE COMMUNICATIONS | (2021) 12:4228 | https://doi.org/10.1038/s41467-021-24240-3 | www.nature.com/naturecommunications ARTICLE ARTICLE Homozygous MTAP deletion in primary human glioblastoma is not associated with elevation of methylthioadenosine Yasaman Barekatain 1,2,3✉, Jeffrey J. Ackroyd1,3, Victoria C. Yan 1,3, Sunada Khadka1,2,3, Lin Wang4, Ko-Chien Chen2,3, Anton H. Poral1, Theresa Tran1, Dimitra K. Georgiou 1, Kenisha Arthur1, Yu-Hsi Lin1, Nikunj Satani 1, Elliot S. Ballato 1, Eliot I. Behr 1,2, Ana C. deCarvalho 5, Roel G. W. Verhaak6, John de Groot7, Jason T. Huse8, John M. Asara9, Raghu Kalluri 2 & Florian L. Muller 1,10✉ Homozygous deletion of methylthioadenosine phosphorylase (MTAP) in cancers such as glioblastoma represents a potentially targetable vulnerability. Homozygous MTAP-deleted cell lines in culture show elevation of MTAP’s substrate metabolite, methylthioadenosine (MTA). High levels of MTA inhibit protein arginine methyltransferase 5 (PRMT5), which sensitizes MTAP-deleted cells to PRMT5 and methionine adenosyltransferase 2A (MAT2A) inhibition. While this concept has been extensively corroborated in vitro, the clinical rele- vance relies on exhibiting significant MTA accumulation in human glioblastoma. In this work, using comprehensive metabolomic profiling, we show that MTA secreted by MTAP-deleted cells in vitro results in high levels of extracellular MTA. We further demonstrate that homozygous MTAP-deleted primary glioblastoma tumors do not significantly accumulate MTA in vivo due to metabolism of MTA by MTAP-expressing stroma. These findings highlight metabolic discrepancies between in vitro models and primary human tumors that must be considered when developing strategies for precision therapies targeting glioblastoma with homozygous MTAP deletion. 1 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-24240-3 G e d t G enomic deletions of tumor-suppressor genes are prevalent drivers of tumor progression in various cancers, but their therapeutic inaccessibility subverts their potential use via precision oncology. Such deletion events often confer less-obvious genetic vulnerabilities that may be therapeutically exploited by identifying their metabolic consequences1,2. Homozygous dele- tion of CDKN2A/B at the 9p21 chromosome is an early clonal event in tumorigenesis3 that is homogeneously distributed in glioblastoma multiforme (GBM), pancreatic adenocarcinoma, and lung cancer4. Within the context of GBM, homozygous 9p21 deletions are found in around 30–50% of all cases5. Of particular interest in developing therapies that capitalize on aberrant tumor metabolism is collateral deletion of the evolutionarily conserved metabolic enzyme methylthioadenosine phosphorylase (encoded by MTAP). Owing to the proximity of MTAP to the CDKN2A tumor-suppressor locus6,7, co-deletion of MTAP may be observed in 80–90% of all tumors harboring homozygous deletion of CDKN2A8 (Supplementary Fig. 1). Results S i High levels of MTA act as an endogenous inhibitor of PRMT5 activity, thereby hindering methylosome formation and sensitizing cells to reduced levels of PRMT5 and WDR77. Therapeutic targeting of PRMT5 in homozygous MTAP-deleted cancer cells has thus been considered a promising strategy to prevent methylosome organization, selectively killing cancer cells9,10,15 (Supplementary Fig. 1). Another approach leverages the relationship between PRMT5 and S-adenosylmethionine (SAM)9,16,17. SAM is the natural substrate of PRMT5, and MTA is a substrate-competitive inhibitor of PRMT5. Lowering levels of SAM would thus exacerbate the inhibitory effect of elevated MTA. Accordingly, inhibition of methionine adenosyltransferase 2A (MAT2A), which generates SAM, has also been considered as a potential therapeutic target in homozygous MTAP-deleted cancers9,16,17 (Supplementary Fig. 1), and this strategy has progressed to an ongoing phase I trial (NCT03435250). To independently investigate the likelihood of MTA’s pre- dominant presence in conditioned media from MTAP-deleted cells, we performed mass spectroscopy on cell pellets and corre- sponding conditioned media (Fig. 1b) from the panel of cell lines with verified MTAP status (Fig. 1c and Supplementary Fig. 3). First, we compared MTA levels from the intracellular and extracellular environments of three MTAP-deleted glioma cell lines (U87, Gli56, and SW1088) with MTAP-WT cell lines (Fig. 1d, e). We observed dramatic increases in MTA levels in conditioned media of MTAP-deleted versus WT cell lines but a much more modest increase of MTA levels in the cell pellets of these respective cell lines. The significant differences in extracellular MTA between MTAP-deleted and WT compared to the minor differences in intracellular MTA levels of the same cells indicate that MTAP-deleted cells secrete MTA (Fig. 1d versus Fig. 1e). To corroborate our findings, we also performed a time-course experiment for MTAP-deleted and MTAP-WT cell lines. Although a time-dependent increase in MTA was apparent in both the cell pellets and conditioned media, the increase was exacerbated in the extracellular profile (Fig. 1f). Comparison of intracellular and extracellular levels of MTA and SAM demon- strated the tight regulation of intracellularly retained versus Both of the aforementioned therapeutic approaches are pre- dicated on the presence of exceedingly high MTA levels in MTAP-deleted cancer cells compared to MTAP-intact tissues. While it may seem natural that homozygous MTAP-deleted primary human tumors should also display this phenotype, recent reports identifying this intriguing vulnerability have not mea- sured MTA levels in primary human tumors9,10,18. Results S i Secretion of MTA by MTAP-deleted cells in culture. By ana- lyzing multiple metabolic profiling studies using independent metabolomic platforms, we observed a discrepancy between reported intracellular MTA levels in homozygous MTAP-deleted cells9,19–25, even within the same cell lines (Supplementary Fig. 2a, b). This discrepancy can be attributed to the extent to which these studies distinguish between intracellular and extra- cellular distribution—which depends on how extensively the cells were washed before metabolite extraction. For instance, in the NCI-60 profiling study by Ortmayr et al.22, which focused exclusively on intracellular metabolites (extensive washing of cell pellets with minimal residual media), there was no statistically significant increase in MTA levels in MTAP-deleted compared to WT cancer cell lines (Supplementary Fig. 2a). However, a colla- boration between Metabolon, Inc. and the NCI using the NCI-60 panel26 did not specify whether the cell pellet was washed and reported a threefold increase in MTA in MTAP-deleted compared to MTAP-WT cell lines (Supplementary Fig. 2b). In contrast to intracellular levels of MTA, re-analysis of one notable study that conducted mass spectrometry (MS) profiling of conditioned media (secreted metabolites) showed >100-fold increases in MTA in MTAP-deleted cell lines compared to intact cell lines (Sup- plementary Fig. 2c; for comparison with lactate, see Supplemen- tary Fig. 2d)27. The micromolar extracellular levels of MTA reported by these studies sharply contrasts the nanomolar to picomolar levels of MTA typically found in conditioned media of MTAP-WT cells24,28. Also, direct measurement of MTA secretion from MTAP-deleted cells into culture medium showed a sig- nificantly higher rate of secretion from MTAP-deleted cell lines than from MTAP-WT lines19,29. y In addition to its broader role in polyamine biosynthesis, MTAP is critically involved in the salvage pathways of both methionine and adenine, catalyzing the conversion of methyl- thioadenosine (MTA) to S-methyl-5-thio-D-ribose-1-phosphate (Fig. 1a). Cancers sustaining homozygous MTAP deletions are thus expected to accumulate MTA; extensive in vitro evidence in diverse cancer cell lines supports this logic9,10 (though methio- nine and cysteine availability in media also influences MTA levels11). Efforts to act on this intriguing metabolic phenotype have identified the inhibitory effect of excess MTA on protein arginine methyltransferase 5 (PRMT5), a key regulator of tran- scription. PRMT5 exerts its regulatory effects when conjugated with WD repeat-containing protein (WDR77) to generate what is known as the methylosome12–14. Homozygous MTAP deletion in primary human glioblastoma is not associated with elevation of methylthioadenosine The ubiquity of this event alongside the poor prognosis of cancers such as GBM has urged the development of novel therapies that capitalize on downstream vulnerabilities conferred by MTAP deletion. secretion of MTA by MTAP-deleted cells. Building on this finding in primary tumors, our data strongly suggest that the abundance of non-malignant MTAP wild-type (WT) stromal cells metabolize the secreted MTA from the homozygous MTAP-deleted GBM cells. As the promise of synthetic therapies that are lethal against homozygous MTAP-deleted cancers is dependent on intracellular accumulation of MTA, our data caution against the expedient translation of the MTAP-deletion-targeted precision therapies to the clinic and strongly contend that more research is needed into the fundamental metabolic differences between model systems and primary tumors. NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-24240-3 ARTICLE Fig. 1 Dramatic elevation of MTA in conditioned medium of MTAP-deleted cells versus WT coupled with mild intracellular elevation. a The methionine salvage pathway (based on KEGG68) with most genes are housekeeping expressed broadly across cell lines and tissues (DepMap, The Human Protein Atlas). SAM S-adenosylmethionine, dcSAM S-adenosylmethionineamine, MTA methylthioadenosine, SPD spermidine, PTN putrescine, MTOB 4- methylthio-2-oxobutanoate, DMTP 1,2-dihydroxy-5-(methylthio)pent-1-en-3-one, MTP 3-(methylthio)propanoate, MTDO-P 5-(methylthio)-2,3- dioxopentyl-phosphate, MTRi-P S-methyl-5-thio-D-ribulose-1-phosphate, 5-MTR-P methylthioribose-1-phosphate. b MTA levels were determined in conditioned media (extracellular) and washed cell pellets (intracellular) of homozygous MTAP-deleted (MTAP−) and wild-type (MTAP+) cells in culture. Color coding reflects the same cell line for pellet/media. c MTAP status of cell lines confirmed by western blot repeated independently twice. d, e Levels of MTA (mean + SD, N = 3 biological replicates) in a panel of glioma cell; washed cell pellet—intracellular (d) and conditioned media—extracellular (e). Note >200-fold increase in MTA in conditioned media of MTAP-deleted versus intact cell lines (***p = 0.0005, unpaired two-tailed Student’s t test with unequal variance), while only marginal elevations are seen for this comparison in the cell pellet. f, g Time course of MTA levels in cell pellet and conditioned media (f) with SAM shown for comparison (g). x-axis: time of harvest after last media change; y-axis: ion counts for each metabolite. Each bar represents one biological replicate. There was a distinct, time-dependent increase in MTA in media of MTAP-deleted cells with a modest increase of MTA in the cell pellet. There was an imbalance of MTA in the pellet versus the media, contrasting what was observed with SAM, which is exclusively intracellular. h Absolute quantification of MTA in cell pellet and conditioned media (mean +/−SD, N = 3 biological replicates). The amount of MTA recovered from conditioned media of MTAP-deleted cells is 200-fold greater than recovered from the cell pellets. In contrast, in MTAP-WT cells, the amount of MTA in conditioned NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-24240-3 ARTICLE Fig. 1 Dramatic elevation of MTA in conditioned medium of MTAP-deleted cells versus WT coupled with mild intracellular elevation. a The methionine salvage pathway (based on KEGG68) with most genes are housekeeping expressed broadly across cell lines and tissues (DepMap, The Human Protein Atlas). SAM S-adenosylmethionine, dcSAM S-adenosylmethionineamine, MTA methylthioadenosine, SPD spermidine, PTN putrescine, MTOB 4- methylthio-2-oxobutanoate, DMTP 1,2-dihydroxy-5-(methylthio)pent-1-en-3-one, MTP 3-(methylthio)propanoate, MTDO-P 5-(methylthio)-2,3- dioxopentyl-phosphate, MTRi-P S-methyl-5-thio-D-ribulose-1-phosphate, 5-MTR-P methylthioribose-1-phosphate. NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-24240-3 b MTA levels were determined in conditioned media (extracellular) and washed cell pellets (intracellular) of homozygous MTAP-deleted (MTAP−) and wild-type (MTAP+) cells in culture. Color coding reflects the same cell line for pellet/media. c MTAP status of cell lines confirmed by western blot repeated independently twice. d, e Levels of MTA (mean + SD, N = 3 biological replicates) in a panel of glioma cell; washed cell pellet—intracellular (d) and conditioned media—extracellular (e). Note >200-fold increase in MTA in conditioned media of MTAP-deleted versus intact cell lines (***p = 0.0005, unpaired two-tailed Student’s t test with unequal variance), while only marginal elevations are seen for this comparison in the cell pellet. f, g Time course of MTA levels in cell pellet and conditioned media (f) with SAM shown for comparison (g). x-axis: time of harvest after last media change; y-axis: ion counts for each metabolite. Each bar represents one biological replicate. There was a distinct, time-dependent increase in MTA in media of MTAP-deleted cells with a modest increase of MTA in the cell pellet. There was an imbalance of MTA in the pellet versus the media, contrasting what was observed with SAM, which is exclusively intracellular. h Absolute quantification of MTA in cell pellet and conditioned media (mean +/−SD, N = 3 biological replicates). The amount of MTA recovered from conditioned media of MTAP-deleted cells is 200-fold greater than recovered from the cell pellets. In contrast, in MTAP-WT cells, the amount of MTA in conditioned media is comparable to that in cell pellet. The modest intracellular accumulation of MTA (sixfold) in the MTAP-deleted cells compared to the WT cells is overshadowed by the increase of extracellular MTA. Significant values are indicated as ****p = 4 × 10−8, ***p = 0.00006, @@@@p = 10−12, and %p = 0.02 using multiple t test with Bonferroni correction. sharp contrast with the MTAP-WT cells, where a comparable amount of MTA was recovered from media (extracellular) and the cell pellet (intracellular). Taken together, our findings agree with previous conclusions 19,29 that homozygous MTAP-deleted cells secret MTA resulting in significant extracellular accumula- tion of MTA. secreted metabolites (Fig. 1f versus Fig. 1g). We further measured the absolute amount (nanomoles) of MTA for a paired sample of cell pellets and conditioned media. Results S i We could not find any previous studies that reported MTA measurements in actual primary human tumors as a function of the MTAP genotype. g yp In this study, we found that highly elevated MTA levels found in MTAP-deleted glioma cell lines in culture cannot be extra- polated to primary GBMs. Our series of metabolomic studies in cell culture and primary tumors demonstrate that MTA accu- mulation in culture is most evident extracellularly, reflecting NATURE COMMUNICATIONS | (2021) 12:4228 | https://doi.org/10.1038/s41467-021-24240-3 | www.nature.com/naturecommunications 2 NATURE COMMUNICATIONS | (2021) 12:4228 | https://doi.org/10.1038/s41467-021-24240-3 | www.nature.com/naturecommunications ARTICLE We next interrogated another human glioma metabolomic profiling dataset from Chinnaiyan et al.44 (Supplementary Fig. 7), where again no genomic data for individual tumors were available, which would have allowed us to determine MTAP- deletion status. Thus, we sought to match the metabolomic data for different glioma grades with the reported frequency of MTAP deletions in them. The frequency of homozygous CDK2NA/MTAP deletion is <3% in grade 2 glioma and <10% in grade 2/3 glioma30, sharply contrasting the frequency of homozygous MTAP deletion, which is as high as 50% in grade 4 glioma (GBM)5,39,41. Thus, we would expect that the elevation of MTA would correspond to this deletion frequency pattern according to tumor grade, with higher MTA levels in GBM and considerably lower levels in grade 2 glioma. However, despite these different frequencies of MTAP deletion, MTA levels were similar in grade 2, grade 3, and grade 4 (GBM) tumors (p = 0.15, single-factor analysis of variance, Supplementary Fig. 7a, b). Importantly, no extreme outliers in MTA were evident. Even the highest MTA levels in individual GBM cases were about twofold higher than the median MTA levels of lower-grade glioma tumors. These data starkly contrast the extreme elevations in 2- HG levels, driven by IDH mutations in gliomas; these elevations were frequent in grade 2 and 3 gliomas but infrequent in GBM (Supplementary Fig. 7c, d). In addition to these MS metabolomic studies, nuclear magnetic resonance (NMR) high-resolution magic-angle spinning (HR-MAS) metabolomic profiles of GBM on a large scale (n > 100 cases) have not detected MTA in MTAP- deleted tumors even though HR-MAS can usually detect metabolites at a minimum concentration of 1 µM45. That MTA could still not be detected in these contexts suggests that <1 µM MTA is present in GBM tumors, which supports our conclusion that there are no glioma tumors, regardless of MTAP deletion status, with extreme elevations in MTA. We conclude that homozygous MTAP-deleted primary GBM tumors do not selectively exhibit significant elevation of MTA as opposed of what was reported in vitro, a conclusion supported by both our own results and corroborated by public domain data. To further confirm the discrepancy in MTA levels in human GBM tumors and cells in culture, we measured MTA levels in a different set of tumors (MDA series) using a different metabolomic platform (Metabolon, Inc.). ARTICLE The MTAP status of tumors was confirmed by immunohistochemistry using a validated MTAP antibody. Consistent with findings from Fig. 2, we did not observe a statistically significant increase in MTA levels between MTAP-deleted and intact human GBM tumors (Supplementary Fig. 5), confirming the discrepancies between the in vitro models and primary human GBM tumors. y p The statistically insignificant elevation of MTA (1.4-fold in absolute MTA levels, and less when correcting for total load and SAM levels) in human GBM tumors is at odds with the multiple- fold-change MTA elevations reported in vitro. Metabolomic data for MTA levels in human tumors are the measure of both intracellular and extracellular MTA. In cell culture, empirical observations indicate that differences in MTA levels between MTAP-deleted and WT cells are most evident in the extracellular rather than intracellular environment (Fig. 1). While we cannot distinguish intracellular and extracellular MTA in primary tumors, we can investigate the effects of MTAP deletion in primary tumors on intracellular PRMT5 activity inhibition. PRMT5 mediates the formation of symmetric dimethylarginine (SDMA); thus, the PRMT5 activity can be assessed by measuring the SDMA levels using the antibody against an SDMA. First, we measured the SDMA levels in MTAP-WT and MTAP-deleted cancer cells in culture (under the same conditions in which we measured MTA accumulation in media) by western blot analysis (Supplementary Fig. 8). As expected, the SDMA levels were lower in MTAP-deleted cells than MTAP-WT, indicating partial inhibition of PRMT5. Also, SDMA levels decreased following MTA treatment (50 and 100 μM). These two data pieces recapitulate the literature and support the antagonistic relation- ship between PRMT5 activity and MTA levels. Then we measured the SDMA levels inside human GBM tumors using the same p y We further sought to test our conclusions by interrogating independent GBM metabolomic datasets in the literature, though we could not find any for which genomic information was also available. Metabolomic analyses of primary human GBM tumors are sparsely reported in the literature. The few studies that have been conducted have favored comparison between tumor populations at different stages or grades, rather than considering the unique metabolic landscape of individual tumors33–37. Supplementary Fig. 6a–f shows the MTA levels among 50 human GBM tumors sorted based on MTAP mRNA levels (data obtained from Supplementary Information of Prabhu et al.38). The frequency of homozygous MTAP deletion in GBM can reach 50%39–41. ARTICLE ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-24240-3 No significant elevation of MTA in homozygous MTAP-deleted human GBMs. While elevated MTA levels in vitro clearly define a promising actionable metabolic vulnerability, none of the studies9,10,15,18 reported on the critical question of whether homozygous MTAP-deleted primary human tumors faithfully replicate the elevation of MTA observed in vitro. In other words, does the metabolic vulnerability mediated by homozygous MTAP deletion hold in primary human tumors, including GBM? To answer this question directly, using our metabolomic profiling data generated with the BIDMC platform, we compared MTA levels between resected human GBM tumors with verified MTAP-deletion status30–32 (Fig. 2a, b). This is the first study to address MTAP deletion’s effects on MTA levels in human tumors. Figure 2c shows that, in 17 GBM tumors (HF series), MTA levels do not differ significantly between homozygous MTAP-deleted and MTAP-intact tumors (~1.4-fold higher median MTA levels in MTAP-deleted tumors; p = 0.20, unpaired 2-tailed t test with unequal variance). When corrected for loading, the median MTA levels were only 1.2- fold higher in MTAP-deleted than in MTAP-intact tumors (p = 0.09; Fig. 2d). Next, we normalized MTA levels to SAM levels for each tumor to account for the methionine salvage pathway’s upregulation (Fig. 2e). Since the levels of SAM were not significantly different between MTAP-deleted and MTAP-WT cells in culture15 (data replotted in Supplementary Fig. 4), SAM is a suitable meta- bolite for normalization purposes. When the MTA levels were corrected to SAM levels in primary human GBM tumors, the dif- ference between MTAP-deleted and MTAP-intact tumors became decisively nonsignificant (1.1-fold higher median MTA levels in MTAP-deleted tumors; p = 0.24; Fig. 2e). This result runs contrary to the expected phenotype in homozygous MTAP-deleted cells in culture. This nonsignificant MTA elevation pales further compared to outliers driven by specific genetic events that fully recapitulate metabolic data recorded intracellularly in cell culture, such as the >100-fold increase in the levels of 2-hydroxyglutarate (2-HG), driven by the point mutation of IDH1 (Fig. 2f). those that are MTAP-intact. The comparison of MTA levels between quartiles is statistically insignificant (p = 0.48, single- factor analysis of variance), which agrees with our conclusion that MTAP-deleted tumors do not show selective elevation of MTA. between quartiles is statistically insignificant (p = 0.48, single- factor analysis of variance), which agrees with our conclusion that MTAP-deleted tumors do not show selective elevation of MTA. NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-24240-3 Figure 1h indicates that the amount of MTA recovered from the conditioned media from MTAP-deleted cells cultured for 3 days is approximately 200 times more than MTA recovered from the cell pellets. This is in 3 ARTICLE Based on an analysis of The Cancer Genome Atlas (TCGA)-GBM tumor42,43 where both mRNA and genomic copy numbers were available, we find that, despite some exceptions, the majority of homozygous MTAP-deleted tumors fall in the two lower quartiles of MTAP expression (Supplementary Fig. 6g). We thus posit that, for data shown in Supplementary Fig. 6a–f, the two lower quartiles of MTAP mRNA expression would include most homozygous MTAP-deleted tumors, while the majority of tumors in the third and fourth quartiles would be URE COMMUNICATIONS | (2021) 12:4228 | https://doi.org/10.1038/s41467-021-24240-3 | www.nature.com/naturecommunications 4 Fig. 2 No significant elevation of MTA and no specific inhibition of PRMT5 activity in homozygous MTAP-deleted primary resected GBM tumors. a GBM tumors (heterogeneous mix of transformed glioma cells and non-malignant stroma) of defined MTAP-deletion status (genomic profiling from Kim et al.31) were evaluated by mass spectrometry for MTA levels and western blot for SMDA. b Genomic copy-number data (dark blue: homozygous deletion, dark red: amplification) around the 9p21 locus. Each strip in the y-axis represents a single tumor at the specific chromosomal location (x-axis). c MTA levels in homozygous MTAP-deleted (MTAP−) versus MTAP-intact (MTAP+) GBM tumors, using the BIDMC mass spectrometric platform. Each bar represents MTA levels for each tumor (N = 1). There is a 1.4-fold increase in the median MTA levels (absolute ion counts) in MTAP-deleted tumors compared to intact tumors (p = 0.20, unpaired 2-tailed t test with unequal variance). d, e Same data but expressed normalized to total ion count for sample loading normalization (d) and as a ratio to SAM levels to account for methionine salvage pathway activity (e). Regardless of normalization, no significant elevation of MTA in MTAP-deleted tumors was evident. In contrast, f tumors with IDH1 mutation stand out by their dramatic elevation of 2-hydroxyglutarate (2-HG), providing a positive control for genomic/metabolic correlation. g To evaluate PRMT5 activity, GBM tumor lysates were immunoblotted for SDMA, repeated once. As a positive control, we assessed SDMA levels in MTAP-deleted U87 glioma cells in culture, alone or treated with exogenous MTA versus MTAP-reconstituted (U87-MTAP) cells. SDMA levels were lower in U87 than in U87-MTAP cells, indicating partial inhibition of PRMT5. SDMA levels further decreased following MTA treatment. Unlike in cultured cells, no specific decrease in SDMA levels was observed in MTAP-deleted versus intact GBM tumors. ARTICLE The presence of myeloid cells in a tumor is confirmed by the myeloid marker IBA1 in tumor lysates but not seen in cells in culture. h MTAP protein levels corrected for loading with GAPDH control (samples derive from the same experiment and gels were processed in parallel). Due to the presence of non-malignant MTAP-expressing cells in the lysate of whole tumors, MTAP protein levels are not zero for homozygous MTAP-deleted tumors; however, on average, they have lower MTAP protein levels compared to intact tumors. NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-24240-3 ARTICLE ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-24240-3 Fig. 2 No significant elevation of MTA and no specific inhibition of PRMT5 activity in homozygous MTAP-deleted primary resected GBM tumors. a GBM tumors (heterogeneous mix of transformed glioma cells and non-malignant stroma) of defined MTAP-deletion status (genomic profiling from Kim et al.31) were evaluated by mass spectrometry for MTA levels and western blot for SMDA. b Genomic copy-number data (dark blue: homozygous deletion, dark red: amplification) around the 9p21 locus. Each strip in the y-axis represents a single tumor at the specific chromosomal location (x-axis). c MTA levels in homozygous MTAP-deleted (MTAP−) versus MTAP-intact (MTAP+) GBM tumors, using the BIDMC mass spectrometric platform. Each bar represents MTA levels for each tumor (N = 1). There is a 1.4-fold increase in the median MTA levels (absolute ion counts) in MTAP-deleted tumors compared to intact tumors (p = 0.20, unpaired 2-tailed t test with unequal variance). d, e Same data but expressed normalized to total ion count for sample loading normalization (d) and as a ratio to SAM levels to account for methionine salvage pathway activity (e). Regardless of normalization, no significant elevation of MTA in MTAP-deleted tumors was evident. In contrast, f tumors with IDH1 mutation stand out by their dramatic elevation of 2-hydroxyglutarate (2-HG), providing a positive control for genomic/metabolic correlation. g To evaluate PRMT5 activity, GBM tumor lysates were immunoblotted for SDMA, repeated once. As a positive control, we assessed SDMA levels in MTAP-deleted U87 glioma cells in culture, alone or treated with exogenous MTA versus MTAP-reconstituted (U87-MTAP) cells. SDMA levels were lower in U87 than in U87-MTAP cells, indicating partial inhibition of PRMT5. SDMA levels further decreased following MTA treatment. Unlike in cultured cells, no specific decrease in SDMA levels was observed in MTAP-deleted versus intact GBM tumors. TURE COMMUNICATIONS | (2021) 12:4228 | https://doi.org/10.1038/s41467-021-24240-3 | www.nature.com/naturecommunications ARTICLE The presence of myeloid cells in a tumor is confirmed by the myeloid marker IBA1 in tumor lysates but not seen in cells in culture. h MTAP protein levels corrected for loading with GAPDH control (samples derive from the same experiment and gels were processed in parallel). Due to the presence of non-malignant MTAP-expressing cells in the lysate of whole tumors, MTAP protein levels are not zero for homozygous MTAP-deleted tumors; however, on average, they have lower MTAP protein levels compared to intact tumors. Fig. 2 No significant elevation of MTA and no specific inhibition of PRMT5 activity in homozygous MTAP-deleted primary resected GBM tumors. a GBM tumors (heterogeneous mix of transformed glioma cells and non-malignant stroma) of defined MTAP-deletion status (genomic profiling from Kim et al.31) were evaluated by mass spectrometry for MTA levels and western blot for SMDA. b Genomic copy-number data (dark blue: homozygous deletion, dark red: amplification) around the 9p21 locus. Each strip in the y-axis represents a single tumor at the specific chromosomal location (x-axis). c MTA levels in homozygous MTAP-deleted (MTAP−) versus MTAP-intact (MTAP+) GBM tumors, using the BIDMC mass spectrometric platform. Each bar represents MTA levels for each tumor (N = 1). There is a 1.4-fold increase in the median MTA levels (absolute ion counts) in MTAP-deleted tumors compared to intact tumors (p = 0.20, unpaired 2-tailed t test with unequal variance). d, e Same data but expressed normalized to total ion count for sample loading normalization (d) and as a ratio to SAM levels to account for methionine salvage pathway activity (e). Regardless of normalization, no significant elevation of MTA in MTAP-deleted tumors was evident. In contrast, f tumors with IDH1 mutation stand out by their dramatic elevation of 2-hydroxyglutarate (2-HG), providing a positive control for genomic/metabolic correlation. g To evaluate PRMT5 activity, GBM tumor lysates were immunoblotted for SDMA, repeated once. As a positive control, we assessed SDMA levels in MTAP-deleted U87 glioma cells in culture, alone or treated with exogenous MTA versus MTAP-reconstituted (U87-MTAP) cells. SDMA levels were lower in U87 than in U87-MTAP cells, indicating partial inhibition of PRMT5. SDMA levels further decreased following MTA treatment. Unlike in cultured cells, no specific decrease in SDMA levels was observed in MTAP-deleted versus intact GBM tumors. The presence of myeloid cells in a tumor is confirmed by the myeloid marker IBA1 in tumor lysates but not seen in cells in culture. ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-24240-3 cerebrospinal fluid from GBM versus normal brain was nonsignificant, with no significant individual outliers. To corroborate the low likelihood of significant MTA secretion into the circulation, we compared MTA levels between venous and arterial plasma downstream and upstream of the glioma, as well as plasma obtained from a dorsal pedal vein of the same patient, control (Supplementary Fig. 11c–e; data obtained from Supple- mentary Information of Xiong et al.48). Metabolites secreted by tumors are carried downstream by venous blood, resulting in high levels of that metabolite in glioma venous compared to arterial plasma. While there were few GBM tumors in this study, given the high frequency of homozygous MTAP deletion in GBM, it is very likely that one or two GBM tumors to be homozygous MTAP-deleted in this dataset. The GBM tumor 5P had the highest MTA ratio of glioma venous to arterial. However, for the same patient, the MTA ratio of the dorsal pedal vein (control) to glioma arterial plasma was also high (Supplementary Fig. 11d), indicating high MTA production in the patient’s leg. Finally, as shown in Supplementary Fig. 11e, no GBM tumor had a significantly higher ratio of MTA in the dorsal pedal vein versus glioma venous plasma than low-grade glioma. These findings indicate that MTA’s secretion out of the tumor into the circulation is not a significant contributor to the discrepancy in the MTA levels between homozygous MTAP-deleted human GBM tumors and cells in culture. (cancer) cells with genetic alterations like homozygous MTAP- deletion and non-transformed non-malignant stromal cells (e.g., myeloid cells). Thus, in homozygous MTAP-deleted tumors, only malignant glioma cells lack MTAP, while stromal cells express normal levels. Such non-malignant MTAP-expressing cells drive the non-zero MTAP expression (mRNA or western blot) in bulk homozygous MTAP-deleted GBM tumors. However, on average, the MTAP-deleted tumors express a lower (but non-zero) amount of MTAP protein levels by the western blot than MTAP-intact (Fig. 2h). The presence of myeloid cells (microglia/macrophages) in a tumor is confirmed by the myeloid marker IBA1 in a western blot of tumor lysates but not seen in cells in culture (Fig. 2g). All tumors show some levels of IBA1, verifying myeloid stromal infiltration. IBA1 levels in MTAP-deleted tumors nicely correlate with residual MTAP levels—i.e., the homozygous MTAP-deleted HF3174 tumor has the lowest IBA1 compared to other homozygous MTAP-deleted tumors, which also expresses the lowest but non-zero MTAP levels. ARTICLE Stromal infiltration drives the in vitro/in vivo discordance in MTA. We next sought to identify the cause of discordance in MTA levels between cells in culture and human GBM tumors. Previous work by Sanderson et al.11, showed that MTAP- deleted cells accumulate less amount of MTA when cultured in restricted methionine (3 μM) or cysteine (6 μM) RPMI than when cultured in the standard formulation with 100 μM methionine and 200 μM cysteine (data replotted in Supplementary Fig. 9). Motivated by this study, we sought to investigate the effects of culturing cells in physiological media on intracellular and extra- cellular levels of MTA . The composition of physiological media better represents the concentration of metabolites in human plasma46 (Supplementary Fig. 10a). Thus, we cultured MTAP- deleted and WT cells in Dulbecco’s Modified Eagle’s medium (DMEM; historic medium) and Plasmax (physiological medium) supplemented with 2.5% fetal bovine serum (FBS) and compared the levels of MTA in the cell pellet and conditioned media. Supplementary Fig. 10b, c shows no significant difference between average MTA levels of MTAP-deleted cells cultured in DMEM versus Plasmax, either extracellular or intracellular. Supplementary Fig. 10d indicates that, just like in DMEM, the MTA levels are highly elevated in conditioned media of MTAP- deleted versus intact cells, while most modest elevations were evident in the cell pellets . These data indicate that, even in physiological media, MTAP-deleted cells secrete MTA. We also investigated the effects of culture medium on SDMA levels of MTAP-deleted and WT glioma cells (Supplementary Fig. 10f). No significant difference was observed in the SDMA levels of MTAP- deleted cells cultured in DMEM versus physiological media, with MTAP-deleted cells having lower SDMA levels. Our findings indicate that MTAP-deleted cells accumulate and extrude MTA when grown in a nutritional environment similar to human plasma, which would more accurately reflect the human situation. However, we do not explicitly rule out the hypothesis that nutrient differences (i.e., extremely low levels of cysteine11) may contribute to the discrepant observation of lack of MTA accu- mulation in primary human tumors with MTAP-deletion. It is already well-established that GBM tumors may have up to 75% (range 25–75%) non-malignant stromal cells, including non- transformed reactive astrocytes, microglia, macrophages, neutro- phils, lymphocytes, endothelial cells, fibroblasts, and axonal and neuronal remnants30,31,49–51. Thus, a homozygous MTAP-deleted GBM tumor is an admixture of non-malignant MTAP-expressing stroma and MTAP-null malignant glioma cells. ARTICLE h MTAP protein levels corrected for loading with GAPDH control (samples derive from the same experiment and gels were processed in parallel). Due to the presence of non-malignant MTAP-expressing cells in the lysate of whole tumors, MTAP protein levels are not zero for homozygous MTAP-deleted tumors; however, on average, they have lower MTAP protein levels compared to intact tumors. gnificant elevation of MTA and no specific inhibition of PRMT5 activity in homozygous MTAP-deleted primary resecte of MTA and no specific inhibition of PRMT5 activity in homozygous MTAP-deleted primary resected GBM tumors. a G correlation between SDMA levels and tumors’ MTAP status, we noticed some variation in SDMA levels between tumors regardless of their MTAP status. Unlike cells in culture, homozygous MTAP-deleted GBM tumors express non-zero MTAP levels in western blot (Fig. 2g). This is because human GBM tumors are comprised of a mix of malignant glioma antibody (Fig. 2g). Unlike cell culture experiments, primary tumors showed no meaningful decrease in levels of SDMA, representing PRMT5 activity, in homozygous MTAP-deleted compared to MTAP-intact human GBM tumors. These data are consistent with the marginal elevation of MTA, intracellular or extracellular, in MTAP-deleted tumors. While we did not find a 5 NATURE COMMUNICATIONS | (2021) 12:4228 | https://doi.org/10.1038/s41467-021-24240-3 | www.nature.com/naturecommunications ARTICLE a Immunohistochemistry (IHC) for MTAP and IBA1 on formalin-fixed paraffin-embedded (FFPE) sections of GBM tumors. b, c IHC was performed with monoclonal anti-MTAP. The slides were developed with EnzMet (black staining indicates MTAP expression) and counterstained with hematoxylin (blue, nuclei) and eosin (pink, cytosol, and extracellular space) on at least 50 cases stained with MTAP and IBA1 and representative of three independent cases are shown. Representative sections of MTAP- positive (b) and an MTAP-negative GBM tumor (c); x20 objective. Note the extensive MTAP-positive staining areas on the background of otherwise non- staining MTAP-negative glioma cells; histopathologic evaluation of morphology indicates that these MTAP-positive cells correspond to stromal components, including microglia, endothelial cells, and activated astrocytes. d Higher magnification view of c golden square. Any MTA secreted by MTAP- deleted glioma cells (blue dashed outline) stands to be phosphorylated by MTAP (producing 5-MTR-P) either released or taken up by MTAP-expressing stromal cells (white dashed outlines). e A representative section of an MTAP-negative GBM tumor stained against IBA1 antibody (myeloid marker), indicating the extensive presence of myeloid cells (microglia/macrophages) inside GBM tumors. f Higher-magnification view of e. Fig. 3 Primary GBM tumors are extensively infiltrated by MTAP-expressing non-malignant stromal cells. a Immunohistochemistry (IHC) for MTAP and IBA1 on formalin-fixed paraffin-embedded (FFPE) sections of GBM tumors. b, c IHC was performed with monoclonal anti-MTAP. The slides were developed with EnzMet (black staining indicates MTAP expression) and counterstained with hematoxylin (blue, nuclei) and eosin (pink, cytosol, and extracellular space) on at least 50 cases stained with MTAP and IBA1 and representative of three independent cases are shown. Representative sections of MTAP- positive (b) and an MTAP-negative GBM tumor (c); x20 objective. Note the extensive MTAP-positive staining areas on the background of otherwise non- staining MTAP-negative glioma cells; histopathologic evaluation of morphology indicates that these MTAP-positive cells correspond to stromal components, including microglia, endothelial cells, and activated astrocytes. d Higher magnification view of c golden square. Any MTA secreted by MTAP- deleted glioma cells (blue dashed outline) stands to be phosphorylated by MTAP (producing 5-MTR-P) either released or taken up by MTAP-expressing stromal cells (white dashed outlines). e A representative section of an MTAP-negative GBM tumor stained against IBA1 antibody (myeloid marker), indicating the extensive presence of myeloid cells (microglia/macrophages) inside GBM tumors. f Higher-magnification view of e. correlation between mRNA levels of MTAP and AIF1 in MTAP- deleted tumors versus intact ones. ARTICLE To verify the diagnosis of homozygous MTAP deletion and contrast this status with the extensive MTAP expression levels in non-malignant tissue, we performed immunohistochemistry on intracranial xenografted gliomas in mice. We first validated an MTAP rabbit monoclonal antibody by demonstrating intense staining in formalin-fixed paraffin-embedded (FFPE) sections of MTAP- WT tumors and loss of staining in MTAP-deleted tumors gener- ated from known MTAP-genotype cell lines (Supplementary Figs. 12–14). Every xenograft established from MTAP-WT (or isogenic MTAP-rescued) cancer cell lines showed intense staining, while all xenografts established from MTAP-deleted cell lines showed no staining. Simultaneously, non-malignant stromal cells such as microglia, lymphocytes, and endothelial cells stained positive for MTAP. Together, these staining results support that this antibody accurately quantifies MTAP levels in FFPE slides. Notable among our stained slides was the significant amount of positive staining in the normal mouse brain and stromal cells, especially given that xenografts typically grow much less invasively and are less populated by stromal cells compared to human GBMs (Supplementary Fig. 15). By extension, true human GBMs are more likely to contain a higher proportion of stromal components such as astrocytes and myeloid cells. Next, we applied this antibody to FFPE primary GBM sections. Of the 60 cases analyzed, we found that approximately 40% of cases showed a complete lack of MTAP staining in cancer cells, which generally corresponds to the expected MTAP-deleted frequency in GBM41; notably, intense staining was observed in non-malignant stromal cells. MTAP-intact cases showed intense, uniform staining in both glioma and stromal cells (Fig. 3, Supplementary Fig. 17). The histology of MTAP-deleted primary GBM cases appeared similar to the MTAP-deleted intracranial xenografted cases, except that the degree of MTAP-positive stromal infiltration was much more significant in the primary human GBM tumors compared to xenografts (Supplementary Another possible explanation could be the extrusion of MTA out of the tumor into the circulation. To investigate this possibility, we interrogated MTA levels in a metabolomic profiling dataset of cerebrospinal fluid from GBM compared and normal brain47. Given that the frequency of homozygous MTAP deletions in GBM is around 50%, we would have expected to observe a higher MTA level in the cerebrospinal fluid from at least some GBM compared to the normal brain (Supplementary Fig. 11a, b). However, the difference in MTA levels between 6 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-24240-3 Fig. 3 Primary GBM tumors are extensively infiltrated by MTAP-expressing non-malignant stromal cells. NATURE COMMUNICATIONS | (2021) 12:4228 | https://doi.org/10.1038/s41467-021-24240-3 | www.nature.com/naturecommunications Discussion We undertook this study in response to the emergence of homozygous MTAP-deleted precision therapy endeavors despite a lack of exploration of the metabolic state of primary tumors. As the therapeutic window of therapies directed at MTAP deletion relies on massive intracellular accumulation of MTA9,10,15,52–54, ensuring that this in vitro phenotype is consistent in human tumors was a natural and urgent area for investigation. Perhaps surprisingly, none of the recent papers documenting MTAP- dependent vulnerabilities reported MTA measurements in pri- mary human tumors or even xenografted tumors of defined MTAP-deletion status, despite the significant efforts that have been built on the supposition that observed elevations in MTA in vitro would be reflected in primary tumors9,10,15,52–54. We took to the literature—keen on finding studies describing MTA levels in distinct, genetically defined homozygous MTAP- deleted primary tumors—but with little success. We could not find any literature reports directly measuring MTA levels in genomically identified MTAP-deleted than MTAP-intact primary human tumors, whether GBM or others. This was quite sur- prising, given that MTA is a metabolite frequently detected in many metabolomic profiling studies. Next, we co-cultured MTAP-deleted glioma cells with macro- phages (RAW-264.7), immortalized normal human astrocytes, and isogenic MTAP-rescued cells (U87-MTAP) and measured MTA levels in conditioned media using 1H-NMR. We observed robust accumulation of MTA in the conditioned media of MTAP- deleted cells but not of MTAP-WT or MTAP-rescued cells (Fig. 4e). When MTAP-deleted U87, SW1080, HT1080, and SKMEL5 cells were co-cultured with MTAP-WT macrophages (Fig. 4e) or normal human astrocytes (Supplementary Fig. 18c), no MTA peaks were detected in the conditioned media by 1H- NMR. Furthermore, the co-culture of U87 parental MTAP- deleted glioma cells with U87 MTAP-rescued cells resulted in the disappearance of MTA peaks in the 1H-NMR spectrum of the conditioned media (Supplementary Fig. 20). These results indicate that secreted MTA from MTAP-deleted cells is consumed (metabolized) by MTAP-WT cells; thus, co-culture of MTAP-deleted with MTAP-WT cell lines abrogates MTA accumulation in conditioned media. We also confirmed secretion and consumption of MTA when MTAP-deleted cells are co- cultured with macrophages in physiological media (Plasmax, Supplementary Fig. 18d). Our findings indicate that MTAP- WT cells in the vicinity of MTAP-deleted cancer cells prevent extracellular accumulation of MTA. To fill this gap, we interrogated our metabolomic datasets generated from different sets of GBM tumors using two inde- pendent metabolic platforms (BIDMC and Metabolon, Inc.). ARTICLE ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-24240-3 deleted and MTAP-WT cancer cell lines. To avoid such problems, we used a different approach to assess MTA levels inside cells indirectly. Since intracellular MTA inhibits PRMT5 activity and subsequently decreases SDMA levels, assessing SDMA levels by western blot is an indirect measurement of intracellular MTA. MTAP-deleted and MTAP-rescued cells were co-cultured with suspended leukemia MV-4-11 (monomyelocytic leukemia, macrophage-like) cells in DMEM. After 3 days, suspended leukemia cells were removed, and adherent cells were washed twice with phosphate-buffered saline (PBS) before they were lysed. On western blot (Fig. 4f), monoculture MTAP-deleted cells had lower SDMA levels, indicating lower PRMT5 activity and higher intracellular MTA levels than co-cultured MTAP-deleted cells. These findings emphasize that the co-culture of MTAP- deleted cells with MTAP-intact cells negates the selective vulnerability caused by MTAP deletion, thereby indicating that MTAP-deletion sensitivity in a heterogeneous human GBM tumor may be attenuated by the presence of stroma in the tumor microenvironment. Having identified MTAP-WT cells’ presence (i.e., stromal cells, which are predominantly myeloid in GBM) inside MTAP-deleted tumors, we hypothesized that excess MTA secreted from homozygous MTAP-deleted cancer cells may be metabolized by non-malignant MTAP-expressing cells present inside tumors. To test this hypothesis, we cultured MTAP-WT macrophages with exogenous MTA and measured MTA levels in conditioned media using proton NMR (1H-NMR). In the Supplementary Fig. 18a, b, no MTA peaks were detected from the conditioned media of MTAP-WT macrophages (RAW-264.7 and MV-4-11) incubated with exogenous MTA compared to media from a control plate (DMEM) or conditioned media from MTAP-deleted leukemia cells (CCRF-CEM) under the same experimental condition. This result indicates that either MTAP-WT cells release functional MTAP enzyme into the media or take up MTA from the media and metabolize it. First, we sought to see whether the release of functional MTAP enzyme by MTAP-WT cells is responsible for eliminating exogenous MTA from conditioned media. We grew MTAP-WT RAW-264.7 (macrophages) cells in DMEM; after 3 days, we collected the conditioned media and spun it down to remove any cells, then we incubated the conditioned media collected from RAW-264.7 cells with 20 μM exogenous MTA for 3 more days. Supplementary Fig. 19a shows that exogenous MTA was not eliminated from the cell-free conditioned media from macrophages. This result indicates that the release of functional MTAP enzymes does not contribute to the disappearance of exogenous MTA from the conditioned media of MTAP-WT cells. ARTICLE Next, we sought to investigate whether exogenous MTA is taken up and metabolized by MTAP-WT cells. Thus, we cultured MTAP-WT macrophages (RAW-264.7) or glioma cells (D423) with labeled methyl tri-deuterated-MTA (D3-MTA) (Fig. 4a). We observed rapid elimination of labeled D3-MTA from the condi- tioned media of MTAP-WT cells followed by deuterium enrich- ment of intracellular MTA, methionine, and SAM (Fig. 4b, c, and Supplementary Fig. 19c, d). Figure 4d shows the deuterium’s fate from the labeled D3-MTA into methionine (methionine salvage pathway) and SAM (polyamine biosynthesis). This result indicates that extracellular MTA can be taken up and metabolized by MTAP-WT cells. ARTICLE However, no positive correlation was observed between MTAP and AIF in MTAP- intact tumors since both glioma and stromal cells express MTAP in MTAP-intact tumors. This figure also indicates that homo- zygous MTAP-deleted GBM tumors, on average, have lower but non-zero levels of MTAP mRNA levels compared to MTAP- intact tumors, supporting our western blot data in Fig. 2g, h and validate that IBA1-positive myeloid cells drive the non-zero MTAP expression in bulk MTAP-deleted GBM tumors. Fig. 15). Supplementary Fig. 16a–c shows the strong correlation between the residual MTAP staining and myeloid content in MTAP-deleted GBM tumors. In homozygous MTAP-deleted tumors, the residual MTAP staining is restricted to stromal cells (no MTAP staining in malignant glioma cells), predominantly of myeloid origin (IBA1-positive). We also interrogate an indepen- dent dataset (TCGA41) for the correlation between mRNA levels of MTAP and AIF1 (the official gene symbol of IBA1, microglia/ macrophages marker) among GBM tumors with known MTAP status. Supplementary Fig. 16d indicates a significant positive 7 NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-24240-3 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-24240-3 Fig. 4 Exogenous MTA is consumed by MTAP-intact cells through the methionine salvage pathway. a MTAP-WT macrophages (RAW-264.7) were cultured with 100 µM methyl tri-deuterated-MTA (D3-MTA). Cells and conditioned media were extracted and prepared for LC-MS and NMR measurements, respectively. b Exogenous D3-MTA rapidly disappears from the media in cultures of macrophages, as the deuterium label (Mass + 3; M + 3) appears in intracellular methionine and SAM (c), consistent with MTAP-dependent metabolism by the methionine salvage pathway (d). Data in panel c are expressed as mean + SD, N = 3 biological replicates. e MTAP-deleted cancer cells accumulate MTA in conditioned media (detected by NMR) while secreted MTA is consumed by MTAP-intact macrophages, abrogating MTA accumulation in the extracellular environment. f Co-culture of MTAP-deleted cancer cell lines with MTAP-intact myeloid cells prevents loss of SDMA, indicating maintained PRMT5 activity, repeated once. MTAP-deleted and reconstituted glioma cells were co-cultured with myeloid leukemia cells (MV-4-11, a monomyelocytic suspension cell line). Before lysate preparation, suspended MV-4-11 myeloid cells were removed, and cancer cells were extensively washed. For each independent MTAP-deleted cell line, SDMA levels increased with myeloid co-culture, indicating restoration of PRMT5 activity. NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-24240-3 ARTICLE nine salvage pathway. a MTAP-WT macrophages (RAW-264.7) were ned media were extracted and prepared for LC-MS and NMR edia in cultures of macrophages, as the deuterium label (Mass + 3; M + ent metabolism by the methionine salvage pathway (d). Data in panel c cells accumulate MTA in conditioned media (detected by NMR) while mulation in the extracellular environment. f Co-culture of MTAP-deleted ng maintained PRMT5 activity, repeated once. MTAP-deleted and 1, a monomyelocytic suspension cell line). Before lysate preparation, ely washed. For each independent MTAP-deleted cell line, SDMA levels onine salvage pathway. a MTAP-WT macrophages (RAW-264.7) were oned media were extracted and prepared for LC-MS and NMR Fig. 4 Exogenous MTA is consumed by MTAP-intact cells through the methionine salvage pathway. a MTAP-WT macrophages (RAW-264.7) were cultured with 100 µM methyl tri-deuterated-MTA (D3-MTA). Cells and conditioned media were extracted and prepared for LC-MS and NMR measurements, respectively. b Exogenous D3-MTA rapidly disappears from the media in cultures of macrophages, as the deuterium label (Mass + 3; M + 3) appears in intracellular methionine and SAM (c), consistent with MTAP-dependent metabolism by the methionine salvage pathway (d). Discussion Our data suggest a statistically insignificant increase in MTA levels between homozygous MTAP-deleted and MTAP-intact tumors. This marginal elevation in MTA levels was at odds with the vast elevations in MTA reported for MTAP-deleted cell lines in vitro9,10,15. To investigate whether the in vitro metabolic vul- nerability posed by MTAP deletion holds in human GBM tumors, we assessed PRMT5 activity by measuring SDMA levels in the same set of human tumors in which we performed metabolomics. Unlike in cell culture experiments, we did not observe lower SDMA levels in homozygous MTAP-deleted tumors compared to intact ones. As an independent verification of our findings, we queried the public domain datasets of metabolomic studies per- formed on human GBM tumors33,44. We did not observe statis- tically significant MTA elevation in GBM tumors with low MTAP expression compared to those with high MTAP expression or GBM tumors with frequent MTAP deletions and grade 2 and 3 glioma tumors with rare MTAP deletions. If MTAP deletions do indeed lead to MTA accumulation in primary human tumors, we would expect to observe dramatically higher MTA levels (on the order of >20-fold, as seen in in vitro intracellular data from We also investigated the effects of co-culturing MTAP-deleted and MTAP-intact cells on PRMT5 activity—intracellular MTA levels—of MTAP-deleted cells. We earlier discussed the dis- crepancies in the magnitudes of reported MTA between MTAP- NATURE COMMUNICATIONS | (2021) 12:4228 | https://doi.org/10.1038/s41467-021-24240-3 | www.nature.com/naturecommunications 8 NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-24240-3 In the lit- erature, we noticed large discrepancies between different studies TURE COMMUNICATIONS | (2021) 12:4228 | https://doi.org/10.1038/s41467-021-24240-3 | www.nature.com/naturecommunications 9 9 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-24240-3 in the magnitudes of reported MTA increases in MTAP-deleted versus MTAP-intact cancer cell lines (Supplementary Fig. 2). Careful analysis of these results in light of our own in vitro data points to MTA secretion out of homozygous MTAP-deleted cells. Thus, the discrepancies between different studies appear to arise from how well intracellular and extracellular metabolite levels are differentiated during sample processing. Secretion of MTA by MTAP-deleted cells in agreement with previous studies19,55 fur- ther corroborate this finding. in the magnitudes of reported MTA increases in MTAP-deleted versus MTAP-intact cancer cell lines (Supplementary Fig. 2). Careful analysis of these results in light of our own in vitro data points to MTA secretion out of homozygous MTAP-deleted cells. Thus, the discrepancies between different studies appear to arise from how well intracellular and extracellular metabolite levels are differentiated during sample processing. Secretion of MTA by MTAP-deleted cells in agreement with previous studies19,55 fur- ther corroborate this finding. data43,60 and DepMap from the Cancer Cell Line Encyclopedia and the Genentech collection61. Where deletion calls were ambiguous (e.g., SF-295), we looked at mRNA expression data or literature western blot data9,10,15,52 to decide what constituted a functionally MTAP-null cell line. In the NCI-60 cell line panel, the following cell lines lack MTAP: NCI-H322, SKMEL5, K562, SF268, MALME3M, ACHN, MDAMB231, OVCAR5, CCRF-CEM, SR, MCF7, and A549. Cell culture and xenograft generation. The cell lines used in this study that are MTAP-WT were D423 (CVCL_1160, H423/D423-MG) and D502 (CVCL_1162, H50262), which were kindly provided by Darrel Bigner62. U343 (CVCL_S471, U343-MG63), LN319 (CVCL_3958, a sub-clone of LN-99264), and NB1 (CVCL_1440) were obtained from the Department of Genomic Medicine/Institute for Applied Cancer Science Cell Bank at MD Anderson. MV-4-11 (CVCL_0064) was purchased from NCI, RAW-264.7 (CVCL_0493) was purchased from ATCC (TIB-71), and immortalized normal human astrocytes was kindly provided by Dr. Seth Gammon (Cancer System Imaging. The MTAP-deleted cell lines U87 (CVCL- 0022), SW1088 (CVCL_1715), and SKMEL5 (CVCL_0527) were obtained from the Department of Genomic Medicine/Institute for Applied Cancer Science Cell Bank at MD Anderson. Gli56 (D. Louis) and CCRF-CEM (CVCL_0207) were purchased from NCI. HT1080 (CVCL_0317) was kindly provided by Dr. Seth Gammon (Cancer Systems Imaging). All cell culture experiments were conducted according to the provider’s instructions and as previously described65. NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-24240-3 All cell lines were confirmed as mycoplasma negative by enzyme-linked immunosorbent assay using the MycoAlert PLUS Detection Kit (Lonza) and were authenticated by short tandem repeat DNA fingerprinting and chromosomal analysis by the Cytogenetics and Cell Authentication Core at MD Anderson. All cells were cultured at 37 °C in a 5% CO2 atmosphere at pH 7.4 in ATCC- suggested media (DMEM) unless stated otherwise. DMEM has 4.5 g/l glucose, pyruvate and glutamine (Cellgro/Corning, #10-013-CV) with 10% (20% for Gli56) fetal bovine serum (Gibco/Life Technologies, #16140-071), 1% g penicillin–streptomycin (Gibco/Life Technologies, #15140-122), and 0.1% amphotericin B (Gibco/Life Technologies, #15290-018). All cell lines were confirmed as mycoplasma negative by enzyme-linked immunosorbent assay using the MycoAlert PLUS Detection Kit (Lonza) and were authenticated by short tandem repeat DNA fingerprinting and chromosomal analysis by the Cytogenetics and Cell Authentication Core at MD Anderson. MD Andersons’s Institutional Animal Care and Use Committee approved all procedures for animal studies. Xenografts with the D423 (MTAP-WT), U87 (MTAP-deleted), U87 pCMV MTAP (MTAP-rescued), NB1 (MTAP-WT), SKMEL5 (MTAP-deleted), and Gli56 (MTAP-deleted) cancer cell lines were generated as detailed below for FFPE sections. For subcutaneous xenografts, 5 million cells were injected in the flanks of a nude athymic mouse (bred by MD Anderson’s Department of Experimental Radiation Oncology). Mice were housed in a room with an ambient temperature between 20 and 22 °C and humidity of 30–70%, in a 12-h light–12-h dark cycle. Tumors were harvested and fixed in 4% phosphate-buffered formalin. Dehydration, paraffin embedding, and tissue sectioning were performed by MD Anderson’s Veterinary Pathology Core. Intracranial glioma cell injections were performed by the MD Anderson Intracranial Injection Core at MD Anderson (Dr. Fred Lang, Director66). Immunocompromised female nude Foxn1nu/nu mice were first bolted. After 2 weeks, the mice were injected with 200,000 cells into the brain through the bolt by the MD Anderson Intracranial Injection Fee-for-Service Core66. Formalin-fixed brains with xenografted glioma tumors were submitted to the Veterinary Pathology Core for embedding and sectioning. p y That MTAP deletions can be leveraged as a point of selective vulnerability in various cancers has spurred much excitement. Our analysis challenges whether the metabolic conditions required for therapies to exploit vulnerabilities associated with elevated MTA/MTAP deletions are present in primary human tumors, giving pause to whether these would translate to the clinic. However, our findings do not rule out that MAT2A and/or PRMT5 inhibitors could prove useful in oncology. NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-24240-3 Merely, such inhibitors would have to achieve a therapeutic window through a mechanism other than MTA accumulation. Indeed, recent research has already pointed toward MTAP-deletion-independent sensitization mechanism to PRMT5 inhibition58. Some GBM tumors in our dataset showed relatively low SDMA levels—sug- gesting low PRMT5 activity—without MTAP deletions (Fig. 2g). Perhaps such tumors with low PRMT5 activity could be suitable candidates for targeted therapies against PRMT5 or MAT2A, provided a reliable molecular marker can be found to identify them. Metabolomic profiling of polar metabolites. Polar metabolites were profiled using the BIDMC platform. The extraction of samples was performed in the house as follows for each specific set of samples. Polar metabolites of GBM tumors were also profiled through fee for service by Metabolon, Inc. (Durham, NC). Targeted MS. A hybrid QTRAP 5500 triple quadrupole mass spectrometer (AB SCIEX), which is coupled to a Prominence UFLC HPLC system (Shimadzu), was used. First, each sample was resuspended in 20 μl high-performance liquid chro- matography (HPLC)-grade water, then 5–7 μl of it was injected into the spectro- meter. The selected reaction monitoring (SRM) method was used for steady-state analyses with 262 endogenous water-soluble metabolites with a dwell time of 3 ms for each SRM, and a total cycle time of 1.55 s, with 10–14 data points acquired for each metabolite. Voltages used for the positive and negative ion modes were +4950 and −4500 V, respectively. Hydrophilic interaction chromatography using a 4.6 mm × 10 cm Amide XBridge column (Waters) at 400 μl/min was used to deliver samples to the spectrometer. Following gradient sequence were used during the measurement: 85% buffer B (HPLC grade acetonitrile) to 42% B from 0 to 5 min, followed by 42% B to 0% B from 5 to 16 min, with no gradient from 16 to 24 min, and finally, gradients were run from 0% B to 85% B from 24 to 25 min, and 85% B was held for 7 min to re-equilibrate the column. Buffer A comprised 20 mM ammonium hydroxide/20 mM ammonium acetate (pH 9.0) in 95:5 water: NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-24240-3 Data in panel c are expressed as mean + SD, N = 3 biological replicates. e MTAP-deleted cancer cells accumulate MTA in conditioned media (detected by NMR) while secreted MTA is consumed by MTAP-intact macrophages, abrogating MTA accumulation in the extracellular environment. f Co-culture of MTAP-deleted cancer cell lines with MTAP-intact myeloid cells prevents loss of SDMA, indicating maintained PRMT5 activity, repeated once. MTAP-deleted and reconstituted glioma cells were co-cultured with myeloid leukemia cells (MV-4-11, a monomyelocytic suspension cell line). Before lysate preparation, suspended MV-4-11 myeloid cells were removed, and cancer cells were extensively washed. For each independent MTAP-deleted cell line, SDMA levels increased with myeloid co-culture, indicating restoration of PRMT5 activity. Fig. 3c in that publication9) in a substantial fraction of GBM tumors. Our study evaluated MTA in bulk GBM tumors, which are a heterogeneous mixture of cancer and stromal cells. One shortcoming of performing metabolomics and western blots on such tumors is that the measured signal (i.e., MTA levels and SDMA levels) derived from both cancer and stromal cells. Although signal derived from cancer cells is diluted by stromal cells, significant metabolic aberrations such as elevation of 2-HG can still be seen in resected bulk tumors (Fig. 2f and Supple- mentary Fig. 7). However, our data of profiling GBM tumors from two different dataset (HF series and MDA series) demon- strate that there are nonsignificant 1.4-fold (p = 0.2) and 1.14- fold (p = 0.9) increase in MTA levels in MTAP-deleted tumors versus intact ones. These numbers are too low to be explained by signal dilution caused by stromal contamination if cell culture data are taken as a reference point. However, our data do not rule the possibility of minor accumulation of MTA in MTAP-deleted glioma cells; just that such accumulation would have to be much less than reported in culture. p In sum, the extreme elevation of MTA in homozygous MTAP- deleted cells in vitro did not extend to our metabolome analyses of primary GBM tumors. A variety of reasons may explain this discrepancy between cell culture and human tumor data. The explanation we favor centers on the observation that MTAP- deleted cells excrete MTA into the media (Fig. 1h). NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-24240-3 The U87 MTAP- rescued cell line was generated using the same procedure we previously used to generate an ENO1-rescued cell line1. The MTAP cDNA was obtained from Life Technologies, sequence-verified, and cloned into a pCMV GFP lentiviral vector using Gateway cloning technology. 293T cells were transfected with viral plasmids using polyethyleneimine. The viral supernatant was collected 72 h after transfection and added to the U87 cell line for infection. g An additional explanation suggested by our findings is that human GBM tumor environments are extensively infiltrated by MTAP-expressing non-malignant stromal cells (Fig. 3 and Sup- plementary Figs. 15 and 16). To investigate the effects of MTAP- WT cells in the vicinity of MTAP-deleted cells on intracellular and extracellular MTA levels, we co-cultured MTAP-deleted cells with MTAP-WT ones. We observed that this co-culture abro- gated the accumulation of MTA in the conditioned media of MTAP-deleted cells. Furthermore, we found that the co-culture of MTAP-deleted cells with suspended MTAP-WT leukemia cells (MV-4-11, myeloid-like) resulted in higher SDMA levels—indi- cating higher PRMT5 activity and lower intracellular MTA levels —compared to the monoculture MTAP-deleted cells. Also, cul- turing MTAP-WT cells with labeled D3-MTA revealed labeling of intracellular MTA, methionine, and SAM, evidencing MTA uptake, consumption, and metabolism by MTAP-WT cells. These findings suggest that the metabolism of MTA by stromal cells is likely the predominant cause of the absence of any detectable increase in MTA in primary GBMs. However, our study does not rule out other factors that could influence MTA accumulation in MTAP-deleted cells, i.e., extreme cysteine deficiency shown by Sanderson et al.11 (though physiological media Plasmax still yielded MTA secretion in MTAP-deleted glioma cells, Supple- mentary Fig. 10). Our findings highlight the metabolic dis- crepancies between in vitro models and primary human tumors. It is already well understood that cell culture conditions do not always accurately reflect physiologic, metabolic conditions11,56,57: our study exemplifies another aspect, stromal infiltration, as a complicating factor for tumor metabolism. Our results suggest more systematic efforts to validate cell culture and xenograft studies on cancer metabolism with primary human tumor data. All cells were cultured at 37 °C in a 5% CO2 atmosphere at pH 7.4 in ATCC- suggested media (DMEM) unless stated otherwise. DMEM has 4.5 g/l glucose, pyruvate and glutamine (Cellgro/Corning, #10-013-CV) with 10% (20% for Gli56) fetal bovine serum (Gibco/Life Technologies, #16140-071), 1% penicillin–streptomycin (Gibco/Life Technologies, #15140-122), and 0.1% amphotericin B (Gibco/Life Technologies, #15290-018). NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-24240-3 Sec- tions were then covered with anti-MTAP rabbit monoclonal (Abcam, #ab126623, clone EPR6892, Lot: GR97816-5, Lot: YI070108C5) in a 1:200 dilution with 2% goat serum in PBS and left to incubate overnight at 4 °C. Sections were then washed in PBS and incubated for 30 min with 1× goat anti-rabbit IgG secondary antibody, poly-horseradish peroxidase conjugate (Invitrogen by Thermo Scientific, Ref: B40962, Lot: 2140280). After being washed in PBS and Tween 20 (Fisher BioR- eagents, #BP337-500), sections were developed using either Impact NOVAred (Vector Laboratories; yields a red to brown color for stain) or using EnzMet (Nanoprobes, #6001, 30 ml; yields a black stain). For NOVAred, slides were then counterstained using hematoxylin; for EnzMet, slides were counterstained with hematoxylin and, optionally, an eosin counterstain. Sections were mounted using Denville Ultra Microscope Cover Glass (#M1100-02) and Thermo Scientific Cytoseal 60 and left to dry overnight. There was an absolute correspondence between the genotype of the xenografts and the MTAP staining by immunohis- tochemistry, with only mouse stromal cells staining positive for MTAP in MTAP- deleted xenografts (Supplementary Figs. 12–14). We thus proceeded to utilize this antibody for scoring homozygous MTAP deletions in human primary GBM FFPE sections. Immunohistochemistry was performed as described for the human xenografted tumors. Immunohistochemistry was performed by utilizing citrate antigen retrieval and blocking in 2% goat serum. Slides were stained against MTAP (Abcam, rabbit monoclonal, #ab126623, Lot: GR97816-5, Lot: YI070108C5, Lot: GR90092-13, overnight at 4 °C) in a 1:250 dilution and developed with 1× goat anti-rabbit IgG secondary, poly-horseradish peroxidase conjugate (Thermo Sci- entific, #B40962) for 30 min at room temperature and developed with NOVAred (Vector Laboratories) or EnzMet (Nanoprobes, #6001, 30 ml) followed by coun- terstaining with hematoxylin or hematoxylin and eosin. The same procedure was used to stain the slides against the IBA1 antibody (Abcam, #ab178846, lot: GR207976-27) in a 1:1000 dilution. Determination of MTA by 1H-NMR in conditioned cell culture media. Condi- tioned media from cells was collected and centrifuged for 10 min to remove cells and cell debris. Post-centrifugation media (2 ml) was transferred to a new tube, where 4 (8 ml) volumes of methanol were added to precipitate proteins. The sample was then vortexed and centrifuged at the highest speed for 10 min, and the supernatant was transferred to a new tube. NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-24240-3 The supernatant containing polar metabolites was concentrated using an Eppendorf Vacufuge Plus67 and submitted to the BIDMC MS core. y trile, pH 9.45) and solvent B (acetonitrile). The flow rate was 150 µl/min. LC gradient was: 0 min, 85% B; 2 min, 85% B; 3 min 80% B; 5 min, 80% B; 6 min, 75% B; 7 min, 75% B; 8 min, 70% B; 9 min, 70% B; 10 min, 50% B; 12 min 50% B; 13 min, 25% B; 16 min, 25% B; 18 min, 0% B; 23 min, 0% B; 24 min, 85% B. MS scans were performed from m/z 70–1000 at 140,000 resolution. For MTA quantification, Secreted metabolites (conditioned media). A total of 1–2 ml of conditioned media was centrifuged at maximum speed for 10 min to remove any unblocked cells or cell debris. Subsequently, 4 volumes of −80 °C pre-cooled methanol for every 1 volume of media was added to make the final concentration of 80% (vol/vol) methanol solution, then vortexed and left at −80 °C for 6–8 h to pre- cipitate serum and extract polar metabolites. Ice-cold methanol media mix was centrifuged at maximum speed (17,000 × g) for 10 min at 4 °C. Following cen- trifugation, the supernatant was separated and dried in the vacuum concentrator (Eppendorf Vacufuge Plus) and then sent to the BIDMC core67. Tracing deuterated MTA. MTAP-WT mouse macrophages (RAW-267.4) and glioma cells (D423) were cultured in a 6-well plate with and without 100 μM deuterated MTA in DMEM for 1 day. Then cell culture media were collected and prepared for NMR studies. Cells were washed with ice-cold 1× PBS (Corning) and placed in dry ice. We then added 200 mM of the extraction buffer (40% methanol: 40% acetonitrile: 20% water) pre-cooled to −20 °C. Cells were scraped off the plate while on dry ice placed in pre-cooled tubes and finally centrifuged. Frozen tumors. GBM tumors were collected under the protocol approved by the institutional review board at MD Anderson Cancer Center (PA15-0940, LAB03- 0320, and LAB03-0221). Under the protocol, informed written consent from patients was obtained. Tumors snap-frozen in liquid nitrogen were kept at −80 °C until extraction and never thawed. Every effort was taken to keep tumors frozen until they were placed in cold methanol. NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-24240-3 acetonitrile. MultiQuant v2.1 software (AB SCIEX) was used to integrate total ion current peak. Jose, CA) coupled to hydrophilic interaction chromatography was used for MTA quantification. LC separation was on an XBridge BEH Amide column (2.1 mm × 150mm × 2.5 mm particle size, Waters, Milford, MA) using a gradient of solvent A (20 mM ammonia acetate, 20 mM ammonium hydroxide in 95:5 water: acetoni- trile, pH 9.45) and solvent B (acetonitrile). The flow rate was 150 µl/min. LC gradient was: 0 min, 85% B; 2 min, 85% B; 3 min 80% B; 5 min, 80% B; 6 min, 75% B; 7 min, 75% B; 8 min, 70% B; 9 min, 70% B; 10 min, 50% B; 12 min 50% B; 13 min, 25% B; 16 min, 25% B; 18 min, 0% B; 23 min, 0% B; 24 min, 85% B. MS scans were performed from m/z 70–1000 at 140,000 resolution. For MTA quantification, deuterium-labeled MTA was used as an internal standard. Data were analyzed using the EI-Maven 7.0 software (Elucidata, LLC., elucidata.io.), and isotope labeling was corrected for the natural abundance. The absolute amount of MTA in the total cell pellet and media (cell pellet approximately 3 M cells, media 3 ml) was calculated from this concentration. The cell density was determined by harvesting cells with trypsin and counting cells using trypan blue. Jose, CA) coupled to hydrophilic interaction chromatography was used for MTA quantification. LC separation was on an XBridge BEH Amide column (2.1 mm × 150mm × 2.5 mm particle size, Waters, Milford, MA) using a gradient of solvent A (20 mM ammonia acetate, 20 mM ammonium hydroxide in 95:5 water: acetoni- Intracellular metabolites from cells in culture (cell pellet). Adherent cancer cells growing in 10-cm plates at 50–90% confluency were harvested for metabo- lomic profiling as follows. First, media was removed (and extracted for its profil- ing), and the plated cells were washed twice with ice-cold 1× PBS (Corning). Cells were then placed in dry ice, and we added 4 ml of 80% methanol pre-cooled to −80 °C. Then they were incubated in the −80 °C freezer for 20 min. Cells were scraped off the plate while on dry ice, placed in pre-cooled tubes, and finally centrifuged at maximum speed, 17,000 × g for 5 min at 4 °C. NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-24240-3 The post-centrifuged supernatant (8 ml) was then vacuum concentrated using the Eppendorf Vacufuge Plus followed by lyophilization for 24 h; the resulting pellet was resuspended in 600 μl of D-DMSO (dimethyl sulfoxide-d6; Alfa Aesar, 99.5%, #A16893-18). Any insoluble material was removed by centrifugation. The clear D-DMSO solution was then placed in a 5-mm NMR tube for NMR analysis. 1H-NMR measurements were performed on a Bruker Avance III HD 500 MHz equipped with a CryoProbe Broadband Observe probe or a Bruker Avance One 600 MHz equipped with a 5-mm TXI probe at MD Anderson Cancer Center. The resulting spectrum was obtained using the zg30 pulse sequence with either 100 or 1024 scans and a relaxation delay equal to 1 s. Spectrum analysis was performed using the Bruker TopSpin 3.1 software. For MTA, the adenosine protons (HMDB0001173), with chemical shifts of 8.15 and 8.35 ppm, were readily detectable in the supernatant of MTAP-deleted media extract, but not MTAP-intact or MTAP-rescued extract, and in the supernatant of co-cultured MTAP-deleted and MTAP-intact media extract. The most intense MTA peak occurred at 2.1 ppm, corresponding to the methyl group protons adjacent to the sulfur. However, in the media extract of MTAP-deleted cells, this peak was obscured by more abundant metabolites. The peaks for the ribose group were of low intensity and not detectable. We validated our observed chemical shifts by spiking sample extracts with pure MTA standard (Sigma-Aldrich, #D5011, 100 mg). Absolute quantification of MTA in the cell pellet and the conditioned media. MTAP-deleted and WT cells were grown in DMEM or Plasmax supplemented with 2.5% FBS. After 3 days, the cell pellet and the conditioned media were harvested for quantification of MTA. Media was collected, spun down to remove any cells, and extracted with 80% methanol. Cells were washed with ice-cold 1× PBS (Corning) and placed in dry ice. Two hundred microliters of the extraction buffer (40% methanol: 40% acetonitrile: 20% water) pre-cooled to −20 °C. Cells were scraped off the plate while on dry ice placed in pre-cooled tubes. The concentration of MTA in the extract of cell pellet and extract of media was determined in LC-MS. A quadrupole-orbitrap mass spectrometer (Q Exactive, Thermo Fisher Scientific, San Western blot. Human snap-frozen GBM tumors were kept at −80 °C (same set of tumors used for metabolomic study). Without thawing, approximately 10 mg of tumor tissue was transferred to a new tube for immunoblotting. NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-24240-3 Frozen tumors were weighed on dry ice without thawing, and we placed them in dry ice pre-cooled Fisher Tube (#02-681- 291) with Qiagen steel beads. Then we added 1 ml of 80% at −80 °C pre-cooled methanol to each tube. Then we shook the tubes with Qiagen TissueLyser for 45 s at room temperature at 28 Hz for multiple rounds. We placed the tubes in the dry ice between each round. After samples became homogenous, the samples’ final volume was proportioned to 50 mg of tissue/2 ml of 80% methanol. After this step, we kept the samples at −80 °C for 24 h and then vortexed each sample and cen- trifuged them at maximum speed (17,000 × g) for 10 min at 4 °C. Then we collected the supernatant and stored it at −80 °C until vacuum concentration. For each study, we used Eppendorf Vacufuge Plus to vacuum-concentrate the same volume of samples. Dried samples were sent to the BIDMC core. Validation of an anti-MTAP rabbit monoclonal antibody for the immunohis- tochemistry. To confirm homozygous deletions MTAP by immunohistochemistry in primary tumor FFPE sections, we first validated a monoclonal antibody for this application by demonstrating staining in FFPE slides of xenografted tumors with a known MTAP genotype. Xenografts generated with the D423 (MTAP-WT), U87 (MTAP-deleted), U87 pCMV MTAP (MTAP-rescued) NB1 (MTAP-WT), application by demonstrating staining in FFPE slides of xenografted tumors with a known MTAP genotype. Xenografts generated with the D423 (MTAP-WT), U87 (MTAP-deleted), U87 pCMV MTAP (MTAP-rescued) NB1 (MTAP-WT), SKMEL5 (MTAP-deleted), and Gli56 (MTAP-deleted) cancer cell lines were fixed in formalin and embedded in paraffin. Immunohistochemistry was performed on coronal sections of mouse brain xenografted with human glioblastoma cells. Tissue was fixed in formaldehyde before being embedded in paraffin. The tissue was sliced to the desired thickness and embedded onto the slide. Slides were left to incubate at 60 °C overnight. To detect MTAP, sections were subjected to antigen retrieval in citrate buffer (1:100 Vector Laboratories Antigen Unmasking Solution [Citrate- Based], H-3300, 250 ml) at high pressure for 10 min. Sections were covered with a blocking buffer of 2% goat serum (Vector Laboratories, S-1000, Normal Goat Serum, 20 ml) in PBS (Quality Biological PBS 10×, pH 7.4, 1000 ml) for 1 h. ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-24240-3 Methods Id tifi ti Identification of cell lines with MTAP-homozygous deletions. Scoring of homozygous deletions, even with robust copy-number data, can be problematic owing to uneven ploidy across cell lines, as well as where deletion boundaries only partially cover the gene yet eliminate its functional expression. Thus, we sought to identify functionally MTAP-null cell lines. 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NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-24240-3 NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-24240-3 dilution, MTAP (Abcam, #ab126623, Lot: GR97816-5, Lot: YI070108C5, Lot: GR90092-13) in a 1:1000 dilution, GAPDH (Sigma-Aldrich, #G9545, lot: 127M4814V) in a 1:5000 dilution, IBA1 (Abcam, #ab178846, lot: GR207976-27) in a 1:1000 dilution, and vinculin (Cell Signaling Technology, #13901) in a 1:5000 dilution. Western blot bands were quantified using ImageJ 1.52q. dilution, MTAP (Abcam, #ab126623, Lot: GR97816-5, Lot: YI070108C5, Lot: GR90092-13) in a 1:1000 dilution, GAPDH (Sigma-Aldrich, #G9545, lot: 18. Kalev, P. et al. MAT2A inhibition blocks the growth of MTAP-deleted cancer cells by reducing PRMT5-dependent mRNA splicing and inducing DNA damage. 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Trends in Cancer 5, 329–332 (2019). 12 NATURE COMMUNICATIONS | (2021) 12:4228 | https://doi.org/10.1038/s41467-021-24240-3 | www.nature.com/naturecommunicatio Acknowledgements This work was financially supported by the following grants to F.L.M.: U.S. National Institutes of Health (NIH) grant 1R21CA226301, the American Cancer Society Research Scholar Award RSG-15-145-01-CDD, the National Comprehensive Cancer Network–Young Investigator Award YIA170032, and the Andrew Sabin Family Foun- dation Fellows Award. The University of Texas MD Anderson Cancer Center/Glio- This work was financially supported by the following grants to F.L.M.: U.S. National Institutes of Health (NIH) grant 1R21CA226301, the American Cancer Society Research Scholar Award RSG-15-145-01-CDD, the National Comprehensive Cancer Network–Young Investigator Award YIA170032, and the Andrew Sabin Family Foun- dation Fellows Award. The University of Texas MD Anderson Cancer Center/Glio- © The Author(s) 2021 © The Author(s) 2021 13 NATURE COMMUNICATIONS | (2021) 12:4228 | https://doi.org/10.1038/s41467-021-24240-3 | www.nature.com/naturecommunications

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A new record of Oxychilus alliarius (Gastropoda: Zonitidae) with the species distribution in the Czech Republic

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Malacologica Bohemoslovaca (2009), 8: 63–65 ISSN 1336-6939 Malacologica Bohemoslovaca (2009), 8: 63–65 ISSN 1336-6939 A new record of Oxychilus alliarius (Gastropoda: Zonitidae) with the species distribution in the Czech Republic JITKA HORÁČKOVÁ1 & LUCIE JUŘIČKOVÁ2 1Department of Ecology, Faculty of Science, Charles University in Prague, Viničná 7, CZ-12844 Prague 2, Czech Republic; e-mail: [email protected] 2Department of Zoology, Faculty of Science, Charles University in Prague, Viničná 7, CZ-12844 Prague 2, Czech Republic; e-mail: [email protected] HORÁČKOVÁ J. & JUŘIČKOVÁ L., 2009: A new record of Oxychilus alliarius (Gastropoda: Zonitidae) with the spe cies distribution in the Czech Republic. – Malacologica Bohemoslovaca, 8: 63-65. Online serial at <http://mol lusca.sav.sk> 3-December-2009. A new finding of the land snail species Oxychilus alliarius was recorded in the Czech Republic. This West Euro- pean species was found in the six isolated sites during the last thirteen years always in western part of Bohemia. This paper brings new information on the distribution of Oxychilus alliarius in the Czech Republic. Key words: Mollusca, Czech Republic, river floodplains, faunistic, Oxychilus alliarius Introduction LOŽEK 2003, HLAVÁČ et al. 2003, HORÁČKOVÁ & DVOŘÁK 2008). The first finding of the species was recorded by LO- ŽEK (1996) in Getsemanka II Nature Reserve in the Brdy Mts. This paper brings new information on its occurrence in the Czech Republic. LOŽEK 2003, HLAVÁČ et al. 2003, HORÁČKOVÁ & DVOŘÁK 2008). The first finding of the species was recorded by LO- ŽEK (1996) in Getsemanka II Nature Reserve in the Brdy Mts. This paper brings new information on its occurrence in the Czech Republic. Oxychilus alliarius (Miller, 1822) (Gastropoda: Zonitidae) is a terrestrial snail, widespread in western and northern parts of Europe. Besides isolated occurrences on the is- lands of the Iceland and British Islands, it is common in the continental part of Europe from West France to North Switzerland, North-West Germany, North Poland, South Norway, Sweden, and Finland. Elsewhere in Scandinavia, it occurs only in greenhouses. It is a mesophilous species of woods, rocks, fields, and occasionally gardens and gre- enhouses (KERNEY et al. 1983). Having the shell width up to 7 mm, it belongs to one of the smallest species of the genus Oxychilus in Central Europe (Fig. 1). It has a ruddy shell and dark grey or black coloured body in contrast to related similar species O. cellarius (light grey or brow- nish coloured body) and O. draparnaudi (blue coloured body). Oxychilus alliarius smells strongly of garlic, hence its species-name. It has been found on six sites (Fig. 2) in the western part of the Czech Republic (JUŘIČKOVÁ & Material and methods O. alliarius was collected during the ongoing malacologi- cal research of the Ohře River floodplain. Mollusc assem- blages were sampled using a standard sampling procedure (CAMERON & POKRYSZKO 2005). One person searched by eye in all microhabitats on the site for 30 minutes. Litter samples were taken from four quadrates (25×25 cm) in the plot of size 10×10 m at each of sites. Forty-seven sites situated on both river banks in the alluvium of the Ohře River were researched. Nomenclature follows JUŘIČKOVÁ et al. (2008) with several up-to-date changes. The shells are deposited in the collection of the first author. Fig. 1. Shell of Oxychilus alliarius (Miller, 1822) from the Ohře River floodplain (West Bohemia); height = 1.9 mm, width = 5.0 mm. Photo: Jitka Horáčková. Fig. 1. Shell of Oxychilus alliarius (Miller, 1822) from the Ohře River floodplain (West Bohemia); height = 1.9 mm, width = 5.0 mm Photo: Jitka Horáčková of Oxychilus alliarius (Miller, 1822) from the Ohře River floodplain (West Bohemia); height = 1.9 mm, width = 5 tka Horáčková. hilus alliarius (Miller, 1822) from the Ohře River floodplain (West Bohemia); height = 1.9 mm, width = 5.0 áčk á 63 The list of sites 49°35'35" N, 13°45'10" E, 6448b, 690 m, Míšov near Rožmitál pod Třemšínem, Nature Reserve of Getsemanka II in the Brdy Mts., mesic submontane deciduous forest, 20 May 1993 (JUŘIČKOVÁ & LOŽEK 2003). 1. 49°35'35" N, 13°45'10" E, 6448b, 690 m, Míšov near Rožmitál pod Třemšínem, Nature Reserve of Getsemanka II in the Brdy Mts., mesic submontane deciduous forest, 20 May 1993 (JUŘIČKOVÁ & LOŽEK 2003). 1. 49°35'35" N, 13°45'10" E, 6448b, 690 m, Míšov near Rožmitál pod Třemšínem, Nature Reserve of Getsemanka II in the Brdy Mts., mesic submontane deciduous forest, 20 May 1993 (JUŘIČKOVÁ & LOŽEK 2003). 2. 49°56'29" N, 13°08'58" E, 6044d, 615 m, Umíř village near Plachtín, cellar in the ruins of a house, 13 speci- mens (9 of them living), 7 July 1996 (JUŘIČKOVÁ & LOŽEK 2003). ) 3. 49°56'53" N, 13°03'13" E, 6044c, 650 m, Kejšovice near Úterý, humid ditch with bushes, 3 living specimens, 21 August 2002 (JUŘIČKOVÁ & LOŽEK 2003). 4. 49°41'29.64" N, 12°44'20.02" E, 6342a, 505 m, Muckov village, farm at north-western outskirts of the village, 4 li- ving specimens, 18 May 2003 (HLAVÁČ et al. 2003). 5. 49°32'44.5" N, 12°36'09.3" E, 6441d, 784 m, a former 3. 49°56'53" N, 13°03'13" E, 6044c, 650 m, Kejšovice near Úterý, humid ditch with bushes, 3 living specimens, 21 August 2002 (JUŘIČKOVÁ & LOŽEK 2003). 4. 49°41'29.64" N, 12°44'20.02" E, 6342a, 505 m, Muckov village, farm at north-western outskirts of the village, 4 li- ving specimens, 18 May 2003 (HLAVÁČ et al. 2003). 5. 49°32'44.5" N, 12°36'09.3" E, 6441d, 784 m, a former village of Pleš, the ruins of a church and an old cemetery, 16 August 2007 – 4 living specimens, 1 May 2008 – 3 li- ving specimens (HORÁČKOVÁ & DVOŘÁK 2008). 6. 50°11'03.10" N, 12°42'54.00" E, 5842a, 391 m, Sta- ré Sedlo near Sokolov, softwood forest with ruins of a building in the alluvium of the Ohře River, 27 July 2008 – 2 living specimens, Jitka Horáčková. 3. 49°56'53" N, 13°03'13" E, 6044c, 650 m, Kejšovice near Úterý, humid ditch with bushes, 3 living specimens, 21 August 2002 (JUŘIČKOVÁ & LOŽEK 2003). 4. 49°41'29.64" N, 12°44'20.02" E, 6342a, 505 m, Muckov village, farm at north-western outskirts of the village, 4 li- ving specimens, 18 May 2003 (HLAVÁČ et al. 2003). 5. The list of sites Oxychilus alliarius was found only at one site (site no. 6, Fig. 2) in the alluvium of the Ohře River at the outskirts of Staré Sedlo village near Sokolov (Karlovy Vary district, West Bohemia). On July 27, 2008, two living specimens were found in a litter sample. The habitat is covered by an alluvial hardwood forest with a rich herb layer dominated by Urtica dioica, Aegopodium podagraria, Gallium apa- rine, Stellaria nemorum, and Rubus caesius. Despite the almost natural present character of the habitat, the building ruins are situated here. Along with Oxychilus alliarius fol- lowing species were present: dominant species Carychium minimum (29 %), Monachoides incarnatus (13 %), Vitrina pellucida (10 %), Perpolita hammonis (10 %) and additi- onal species as Arion lusitanicus, Eucobresia diaphana, Arianta arbustorum, Cepaea hortensis, Limax cinereo- niger, Malacolimax tenellus, Discus rotundatus, Urtici- cola umbrosus, Alinda biplicata, Semilimax semilimax, Succinea putris, Trochulus hispidus, Columella edentula, Carychium tridentatum, Vallonia costata, Cochlicopa lu- brica, Punctum pygmaeum, Vertigo pygmaea, Zonitoides nitidus. This site is the northernmost and lowest (391 m) locality of O. alliarius in the Czech Republic. Every other sites were situated more southern at altitudes above 500 m. The scattered localities in western Bohemia related to the distribution of this species in western Europe. The nearest abroad sites are known in Silesia and Saxony (East Ger- many). Except the first finding of O. alliarius from Getse- manka II Nature Reserve (LOŽEK 1996), the other Czech sites support a synantropic character of this species. List of known sites of Oxychilus alliarius in the Czech Republic is given. Data in the site list as follows: number of the site, geographical co-ordinates in WGS-84 system, quadrate number of faunistic mapping according to PRUNER & MÍKA (1996), elevation (m a.s.l.), name of the nearest settlement, descripition of the habitat, date of the research, number of living individuals or empty shells. g p y 1. 49°35'35" N, 13°45'10" E, 6448b, 690 m, Míšov near Rožmitál pod Třemšínem, Nature Reserve of Getsemanka II in the Brdy Mts., mesic submontane deciduous forest, 20 May 1993 (JUŘIČKOVÁ & LOŽEK 2003). 2. 49°56'29" N, 13°08'58" E, 6044d, 615 m, Umíř village near Plachtín, cellar in the ruins of a house, 13 speci- mens (9 of them living), 7 July 1996 (JUŘIČKOVÁ & LOŽEK 2003). 1. The list of sites – IV. New data for the southern part of the Český Les Mts.]. – Malacologica Bohemoslovaca, 7: 81–92, Český Les Mts.]. – Malacologica Bohemoslovaca, 7: 81–9 online serial at <http://mollusca.sav.sk> 1-Oct-2008. JUŘIČKOVÁ L. & LOŽEK V., 2003: Oxychilus alliarius (Gastropoda: Zonitidae) in the Czech Republic. – Acta Soc. Zool. Bohem., 67: 183–184. JUŘIČKOVÁ L., HORSÁK M., BERAN L. & DVOŘÁK L., 2008: Check- -list of the molluscs (Mollusca) of the Czech Republic. – http:// mollusca.sav.sk/malacology/checklist.htm (last update: 26 Au- gust 2008). Acknowledgement The research was supported by the project of the Grant Agency of Charles University GA UK no. 40007 and MSMT project 0021620828. KERNEY M.P., CAMERON R.A.D., JUNGBLUTH J.H., 1983: Die Land- schnecken Nord- und Mitteleuropas. – Hamburg-Berlin: Paul Parey, 384 pp. The list of sites 49°32'44.5" N, 12°36'09.3" E, 6441d, 784 m, a former village of Pleš, the ruins of a church and an old cemetery, 16 August 2007 – 4 living specimens, 1 May 2008 – 3 li- ving specimens (HORÁČKOVÁ & DVOŘÁK 2008). 5. 49°32'44.5" N, 12°36'09.3" E, 6441d, 784 m, a former village of Pleš, the ruins of a church and an old cemetery, 16 August 2007 – 4 living specimens, 1 May 2008 – 3 li- ving specimens (HORÁČKOVÁ & DVOŘÁK 2008). 6. 50°11'03.10" N, 12°42'54.00" E, 5842a, 391 m, Sta- ré Sedlo near Sokolov, softwood forest with ruins of a building in the alluvium of the Ohře River, 27 July 2008 – 2 living specimens, Jitka Horáčková. 6. 50°11'03.10" N, 12°42'54.00" E, 5842a, 391 m, Sta- ré Sedlo near Sokolov, softwood forest with ruins of a building in the alluvium of the Ohře River, 27 July 2008 – 2 living specimens, Jitka Horáčková. Fig. 2. Known distribution of Oxychilus alliarius in the Czech Republic. Fig. 2. Known distribution of Oxychilus alliarius in the Czech Republic. 64 [Molluscs of the Český Les Mts. – III. Kateřinská kotlina and northern part of Čerchovský les (Western Bohemia)]. – Silva Gabreta, 9: 145–166. [Molluscs of the Český Les Mts. – III. Kateřinská kotlina and northern part of Čerchovský les (Western Bohemia)]. – Silva Gabreta, 9: 145–166. The populations of Umíř village (site no. 2) became com- pletely extinct in 2000 (JUŘIČKOVÁ & LOŽEK 2003). The re- peatedly monitored population in Muckov village (2008, 2009; site no. 4) was probably extinct too. , HORÁČKOVÁ J. & DVOŘÁK L., 2008: Měkkýši Českého lesa – IV. N é úd j již í čá Č kéh l [M ll HORÁČKOVÁ J. & DVOŘÁK L., 2008: Měkkýši Českého lesa – IV. Nové údaje pro jižní část Českého lesa [Molluscs of the Český Les Mts. – IV. New data for the southern part of the Nové údaje pro jižní část Českého lesa [Molluscs of the Český Les Mts IV New data for the southern part of the O. alliarius has the eastern boundary of its distribution in the Czech Republic. Although this species is very com- mon in the main area of its distribution, populations on the distribution boundary seem to be more susceptible to disturbance. j p j [ of the Český Les Mts. – IV. New data for the southern part of the of the Český Les Mts. References LOŽEK V., 1996: Oxychilus alliarius v Čechách [Oxychilus allia- rius in Bohemia]. – Živa, 44: 76. CAMERON R.A.D. & POKRYSZKO B. M., 2005: Estimating the spe- cies richness and composition of land mollusc communities: problems, consequences and practical advice. – Journal of Conchology, 38: 529–548. PRUNER L. & MÍKA P., 1996: Seznam obcí a jejich částí v Čes- ké republice s čísly mapových polí pro síťové mapování fauny [List of settlements in the Czech Republic with associated map field codes for faunistic grid mapping system]. – Klapalekiana, 32 (Suppl.): 1–115 (in Czech). HLAVÁČ J.Č., BERAN L., DVOŘÁK L., HORSÁK M., JUŘIČKOVÁ L. & VRABEC V., 2003: Měkkýši Českého lesa – III. Kateřinská kotlina a severní část Čerchovského lesa (západní Čechy) 65 65

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Циклы-утки в системе Льенарда

Vestnik Samarskogo gosudarstvennogo tehničeskogo universiteta. Seriâ: Fiziko-matematičeskie nauki/Vestnik Samarskogo gosudarstvennogo tehničeskogo universiteta. Seriâ Fiziko-matematičeskie nauki

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ЦИКЛЫ-УТКИ В СИСТЕМЕ ЛЬЕНАРДА Рассматривается возможность применения теории Мельникова о предельных циклах слабо-возму- щенных двумерных дифференциальных систем к изучению циклов-уток в сингулярно-возмущенной системе Льенарда. Находится асимптотическое разложение первого порядка значения параметра, при котором в системе Льенарда существует цикл-утка. абота посвящена изучению циклов-уток в двумерной сингулярно-возмущенной системе (1) ( ), ( , ), x y f x y g x y ε α ′ ′ = − = − (1) ( ), x y f x ε ′ = − (1) ( ), ( , ), x y f x y g x y ε α ′ ′ = − = − е 0 ε > - малый параметр, α - параметр, а ( ) f x и ( , ) g x y - аналитические функции. где 0 ε > - малый параметр, α - параметр, а ( ) f x и ( , ) g x y - аналитические функции. Если функция ( , ) g x y зависит только от переменной x , то систему (1) можно переписать в виде дифференциального уравнения второго порядка Если функция ( , ) g x y зависит только от переменной x , то систему (1) можно переписать в виде дифференциального уравнения второго порядка ( ) ( ) 0 x f x x g x ε α ′′ ′ ′ + + − = , (2) ( ) ( ) 0 x f x x g x ε α ′′ ′ ′ + + − = , (2) ( ) ( ) 0 x f x x g x ε α ′′ ′ ′ + + − = , ( ) ( ) 0 x f x x g x ε α ′′ ′ ′ + + − = , (2) известного под названием сингулярно-возмущенного уравнения Льенарда [1]. Поэтому систему (1) будем называть системой Льенарда. График функции ( ) y f x = называется медленной кривой системы (1). Если медленная кривая имеет S -образную форму, то в системе (1) при некоторых значениях параметра α могут появиться релаксационные циклы. При 0 ε → релаксационные циклы стремятся к кривым, состоящим из участков быстрого движения и устойчивых дуг медленной кривой. Но в некоторых случаях система (1) может иметь также решения, фазовые кривые которых при 0 ε → стремятся к кривым, содержащим неустойчивые дуги медленной кривой. Такие решения называются решениями-утками [2]. Если решение-утка является предельным циклом, то это решение называется циклом-уткой. УДК 517.927 УДК 517.927 Общероссийский математический портал С. В. Богатырев, Циклы-утки в системе Льенарда, Вестн. Сам. гос. техн. ун- та. Сер. Физ.-мат. науки, 2001, выпуск 12, 36–39 DOI: 10.14498/vsgtu59 DOI: 10.14498/vsgtu59 Использование Общероссийского математического портала Math-Net.Ru подразумевает, что вы прочитали и согласны с пользовательским соглашением http://www.mathnet.ru/rus/agreement Параметры загрузки: IP: 195.242.1.95 24 октября 2024 г., 08:16:49 Параметры загрузки: IP: 195.242.1.95 24 октября 2024 г., 08:16:49 Тогда замена переменных Тогда замена переменных 0 0 0 0 , , ( , ) x x y y g x y ξ η α β = + = + = + (4) приведет систему (1) к виду ( ), ( , ), p q εξ η ξ η β ξ η ′ ′ = − = − (5) где 0 0 0 0 0 0 ( ) ( ) ( ), ( , ) ( , ) ( , ). p f x f x q g x y g x y ξ ξ ξ η ξ η = + − = + + − (6) Из (3) и (6) следует, что (0) 0, (0) 0, (0) 0, (0,0) 0, (0,0) 0 p p p q qξ ′ ′′ ′ = = ≠ = ≠ . (7) Из (7) следует, что для функций ( ) p ξ и ( , ) q ξ η имеют место следующие разложения: нных 0 0 0 0 , , ( , ) x x y y g x y ξ η α β = + = + = + (4) (4) приведет систему (1) к виду (5) 0 0 0 0 0 0 ( ) ( ) ( ), ( , ) ( , ) ( , ). p f x f x q g x y g x y ξ ξ ξ η ξ η = + − = + + − (6) следует что (6) Из (3) и (6) следует, что (0) 0, (0) 0, (0) 0, (0,0) 0, (0,0) 0 p p p q qξ ′ ′′ ′ = = ≠ = ≠ . (7) дует, что (0) 0, (0) 0, (0) 0, (0,0) 0, (0,0) 0 p p p q qξ ′ ′′ ′ = = ≠ = ≠ . (7) (7) Из (7) следует, что для функций ( ) p ξ и ( , ) q ξ η имеют место следующие разложения: Из (7) следует, что для функций ( ) p ξ и ( , ) q ξ η имеют место следующие разложения: 2 1 ( ) , ( , ) , k k n k kn k k n p p q q ξ ξ ξ η ξ η ∞ ∞ = + ≥ = = ∑ ∑ (8) (8) причем 2 0 p ≠ и 10 0 q ≠ . ЦИКЛЫ-УТКИ В СИСТЕМЕ ЛЬЕНАРДА Известно [3], что если медленная кривая имеет точку складки, через которую с ненулевой скоростью проходит особая точка системы (1) при прохождении параметром α некоторого значения 0 α , то при некотором значении 0 ( ) O α α ε = + система (1) будет иметь траекторию- утку. Для системы (1) эти условия выполняются в том случае, когда функции ( ) f x и ( , ) g x y удовлетворяют условиям у 0 0 0 0 0 : ( ) 0, ( ) 0, ( , ) 0, x x f x f x g x y ′ ′′ ′ ∃ = ≠ ≠ (3) (3) где 0 0 ( ) y f x = . В дальнейшем мы будем считать, что эти условия выполнены. Тогда замена переменных Тогда замена переменных Таким образом, систему (5) можно переписать в виде причем 2 0 p ≠ и 10 0 q ≠ . Таким образом, систему (5) можно переписать в виде 2 2 10 01 3 2 , . k k n k kn k k n p p q q q εξ η ξ ξ η β ξ η ξ η ∞ ∞ = + ≥ ′ ′ = − − = − − − ∑ ∑ (9) (9) Доказательство существования циклов-уток в системе (1) может быть получено с помощью, так называемой, теории Мельникова. После опубликования работы [4] зарубежные математики называют теорией Мельникова метод исследования дифференциальных систем, близких к Доказательство существования циклов-уток в системе (1) может быть получено с помощью, так называемой, теории Мельникова. После опубликования работы [4] зарубежные математики называют теорией Мельникова метод исследования дифференциальных систем, близких к 36 гамильтоно-вым системам, с помощью техники регулярных возмущений. Обычно предполагается, что гамильтонова система имеет семейство специальных решений, например, периодических орбит. Тогда регулярное малое возмущение гамильтоновой системы может привести к разрушению всего семейства специальных решений. Но при этом одно, или несколько специальных решений могут выжить. Под теорией Мельникова понимают метод нахождения этого выжившего решения. Специальное решение невозмущенной системы называют порождающим, если оно выживает в возмущенной системе. Существует большое количество версий теории Мельникова. В настоящей работе будем пользоваться вариантом теории Мельникова, полученным в работе [5]. В этой работе изучались предельные циклы системы ( ) ( , ), z f z g z ε ε ′ = + (10) (10) где 0 ε > - малый параметр, 2 z R ∈ , a ( ) f z и ( , ) g z ε - аналитические функции. Предполага- лось, что система (10) при 0 ε = имеет однопараметрическое семейство периодических орбит : ( ), (0, ) h h t h γ Γ ∈ ∞ периода h T . Было показано, что порождающую периодическую орбиту мож- но найти с помощью, так называемой, функции Мельникова. где 0 ε > - малый параметр, 2 z R ∈ , a ( ) f z и ( , ) g z ε - аналитические функции. Предполага- лось, что система (10) при 0 ε = имеет однопараметрическое семейство периодических орбит : ( ), (0, ) h h t h γ Γ ∈ ∞ периода h T . Было показано, что порождающую периодическую орбиту мож- но найти с помощью, так называемой, функции Мельникова. Тогда замена переменных фу О п р е д е л е н и е 1. [5] Функцией Мельникова возмущенной системы (10) вдоль цикла : ( ) h h t γ Γ периода h T соответствующей невозмущенной системы называется функция 0( , ( ( ))) 0 ( ) ( )( ( ),0) , t h h T f s ds h M h e f g t dt γ γ − ∇ ∫ = ∧ ∫ (11) (11) где произведение x y ∧ векторов 2 ,x y R ∈ определяется равенством 1 2 1 2 x y x y y x ∧ = − . изведение x y ∧ векторов 2 ,x y R ∈ определяется равенством 1 2 1 2 x y x y y x ∧ = − . Т е о р е м а 1. [5] Если существует такое 0 (0, ) h ∈ ∞, что е о р е м а 1. [5] Если существует такое 0 (0, ) h ∈ ∞, что Т е о р е м а 1. [5] Если существует такое 0 (0, ) h ∈ ∞, что 0 0 0 ( ) 0, ( ) 0, M h M h ′ = ≠ (12) (12) то для всех достаточно малых 0 ε ≠ система (10) имеет единственный гиперболический пре- дельный цикл в ( ) O ε -окрестности орбиты 0h Γ . Если 0 ( ) 0 M h ≠ , то для всех достаточно ма- лых 0 ε ≠ система (10) не имеет циклов в ( ) O ε -окрестности орбиты то для всех достаточно малых 0 ε ≠ система (10) имеет единственный гиперболический пре- дельный цикл в ( ) O ε -окрестности орбиты 0h Γ . Если 0 ( ) 0 M h ≠ , то для всех достаточно ма- лых 0 ε ≠ система (10) не имеет циклов в ( ) O ε -окрестности орбиты Отметим, что похожий результат (теорему о циклах слабо-возмущенных гамильтоновых систем) был намного раньше доказан Понтрягиным [6,7]. В настоящей работе будет показано, как с помощью теоремы 1 получить доказательство теоремы о существовании циклов-уток у системы (1). Кроме этого будет найдена аппрок- симация первого порядка значения параметра α , при котором циклы-утки существует. Отметим, что похожий подход для доказательства существования циклов-уток применялся в работах [8,9]. В [10] похожий подход был применен для нахождения первого члена разложения значения параметра, соответствующего циклу-утке в системе Ван-дер-Поля. Прежде всего преобразуем систему (9) к виду (10). Это преобразование может быть выпол- нено с помощью нормирующей замены переменных 2 , , , , . u v t ξ ε η ε ετ β εδ ε µ = = = = = (13) (13) В новых переменных система (9) примет вид В новых переменных система (9) примет вид 2 3 2 2 2 2 3 10 01 20 ( ), ( ) ( ). u v p u p u O v q u q v q u O µ µ µ δ µ ′ ′ = − − + = − + − − + (14) В новых переменных система (9) примет вид 2 3 2 2 2 2 3 10 01 20 ( ), ( ) ( ). u v p u p u O v q u q v q u O µ µ µ δ µ ′ ′ = − − + = − + − − + (14) 2 3 2 2 2 2 3 10 01 20 ( ), ( ) ( ). Т е о р е м а 1. [5] Если существует такое 0 (0, ) h ∈ ∞, что u v p u p u O v q u q v q u O µ µ µ δ µ ′ ′ = − − + = − + − − + (1 (14) Система (14) является слабым возмущением системы Система (14) является слабым возмущением системы 2 2 10 , . u v p u v q u ′ ′ = − = − (15) 2 2 10 , . u v p u v q u ′ ′ = − = − (15) Систему (15) нетрудно проинтегрировать. Все фазовые траектории этой системы лежат на кри- вых, являющихся решениями семейства уравнений Систему (15) нетрудно проинтегрировать. Все фазовые траектории этой системы лежат на кри- вых, являющихся решениями семейства уравнений 2 10 2 2 10 2 2 2 1 exp . 2 q p v u v h const q p p     − − − = ≡       (16) (16)   Элементарный анализ уравнений (16) позволяет доказать следующий результат. Элементарный анализ уравнений (16) позволяет доказать следующий результат Л е м м а 1. Если 10 0 q > , то кривая (16) является замкнутой для всех 10 ( / 2 h q p ∈− Во всех остальных случаях кривая (16) не будет замкнутой ни для каких значений h. Л е м м а 1. Если 10 0 q > , то кривая (16) является замкнутой для всех 2 10 2 ( / 2 ,0) h q p ∈− . В (16) б д й д й h Л е м м а 1. Если 10 0 q > , то кривая (16) является замкнутой для всех 10 ( / 2 h q p ∈ сех остальных случаях кривая (16) не будет замкнутой ни для каких значений h. тальных случаях кривая (16) не будет замкнутой ни для каких значений h. Таким образом, при описанных в лемме 1 значениях параметра h (эти значения мы будем называть допустимыми) система (15) имеет однопараметрическое семейство периодических орбит : ( ) h h t γ Γ , форма которых определяется уравнением (16). Поэтому для изучения 37 предельных циклов системы (14) можно применить теорему 1. Прежде всего найдем функцию Мельникова (11) для системы (14). Т е о р е м а 1. [5] Если существует такое 0 (0, ) h ∈ ∞, что Для этого в двойном интеграле из формулы (17) сделаем замену переменных будет лежать предельный цикл системы (9). Этот цикл будет циклом уткой системы (9). Теперь найдем первый член разложения решения ( ) h δ уравнения ( , ) 0 M h δ = . Для этого в двойном интеграле из формулы (17) сделаем замену переменных ( ) ( ) / / ( ) ( ) 10 2 10 2 / , / . u q p x v q p y = = Тогда для функции ( , ) M h δ получим представление Тогда для функции ( , ) M h δ получим представление ( ) ( ) ( ) 10 2 2 10 3 10 2 20 10 01 2 10 01 2 2 2 2 ( , ) 3 2 2 2 H y q q M h e p q p q x q q y p q q dxdy p p δ δ − Ω   = − − + +     ∫∫ , где H Ω - область, ограниченная кривой ( ) 2 2 1/ 2 y e x y H − − − = , ( 2 10 / H p h q = ). Т е о р е м а 1. [5] Если существует такое 0 (0, ) h ∈ ∞, что Описание этой функ-ции и ее свойств можно найти также в обзорной статье [12], помещенной в Share Library пакета аналитических вычислений Maple V. где через 0 W и 1 W− обозначены две вещественные ветви функции Ламберта W [11], которая определяется как функция, обратная к отображению w w we a . Описание этой функ-ции и ее свойств можно найти также в обзорной статье [12], помещенной в Share Library пакета аналитических вычислений Maple V. p 12], что при 0 h → имеют место следующие асимптотические разложения: p Известно [12], что при 0 h → имеют место следующие асимптотические разло 2 2 0 1 ( ) ( ), ( ) ln | | ln | ln | || (ln | ln | ||) W h h h o h W h h h o h − = − + = − + ( ) 1/ R h ε лишь для exp( 1/ ) h ε Таким образом доказана следующ 2 2 0 1 ( ) ( ), ( ) ln | | ln | ln | || (ln | ln | ||). W h h h o h W h h h o h − = − + = − + ) ~1/ h ε лишь для ~ exp( 1/ ) h ε − . Таким образом, доказана следующая теорема. Т е о р е м а 2. Для каждого допустимого и достаточно маленького значения параметра h ( ~ exp( 1/ )) h ε − найдется такое значение ( ) h β εδ = , где ( ) h δ является решением уравнения ( , ) 0 M h δ = , что в некоторой ( ) O ε - окрестности кривой 2 10 2 2 10 2 2 2 1 exp 2 q p h q p p η ξ η ε ε ε     − − − =       (19) (19) будет лежать предельный цикл системы (9). Этот цикл будет циклом-уткой системы (9). Теперь найдем первый член разложения решения ( ) h δ уравнения ( , ) 0 M h δ = . Для этого в двойном интеграле из формулы (17) сделаем замену переменных будет лежать предельный цикл системы (9). Этот цикл будет циклом-уткой системы (9). Теперь найдем первый член разложения решения ( ) h δ уравнения ( , ) 0 M h δ = . Т е о р е м а 1. [5] Если существует такое 0 (0, ) h ∈ ∞, что С помощью несложных преобразований получим   2 3 2 01 20 3 10 2 ( , ) exp ( ) h p M h v q v q u du p u dv q δ δ Γ    = − − − + =      ∫ ( ) ( ) 2 2 3 10 2 20 2 01 2 10 01 10 10 2 1 exp 3 2 2 2 , h p v p q p q u p q v p q q dudv q q δ Ω    − − − + +      ∫∫ (17) (17) где h Ω является областью, ограниченной замкнутой кривой h Γ . В рассматриваемом случае функция Мельникова зависит от двух параметров h и δ . Легко видеть, что для любого допустимого значения h уравнение ( , ) 0 M h δ = имеет един- ственное решение относительно ( ) h δ δ = . Это означает, что каждая замкнутая кривая h Γ будет порождающей орбитой системы (15) при некотором значении параметра δ , то есть в некоторой ее ( ) O µ -окрестности будет лежать предельный цикл системы (14). Каждому предельному циклу системы (14) соответствует через замену переменных (13) предельный цикл системы (9). Но не каждый из этих предельных циклов системы (9) будет ее циклом-уткой. Если через ( ) R h обозначить размер замкнутой кривой (16), то размер соответствующего предельного цикла системы (9) будет равен 3/2 ( ) ( ) R h O ε ε + . Для того, чтобы этот предельный цикл был циклом-уткой системы (9) необходимо выполнение условия ( ) ~1/ R h ε . Кривая h Γ расположена симметричным образом относительно вертикальной оси Ov . Ее размер ( ) R h можно охарактеризовать разностью ординат точек, в которых эта кривая пересе- кает ось Ov . Эта разность равна 2 2 10 2 2 0 1 2 10 10 2 2 ( ) , 2 q p p R h W h W h p eq eq −       = −               (18) (18) где через 0 W и 1 W− обозначены две вещественные ветви функции Ламберта W [11], которая определяется как функция, обратная к отображению w w we a . Т е о р е м а 1. [5] Если существует такое 0 (0, ) h ∈ ∞, что H H H y y y I x e dxdy I ye dxdy I e dxdy − − − Ω Ω Ω = = = ∫∫ ∫∫ ∫∫ 2 2 2 2 2 1 0 , , . H H H y y y I x e dxdy I ye dxdy I e dxdy − − − Ω Ω Ω = = = ∫∫ ∫∫ ∫∫ Область H Ω в плоскости переменных x и y является криволинейной трапецией, ограничен- ной слева и справа кривыми 2 1/ 2 y x He y = ± + + , а снизу и сверху соответственно прямыми ( ) 0 1 (2 / ) / 2 y W H e = − + и ( ) 1 1 (2 / ) / 2 y W H e − = − + . Это позволяет свести двойные интегралы 0I , 1I и 2I к однократным интегралам и найти их асимптотические разложения при 0 H → : 0 1 2 1 1 1 2 (1), 2 (1), 2 (1). 4 16 16 I e o I e o I e o π π π = + = + = + Отсюда и из представления (20) функции ( , ) M h δ получим первый член асимптотического раз- ложения решения ( ) h δ уравнения ( , ) 0 M h δ = при 0 h → вида Отсюда и из представления (20) функции ( , ) M h δ получим первый член асимптотического раз- ложения решения ( ) h δ уравнения ( , ) 0 M h δ = при 0 h → вида ( ) 2 10 3 10 2 20 01 2 3 2 ( ) 3 2 2 (1). 8 q h p q p q q p o p δ = − − + + (21) ( ) 2 10 3 10 2 20 01 2 3 2 ( ) 3 2 2 (1). 8 q h p q p q q p o p δ = − − + + (21) (21) Этот результат позволяет сформулировать следующую теорему об асимптотическом жении значения параметра α , при котором в системе (1) имеется цикл-утка. Т е о р е м а 3. Т е о р е м а 1. [5] Если существует такое 0 (0, ) h ∈ ∞, что Если система (1) удовлетворяет условиям (3) и, кроме этого 0 0 ( , ) 0 x g x y ′ > , то найдется такое значение параметра α , при котором эта система будет иметь цикл- утку. Первые два члена асимптотического разложения этого значения параметра α при 0 ε → могут быть вычислены по формуле 2 2 3 2 ( ), 2 x x xx y g g f g f g f g O f α ε ε ′   ′′′ ′ ′′ ′′ ′′ ′ = − − + +   ′′ (22) (22) где значения функций f , g и их производных вычисляются в точке 0 0 ( , ) x y . В качестве примера рассмотрим систему Ван-дер-Поля (23) 3 ( /3 ), . x y x x y x ε α ′ ′ = − − = − 3 ( /3 ), . x y x x y x ε α ′ ′ = − − = − (23) Медленная кривая этой системы имеет две точки складки (1, 2/3) − и ( 1,2/3) − . Для обоих точек выполнены условия теоремы 3. Таким образом, система (23) имеет два семейства циклов-уток, соответствующих этим точкам складки. С помощью формулы (22) для первой точки получим 2 1 /8 ( ) O α ε ε = − + , а для второй - 2 1 /8 ( ) O α ε ε = −+ + . Эти разложения совпадают с разложе- ниями, полученными в [2]. Т е о р е м а 1. [5] Если существует такое 0 (0, ) h ∈ ∞, что Отсюда имеем ( ) ( ) ( ) 10 10 3 10 2 20 2 10 01 1 2 10 01 0 2 2 2 2 ( , ) 3 2 2 2 , q q M h p q p q I q q I p q q I p p δ δ   = − − + +     (20) ( ) ( ) ( ) 10 2 2 10 3 10 2 20 10 01 2 10 01 2 2 2 2 ( , ) 3 2 2 2 H y q q M h e p q p q x q q y p q q dxdy p p δ δ − Ω   = − − + +     ∫∫ , ( ) ( ) ( ) 10 2 2 10 3 10 2 20 10 01 2 10 01 2 2 2 2 ( , ) 3 2 2 2 H y q q M h e p q p q x q q y p q q dxdy p p δ δ − Ω   = − − + +     ∫∫ , е H Ω - область, ограниченная кривой ( ) 2 2 1/ 2 y e x y H − − − = , ( 2 10 / H p h q = ). Отсюда имеем ( ) ( ) ( ) 10 10 3 10 2 20 2 10 01 1 2 10 01 0 2 2 2 2 ( , ) 3 2 2 2 , q q M h p q p q I q q I p q q I p p δ δ   = − − + +     (20) ( ) ( ) ( ) 10 10 3 10 2 20 2 10 01 1 2 10 01 0 2 2 2 2 ( , ) 3 2 2 2 , q q M h p q p q I q q I p q q I p p δ δ   = − − + +     (20) (20) 38 2 2 2 2 2 1 0 , , . g q y g 12. Corless R. M., Gonnet G. H., Hare D. E. G., Jeffrey D. J., Knuth D. E. On the Lambert $W$ function // Share Library Maple V. P.1-32. БИБЛИОГРАФИЧЕСКИЙ СПИСОК 1. Lins A., de Melo W., Pugh C. On Lienard's Equation. Lecture Nites in Mathematics. Vol.597. Springer-Verlag. New York, 1977. Р. 335. 1. Lins A., de Melo W., Pugh C. On Lienard's Equation. Lecture Nites in Mathematics. Vol.597. Springer-Verlag. New York, 1977. Р. 335. 2. Звонкин А. К., Шубин М. А. Нестандартный анализ и сингулярные возмущения обыкновенных дифференциаль- ных уравнений // Успехи матем. наук. 1984. Т.39. №.2. С.77-127. 2. Звонкин А. К., Шубин М. А. Нестандартный анализ и сингулярные возмущения обыкновенных дифференциаль- ных уравнений // Успехи матем. наук. 1984. Т.39. №.2. С.77-127. 3. Арнольд В. И., Афраймович В. С., Ильяшенко Ю. С., Шильников Л. П.Теория бифуркаций // Итоги науки и техн. Соврем. пробл. матем. Фунд.напр. 5: Динамические системы - 5. М.: ВИНИТИ, 1986. С.5-219. 3. Арнольд В. И., Афраймович В. С., Ильяшенко Ю. С., Шильников Л. П.Теория бифуркаций // Итоги науки и техн. Соврем. пробл. матем. Фунд.напр. 5: Динамические системы - 5. М.: ВИНИТИ, 1986. С.5-219. ьников В. К. Об устойчивости центра для периодических по времени возмущений // Труды московского ческого общества. 1963. Т.12. С.1-57. 5. Blows T. R., Perko L. M. Bifurcation of limit cycles from centers and separatrix cycles of planar analytic systems // SIAM Rev. 1994. V.36. № 3. P. 341. 6. Баутин Н.Н., Леонтович Е. А. Методы и приемы качественного исследования динамических систем на плоскости. М.: Наука, 1976. 420 с. 7. Арнольд В. И., Ильяшенко Ю. С. Обыкновенные дифференциальные уравнения // Итоги науки и техн. Соврем. пробл. матем. Фунд. напр. 1: Динамические системы - 1. М.: ВИНИТИ, 1985. С. 7-149. у р Мищенко Е. Ф., Колесов А. Ю. Асимптотическая теория релаксационных колебаний // Труды МИАН. М Н. 1991. Т. 197. С. 3-84. 9. Мищенко Е. Ф., Колесов Ю. С., Колесов А. Ю., Розов Н. Х. Периодические движения и бифуркационные процессы в сингулярно возмущённых системах. М.: Наука. 1995. 330 с. р у р у у 10. Freire E., Gamero E., Rodriguez-Luis A. J. First--order approximation for canard periodic orbits in a van der Pol electronix oscillator // Applied Mathematics Letters. 1999. V. 12. P.73-78. 11. Wright E. M. Solution of the equation z ze a = . Proc. Roy. Soc. Edinburgh.(A). 1959. V.65. P.193-203. 12. Corless R. M., Gonnet G. H., Hare D. E. G., Jeffrey D. J., Knuth D. E. On the Lambert $W$ function // Share Library Maple V. P.1-32.

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Dr. ~immenauer. ~3effere Derr6~igun~ un~ 11nterNnft ~er ~o[5[?auer im I~albe, eitt ~lTitte[ 5ur a b w d ? r bes arbeitermangeis im E)oI$1?auerei= ~3etrieb. ~on ~or~meifie~RnHmann in ~armf~abt. UnSer ~orff~i~er ~etrteb iff voie ieber cmbere ~emerroebetrieb I~aupt+~t~i~ abt)~ingig ~on bet ~InbaD~bet 9irbeiter, roe~e un~ ~u~ ~erfiigun~ ffe~en, ~omie vort t~erert ~eifftmg~f~igMt. | hie ~orrtmiTffgOaft fig) in ben neuen Na~nen etneg intengoen Netrtela~ Bemegert, ~ollen 9.Iu~-- ~ef[ere ~er~[iigunO unb lInter~nft bet @ola~aue~ im ~a~be ~c. 565 [orntun~, ~u~d~fodtungen, ~u~afmngen unb ~auterunge., fom[e RuRu~en unb Ru[ttt~pgege ha6) ben neueren ~n[$auungert Bur ~u~fti~rurtg ~ommen, {o i~t ~ier~u ~o~ alIem dne au~rei(Oenbe 9.In~@[ tti4t~ger 9A~:~eiter niiti~. ,~n f~t~D~e~c8eit mar bin:an ~ein ~angeL ~m ~aufe bet 8dten iR ba6 afaev anber~ fiemorben. 5Die ~nbuFtde lmb bie ~3auunternef)munfien ~aben einen fail: ~ieje@aften ~ufi4mun~ ~enommen unb bd6/ifti~en bementfpredjenb auc~ eine rie~engr@e 9.In8@[ ~on 9A~bdlern. ~)er in-buftgelIer~ ~da~/ifti~mIg nei~en bie 9~r~eite~ ~erne ~u, roei~ ~e~tere ~o~ nenber unb bequemer, meiften6 aucb ~et~ter, d~ bie raubere ~dbarbeit JR. ~ie ~orftvermaftung ~at info[~ebe~en mit bet 3eit hi@ nut ~ide i~:e~ frfi~even ~,beiter unb barunte~ oft bie tilct)ti~fiert ~oedoren, foaberrt fie ift, ma~ ~e~r nad)tei~ig empfunben with, nidjt im ftanbe geme~en, junge gr~fte fidj l)erartsie~en ~u l~nnert. ~ ift be~d~ e~enjo gered)t-{ertigt, wie an bye 8eit, bag bie ~m:[iverroaftung im ra@~edianbene~ ~nte~:ege br ~albeigentiime~c~ unb bet ~albmirgd)a[t lid} nctc~ ~ttte[n unb ~egen um[ie~t, au~ benen fie ba~ ~erforene mieber ~tt er[angen unb - - in Ron~u~ren8 mit bet ~nbu~trie -- aud} fii~: [i~ feff~u~dten ~ennas. 9~m empgnb~id}ften flit hie ~offt~erroaftun~ tritt bet 9.1rbeitermangef ira &o[~auerei-,YSetrieb in bie ~gcbeinun~. ~in eigentIi~er ~o~auer| role in f~iibeve~ 3dr, egiftiert beute taurn me~r. ~a~ &o[~auert ift ~idenort~ sum ~iic~enbti~ev ffir avladt~[ole~nbufirie--~rl3eiter gemovben. ~)ie ~ncmsielIert ~adjteife, mef~e fig) f)ie~au~ fitr ben ~afbbefit~er er-geben, unb hie roirt{cbafflk~en ~miedg~eiten, roelc~e barau~ far hie ~orfl~e~ma[tung ~eftgtiemy finb grog. ~n ~bmeD berMben ~at man nun sun~td)ft ble &o~s~auerfObne einer 91e~uIierung unlermorfen unb hie= ldben auf eine &@e geb~:ac~t, auf bet hie 9~:beiter unte~ giinfti~ert ~rbeit~:~3erO/i~tni~en w@~ i~e 91e~ntm~ ftnben ~nnen. ~tir unoart= fti~e ~:beit~--~erb~i[tniffe ~a~en hie alI~emeinen ~@nf~i~e jebo~ nidj~ unb e~ eriibrigt be~afb, bie[e S/~rte auct) ben ahtormen ~er~/Rtniffert, beam. ben ~d)wieriste[ten bet ff)iebe an~upa~en, unb ~o ~um ~Beifioid ni~t far a[[e ~c~[/ige ~[eic~e Sf)I)ne au~uroerfen, ~onbern ben ~ot~= ~ctuem flit ,~iebe in roeniger ~@nenben ~ieb~orten (~tirr~olsfd)[~gen, ~bieMorte rait ~ormiegenbem ~rgebni~ an ~eifi~, ober | mit lkntermu~ =c.) hi, here ~@ne 8u oermilli~en. ~o mid)ti~ bet | ~ur S@ne~b@ung nun au~ flit bie ~d~erung bet ~beit~:~Ser~{iftni~e im ~oI~f)auerd~Bet~:ie5 roa~, ~o mirb bet~efbe bo~ ben erbofften ~rfo[~ erft bann r)aroen, roenn man ie~t f~d) ~u bet ~r~xdfun~ bet ~roeiten ~@na~me, be~ ~irbrge far ben ~o[~bauer ent-i~Hegt. ~eiftet hie ~orftoermdtung ~ierin nid)t ba~dbe, ober gar meniger, 566 ~ugman~: d~ bet it~bu~i~iel~e~l~SeitgeSer,[o ndge~ bie ~oI~auer, tm~ role ~or~er, bottS)in, unb e~ mi~:b un~ t~um gdingett hie e~Iorber~i/~e 9.Insd)~ ~o[~-t)auer ~tt ~a[ten, 8e[~rodge benn, junge, tfig)tige ~Id~eite~: ~u gemtnnen, ~e~an~usie~e~ tmb an unfer ~dbintere~e ~u ~nfipfen. :Die ~telIe, art bet roir in bider goingaSt ein~u0~ei~ert I)agen, fmbet man {o~m, menn man ba~ ~dgenbe 15eberfft. ~)ie im mtnte~It~oettg~o~aua'd~etrie~e 6d~{i~tigte~ 9Ir~eite~ t)a~ett eine {e~r angefirengte g~titigMt. {~/ilIenbe~ ~)o[~e~, {omie ~gretgen be~ M~en au~ ben, o~t bu~@ tlntermu~ unb | ~erroa~{enen, &o~8~a3Iftgen lagen hie g)oi~auer=~Ir~eiten a~ ~:ea3t {~roere er%3dnen. ~agei mft~en hie ~d~etter r ~onate ~inbur~ i'~ttnbig hie 11ngi[ben be~: minte~:~i~en ~itte~un~ ~d mdri ~:m[id)er RMbung au~)a~len. ~ fSgt be~l~a[ra bo~apdtungiinff{g in hie ~agiCoaIe, menn bte &ot~aue~, mie ba~ gegenmSrAig bet {~alI ifi, {eine gdunb~dt~gem~ge, i~er ~m ~i~engenben 5g~ttig~dt im ~vden (~inte~) ~ngeme~ene ~aDun~ ~u f{a5 rid)men. ~e~ &ofS~auer~ t~ig~i4e Rofi ~ei'{e~t gegenmg~tig in Raffee, ioroie in ~u~(i ~on meff~ Iii. ~udit/It. ~)ieie geringme~t[ge Rort roi~b t~tg~i4 ~ut~l:o~ im ~reien I~d ~inb, ~ n e e ober {Regen, o~ {n bu~@= nSfiten R[eibertt eingenommen. ~ iFt dnleu~tenb unb ~u(O M~t ~tt ~eoMa~Ien, roie I~ei fo~@en ~rnft~un~..ySerD~tni~ett hie Rr/ffte, unb ban,it hie gei[tung~f/il)igteit bet g~o~8~uer, gegen ~itte bet | d>he,men, au@ {~* meite~ ma~rsune~men, roie bte g)oI~auer o~t magen: obe~ ~:Dumati~O~eran~merben unb e~ s $e~ ~{iu~g ~eit~eBen~IaldBen. &ie~ ~ n b d ~u f4affe~ i~t a~ge~e~en ~on ~etrt menf~Ii~Oe,~ ~rm/iflungen -- im ~nterege be~ ~rgeit~ebev~ gdegm unb bar~ bei ben }eutiflen @de}en ftSer Rrmffen, unb ,~n~a~ibit~it~-~t~orge ge~abesu a~ gffli@t be~ | ange~e~e~ toe,ben. ~ott bide, ~m~tgungen gdettee, rourbe f/~m~ ~oi~ 8rod ,~rett ira frf~Dren ~tenft6eStd beg llnter~ei@netert baau~ ~ingemidt, bag bie g)oIS~)auer [i~ b~Bu ~erffe~ett m~@letb mittag~ ~Iei$~ in ether ~nl~oe im ~a~be agStffog)ett, f~a~ gefaSa~ au~ ~on einer {Rotte ~ur ~olIen gufrieben~dt bet ~lSeiter. @in ~meiter ~e~iu@ ma~ in meinem je~igen ~ienrt~aesi~ be~ Dger~6ffierd ~armf/abt im ~er-~Iogenen ~inter get 19 &o[~auertt attf foIgenbe ~hat im @ang. ~ie @olS~aue~ ~etten F{@~ha@ eigene~ ~aD~ in ~met @ruppen geteiIt. ~ebe berMbett e~idt auf Roftett be~ ~dbdgetttiimerg etnen Ro~top~ unb ehten ei~e~nen ~)rdlug. :~er ~ld~eitgdaer ftellte meiter~tn, ~un~i~Ft nut ~e~N~gmei~e unb um ben ~e~{tt~) in ~ang ~u 6ringen, ffir iebe Nrul~ve eine @IrlSeilefin,rod~e ~ager I)oItg {~euer anmaa~te, Ra~to~dn iff)/i~te, ata~o~I)te unb Nate~ Nfi~te. ~{e grt~eite~in ~am gegett 9 ~ = morgen~ uub ging .gegen 3 11~ mieber we~. | Mien bMe ~Idaeiten einem - Nef~ere ~ed@i~ung unb 11rtterNnft bet @oIs~aner im ~albe :c. 567 ~dodter au~ bet ~ruppe iiSmrctgen unb ba~iiv drte ~3ev~titurt~ vort 50~60 ?~f. art ben ~Det~e~enbert pro 9.IS~od~ttn~ vermillifit roerben. ~ir 92rbeitr fiel~fenrid) i~r ~ e ~ i v ~ unb bie ~uppensu~i~e (~ar~to~dn, ~ei~, {~rb[en unb ~inien), [omie bct~ ~Mid). ~e~tereg murbe yon ieber @~uppe in einem ~tfi/~ attgdauft unb im 6Jansen ab~dD~)t. ;~ie Rof~m fii~ ~eif~ unb | roet~ugen ~)te~ pro ~ann unb ~2@~sete ca. 20 roi~ 25 ~f. ~enMben ~e,ra~ mft~en hie ~:beiter au4 fiiv ~m:ft unb Ra~ee au~eben; fie ~eben d$o lbeim ~5~o4en nidjt teuerer, ~tSer ge~iinber unb fi:~i~iger. ~iefe~ llmftanb rahab ~meifeI~o~ 5aIjnb~ed~ertb fftr bie meite~e ~erS~:eitung bet neuen ~irt~igOmng Mrt. ~ie ~ie~igert ~:l~eiter, md~e i@ un~ern lid) ~u ~em ~e~u@e ~er~,ctrtben ~aben, mugten hie ~3or~ii~e bte~e~ ~n@rung~a~t to ~nel~ ~u i~t~en, ba~ na~ a@ ~:agen un~err igaggen bortiaen &o~auer bert ~ u n ~ ncte~ ether g{ei~en Strt~idjtung ~u er.eennert ~aben, bern au6) eni~prod~en mu~be. ~6) fain iiber~eu~b ba~ rod*ere ~3e~:{uc~ein anberen Dber~/Miereiert art einem g~et4 be~ie, bigenben ~e~t6tat ~iiIjren merben, menn man bie R~ug~ett geS~attggt, bie @inrid)tung pro primo nut ba ein~ufiI~ren, too hie ~Ra@d~e bet bi~o !)efiflen ~ediJ[fi~ung bur~fi@iger ~iir hie 9.Irbeite~ Stt ~nge ~ie~en. | ~eif~ ~ann mart ctu~ Ron~er~en m@~em ~te ~3e~:~@i~ung ~iemit ift biI~i~e~ unb fabort [i~r 4= bi~ lO Sf. p~:o ~ann ~u laemed-Reiligen. 7)ie ~iefigen ~beiter Ie~nten abet hie Ronfer~en ~o entf4ieben ab, bag dn ~erfuc~ ~iermit 5i~ b@in unteda~ieb. ~nbd~en finb an ~rten, mo ba~ {~iei~a3 meni~er Ieiabt ~tt ~gaben tit, roir ~ier, ober mo man auf ~ilIi{t~ett ~eDn wig hie Ron~e~en{ul~pert ~eD am ~IaD. ~Reben begever ~edSffigung bet &oIbl)aue~: biJ~fte ~ur ~r~a~turtg bet ~efunb~)eit unb ~eifiung~f@i~dt bet ~(rbeiter etne lroffene, marine llnter-Nnft im ~dbe ffir biefdNn Su edtreben iein. ~ mug bern 'Xrbet,e~ rn/~g~i~ genm~)t merben, Mne ~@~seiten ~et urtgfmf~ige~ ~itterttng im gei~fi~ten erm/ivmten ~Raume dnsune~men, ~omie Bier RMber ~u roeffoMn, ober bu~4ne@e RMbung~ffiigr 8u troc~nm unb ft~er~)aupt ~4uit ~u finben gegen vori~bergeDnbe ~itterung~me~M. ~d ~Ie@tem ~ette~ ~ann bann ~ar oft roeiter gea~bdtet unb mandoe~ 2rbeit~tag gemonnen merben. 9~ 9eeignete llntedunft@elle br,r~en Su~ammenle@a~e~retter~ bau~djen Su ro~i~kn Mn, in beren ~itte man dnen Dfen cmbrin~t. ,~m ~a~m~'t/ibter s ift dn Ma~e~ &~u~djen idt ~ergogenem ~inte~ in ~enu~ung. ~a~Mbe ift in bert ~3imenfionen ~,60 m ~iebe~feite, 4,50 m :gang[rite unb ~,20 m &~e, (ver~Mdje umfte~enbe 3ei~-hung) io angefertigt, bag e~ ~et@t au~einanber genommen unb franC-pro:tim n~e~ben ~ann. ~u bieiem ~mec~e jinb hie be~bert ~/ing~m/inbe, ioroie ba~ ~adj au~ 568 ~ulImanrt: ~e[[ere ~er~fi[iigurtg unb 1;Inte~hxrtftbet g)ols~aue~im ~albe. je ~roei gei{en ~ergdlelIt. ~3ie ~neinmtbe~:fagun~ bet gei~r e~fo~gt bura3 R[ammenl IaeSm. buva3 ~anbet{en mit ~gOrauben. 8urn .~Iuf- ober ~t~-~Iagett genitgm 4--5 ~ann. ~3ie ~re/lmcfugen flnb bur/~ aufgenagdte ~eirien gebe/~t; dne ~punbuttg bet ~B~etter emp~eI)R fi~ megen bet ~ fair be~ 11nbidjtroerbm~ ni/~t. ~){e ~3eba~ung au~ ~3~ette~n i~i be~: ~begamg be~ ~3ad}e~ mit ~)ac~pappe vor~u~ieI)en, me~[ bie ~e~te~e ~gt etrter traneporta~[en ~i~tte ~u fe~: bet ~3e{a3~tbigttng au~ge~e~t i~. Ne, i~bigte ~3ctg)pappe erid)me~t d}e~ ben ~agerabgu~ unb gie~t babttrd~ ~eran[af[ung ~um ~urd)bringen bN ~ageN. ~)ag ~retterba~ teert man unb wieber~o[t bie~e~ g~finbH~) jeben ~erbrt. ~)ie ~Iugenfeiten beg &~iug/~en~ me~be~ ~mec~mSgig mit ga~boHnemn angefirtd0en. ~ n tnnert)alb be~ g)~ue~en~, in begen ~itte, aufgeRelIte~ ~fen ermSgIi4t bie ~groffnung nailer R~etbe~, roeI/~e ~roegm/igiger ~ei$e an ben | ro~inben art a~ne~mbare~c ~eirte auf: ~/itt~@en ~eict)t fil~ ~5 ~antt; e~ foffet, ~on 8immermetller g{bam Rrid~er in ~)~rm[~abt angefe~figt, I50 ~r162o~ne ~fen, unb ~It ca. 10 ~i~ 15 ~a~:e att~. ~)ie j~i~rIit~en ~tgrml~ung~, unb gtepm:atuffo[ten merben [i~) auf ca. 15--~0 ~ ~e: Iaufen; fie [inb gering, unb e~ Iggt [i~ a[~ [i/~mc anne~men, bar3 fie gut ~entieren me,ben. ~eIfe att~ ~egeItucI) emp~el}Iet~ ~6) ~fi~ bert gjo[~%aue~ei,~ei~deb meiner 9,tnfi6)t had} meniger, al~ YS~:ette~ftu~dOen, mei[ ba~ ~ud} be~ ~roftmetter Iei/~t ~riia~ig roi~b 1rob roeiI me~te~in fi~ in 8elten ~eirt ~fen au~ftelIel~ [~t. 9cite finb meIp: fiI~ grbeitev geelgnet, hie in bNe~er ~a~re~eit, aI~ hie ~oI~Daue~ arbeiten mann. ~ a n Nnn hie ~e~te beg~aX5 ffi~: ben Ru[tur~ettSe5 irt 9.Iu~fi6)t he, men. ~um ~[tt[[e iei rtodj mitgeteiIt, ba~ hie ~irma gCebriibe~ ~Sbe~ ~u ~armfiabt einen ~a[b~o6)t)erb ~onftruiert unb eirte Rog)pIatte ge.[erfigt ~at, mei6je fie3 beibe a~ ~meffent~pred)enb ge~etgt baben. ~er ~rei~ be~ Rodj~erbe~ 15et~igt ca. ~6 ~ unb be~ Ro~pIatte ttngefg~r 13 d . 

https://openalex.org/W1969748709

https://emerita.revistas.csic.es/index.php/emerita/article/download/764/806/825

Spanish; Castilian

<i>Quem tu, Melpomene</i>: the poet’s lowered voice (<i>C</i>. IV 3)

Emérita/Emerita

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Emerita, Vol. 50, núm. 2 (1982) Emerita, Vol. 50, núm. 2 (1982) Víctor A. Estévez (c) Consejo Superior de Investigaciones Científicas Licencia Creative Commons 3.0 España (by-nc) http://emerita.revistas.csic.es (c) Consejo Superior de Investigaciones Científicas Licencia Creative Commons 3.0 España (by-nc) (c) Consejo Superior de Investigaciones Científicas Licencia Creative Commons 3.0 España (by-nc) (c) Consejo Superior de Investigaciones Científicas Licencia Creative Commons 3.0 España (by-nc) (c) Consejo Superior de Investigaciones Científicas Licencia Creative Commons 3.0 España (by-nc) (c) Consejo Superior de Investigaciones Científicas Licencia Creative Commons 3.0 España (by-nc) (c) Consejo Superior de Investigaciones Científicas Licencia Creative Commons 3.0 España (by-nc) (c) Consejo Superior de Investigaciones Científicas Licencia Creative Commons 3.0 España (by-nc) (c) Consejo Superior de Investigaciones Científicas Licencia Creative Commons 3.0 España (by-nc) http://emerita.revistas.csic.es (c) Consejo Superior de Investigaciones Científicas Licencia Creative Commons 3.0 España (by-nc) (c) Consejo Superior de Investigaciones Científicas Licencia Creative Commons 3.0 España (by-nc) (c) Consejo Superior de Investigaciones Científicas Licencia Creative Commons 3.0 España (by-nc) (c) Consejo Superior de Investigaciones Científicas Licencia Creative Commons 3.0 España (by-nc) (c) Consejo Superior de Investigaciones Científicas Licencia Creative Commons 3.0 España (by-nc) (c) Consejo Superior de Investigaciones Científicas Licencia Creative Commons 3.0 España (by-nc) (c) Consejo Superior de Investigaciones Científicas Licencia Creative Commons 3.0 España (by-nc) (c) Consejo Superior de Investigaciones Científicas Licencia Creative Commons 3.0 España (by-nc) http://emerita.revistas.csic.es (c) Consejo Superior de Investigaciones Científicas Licencia Creative Commons 3.0 España (by-nc) (c) Consejo Superior de Investigaciones Científicas Licencia Creative Commons 3.0 España (by-nc) (c) Consejo Superior de Investigaciones Científicas Licencia Creative Commons 3.0 España (by-nc) (c) Consejo Superior de Investigaciones Científicas Licencia Creative Commons 3.0 España (by-nc) (c) Consejo Superior de Investigaciones Científicas Licencia Creative Commons 3.0 España (by-nc) (c) Consejo Superior de Investigaciones Científicas Licencia Creative Commons 3.0 España (by-nc) http://emerita.revistas.csic.es http://emerita.revistas.csic.es http://emerita.revistas.csic.es http://emerita.revistas.csic.es http://emerita.revistas.csic.es http://emerita.revistas.csic.es http://emerita.revistas.csic.es http://emerita.revistas.csic.es http://emerita.revistas.csic.es http://emerita.revistas.csic.es http://emerita.revistas.csic.es http://emerita.revistas.csic.es (c) Consejo Superior de Investigaciones Científicas Licencia Creative Commons 3.0 España (by-nc) http://emerita.revistas.csic.es (c) Consejo Superior de Investigaciones Científicas Licencia Creative Commons 3.0 España (by-nc) http://emerita.revistas.csic.es http://emerita.revistas.csic.es (c) Consejo Superior de Investigaciones Científicas Licencia Creative Commons 3.0 España (by-nc) http://emerita.revistas.csic.es http://emerita.revistas.csic.es http://emerita.revistas.csic.es (c) Consejo Superior de Investigaciones Científicas Licencia Creative Commons 3.0 España (by-nc) http://emerita.revistas.csic.es http://emerita.revistas.csic.es (c) Consejo Superior de Investigaciones Científicas Licencia Creative Commons 3.0 España (by-nc) http://emerita.revistas.csic.es http://emerita.revistas.csic.es http://emerita.revistas.csic.es (c) Consejo Superior de Investigaciones Científicas Licencia Creative Commons 3.0 España (by-nc) http://emerita.revistas.csic.es (c) Consejo Superior de Investigaciones Científicas Licencia Creative Commons 3.0 España (by-nc) http://emerita.revistas.csic.es (c) Consejo Superior de Investigaciones Científicas Licencia Creative Commons 3.0 España (by-nc) http://emerita.revistas.csic.es (c) Consejo Superior de Investigaciones Científicas Licencia Creative Commons 3.0 España (by-nc) http://emerita.revistas.csic.es http://emerita.revistas.csic.es http://emerita.revistas.csic.es http://emerita.revistas.csic.es

W4240272408.txt

http://vital.lib.tsu.ru/vital/access/services/Download/vtls:000782832/SOURCE1

ru

EVOLYuTsIYa STRUKTURNO-FAZOVOGO SOSTOYaNIYa GETEROGENNOGO NANOKRISTALLIChESKOGO SPLAVA Ti-50,9 at. % Ni PRI MEKhANIChESKOM TsIKLIROVANII

Mezhdunarodnaya konferentsiya "Fizicheskaya mezomekhanika. Materialy s mnogourovnevoy ierarkhicheski organizovannoy strukturoy i intellektual'nye proizvodstvennye tekhnologii" : tezisy dokladov

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ТЕЗИСЫ ДОКЛАДОВ МЕЖДУНАРОДНАЯ КОНФЕРЕНЦИЯ «Физическая мезомеханика. Материалы с многоуровневой иерархически организованной структурой и интеллектуальные производственные технологии», посвященная 90-летию со дня рождения основателя и первого директора ИФПМ СО РАН академика Виктора Евгеньевича Панина в рамках Международного междисциплинарного симпозиума «Иерархические материалы: разработка и приложения для новых технологий и надежных конструкций» 5–9 октября 2020 года Томск, Россия Томск Издательство ТГУ 2020 Секция 4. Научные основы разработки материалов с многофазной иерархически организованной структурой, в том числе для экстремальных условий эксплуатации DOI: 10.17223/9785946219242/218 ЭВОЛЮЦИЯ СТРУКТУРНО-ФАЗОВОГО СОСТОЯНИЯ ГЕТЕРОГЕННОГО НАНОКРИСТАЛЛИЧЕСКОГО СПЛАВА Ti-50,9 ат. % Ni ПРИ МЕХАНИЧЕСКОМ ЦИКЛИРОВАНИИ Полетика Т.М., Гирсова С.Л. Институт физики прочности и материаловедения СО РАН, Томск Основными требованиями к нанокристаллическим (НК) сплавам TiNi, используемым в медицине, являются стабильность механических характеристик и циклическая долговечность. В этой связи особый интерес вызывают гетерогенные НК материалы со структурной неоднородностью, отличающиеся высоким сопротивлением усталости и циклической стабильностью. Среди них - НК сплавы TiNi с зеренно/субзеренной структурой, формирующейся после умеренной холодной деформации прокаткой. В работе представлены результаты исследования структуры, фазового состава и деформационного поведения гетерогенного НК сплава TiNi при циклическом деформировании. Исследовали образцы нанокристаллического сплава Ti-50,9 ат.% Ni после низкотемпературного отжига при 300С, 1 час. Последовательность мартенситных превращений после отжига: B2↔R↔B19′ (TR = 22С, MS = -50С, MF = -134, AS = -37С, AF = -14С). Сплав имел зеренно/субзеренную преимущественно B2-аустенитную структуру, представляющую смесь нанозерен с высокоугловыми границами и субзерен, образующих области субмикронного и микронного размеров. Средний размер зерен/субзерен составлял 70 нм. Структурные исследования проводили методом ПЭМ на микроскопе JEM 2100 JEOL. Диаграммы «нагружения-разгрузки» при одноосном растяжении образцов получали при комнатной температуре со скоростью 4×10-5с-1 на испытательной машине LFM-125. Установлено, что зеренно/субзеренная структура способствует неоднородному развитию процесса низкотемпературного старения с выделением частиц Ti3Ni4 в объеме НК материала. Наличие дислокаций внутри субзерен и нанозерен является условием реализации распада B2-аустенита при низкотемпературном старении. Частицы Ti3Ni4 размером менее 5 нм выделяются преимущественно в субструктуре и не обнаружены в бездислокационных нанозернах. Декорирование дислокаций когерентными частицами Ti3Ni4 способствует росту дислокационного предела текучести B2-аустенита и реализации сверхэластичности с нулевой остаточной деформацией после 10 циклов «нагружения-разгрузки». Показано, что различие структурно-фазового состояния элементов структуры (нанозѐрен и групп субзѐрен), их упругих и пластических свойств, а также неоднородность распределения в объеме материала является одной из причин реализации мультистадийности превращений B2↔R↔B19′. Установлено, что мартенситные превращения в процессе нагружения начинаются в областях субструктуры с формирования нанодоменной R-фазы. После 10 циклов «нагружениия-разгрузки» в образце наблюдается формирование из Rнанодоменов пластин, пересекающих субмикронные области субструктуры. Установлено, что наличие в субструктуре малоугловых границ, наночастиц Ti 3Ni4 и дислокаций не является препятствием для движения пластин B19′-мартенсита, в то же время, они тормозятся большеугловыми границами нанозерен. Показано, что структурная неоднородность нанокристаллического сплава TiNi способствует одновременному вовлечению в деформацию нано- и субмикроструктурных уровней, что способствует эффективной аккомодации высоких внутренних напряжений при деформировании и является одним их механизмов повышения сопротивления усталости материала в условиях сверхэластичности. Работа выполнена в рамках государственного задания ИФПМ СО РАН, проект III.23.2.2 и проекта РФФИ №16-58-00143 Бел_а. 340 

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MODULATORY EFFECTS OF L-CARNITINE ON TAMOXIFEN TOXICITY AND ONCOLYTIC ACTIVITY: IN VIVO STUDY

˜Al-œAzhar Journal of Pharmaceutical Sciences/Al-Azhar Journal of Pharmaceutical Sciences

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ABSTRACT The aim of this study was to investigate the protective effect of L-carnitine (L-CAR) in tamoxifen (TAM)–induced toxicity and antitumor activity.Adult female rats were randomly divided into 4 groups. Group I was served as control, groups II and III were injected with (10mg/kg, P.O) TAM and L-CAR (300 mg /kg, i.p.) respectively while, group IV was treated with both compounds.The treatment continued daily for 28 days. Administration of TAM resulted in significant increase in serum lipid profiles, liver enzymes and bilirubin level. In addition, TAM produced significant increase in lipid peroxides (LPO) level accompanied with significant decrease in superoxide dismutase (SOD) activity of hepatic and uterus tissues and significant decrease in glutathione (GSH) content of uterus tissue. Administration of L-CAR prior to TAM treatment decreased significantly serum lipids and liver enzymes and significantly increased SOD activity in liver and uterus tissues compared to TAM treated group. Furthermore, it restored LPO and GSH levels in uterus tissue. On the other hand, the apoptotic markers of caspases 9 and 3 were not detected in liver of all the treated groups. Histopathologically, alterations in the liver and uterus structures after TAM treatment which was attenuated by L-CAR administration. The antitumor effect and survival of the combined treatment of Ehrlich Ascites Carcinoma (EAC)-bearing mice was less than each one alone. In addition, L-CAR interestingly increased survival rate of EAC-bearing mice more than TAM treated group. In conclusion, L-CAR has beneficial effects regarding TAM toxicity; however, it interferes with its antitumor effect. Key words: Tamoxifen, L-carnitine, organ toxicity, antitumor activity, antioxidants Amel B. Ibrahima,b, Samia A. Shoumanc, Heba H. Mansourd, Amany A. Eissaa and Seham M. Abu El Nourd Amel B. Ibrahima,b, Samia A. Shoumanc, Heba H. Mansourd, Amany A. Eissaa an Seham M. Abu El Nourd aDepartment of Pharmacology and Toxicology, Faculty of Pharmacy, Helwan University, Egypt. bDepartment of Pharmacology, Faculty of Medicine, Zawia University, Libya.cCancer biology department, Pharmacology unit, National Cancer Institute, Cairo University, Egypt.dHealth Radiation Research department, National Center for Radiation Research and Technology,Atomic Energy Authority, Cairo, Egypt aDepartment of Pharmacology and Toxicology, Faculty of Pharmacy, Helwan University, Egypt. bDepartment of Pharmacology, Faculty of Medicine, Zawia University, Libya.cCancer biology department, Pharmacology unit, National Cancer Institute, Cairo University, Egypt.dHealth Radiation Research department, National Center for Radiation Research and Technology,Atomic Energy Authority, Cairo, Egypt aDepartment of Pharmacology and Toxicology, Faculty of Pharmacy, Helwan University, Egypt. bDepartment of Pharmacology, Faculty of Medicine, Zawia University, Libya.cCancer biology department, Pharmacology unit, National Cancer Institute, Cairo University, Egypt.dHealth Radiation Research department, National Center for Radiation Research and Technology,Atomic Energy Authority, Cairo, Egypt aDepartment of Pharmacology and Toxicology, Faculty of Pharmacy, Helwan University, Egypt. bDepartment of Pharmacology, Faculty of Medicine, Zawia University, Libya.cCancer biology department, Pharmacology unit, National Cancer Institute, Cairo University, Egypt.dHealth Radiation Research department, National Center for Radiation Research and Technology,Atomic Energy Authority, Cairo, Egypt Az. J. Pharm Sci. Vol. 45, March, 2012 Az. J. Pharm Sci. Vol. 45, March, 2012 56 2.1.2. Drug TAM citrate was a kind gift from Medical Union Pharmaceuticals Company (MUP), Cairo, Egypt. It is obtained as white powder soluble in sterile water and was diluted to the required concentration before use. Az. J. Pharm Sci. Vol. 45, March, 2012 addition, TAM leads to oxidative liver damage and has been elucidated to be a hepatocarcinogen in rodents which is due to overproduction of oxygen radical during TAM metabolism (Senkus-Konefka et al., 2004). It was demonstrated that TAM caused DNA- adducts in rat liver and in the endometrium (Chao et al., 2011; Wang et al., 2009). L-CAR (L-trimethyl-3-hydroxy-ammoniabutanoate) is quaternary ammonium compound biosynthesized from amino acids lysine and methionine (Gulcin, 2006). It plays anessential role in human intermediary metabolism (Doberenz et al., 2007). The most prominent function lies in its role in the transport of activated long-chain fatty acids from the cytosol to the mitochondrial matrix where β-oxidation takes place and production of cellular energy (Vamos et al., 2010). L-CAR has antioxidant properties and can reduce oxidative stress and acts as a free-radical scavenger. L-CAR has protective effects against acute and chronic doxorubicin toxicity in rat (Sayed-Ahmed, 2010). In addition, it significantly inhibited preneoplastic lesions and hepatocarcinogenesis in animals (Hoang et al., 2007).Therefore, the current study was designed to test the ability of L-CAR to protect liver and endometrial tissues of TAM–treated rats from toxicity without impediments the antitumor effect. 2.1.1. Animals Adult female Wistar albino rats weighing 120–170 g were obtained from the Egyptian Organization for Biological Products and Vaccines (VACSERA, Giza, Egypt) and female Swiss albino mice weighing 18-20g were obtained from animal facility, Pharmacology unit, National Cancer Institute (NCI), Cairo University, Egypt. Animals were kept under standard conditions and were allowed free access to a standard requirement diet and water ad.Libitum. Animals were kept under a controlled lighting condition (light: dark, 13 h: 11 h). The animals' treatment protocol has been approved by the animal care committee of the National Cancer Institute, Cairo University, Egypt. 2.1.3. Chemicals L-CAR was a generous donation from the Pharmacology Unit, National Cancer Institute (NCI), Cairo University, Egypt. All other chemicals and solvents used were of the highest purity grade available. 1- INTRODUCTION Breast cancer is the leading cause of cancer deaths in women worldwide (Buijs et al., 2008). Tamoxifen (TAM), 1-[4-(2-dimethyl-aminoethoxy) phenyl] - 1, 2-diphenyl-1- butene), a non-steroidal antiestrogen drug, is selective estrogen receptor modulator used in prevention and treatment of all stages of hormone-responsive breast cancer (Matsuoka et al., 2009). Beside long treatment with TAM and its widespread uses, attention has been focused on its adverse effects, particularly liver and endometrium toxicity (Stanley et al., 2001). According to studies, adjuvant TAM significantly increases women’s risk of subsequently developing endometrial carcinoma (Singh et al., 2007). Fatty liver was observed in more than 30% of breast cancer patients who received TAM treatment (Albukhari et al., 2009). Hepatotoxicity has been described with toxic hepatitis, multifocal hepatic fatty infiltration, submassive hepatic necrosis and cirrhosis (Parvez et al., 2006). In Az. J. Pharm Sci. Vol. 45, March, 2012 57 2.2.1.6.. Determination of tissue reduced glutathione Reduced glutathione (GSH) was determined according to the methods of Ellman (1959), it is based on the reduction of Ellman’s reagent [5,5'-dithio-bs- (2-nitrobenzoic acid)] by SH groups to form 1mole of 2-nitro-5- mercaptobenzoic acid per mole of SH. The optical density was measured at 412 nm against a reagent blank and the results were expressed as μmol/g tissue. Az. J. Pharm Sci. Vol. 45, March, 2012 Az. J. Pharm Sci. Vol. 45, March, 2012 group IV were injected with L-CAR (300mg/kg, i.p.) 1 hour later; rats were treated with TAM (10mg/kg, P.O). The treatment schedule wascontinued once/day for 28 consecutive days. group IV were injected with L-CAR (300mg/kg, i.p.) 1 hour later; rats were treated with TAM (10mg/kg, P.O). The treatment schedule wascontinued once/day for 28 consecutive days. Twenty-four hours after the last treatment, animals were anesthetized after exposure to ether in desiccators kept in a well-functioning hood. Blood samples were collected by heart puncture and serum samples were separated. Livers and uterus were quickly excised, washed with saline, blotted with a piece of filter paper and homogenized using a Branson sonifier (250, VWR Scientific, Danbury, CT, USA). 2.2.1.4. Determination of tissue superoxide dismutase (SOD) Superoxide dismutase (SOD) activity in liver and uterus homogenates was determined according to the method of Minami and Yoshikawa (1979). This method is based on thegeneration of superoxide anions by pyrogallol autoxidation, detection of generatedsuperoxide anions by nitro blue tetrazolium (NBT) formazan color development andmeasurement of the amount of generated superoxide anions scavenged by SOD (theinhibitory level of formazan color development). The formazan color developed wasdetermined spectrophotometrically (Spectronic 501, Shimadzu, Japan). Enzymaticactivity was expressed inform of μg/g of tissue. 2.2.1.3. Determination of liver function test Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activities weredetermined according to the method of Zilva and Pannall (1979) usingspectrophotometric kit (Spectrum diagnostics, Cairo, Egypt). Total and directbilirubin levels were determined according to the method of Malloy and Evelyn (1937). Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activities weredetermined according to the method of Zilva and Pannall (1979) usingspectrophotometric kit (Spectrum diagnostics, Cairo, Egypt). Total and directbilirubin levels were determined according to the method of Malloy and Evelyn (1937). 2.2.1.5. Determination of tissue lipid peroxidation Malondialdehyde (MDA) levels in liver and uterus tissues homogenates were determined spectrophotometrically using the method of Buege and Aust (1978). MDA content was measured at 535 nm. The results were expressed as nmol/g tissue. 2.2.1.2. Determination of lipid profile Lipid profile was determined using a standard commercial kit (Spectrum diagnostics,Cairo, Egypt). Serum triglycerides (TG), total cholesterol (CH), high- densitylipoprotein-cholesterol (HDL) were determined according to the methods describedby Tietz et al. (1959), Ellefson and Caraway (1976) and Warnick and Wood (1995), respectively. Low-density lipoprotein-cholesterol (LDL) was calculated bysubtracting the HDL-cholesterol from total cholesterols described by Terpstra et al.(1982). 2.2.1.1. Experimental design Twenty four female rats were divided randomly into four groups (6 animals each). Rats of group I was administeredwith 0.5 ml of normal saline and served as control group. Rats of group II were administered TAM (10mg/kg, P.O) (Perumal et al., 2005).Rats of group III were injected with L-CAR (300mg/kg, i.p.) (Muthuswamy et al., 2006). Rats of 58 2.2.1.8. Determination of DNA fragmentation: agarose gel electrophoresis According to the method of Katoh et al. (1996)the liver tissue was homogenized and lysed in a cold lysis buffer (10 mMTris– HCl, 5mM disodium EDTA, and 0.5% Triton X- 100, pH 8.0) for 10 min at 4 C. The DNA was sequentially extracted twice using half volumes of phenol/chloroform and incubated at 55 C for 10 min. After centrifugation at 3000 rpm for 20 min, the upper layer was incubated with proteinase K at 37 C for 60 min followed by incubation with ribonuclease at 37 C for 60 min. The DNA was precipitated by adding 10 M ammonium acetate and 100% ethanol and maintained at _20 °C overnight. DNA was collected by centrifugation at 15,000 x g for 20 min, air-dried, and resuspended in TE buffer (10 mMTris–HCl, 5mM EDTA, pH 7.4). Agarose gel electrophoresis (Yokozawa and Dong, 2001) was carried out for the analysis of DNA fragmentation. The resulting DNA preparations were electrophoresed through a 1.4% agarose gel containing ethidium bromide using TBE buffer (Tris-boric acid–EDTA buffer, pH 8.3) at 40 V for 5 h. Equal quantities of DNA (based on optical density measurements at 260 nm) were loaded in each lane, and a molecular DNA marker was used as a molecular mass standard. DNA fragmentation was visualized and photographed under ultraviolet illumination. 2.2.1.9. Determination of apoptotic markers: western blotting The livers were homogenized with phosphate buffer saline (PBS) followed by centrifugation at 11000 rpm 4˚C for 15 min. Protein content in the resulting supernatant was determined using Bradford reagent (Thermo scientific, USA). Equal volume of supernatant was mixed with 1x loading buffer and 5 µl of β-Mercaptoethanoland boiled for 5 min. Proteins were separated by sodium dodecyl sulfatepolyacrylamidegel electrophoresis (SDS– PAGE) and then electrotransferred onto polyvinylidene fluoride (PVDF) membrane using semidry transfer apparatus (Biometra, Germany).The membrane was blocked according to manufacture instruction of chromogenic western max detection kit (Ameresco, USA). After blocking with dilution buffer plus 1% Tween-20 (DBT) plus 1%bovine serum albumin (BSA), the membrane blots was incubated with indicated primary antibody (in DBT) at4˚C over night then washed three times with DBT buffer, and incubated with horseradish peroxidase-conjugated secondary antibody for 4 h at room temperature. After washing three times with DBT buffer, the protein bands were visualized by 3,3ʼ-Diaminobenzidine (DAB) chromogen which gives brown precipitate at the reaction site, specific protein bands on these transferred membranes were detected using the following antibodies:Purfied antiβ-actin antibody was obtained from(Biolegend, USA) and anti-mousecaspase 3 monoclonal antibody were obtained from (Bioscience, USA).Relative expression of proteins was evaluated by normalizing the expression of proteins with quantitative housekeeping protein β-actin (Salami and Karami-Tehrani, 2003). 2.2.2. Histopathological study The samples were fixed in 10% neutral buffered formalin, dehydrated through alcohols, cleared in xylene and then embedded in paraffin wax. Sections (5 mm thick) were stained with haematoxylin and eosin (Albukhari et al., 2009). Az. J. Pharm Sci. Vol. 45, March, 2012 spectrophotometrically at 540 nm. The absorbance at 540 nm was measured using a plate reader. The levels of NO(x) were expressed as nmol/g wet tissue in homogenate. 2.2.1.7. Determination of tissue total nitrate/nitrite (NO(x)) Total nitrate/nitrite (NO(x)) was measured as stable end product, nitrite, according to the method of Miranda et al. (2001). The assay is based on the reduction of nitrate by vanadium trichloride combined with detection by the acidic Griess reaction. The diazotization of sulfanilic acid with nitrite at acidic pH and subsequent coupling with N-(10- naphthyl) ethylenediamine produced an intensely colored product that is measured Az. J. Pharm Sci. Vol. 45, March, 2012 59 Az. J. Pharm Sci. Vol. 45, March, 2012 after cell inoculation, 40 mice wereclassified into four groups.GroupI; animals were injected with 0.2 ml of saline and servedas control.GroupII; animals were injected once with TAM (10 mg/kg, i.p.). GroupIII; animals were injected once with L-car (300 mg/kg, i.p.). GroupIV; animals were injected once with L-car (300mg/kg, i.p.) followed by TAM in a dose of (10 mg/kg, i.p.) with an in-between interval of 30 min. The change in the percent survival of animals was recorded daily during a period of 45 days following treatment. Percent survival= number of living animals/10 X100. 2.2.3.2. Tumor volume: Forty female mice were used and solid tumor was transplanted subcutaneous in the right thigh of the lower limb of each mouse. Mice with a palpable tumor mass (100m ) that developed within 7 days after implantation were divided into 4 groups: Group I: ten mice were injected intraperitoneally four times every other day with normal saline and served as control group. Group II: ten mice were injected four times every other day with TAM (10mg/kg, i.p.). GroupIII: ten mice were injected four times every other day with L- car (300mg/kg, i.p.). Group IV: ten mice were injected with L-car (300mg/kg, i.p.), 30 min later, injected with TAM (10mg/kg, i.p.)four times every other day. The change in tumor volume was measured every other day using a Vernir caliper and calculated by following formula according to Osman et al. (1993). TumourVolum (m )= 4 X (B/2)/ 3. Where A and B were denote the minor and major tumor axis,respectively. 2.3. Statistical analysis Differences between obtained values (mean±S.E.M. n = 6) were carried out by one way analysis of variance (ANOVA) followed by the Tukey–Kramer multiple comparison test. A p value of 0.05 or less was taken as a criterion for a statistically significant difference. 2.2.3.1. Percentage survival of animals Forty female albino mice inoculated with Ehrlich Ascites Carcinoma (EAC) cells (2.5 X 106cells/0.1ml). Each mouse was injected i.p. with EAC cells. Twenty-four hours 60 3.2. Effect on serum liver function AST, ALT, total bilirubin and direct bilirubin of rats treated with tamoxifen were significantly elevated by (20.85%, 86.57%, 104.65%and 172.09%), respectively compared to the control group. On other hand, treatment with both L-CAR and TAM significantly decreased AST (19.12%), ALT (54.78%), total bilirubin (45.08%) and direct bilirubin (28.21%), as compared to the TAM treated group, which still significantly increased compared to the control group (Table 2). Table (2): Effect of Tamoxifen (TAM), L-carnitine (L-CAR) and their combination on the level of aspartate aminotransferase (AST), alanine aminotransferase (ALT), total bilirubin and direct bilirubin in serum of female rats. : Effect of Tamoxifen (TAM), L-carnitine (L-CAR) and their combination on the level of aspartate aminotransferase (AST), alanine aminotransferase (ALT), total bilirubin and direct bilirubin in serum of female rats. Direct bilirubin (mg/dl) Total bilirubin (mg/dl) ALT (U/l) AST (U/l) Treatment 0.43±0.03 1.29±0.04 55.53±9.04 65.61±2.08 Control 1.17±0.02 a 2.64±0.09 a 103.60±4.31 a 79.29±3.02 a TAM 0.41±0.04 1.34±0.09 48.16±5.51 66.77±1.54 L-CAR 0.84±0.08 ab 1.45±0.08 b 46.85±8.31 b 64.13±4.44 b L-CAR+TAM Az. J. Pharm Sci. Vol. 45, March, 2012 The values are expressed as mean ± S.E.M of 6 rats/ group. P value is significant 0.05 using ANOVA followed by Tukey-Kramer as a post ANOVA test. a,b Significantly different from the control group and TAM treated group respectively. TAM (10mg/kg, P.O) and L-CAR (300mg/kg, i.p.) were administered for 28 consecutive days. In the groups treated with two drug regimens, the animals were pretreated with L- CAR 1 hour prior to TAM and the administration was continued as described above. days. In the groups treated with two drug regimens, the animals were pretreated with L- CAR 1 hour prior to TAM and the administration was continued as described above. 3.1. Effect on serum lipid profile Treatment of rats with TAM(10mg/kg for 28 days) produced significant increase in serum levels of TG, Ch and LDL by (85.36%, 40.78% and 129.07%); respectively as compared to the control group. The combined effect of L-CAR and TAM resulted in significant decrease in levels of TG by (25.88%) as compared to TAM group but it is significantly high in compared to control group. On the other hand, the levels of CH and LDL was significantly decreased and reached to normal level (Table 1). Table (1): Effect of Tamoxifen (TAM), L-carnitine (L-CAR) and their combination on the level of triglycerides (TG), cholesterol (CH), high density lipoprotein (HDL) and low density lipoprotein (LDL) in serum of female rats. LDL (mg/dl) HDL (mg/dl) CH (mg/dl) TG (mg/dl) Treatment 35.60±2.16 52.49±3.35 87.39±7.47 62.16±5.02 Control 81.55±8.08 a 41.48±2.63 123.03±11.41 a 115.22±6.41 a TAM 35.95±4.51 50.93±4.55 86.88±7.48 75.17±5.69 L-CAR 36.99±3.66 b 50.79±4.81 87.79±6.63 b 85.41±3.79 ab L-CAR+TAM Table (1): Effect of Tamoxifen (TAM), L-carnitine (L-CAR) and their combination on the level of triglycerides (TG), cholesterol (CH), high density lipoprotein (HDL) and low density lipoprotein (LDL) in serum of female rats. Table (1): Effect of Tamoxifen (TAM), L-carnitine (L-CAR) and their combination on the level of triglycerides (TG), cholesterol (CH), high density lipoprotein (HDL) and low density lipoprotein (LDL) in serum of female rats. y p p LDL (mg/dl) HDL (mg/dl) CH (mg/dl) TG (mg/dl) Treatment 35.60±2.16 52.49±3.35 87.39±7.47 62.16±5.02 Control 81.55±8.08 a 41.48±2.63 123.03±11.41 a 115.22±6.41 a TAM 35.95±4.51 50.93±4.55 86.88±7.48 75.17±5.69 L-CAR 36.99±3.66 b 50.79±4.81 87.79±6.63 b 85.41±3.79 ab L-CAR+TAM 61 Az. J. Pharm Sci. Vol. 45, March, 2012 3.3 Oxidative status markers Figure1A-D shows the effects of TAM, L-CAR and their combination on the activity of SOD, level of LPO, GSH content and level of NO(x), respectively, in liver and uterus tissues. TAM treatmentinduced a significant decrease in the activity of SOD by (30.06, 20.31%)in liver and uterus, respectively. Administration of L-CAR prior to TAM induced significant increase in SOD activity (44.8%) in liver tissue and no significant change in uterus compared to the TAM treated group (Figure1-A). Treatment with TAM showed significant increase by (102.55, 118.44%) in LPO levels of liver and uterus,respectively. Treatment with both L-CAR and TAM induced no significant decrease in LPO level of liver compared to the TAM treated group while induced significant decrease in the level of LPO (34.63%)in uterus compared to the TAM treated group, however, the value is still high significant when compared with control group (Figure 1-B). Administration of TAM resulted in a significant decrease by (62.38%) in uterus GSH level and no significant change in liver (Figure 1-C). On other hand, administration of L- CAR prior to TAM induced significant increase(100%) in GSH level of uterus compared to the TAM treated group, however, the value is significantly decrease compared to the control group while the value of GSH still high significant in liver compared to the control group. Treatment of animals with TAM treatment induced non- significant change in level of NO(x) in the liver and uterus compared to the control group. Treatment with both L-CAR Az. J. Pharm Sci. Vol. 45, March, 2012 62 and TAM induced significant increase (29.26%) in the level of NO(x)in uterus tissue compared to TAM treated group (Figure 1-D). B A D C Figure (1): Effect of Tamoxifen (TAM), L-carnitine (L-CAR) and their combination on (A)superoxide dismutase expressed as µg/g tissue (SOD) activity, (B) lipid peroxides expressed as nmol/g tissue (LPO) level, (C) reduced glutathione expressed as μmol/g tissue (GSH) level, (D) total nitrate/nitrite expressed as nmol/g tissue (NO(x)) level in female rats for 28 consecutive days. Data are presented as % of control ± S.E.M of 6 rats/ group. a,b Significantly different from the control group and TAM treated group at p 0.05 using ANOVA followed by Tukey- Kramer as a post ANOVA test. TAM (10mg/kg, P.O) and L-CAR (300mg/kg, i.p.) were administered for 28consecutive and TAM induced significant increase (29.26%) in the level of NO(x)in uterus tissue compared to TAM treated group (Figure 1-D). and TAM induced significant increase (29.26%) in the level of NO(x)in uterus tissue compared to TAM treated group (Figure 1-D). B A D C D D C D C Figure (1): Effect of Tamoxifen (TAM), L-carnitine (L-CAR) and their combination on (A)superoxide dismutase expressed as µg/g tissue (SOD) activity, (B) lipid peroxides expressed as nmol/g tissue (LPO) level, (C) reduced glutathione expressed as μmol/g tissue (GSH) level, (D) total nitrate/nitrite expressed as nmol/g tissue (NO(x)) level in female rats for 28 consecutive days. g y Data are presented as % of control ± S.E.M of 6 rats/ group. a,b Significantly different from the control group and TAM treated group at p 0.05 using ANOVA followed by Tukey- Kramer as a post ANOVA test. Data are presented as % of control ± S.E.M of 6 rats/ group. a,b Significantly different from the control group and TAM treated group at p 0.05 using ANOVA followed by Tukey- Kramer as a post ANOVA test. TAM (10mg/kg, P.O) and L-CAR (300mg/kg, i.p.) were administered for 28consecutive days. In the groups treated with two drug regimens, the animals were pretreated with L-CAR 1 hour prior to TAM and the administration was continued as described above. TAM (10mg/kg, P.O) and L-CAR (300mg/kg, i.p.) were administered for 28consecutive days. In the groups treated with two drug regimens, the animals were pretreated with L-CAR 1 hour prior to TAM and the administration was continued as described above. 3.5. Effect on the level of caspase 3 proteins The level of caspase 3 proteins were determined by western immunoblotting analysis. As it is observed there was no marked alteration in the level of caspase 3 proteins by TAM (Figure 3). While, L-CAR alone and with TAM induced activation in level of caspase 3. Figure (3): Effect of TAM, L-CAR and their combination on caspase 3 in hepatic tissue using western blot technique. TAM (10mg/kg, P.O) and L-CAR (300mg/kg, i.p.) were administered for 28consecutive days. In the groups treated with two drug regimens, the animals were pretreated with L-CAR 1 hour prior to TAM and the administration was continued as described above. Figure (3): Effect of TAM, L-CAR and their combination on caspase 3 in hepatic tissue using western blot technique. TAM (10mg/kg, P.O) and L-CAR (300mg/kg, i.p.) were administered for 28consecutive days. In the groups treated with two drug regimens, the animals were pretreated with L-CAR 1 hour prior to TAM and the administration was continued as described above. Az. J. Pharm Sci. Vol. 45, March, 2012 M 1 2 3 4 Figure (2): Gel electrophoresis of DNA in hepatic tissue, M: molecular weight marker, lane 1: liver DNA of normal control female rat, lane 2: liver DNA of female rat treated with TAM (10mg/kg,P.O), lane 3: liver DNA of female rat treated with L-CAR (300mg/kg, i.p.), lane 4: liver DNA of female rat treated with L-CAR 1 hour prior to tamoxifen for 28 consecutive days. In the groups treated with two drug regimens, the animals were pretreated with L-CAR 1 hour prior to TAM and the administration was continued as described above. 3 5 Effect on the level of caspase 3 proteins M 1 2 3 4 3.4. DNA fragmentation Figure (2) shows the gel electrophoresis of DNA in liver tissue of female rat treated with TAM (10 mg/kg, P.O) and/or L-CAR (300mg/kg, i.p.) for 28 consecutive days did not show any change indicating no DNA fragmentation. 63 Az. J. Pharm Sci. Vol. 45, March, 2012 M 1 2 3 4 Figure (2): Gel electrophoresis of DNA in hepatic tissue, M: molecular weight marker, lane 1: liver DNA of normal control female rat, lane 2: liver DNA of female rat treated with TAM (10mg/kg,P.O), lane 3: liver DNA of female rat treated with L-CAR (300mg/kg, i.p.), lane 4: liver DNA of female rat treated with L-CAR 1 hour prior to tamoxifen for 28 consecutive days. In the groups treated with two drug regimens, the animals were pretreated with L-CAR 1 hour prior to TAM and the administration was continued as described above. 3.6. Histopathological findings (I,j): photomicrograph of rat endometrium of TAM treated c b a f e d i h g l k j V v P H H v S P A ِ D V H EP S G EP G S EP G G S EP EP G S S G EP c b v P H a b b e P A ِ D c f V H c a d d e h i h EP G Figure (4): Effec i h g l k j EP S G EP G S EP G G S EP EP G S S G EP i S EP G G g EP G S g k 3.6. Histopathological findings The protection effect of L-CAR against TAM-induced hepatotoxicity was further confirmed by conventional histopathological examination. Liver sections in the control group showed branching cords of hepatocytes are radiating from the central vein (v). The hepatocytes (H) are having vesicular nuclei and some binucleated cells are separated by sinusoids (S) lined by flat endothelial cells and kupffer cells Figure 4 (a). In Figure 4 (d) Liver section of rat liver treated with L-CAR group showing similar histological structure as control. Figure 4(b, c) photomicrograph of rat liver of TAM treated group showing most of hepatocytes (H) around dilated and congested portal vein (P) with empty cytoplasm and dark nuclei, thickening and congested central vein (v), and some hepatocytes (H) appear with cytoplasmic vacuolation. In combined group L-CAR and TAM showed relatively normal of hepatocytes around the portal vein (p), bile duct (b) and hepatic artery (A). However, the remaining cells are moderately swollen and vacuolated. Figure 4 (e, f). 64 Az. J. Pharm Sci. Vol. 45, March, 2012 Figure (4): Effect l-carnitine pretreatment on TAM-induced hepatic and endometrium toxicities in female rats. (a): liver section of the normal control group did not show histopathological changes. (d): photomicrograph of rat liver of treated L-carnitine group showing similar histological structure as control. (b, c): photomicrograph of rat liver of TAM treated group showing most of hepatocytes (H) around dilated and congested portal vein (p) with empty cytoplasma and dark nuclei, thickening and congested central vein (v) .and some hepatocytes (H) appear with cytoplasmic vacuolation. (e,f) : photomicrograph of rat liver of L-carnitine+TAM treated group showing relatively normal of hepatocytes around the portal vein (p) ,bile duct (b) and hepatic artery (A) and remarkably healthy hepatocytes. (g): photomicrograph of rat endometrium of control group did not show histopathological changes. (h): photomicrograph of rat endometrium of L-carnitine treated group showing no histological changes were seen as compared to the control group. 3.7.1.Percent survival of animals Figure (5) showed the percent survival of Ehrlich Ascites Carcinoma EAC-bearing mice treated with TAM(10mg/kg single i.p.) and/or L-CAR (300mg/kg single i.p.).At day 23 of the experiment, the percentage of survival was 0% in control untreated tumor-bearing mice, while at day 24 of experiment, the percentage of survival was 0% in TAM treated group. On the other hand, at day 41, the percentage of survival was 0% in mice treated with L-CAR, whereas at day 19, the percentage of survival was 0% in mice treated with L-CAR and TAM. Figure (5): Effect of Tamoxifen (10mg/kg), L-carnitine (300mg/kg) and their combination on the percentage survival rate of EAC-bearing mice. Figure (5): Effect of Tamoxifen (10mg/kg), L-carnitine (300mg/kg) and their combination on the percentage survival rate of EAC-bearing mice. Figure (5): Effect of Tamoxifen (10mg/kg), L-carnitine (300mg/kg) and their combination on the percentage survival rate of EAC-bearing mice. Figure Figure (4): Effect l-carnitine pretreatment on TAM-induced hepatic and endometrium toxicities in female rats. (a): liver section of the normal control group did not show histopathological changes. (d): photomicrograph of rat liver of treated L-carnitine group showing similar histological structure as control. (b, c): photomicrograph of rat liver of TAM treated group showing most of hepatocytes (H) around dilated and congested portal vein (p) with empty cytoplasma and dark nuclei, thickening and congested central vein (v) .and some hepatocytes (H) appear with cytoplasmic vacuolation. (e,f) : photomicrograph of rat liver of L-carnitine+TAM treated group showing relatively normal of hepatocytes around the portal vein (p) ,bile duct (b) and hepatic artery (A) and remarkably healthy hepatocytes. (g): photomicrograph of rat endometrium of control group did not show histopathological changes. (h): photomicrograph of rat endometrium of L-carnitine treated group showing no histological changes were seen as compared to the control group. (I,j): photomicrograph of rat endometrium of TAM treated group showing degeneration surface epithelium (EP), partially ulcerated, edematous stroma (S) contains tortuous glands (G), hyperplasia stroma (S) contain cells with deeply stained Az. J. Pharm Sci. Vol. 45, March, 2012 65 nuclei and endometrial glands (G) within its wide lumen. (k, l): photomicrograph of rat endometrium of L-carnitine+TAM treated group showing recovery of the surface epithelium (EP) nearly normal cellular stroma (S). (1-6, 10) X400; (7-9, 11, 12) X100. H & E. Photomicrograph of rat endometrium of control group showing surface columnar epithelium (EP) and highly cellular stroma (S) contains numerous corkscrew endometrial glands (G) Figure 4 (g). Section of rat endometrium of L-CAR treated group showing no histological changes were seen as compared to the control group Figure 4 (h). photomicrograph of rat endometrium of TAM treated group showing degeneration surface epithelium (EP), partially ulcerated and edematous stroma (S) contains tortuous glands (G), hyperplasia stroma (S) contain cells with deeply stained nuclei and endometrial glands (G) within its wide lumen Figure 4 (i, j). photomicrograph of rat endometrium of L-CAR with TAM treated group showing recovery of the surface epithelium (EP) nearly normal cellular stroma (S) and most of the endometrium histological architure Figure 4 (k, l). Az. J. Pharm Sci. Vol. 45, March, 2012 66 CAR in the combined group resulted in significant decrease in tumor volume compared to control group. 0 100 200 300 400 500 Control TAM L-CAR L-CAR+TAM a a Treatment Tumor Volume (mm3) Figure (6): Effect of TAM, L-CAR and their combination on the tumor volume of EAC- bearing mice. Data are expressed as mean ± S.E.M of 6 mice/ group. P value is significant 0.05 using ANOVA followed by Tukey-Kramer as a post ANOVA test. a Significantly different from the control group. TAM (10mg/kg, i.p.) and L- CAR (300mg/kg, i.p.) were administered four times. In the groups treated with two drug regimens, the animals were pretreated with L-CAR 1 hour prior to TAM and the administration was continued as described above. 0 100 200 300 400 500 Control TAM L-CAR L-CAR+TAM a a Treatment Tumor Volume (mm3) Figure (6): Effect of TAM, L-CAR and their combination on the tumor volume of EAC- bearing mice. Data are expressed as mean ± S.E.M of 6 mice/ group. P value is significant 0.05 using ANOVA followed by Tukey-Kramer as a post ANOVA test. a Significantly different from the control group. TAM (10mg/kg, i.p.) and L- CAR (300mg/kg, i.p.) were administered four times. In the groups treated with two drug regimens, the animals were pretreated with L-CAR 1 hour prior to TAM and the administration was continued as described above. 3.7.2.Tumor volume Figure (6) showed the effect of TAM, L-CAR and their combination on the growth of solid Ehrlich carcinoma. The tumor volume of control group showed progressive increase, whereas treatment of mice four times every other day with TAM (10mg/kg, i.p.) resulted in significant decrease in tumor volume as compared to control group. L-CAR alone induced non-significant decrease in tumor volume compared to control group. Pretreatment with L- 4. DISCUSSION TAM is an anti-estrogenic drug widely used for the treatment of all stages of breast cancerin spite of its hepaticand endometrium toxicities in rats (Karki et al., 2000; Stanley et al., 2001) and human (Akcay et al., 2000). In the present study TAM induced a significant increase in TG, CH and LDL. Our results are in agreement with previous studies (Akhondi- Meybodi et al., 2011; Lee et al., 2010). They reported that,the increased lipid profile after TAM administration was probably due to reduction in hepatic ability of lipid β-oxidation that ultimately enhanced hepatic fat content and hypertriglyceridaemia and increase in the rate of CH biosynthesis in liver by TAM.Increase in TG level within normal range in most of patient while, severe hypertriglyceridemia developed in small number of them was reported in postmenopausal patients receiving adjuvant TAM (Liu and Yang, 2003). However, lower CH and LDL levels were detected by TAM in ovariectomized rats (Lundeen et al., 1997) and in postmenopausal women (Love et al., 1994). Our study demonstrated a significant increase in serum activity of AST, ALT and level of bilirubin in TAM treated rats. Our results are in agreement with the previous findings of Jain et al. (2011) and Kumarappan et al. (2011) who reported similar elevation in liver function. TAM has much higher affinity for hepatic tissue, its active metabolite, 4- hydroxytamoxifen induces conformational changes in the lipid bilayers of cell membranes an effect which can lead to an increase in the membrane permeability and facilitates the passage of cytoplasmic enzymes outside the cells leading to the increase in the Az. J. Pharm Sci. Vol. 45, March, 2012 67 aminotransferase activities in blood (Albukhari et al., 2009). L-CAR in this study significantly decreased the levels of TG, CH, LDL, and significantly inhibited the rise in liver function compared to TAM treated group. L-CAR is an essential cofactor in the transport of long-chain fatty acids from the cytosol to mitochondria for subsequent β- oxidation and production of cellular energy. In addition, it exhibits antioxidant and antiapoptotic activity (Muthuswamy et al., 2006). It is very effective in minimizing age- associated disorders, which free radicals are the major cause (Gulcin, 2006; Gomez- Amores et al., 2006). Previous study reported restoration of lipid profiles by L-CAR in rats (Sidoriak and Volgin, 1996) and in premenopausal women (Lofgren et al., 2005). 4. DISCUSSION In consistent with previous studies (Albukhari et al., 2009; Jain et al., 2011; Tabassum et al., 2006), our study showed a significant increase in LPO with significant decrease in antioxidant levels in hepatic and endometrium tissue after TAM administration. TAM impaired beta oxidation of fatty acids (Parvez et al., 2006) and mitochondrial functions as it acts as an uncoupling agent and a powerful inhibitor of mitochondrial electrontransport chain eventually results in mitochondrial oxidativedamage (Parvez et al., 2008). TAM decreased the activities of catalase, glutathione peroxidase and SOD and content of GSH in liver,the decreased glutathione peroxidase activity leads to H2O2 accumulation in the liver, which in turns inactivates SOD (Kakkar et al., 1997). The decreased antioxidant power of TAM-intoxicated rats could be attributed toimpairment of hexose monophosphate (HMP) shunt and thereby reduced NADPHavailability and the ability to recycle substances as GSSG to GSH is decreased (Stanley et al., 2001). However, Perumal et al. (2005) have shown that TAM inhibited LPO triggered by freeradicals in 7, 12 dimethyl Benz (a) anthracene (DMBA) induced peroxidative damagein rat mammary carcinoma. Reduction in levels of LPO and elevation in antioxidants in L-CAR treated rats suggested that L CAR scavenges free radicals generated during oxidative stress. L-CAR had an effective superoxide anion radical scavenging, hydrogen peroxide scavenging, total reducing power and metal chelating on ferrous ions activities (Gulcin, 2006; Kalaiselvi and Panneerselvam, 1998). Nitric oxide (NO(x)) has been implicated in various aspects of cancer biology, including both pro and anti- tumor functions (Ostad et al., 2009). In our study neither TAM nor L-CAR alone had an effect on NO(x) level; however, significant increase in NO(x) in uterus tissue was observed when we use two drugs in combinations. We assume that this dual modulatory effects were not notable until both drugs were used resulting in significant increase in NO(x). On the other hand, enhanced NO(x) production was found by TAM (Loo et al., 1998) and L-CAR (Bueno et al., 2005) in serum of rats and in breast cancer tissue (Erbas et al., 2007). Our study showed no change in the expression of caspases 3 and intact genomic DNA was observed in normal hepatic tissue treated with TAM. The concept that TAM has favorable antineoplastic effect without any adverse effect on human normal breast tissue was indicated in benign biopsies of patients receiving TAM daily (de Lima et al., 2003; Walker et al., 1991). Az. J. Pharm Sci. Vol. 45, March, 2012 68 break by maintaining thiol-containing compound and improving the glutathione redox status, in addition, L-CAR decreased the levels of oxidative stress mediated DNA damage during aging in rats. However, our results indicated histopathological changes in architecture of the liver and uterus in TAM treated group which might be attributed to impaired β-oxidation of fatty acids and generation of ROS (Albukhari et al., 2009). It was found that TAM induced DNA adduct formation in rat liver and uterus through metabolic activation to α- hydroxytamoxifen which is sulfonated, forming an electrophilic carbocation that is capable of reacting with DNA yields a DNA adduct (Costa et al., 2007). L-CAR clearly ameliorated the histopathological changes induced by TAM and showed relatively normal and recovery of liver and uterus tissues. These findings support previous results indicating the antioxidant activity exhibited by L-CAR. In the present study, we have tested the effect of administration of TAM against induction of mammary carcinoma in female mice. Our study demonstrated that TAM decreased tumor volume and increased survival rate compared with the untreated control. Gabri et al. (2004) reported a significant reduction in tumor volume and a significant increase in the mean survival time of EAC-bearing animals in TAM pretreated group. L-CAR as amonotherapy had an inhibitory effect on tumor volume (Sayed-Ahmed et al., 1999). Niang and Melka (2000) reported that Acetyl-L-carnitine prolonged the survival of micewith the ascitic form of leukaemia L1210 and L-CAR treatment prolonged survival ofrats with adriamycin-induced heart failure by improving the myocardial metabolism of fatty acids (Kawasaki et al., 1996). The combined effect of TAM and L-CAR on the survival in our study was less than the individual drug. It seems that L-CAR antagonize both the antitumor effect and survival of TAM upon administration of both drugs together. The antitumor effect and survival of the combined drug was less than each one alone. Interestingly L-CAR increased survival rate of EAC-bearing mice more than TAM treated group. The antagonistic effect of L-CAR with TAM may be due to interference with TAM metabolism, increase its excretion (Olszowy et al., 2006) or may be due to drug interaction which affects the cytotoxic effect of TAM. Also, this effect might be due to the antioxidant activity of L-CAR (Gulcin, 2006). 5. CONCLUSION: This study illustrates that although L-CAR ameliorates TAM-induced hepatic and endometrium toxicities by preventing oxidative stress and enhancingantioxidant enzymes, it interferes with its antineoplastic activity. Declaration of interest: The authors report no conflicts of interest. The authors are responsible for the content and writing of the paper. 4. DISCUSSION Moreover, no change in the expression of caspase-3 in MCF-7 cell by TAM however, it exerts a significant caspase-3 dependent apoptosis in ER-MDA-MB468cells and co-incubation with caspase-3 inhibitor abolished the apoptosis was found (Salami and Karami-Tehrani, 2003). L-CAR was reported as an important anti-apoptotic mediator (Moretti et al., 2002). L-CAR was reported to induce apoptosis in hepa1c1c7 cells by regulating Fas ligands and inhibiting the expression of Bcl-2 and inducing the up-regulation of caspase-9 and caspase-3 (Fan et al., 2009). 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Vol. 45, March, 2012 تأثيز ات ل-كارًتيي الوعدلة لسوية تاهىكسيفيي و كذلك لٌشاطه الوضاد لألورام: دراسة داخل جسن الحى  ,*أهل بلقاسن إبزاهين, **ساهية عبدالسويع شىهاى ,*أهاًى على عيسى, ***هبة حسٌى هٌصىر ***سهام هحود أبى ًىر  قسى االدٔيت, كهيت انطب, خبيؼت انضأيت , نيبيب. .ٌ*قسى أدٔيت ٔسًٕو, كهيت انصيذنت, خبيؼت حهٕا .** قسى بيٕنٕخيب االٔساو, ٔحذة األدٔيت, يؼٓذ انقٕيٗ ألٔساو, خبيؼت انقبْشة ***نبحٕد ٔحكُٕنٕخيب اإلشؼبع ,ْيئت انطبقت انزسيت قسى انبحٕد انصحيت اإلشؼبػيت , انًشكض انقٕي تأثيز ات ل-كارًتيي الوعدلة لسوية تاهىكسيفيي و كذلك لٌشاطه الوضاد لألورام: دراسة داخل جسن الحى  ,*أهل بلقاسن إبزاهين, **ساهية عبدالسويع شىهاى ,*أهاًى على عيسى, ***هبة حسٌى هٌصىر ***سهام هحود أبى ًىر  ,*أهل بلقاسن إبزاهين, **ساهية عبدالسويع شىهاى ,*أهاًى على عيسى, ***هبة حسٌى هٌصىر ***سهام هحود أبى ًىر  قسى االدٔيت, كهيت انطب, خبيؼت انضأيت , نيبيب. ٌ*قسى أدٔيت ٔسًٕو, كهيت انصيذنت, خبيؼت حهٕا .** قسى بيٕنٕخيب االٔساو, ٔحذة األدٔيت, يؼٓذ انقٕيٗ ألٔساو, خبيؼت انقبْشة ش ٕ ٗ أٔس و ٓ أٔي أس و ٔ ى بيٕ ٕ ي *** قسى انبحٕد انصحيت اإلشؼبػيت , انًشكض انقٕيٗ نبحٕد ٔحكُٕنٕخيب اإلشؼبع ,ْيئت انطبقت انزسيت. أٌ انٓذف يٍ ْزِ انذساست االسخذالل ٔ انخؼش ٗف ػه ان خأثيش ٗانٕقبئ ل- ٍكبسَخيٍ ػهٗ انسًيت انًحذثت بذٔاء انخبيٕكسيفي ٌٔ فٗ ػالج انسشطب. ٔ نخحقيق األْذاف انسببقت فقذ حى حقسيى اندشراٌ انبيضبء انٗ اسبغ يدًٕػبث حخكٌٕ كم يدًٕػت يٍ سخ ت :ٗخشراٌ حى حقُٓب ػهٗ انُحٕ انخبن- انًدًٕػت االٔنٗ حى اػطبء يحهٕل يهحٗ ك ًدًٕػت ضببطت ٔ فٗ انًدًٕػت ٍانخبَيت انخبيٕكسيفي ( 01يدى / كدى فًٕيب) ٔ فٗ انًدًٕػت انثبنثت ل- ( ٍكبسَخي011 )َٕٗيدى / كدى فٗ انخدٕيف انبشيخ ٔ انشابؼت ببل- كبسَخيٍ ثى انخبيٕكسيفيٍ بؼذ سبػت ٔاحذة) ٔاسخًشث ْزِ انذساست نًذة28 . ٗيٕيب ػهٗ انخٕان ٔقذ اظٓشث َخبئح ْزِ انذساست اٌ انخبيٕكسيفيٍ قذ سبب صيبدة في يؼذل انذٌْٕ, َشبط اَضيًبث ٔ انكبذ ٍيسخٕيبث انبيهيشٔبي. ْزا ببالضبفت اني اَّ سببٗصيبدة ف ٍانهيبيذ بشٔكسيذاش ٔاَخفبض في َشبط اَضيى انسٕبش اكسـيذ ديسًيٕحيض في اَسدت ٔ انكبـــذ ٔانشحـى ٗاَخفبض يهحٕظ ف انٗدهٕحبثيٌٕ انًخخضل ف اَسدت انشحى. يٍ َبحيت اخشٖ اثبخج َخبئح ْزِ انذساست اٌ اسخخذاو ل- كبسَخيٍ سبػت قبم انخبيٕكسيفيٍ قذ قهم يٍ انسًيت انًحذثت بٕاسطت انخبيٕكسيفيٍ ٔرانك بخقهيم كم يٍ يؼذل ,ٌْٕانذ َشبط اَضيًبث انكبذ, ٗٔ صيبدة يهحٕظت ف َشبط اَضيى انسٕبش اكسيذ د يسًيٕحيض في اَسدت انكبـــذ ٔانشحـى يقبسَت ببسخخذاو دٔاء انخبيٕكسيفيٍ فقظ. كًب ادٖ انٗ ححسٍ يسخٕيبثٍانهيبيذ بشٔكسيذاش ٔ ان دهٕحبثيٌٕ انًخخضل في اَسدت انشحى ٖٔ يٍ َبحيت اخشٖ نى يحذد ا حُشيظ فٗ يسخٕٖ حكٕيٍ كسبيس 9 ٔ 0 فٗ كم انًدًٕػبث. Az. J. Pharm Sci. Vol. 45, March, 2012 72 Senkus-Konefka, E., Konefka, T., Jassem, J., (2004): The effects of tamoxifen on the femalegenital tract. Cancer Treatment Reviews 30, 291-301. Sidoriak, N.G., Volgin, D.V., (1996): Effect of L-carnitine on lipid peroxidation and lipidcomposition in blood serum in hemic hypoxia.UkrBiokhimZh. 68, 54-61. Singh, M.N., Stringfellow, H.F., Paraskevaidis, E., Martin-Hirsch, P.L.,Martin,F.L., (2007): Tamoxifen: Important considerations of a multi-functional compound with organ-specific properties. Cancer Treatment Reviews 33, 91-100. Stanley, L.A., Carthew, P., Davies, R., Higginson, F., Martin, E., Styles, J.A., (2001): Delayedeffects of tamoxifen in hepatocarcinogenesis-resistant Fischer 344 rats as compared with susceptible strains.Cancer Lett. 171, 27-35. Tabassum, H., Rehman, H., Banerjee, B.D., Raisuddin, S., Parvez, S., (2006): Attenuation of tamoxifen-induced hepatotoxicity by taurine in mice.Clinica.Chimica.Acta. 370, 129-136. Terpstra, A.H.M., Sanchez-Muniz, F.J., West, C.E., Woodward, C.J.H., (1982): The density profile and cholesterol concentration on serum lipoprotein in domestic and laboratory animals. Comp.Biochem. Physiol. 71B, 669-673. Tietz, N.W., Boden, T., Stepleton, J.D., (1959): An improved method for determination of lipase in serum. Am. J.Clin.Pathol.31, 148. Vamos, E., Voros, K., Vecsei, L., Klivenyi, P., (2010): Neuroprotective effects of L-carnitine in a transgenic animal model of Huntington’s disease. Biomedicine &Pharmacotherapy 64, 282-286. Walker, K.J., Price-Thomas, J.M., Candlish, W., Nicholson, R.I., (1991): Influence of the antiestrogen tamoxifen on normal breast tissue. Br. J. Cancer.64,764–768. Wang, S., Wang, L., Yin, J., Wang, Z., Fu, P.P., Yu, H., (2009): Light-induced toxic effects of tamoxifen: A chemotherapeutic and chemopreventive agent. J.Photochem.Photobiol.201, 50-56. Warnick, G., Wood, P., (1995): National Cholesterol Education Program Recommendation for Measurement of High –Density Lipoprotein Cholesterol:Executive Summary. Clin. Chem. 41, 1427-1433. Yokozawa, T., Dong, E., (2001): Role of ginsenoside-Rd in cisplatin induced renal injury: special reference to DNA fragmentation. Nephron. 89, 433-438. Zilva, J.F., Pannall, P.R., (1979): Plasma enzyme in diagnosis in clinical chemistry in: diagnosis and treatment. lioydluke London chap 17,338. 73 Az. J. Pharm Sci. Vol. 45, March, 2012 Az. J. Pharm Sci. Vol. 45, March, 2012 ٔ قذ أكذث ًانذساست ان دٓشيت نألَسدت انكبذ ٔانشحى ْزِ انُخبئح انبيٕكيًيبئيت. ٔ قذ أظٓشث انذساست أٌ ٔ يؼذل انبقبء فبػهيت ػالج ٌانسشطب نٍهخبيٕكسيفي ٔ ل ٍكبسَخي يؼب ٗفٔ ٗانفئشاٌ انبيضبء انحبيهت نخاليب سشطبٌ أسنيخ األسخسقبئ أسنيخ انصهب اقم يٍ كم يًُٓب ػهٗ حذة. ٖبيًُب اد ل- ٍكبسَخي انٗ صيبدة فٗ يؼذل انبقبء يقبسَت ببسخخذاو دٔاء انخبيٕكسيفيٍ فقظ . ِيٍ ْز انذساست َسخخهص اٌ دٔاء ل كبسَخيٍ قهم يٍ حبثيش انخبيٕكسيفيٍ انسًي ٔنكٍ يدب اٌ يكٌٕ اسخخذايّ يحذٔدا َظشا ٌنخقهيهّ يٍ فبػهيت انخبيٕكسيفيٍ في ػالج انسشطب

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Voluntary Medical Male Circumcision Programs Can Address Low HIV Testing and Counseling Usage and ART Enrollment among Young Men: Lessons from Lesotho

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Abstract doi:10.1371/journal.pone.008 Editor: Nathan Ford, World Health Organization, Switzerland Editor: Nathan Ford, World Health Organization, Switzerland Received August 13, 2013; Accepted November 5, 2013; Published May 6, 2014 Received August 13, 2013; Accepted November 5, 2013; Published May 6, 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. Funding: This work has been supported by the President’s Emergency Plan for AIDS Relief (PEPFAR) through the U.S. Agency for International Development (USAID) under the terms of Cooperative Agreement No. GHS-A-00-08-00002-000. The funder played a significant technical role in study analysis, decision to publish and preparation of the manuscript. USAID staff did not play a role in data collection. The views expressed in this manuscript do not represent USAID or U.S. Government opinions. Competing Interests: The authors have declared that no competing interests exist. * E-mail: [email protected] Virgile Kikaya1*, Laura Skolnik1, Macarena C. Garcı´a2, John Nkonyana3, Kelly Curran4,5, Tigistu Adamu Ashengo4,5 1 Jhpiego, Maseru, Lesotho, 2 Lesotho PEPFAR Program, U.S. Agency for International Development, Maseru, Lesotho, 3 Ministry of Health, Maseru, Lesotho, 4 Jhpiego, Baltimore, Maryland, United States of America, 5 Department of International Health, Johns Hopkins University Bloomberg School of Public Health, Baltimore, Maryland, United States of America Abstract Background: Early diagnosis of HIV and treatment initiation at higher CD4 counts improves outcomes and reduces transmission. However, Lesotho is not realizing the full benefits of ART because of the low proportion of men tested (40%). Public sector VMMC services, which were launched in district hospitals in February 2012 by the Lesotho MOH supported by USAID/MCHIP, include HIV testing with referral to care and treatment. The objective of this study was to better understand the contribution of VMMC services to HIV diagnosis and treatment. Methods: VMMC clients diagnosed with HIV were traced after 6 months to ascertain whether they: (1) presented to the referral HIV center, (2) had a CD4 count done and (3) were enrolled on ART. Linkages between VMMC and HIV services were assessed by comparing the proportion of HIV-infected males referred from VMMC services with those from other hospital departments. Results: Between March and September 2012, 72 men presenting for VMMC services tested positive for HIV, representing 65% of the total male tests at the hospital; 45 of these men (62.5%) received an immediate CD4 count and went to the HIV referral site; 40 (89%) were eligible for treatment and initiated ART. 27 clients did not have a CD4 count due to stock-out of reagents. Individuals who did not receive a CD4 count on the same day did not return to the HIV center. Conclusion: All VMMC clients testing positive for HIV and receiving a CD4 count on the testing day began ART. Providing VMMC services in a district hospital offering the continuum of care could increase diagnoses and treatment uptake among men, but requires an investment in communication between VMMC and ART clinics. In high HIV prevalence settings, investing in PIMA CD4 devices at integrated VMMC clinics is likely to increase male ART enrolment. ation: Kikaya V, Skolnik L, Garcı´a MC, Nkonyana J, Curran K, et al. (2014) Voluntary Medical Male Circumcision Programs Can Addres unseling Usage and ART Enrollment among Young Men: Lessons from Lesotho. PLoS ONE 9(5): e83614. Voluntary Medical Male Circumcision Programs Can Address Low HIV Testing and Counseling Usage and ART Enrollment among Young Men: Lessons from Lesotho ya1*, Laura Skolnik1, Macarena C. Garcı´a2, John Nkonyana3, Kelly Curran4,5, Results Males tested for HIV through VMMC services represented an important proportion of all males who were tested at Mafeteng district hospital from February to December 2012. Of the 2,941 males, ages 15–49, tested at Mafeteng hospital, 1,906 (65%) were tested at the VMMC clinic. Four hundred forty six (446) males tested HIV-positive at the hospital of which 72 (16%) were tested at the VMMC site. The VMMC program in Lesotho is implemented by the Lesotho Ministry of Health (MOH) with support from the Maternal and Child Health Integrated Program (MCHIP) led by Jhpiego and funded by the U.S. Agency for International Development (USAID). Consistent with the Lesotho National Health Strategic Plan, integrated public-sector VMMC services were launched in district hospitals in February 2012. These services are provided at no cost for all male clients presenting at VMMC clinics. Demographic information on VMMC clients who tested HIV- positive is included in table 1. Approximately 80% of VMMC clients who tested HIV-positive reside in Mafeteng district followed by 17% in Maseru district. There were about equal numbers of single and married clients, 47% and 49% respectively. Half of the clients were employed. In the first 10 months of operation, it became clear that men in Lesotho were highly motivated to seek VMMC services. Between February and December 2012, more than 9,500 men (of whom 75% were between the ages of 15 and 24) were medically circumcised at one of six district hospitals in Lesotho, compared with only 400 men in the year before the launch of this national program [10]. Furthermore, 97% of clients presenting for VMMC services agreed to HIV testing; 5% of these men tested positive for HIV. Based on this performance, program implementers decided to retrospectively conduct secondary analysis of data collected at one hospital to further explore the linkages between VMMC, HTC and ART enrollment. In this study, we collected data on VMMC clients who tested HIV-positive in order to determine the outcome in terms of enrolment in HIV treatment and care. The 72 VMMC clients testing positive were referred to the ART clinic for a baseline CD4 count (Figure 1). Forty-five (62.5%) of these clients received their CD4 count on the same day as being tested for HIV and receiving a positive result. Introduction and treatment. Yet early identification of HIV and enrollment in HIV care and treatment has been proven not only to improve health outcomes for HIV-infected individuals, but also to reduce HIV transmission. HIV prevalence in Lesotho is among the highest in the world, with an estimated 23% of adults infected [1]. In Lesotho, an estimated 40% of men ages 15–49 have ever received an HIV test, compared to 68% of women [1]. Furthermore, Lesotho health survey data demonstrate that men are far less likely than women to know their HIV status or to have undergone an HIV test in the previous year. Studies have shown that men are more likely than women to start an antiretroviral therapy (ART) at a later stage with a more advanced HIV disease [2–3]. In addition, linkages between HIV testing and counseling (HTC) and ART services are generally weak, and clients tested in HTC programs often cannot be traced or followed up to ensure that they have enrolled in care In March 2007, WHO and UNAIDS issued guidance urging countries with high HIV prevalence and low male circumcision rates to incorporate male circumcision into their comprehensive HIV prevention programs. This recommendation was based on three randomized controlled trials that determined unequivocally that voluntary medical male circumcision (VMMC) reduces female to male HIV transmission by approximately 60% [4–6]. Post-trial surveillance also suggests that risk compensation has not been a problem in the clinical trial sites and that the population- level prevention benefit of VMMC has increased with time [7,8]. May 2014 | Volume 9 | Issue 5 | e83614 PLOS ONE | www.plosone.org 1 May 2014 | Volume 9 | Issue 5 | e83614 VMMC Can Increase ART Enrollment among Young Men VMMC Can Increase ART Enrollment among Young Men Mathematical modeling studies suggest that scaling up VMMC to reach 80% coverage in 13 priority countries by 2015 could avert 3.36 million HIV infections by 2025 [9]. VMMC services are designed to be part of a comprehensive package that includes HTC, STI screening, condom promotion and health education. In countries such as Lesotho, where rates of HIV testing and ART enrollment are low especially among men, it is important to consider whether VMMC services could be an effective strategy to attract men to HTC and link them to care and treatment. Introduction waived by the IRB as it involved secondary data analysis of a pre- existing, de-identified/de-linked, not publicly available data set, and as investigators were not involved in the original data collection. Methods Data were drawn from Lesotho’s first public facility to be equipped to provide VMMC: Mafeteng government district hospital, located about 80 km from Maseru. The number of clients accessing HTC and the percentage of positive results were extracted for all clients presenting for VMMC services between February and December 2012. HTC hospital statistics were reviewed to quantify the percentage of males 15–49 who tested at the VMMC clinic in comparison to other hospital department. VMMC clients testing positive for HIV were traced to ascertain whether they (1) presented at the ART clinic, (2) had received a CD4 count and (3) enrolled in treatment. Missed clients were followed through phone calls. Data were collected from the ART clinic in an effort to retrieve HIV-positive clients who enrolled in care and treatment. All data were collected from the hospital registers by the counselor in charge. Table 1. Demographics of clients who tested HIV-positive at the Mafeteng VMMC site (n = 72). n % District of origin Mafeteng 57 (79.1%) Maseru 12 (16.7%) Thaba Tseka 1 (1.4%) Mohales’hoek 1 (1.4%) Berea 1 (1.4%) Marital status Single 34 (47.2%) Married 35 (48.6%) Divorced 2 (2.8%) Widowed 1 (1.4%) Employment status Employed 36 (50.0%) Unemployed* 36 (50.0%) *Includes students. doi:10.1371/journal.pone.0083614.t001 May 2014 | Volume 9 | Issue 5 | e83614 Table 1. Demographics of clients who tested HIV-positive at the Mafeteng VMMC site (n = 72). Table 1. Demographics of clients who tested HIV-positive at the Mafeteng VMMC site (n = 72). Results The remaining 27 clients were unable to receive an immediate CD4 count due to a stock-out of reagents at Mafeteng district hospital between July and September 2012. These clients were referred to other district hospitals for the CD4 count or were requested to return at a later date. The mean CD4 count result was 302 (195–685). Among the 45 clients who received a CD4 count, 40 (88%) were eligible for treatment (i.e., CD4 count less than 350, as per Lesotho national guidelines). All clients who were eligible for treatment were enrolled and followed up. Figure 1 presents the algorithm for VMMC clients who tested HIV-positive. Methods The 27 clients who did not receive a CD4 count the same day as HIV diagnosis did not return to Mafeteng hospital for this service at a later date, as per ART register review. These clients were traced through phone calls. Sixteen could not be contacted Ethics statement This work has a non-human subjects research determination notice from the Johns Hopkins School of Public Health IRB. The Ministry of Health of Lesotho does not require IRB review for secondary program data analysis as long as data are not identifiable. All VMMC clients provided written informed consent before undergoing HIV testing as well as VMMC procedures as per national guidelines. As VMMC services are integrated into hospital services, all patient files are kept on site and only accessed by hospital providers. However, there was no separate informed consent collected specifically for this review. This research was PLOS ONE | www.plosone.org 2 VMMC Can Increase ART Enrollment among Young Men Figure 1. Flow chart describing ART enrolment outcome of VMMC clients who tested HIV-positive. All clients who received a CD4 count and who were eligible while seeking VMMC services were initiated on ART. Among the 27 clients who did not receive a CD4 count while seeking VMMC services, only four were enrolled in treatment. doi:10.1371/journal.pone.0083614.g001 Figure 1. Flow chart describing ART enrolment outcome of VMMC clients who tested HIV-positive. All clients who received a CD4 count and who were eligible while seeking VMMC services were initiated on ART. Among the 27 clients who did not receive a CD4 count while seeking VMMC services, only four were enrolled in treatment. doi:10.1371/journal.pone.0083614.g001 A number of studies are exploring how newly HIV diagnosed individuals are linked to care and treatment. Two studies discussed by Gardner et al. show that 73% and 64% of a cohort of newly identified HIV-positive individuals were enrolled in HIV care and treatment at one year and three months after diagnosis, respectively [13]. The same study showed that approximately 75% of a cohort of individuals who tested HIV-positive were successfully linked to HIV care within six to 12 months, and 80% to 90% of the cohort after three to five years. At Mafeteng hospital, an estimated 68% of all HIV-positive VMMC clients were linked to care at the time of the study. Several factors can influence the proportion of individuals who are linked to care and treatment. WHO recommends that linkage to care and treatment be more rigorously evaluated [14] and that good practices that can improve such linkages be identified. Ethics statement According to WHO recommendations, these good practices include but are not limited to providing on-site or immediate CD4 testing with same-day results; assisting with transport if the ART site is far from the HTC site; and involving community outreach workers to identify the people lost to follow-up [14]. This study identified additional elements that might influence linkages in the context of VMMC services being implemented in a hospital setting in Lesotho, including availability of CD4 count laboratory tests, which is consistent with WHO recommendations. However, additional elements such as the attitude of services providers, the routine review of information and weaknesses in the national supply chain were identified as elements influencing the effectiveness of linkages to care and treatment. because their phone numbers were not available. Among the 11 clients contacted, four reported that they received a CD4 count elsewhere and had enrolled in treatment; seven reported that they had not received a CD4 count. Acknowledgments The national scale-up of VMMC services would not have been possible without the strong leadership and commitment of key personnel in the Ministry of Health, particularly the Director General of Health Services, Dr. M. Moteetee, and her commitment to improving the lives of the Basotho people. Special thanks to Mr. Tsasanyane, counselor at Mafeteng hospital, whose incredible work has inspired this study. Finally, the authors would like to thank the MCHIP Publications Department for their work on editing and formatting this document. Monitoring and evaluation of VMMC clients’ clinic data VMMC providers at Mafeteng hospital maintain detailed data statistics on clients’ HIV testing and diagnosis. The records are then integrated with hospital data. For this study, the VMMC counselor cross-referenced data with information at the ART clinic to identify VMMC clients who were not enrolled in HIV care and treatment and had not yet returned. Counselors then attempted to contact these clients. This review highlights the need to ensure routine data review and analysis of the VMMC and the ART clinic data sets. Author Contributions Conceived and designed the experiments: VK LS KC. Analyzed the data: VK LS MG JN KC TA. Wrote the paper: VK LS MG JN KC TA. Conceived and designed the experiments: VK LS KC. Analyzed the data: VK LS MG JN KC TA. Wrote the paper: VK LS MG JN KC TA. CD4 count on site CD4 count on site The findings from the secondary analysis of programmatic data strongly suggest that clients testing positive for HIV should be offered a CD4 count immediately following diagnosis. This study shows that when this occurs, clients are more likely to enroll in HIV care and treatment. Receiving a CD4 count immediately provides an opportunity for clients to be counseled on HIV care and treatment, to be enrolled into services and increases adherence outcomes. VMMC clients who did not get a CD4 count in the VMMC clinic after HIV-positive diagnosis were less likely to either return or to get a CD4 count elsewhere. This finding demonstrates the importance of having a CD4 count available at the point of care, preferably within the VMMC clinic. Given that VMMC clients return for follow-up care after 48 hours and again within seven days from the date of procedure, VMMC services provide an opportunity to ensure that men are effectively linked to HIV treatment and care services—especially in Lesotho, where VMMC services are offered and integrated within hospital services. In Mafeteng, the laboratory is informed of routine days for VMMC services and VMMC hospital-based campaigns and collaborates closely with VMMC providers. Ensuring that other hospital units are aware and oriented on VMMC services and that they recognize VMMC service provision as a catalyst for increased HIV testing among Basotho men is essential to strengthen linkages to HIV prevention, care and treatment. The review of VMMC program data identified stock-outs of essential HIV-related commodities, as a challenge to the provision of services to clients attending VMMC programs. In Lesotho, because of a stock-out of CD4 reagents, dozens of clients who tested positive for HIV through the VMMC program were unable to receive a CD4 count at the time of their visit and did not come back for it later. This fact highlights the need to optimize linkages between VMMC services and HIV care and treatment by also strengthening supply chain management systems. Conclusion All VMMC clients who tested positive for HIV and who received a CD4 count on the day of diagnosis were initiated on ART. The provision of VMMC within an integrated service delivery setting such as a district hospital has contributed to the increase of HIV testing among men in Lesotho. Moreover, the VMMC program in Lesotho has the potential to increase HIV- positive men’s enrollment in ART given that linkages with HIV care and treatment have been strengthened. The successful linkages between VMMC clinic clients to HIV care and treatment services provides an important opportunity given Lesotho’s high HIV prevalence and low rates of HIV testing among men. The Mafeteng district hospital model, which consists of integrated services and effective hospital department communica- tions, provides a successful framework to be replicated elsewhere. Study findings serve as a foundation for further analysis of the potential of VMMC services as an entry point to care and treatment for HIV-positive men in other hospital settings. Hospital staff VMMC services in Lesotho are delivered by a team of medical doctors, nurses and HTC counselors. At Mafeteng hospital, the success of the referral and linkage program is due largely to having hospital staff trained in both ART and VMMC services. Nurses draw blood for patients’ CD4 count at the VMMC site and take it to the laboratory. The VMMC counselor, who works at both the VMMC site and the ART clinic, has experience in countrywide HTC campaigns and proactively ensures that nurses from the ART clinic are available to collect blood samples on VMMC clinic days. The counselor, when necessary will ensure physical referral (escorting) to the ART clinic. VMMC providers also foster positive communication with the laboratory and other services to ensure on-time CD4 count results are available to VMMC clients. However, improving VMMC–ART linkages will require an investment in follow-up with clients and communication between VMMC and ART clinics. In high HIV prevalence settings, investing in PIMA CD4 devices at integrated VMMC clinics will also likely increase male enrolment in care and treatment services, as was evident in this study. 7. Mattson C, Campbell R, Bailey R, Agot K, Ndinya-Achola J, et al. (2008) Risk compensation is not associated with male circumcision in Kisumu, Kenya: a multi-faceted assessment of men enrolled in a randomized controlled trial. PLoS ONE 3(6): e2443. doi:10.1371/journal.pone.0002443. 8. Gray R, Kigozi G, Kong X, Ssempiija V, Makumbi F, et al. (2012) The effectiveness of male circumcision for HIV prevention and effects on risk behaviors in a posttrial follow-up study. AIDS 26(5): 609–615. Discussion The 2012 WHO strategic HTC program framework empha- sizes the importance of ensuring linkages between HTC programs and prevention, treatment, care and support services [11]. VMMC services in Lesotho, an evidence-based biomedical intervention, include HIV testing as well as an increased focus on ensuring linkages with care and treatment as part of the package of services that are offered to clients. The HIV continuum of care model provides a framework to identify issues and opportunities for improving HIV services delivery [12]. The two first steps of the model are HIV diagnosis and linkages to care and treatment. VMMC services implemented in a hospital setting provide an opportunity to effectively increase HIV testing among Basotho men and to ensure that linkages to HIV care and treatment are strengthened. The provision of VMMC services in Lesotho has contributed to an increasing number of men who know their HIV status. As per the data reviewed at Mafeteng hospital, 65% of men who tested for HIV were tested at the VMMC clinic. In Lesotho, this contribution to testing services is of tremendous importance as the 2009 DHS suggests that almost 60% of men have never tested compared to 30% for women (age 15–59) [1]. In Lesotho, VMMC services represent a non-negligible opportunity to get men to know their status. May 2014 | Volume 9 | Issue 5 | e83614 3 PLOS ONE | www.plosone.org VMMC Can Increase ART Enrollment among Young Men 4. Auvert B, Taljaard D, Lagarde E, Sobngwi-Tambekou J, Sitta R, et al. (2005) Randomized controlled intervention trial of male circumcision for reduction of HIV infection risk: the ANRS 1265 trial. PLoS Med 2(11): 298. 6. Gray R, Kigozi G, Serwadda D, Makumbi F, Watya S, et al. (2007) Male circumcision for HIV prevention in men in Rakai Uganda: a randomized trial. Lancet 369: 657–666. 5. Bailey R, Moses S, Parker CB, Agot K, Maclean I, et al. (2007) Male circumcision for HIV prevention in young men in Kisumu, Kenya: a randomized controlled trial. Lancet 369: 643–656. 5. Bailey R, Moses S, Parker CB, Agot K, Maclean I, et al. (2007) Male circumcision for HIV prevention in young men in Kisumu, Kenya: a randomized controlled trial. Lancet 369: 643–656. 6. Gray R, Kigozi G, Serwadda D, Makumbi F, Watya S, et al. (2007) Male circumcision for HIV prevention in men in Rakai Uganda: a randomized trial. Lancet 369: 657–666. 7. Mattson C, Campbell R, Bailey R, Agot K, Ndinya-Achola J, et al. (2008) Risk compensation is not associated with male circumcision in Kisumu, Kenya: a multi-faceted assessment of men enrolled in a randomized controlled trial. PLoS ONE 3(6): e2443. doi:10.1371/journal.pone.0002443. 8. Gray R, Kigozi G, Kong X, Ssempiija V, Makumbi F, et al. (2012) The effectiveness of male circumcision for HIV prevention and effects on risk behaviors in a posttrial follow-up study. AIDS 26(5): 609–615. 9. Njeuhmeli E, Forsythe S, Reed J, Opuni M, Bollinger L, et al. (2011) Voluntary medical male circumcision: modeling the impact and cost of expanding male circumcision for HIV prevention in Eastern and Southern Africa. PLoS Med 8(11): e1001132. doi:10.1371/journal.pmed.1001132. p 13. Gardner EM, McLees MP, Steiner JF, del Rio C, Burman WJ (2011) The Spectrum of Engagement in HIV Care and its Relevance to Test-and-Treat Strategies for Prevention of HIV Infection. Clinical Infectious Disease 52 (6) p. 793–800. 11. World Health Organization (2012) Service delivery approaches to HIV testing and counselling (HTC): a strategic HTC policy framework. Geneva: WHO. 12. US department of Health and Human services (2013) HIV/AIDS care continuum. [Online] Available: http://www.aids.gov/federal-resources/ policies/care-continuum/. Accessed October 22, 2013. 14. World Health Organization (2013) Consolidated guidelines on the use of antiretroviral drugs for treating and preventing HIV infection. Geneva: WHO. ( ) j p 10. Lesotho Ministry of Health (2012) Unpublished program data VMMC Can Increase ART Enrollment among Young Men References 5. Bailey R, Moses S, Parker CB, Agot K, Maclean I, et al. (2007) Male circumcision for HIV prevention in young men in Kisumu, Kenya: a randomized controlled trial. Lancet 369: 643–656. 1. Ministry of Health and Social Welfare (MOHSW) and ICF Macro (2010) Lesotho Demographic and Health Survey 2009. Maseru, Lesotho: MOHSW and ICF Macro. 6. Gray R, Kigozi G, Serwadda D, Makumbi F, Watya S, et al. (2007) Male circumcision for HIV prevention in men in Rakai Uganda: a randomized trial. Lancet 369: 657–666. 2. Druyts E, Dybul M, Kanters S, Nachega J, Birungi J, et al. (2013) Male gender and the risk of mortality among individuals enrolled in antiretroviral treatment programs in Africa: A systematic review and meta-analysis. AIDS 27(3): 417– 425. 3. Mills EJ, Bakanda C, Birungi J, Chan K, Hogg RS, et al. (2011) Male gender predicts mortality in a large cohort of patients receiving antiretroviral therapy in Uganda. J Int AIDS Soc 14:52. 4. Auvert B, Taljaard D, Lagarde E, Sobngwi-Tambekou J, Sitta R, et al. (2005) Randomized controlled intervention trial of male circumcision for reduction of HIV infection risk: the ANRS 1265 trial. PLoS Med 2(11): 298. 4. Auvert B, Taljaard D, Lagarde E, Sobngwi-Tambekou J, Sitta R, et al. (2005) Randomized controlled intervention trial of male circumcision for reduction of HIV infection risk: the ANRS 1265 trial. PLoS Med 2(11): 298. PLOS ONE | www.plosone.org May 2014 | Volume 9 | Issue 5 | e83614 4 9. Njeuhmeli E, Forsythe S, Reed J, Opuni M, Bollinger L, et al. (2011) Voluntary medical male circumcision: modeling the impact and cost of expanding male circumcision for HIV prevention in Eastern and Southern Africa. PLoS Med 8(11): e1001132. doi:10.1371/journal.pmed.1001132. 10. Lesotho Ministry of Health (2012) Unpublished program data. 11. World Health Organization (2012) Service delivery approaches to HIV testing and counselling (HTC): a strategic HTC policy framework. Geneva: WHO. VMMC Can Increase ART Enrollment among Young Men May 2014 | Volume 9 | Issue 5 | e83614 PLOS ONE | www.plosone.org PLOS ONE | www.plosone.org 5

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A Conserved Role for the NAM/miR164 Developmental Module Reveals a Common Mechanism Underlying Carpel Margin Fusion in Monocarpous and Syncarpous Eurosids

Frontiers in plant science

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ORIGINAL RESEARCH published: 13 January 2016 doi: 10.3389/fpls.2015.01239 Keywords: Arabidopsis thaliana, Medicago truncatula, CUP SHAPED COTYLEDON, NO APICAL MERISTEM, miR164, gynoecium, carpel, syncarpy A Conserved Role for the NAM/miR164 Developmental Module Reveals a Common Mechanism Underlying Carpel Margin Fusion in Monocarpous and Syncarpous Eurosids Aurélie C. M. Vialette-Guiraud1, Aurélie Chauvet1, Juliana Gutierrez-Mazariegos1, Alexis Eschstruth2, Pascal Ratet2 and Charles P. Scutt1* 1 Laboratoire de Reproduction et Développement des Plantes, UMR 5667, Centre National de la Recherche Scientifique – Institut National de la Recherche Agronomique – Université de Lyon, Ecole Normale Supérieure de Lyon, Lyon, France, 2 Institute of Plant Sciences Paris-Saclay, Centre National de la Recherche Scientifique – Institut National de la Recherche Agronomique – Université de Paris Sud, Orsay, France The majority of angiosperms are syncarpous- their gynoecium is composed of two or more fused carpels. In Arabidopsis thaliana, this fusion is regulated through the balance of expression between CUP SHAPED COTYLEDON (CUC) genes, which are orthologs of the Petunia hybrida transcription factor NO APICAL MERISTEM (NAM), and their post-transcriptional regulator miR164. Accordingly, the expression of a miR164- insensitive form of A. thaliana CUC2 causes a radical breakdown of carpel fusion. Here, we investigate the role of the NAM/miR164 genetic module in carpel closure in monocarpous plants. We show that the disruption of this module in monocarpous flowers of A. thaliana aux1-22 mutants causes a failure of carpel closure, similar to the failure of carpel fusion observed in the wild-type genetic background. This observation suggested that closely related mechanisms may bring about carpel closure and carpel fusion, at least in A. thaliana. We therefore tested whether these mechanisms were conserved in a eurosid species that is monocarpous in its wild-type form. We observed that expression of MtNAM, the NAM ortholog in the monocarpous eurosid Medicago truncatula, decreases during carpel margin fusion, suggesting a role for the NAM/miR164 module in this process. We transformed M. truncatula with a miR164- resistant form of MtNAM and observed, among other phenotypes, incomplete carpel closure in the resulting transformants. These data confirm the underlying mechanistic similarity between carpel closure and carpel fusion which we observed in A. thaliana. Our observations suggest that the role of the NAM/miR164 module in the fusion of carpel margins has been conserved at least since the most recent common ancestor of the eurosid clade, and open the possibility that a similar mechanism may have been responsible for carpel closure at much earlier stages of angiosperm evolution. We combine our results with studies of early diverging angiosperms to speculate on the role of the NAM/miR164 module in the origin and further evolution of the angiosperm carpel. Edited by: Rainer Melzer, University College Dublin, Ireland Reviewed by: Stefan De Folter, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional, Mexico Barbara Ambrose, The New York Botanical Garden, USA *Correspondence: Charles P. Scutt [email protected] Specialty section: This article was submitted to Plant Evolution and Development, a section of the journal Frontiers in Plant Science Received: 03 October 2015 Accepted: 20 December 2015 Published: 13 January 2016 Citation: Vialette-Guiraud ACM, Chauvet A, Gutierrez-Mazariegos J, Eschstruth A, Ratet P and Scutt CP (2016) A Conserved Role for the NAM/miR164 Developmental Module Reveals a Common Mechanism Underlying Carpel Margin Fusion in Monocarpous and Syncarpous Eurosids. Front. Plant Sci. 6:1239. doi: 10.3389/fpls.2015.01239 Specialty section: This article was submitted to Plant Evolution and Development, a section of the journal Frontiers in Plant Science Specialty section: This article was submitted to Plant Evolution and Development, a section of the journal Frontiers in Plant Science Received: 03 October 2015 Accepted: 20 December 2015 Published: 13 January 2016 Edited by: Rainer Melzer, University College Dublin, Ireland INTRODUCTION miR164 triple mutants or CUC2g-m4 transformants, the two carpels of the A. thaliana gynoecium emerge separately and remain unfused and open throughout development. The female whorl, or gynoecium, of the angiosperm flower consists of one or more carpels which enclose the ovules. In apocarpous gynoecia, the carpels remain separate throughout development, while in syncarpous gynoecia, they are fused together, either from their inception (congenital fusion), or from a later developmental stage (post-genital fusion). If only one carpel is produced per flower, the gynoecium is termed monocarpous. Carpels in apocarpous or monocarpous gynoecia may emerge from the floral meristem with their margins already fused together, in which case they are described as ascidiate (bottle-shaped), or may emerge with unfused margins that subsequently fuse by folding, in which case they are described as plicate. In addition to their role in carpel development, studies of NAM orthologs in eudicots show these factors to be involved in meristem formation and cotyledon development (Souer et al., 1996; Aida et al., 1997, 1999; Takada et al., 2001; Weir et al., 2004), leaf development (Ishida et al., 2000; Nikovics et al., 2006; Blein et al., 2008), ovule development (Galbiati et al., 2013; Kamiuchi et al., 2014) and phyllotaxy (Peaucelle et al., 2007). These transcription factors are expressed at organ margins and tissue boundaries, and their down-regulation by miR164 consequently facilitates organ outgrowth and/or developmental fusion. The action of the NAM/miR164 module in the A. thaliana leaf margin has been modeled and found to generate, via effects on the auxin efflux carrier PINFORMED1 (PIN1), an alternating series of auxin maxima and minima that, respectively, generate regions of higher and lower marginal growth (Bilsborough et al., 2011). Syncarpy is believed to confer several selective advantages over apocarpy, including a larger landing platform for pollinating insects, a compitum (a common intersection in the route for pollen tube growth), and larger fruits with more sophisticated mechanisms for seed dispersal. Mapping of character states onto angiosperm phylogeny indicates that syncarpy has arisen at least 17 times in the angiosperms, while the evolution of apocarpy from syncarpy is much less frequent (Armbruster et al., 2002). ) In this work, we hypothesized that the role of the NAM/miR164 module in syncarpous fusion in A. thaliana might reflect a more general role in the fusion of carpel margins in angiosperms. Citation: Vialette-Guiraud ACM, Chauvet A, Gutierrez-Mazariegos J, Eschstruth A, Ratet P and Scutt CP (2016) A Conserved Role for the NAM/miR164 Developmental Module Reveals a Common Mechanism Underlying Carpel Margin Fusion in Monocarpous and Syncarpous Eurosids. Front. Plant Sci. 6:1239. doi: 10.3389/fpls.2015.01239 January 2016 | Volume 6 | Article 1239 1 Frontiers in Plant Science | www.frontiersin.org Evolution of Carpel Margin Fusion in Eurosids Vialette-Guiraud et al. INTRODUCTION Consequently, we tested the role of this module in the closure of monocarpous gynoecia produced both in A. thaliana aux1-22 mutants, which are null mutants of the AUX1 auxin influx transporter (Bennett et al., 1996) and in a wild-type genetic background of the distantly related eurosid M. truncatula. From the results of these experiments, we conclude that the NAM/miR164 module has conserved a role in carpel margin fusion, at least since the most recent common ancestor (MRCA) of living eurosids. A detailed comparison of gene expression patterns suggests that fine-tuning of the NAM/miR164 module may regulate species–specific differences in the timing of carpel margin fusion. Accordingly, we discuss the possibility that the activity of the NAM/miR164 module may be conserved in carpel development throughout the angiosperms, while subtle modulations to this mechanism may determine the distinction between congenital and post-genital carpel margin fusion events in specific angiosperm groups. We further speculate on mechanisms acting upstream of the NAM/miR164 module that may have contributed to the origin of the carpel in the first flowering plants. y y The model angiosperm Arabidopsis thaliana possesses a syncarpous gynoecium of two congenitally fused carpels. These organs emerge from the center of the floral meristem as a single dome of cells, within which a central slot-like cavity forms as the gynoecium begins to elongate (Smyth et al., 1990). The positions of the carpel margins within the gynoecium wall only become apparent at a later stage, when this structure undergoes differentiation into valve and abaxial replum tissues. Meristematic activity from the abaxial replum then generates the adaxial replum, or septum, which grows inward to divide the ovary into two chambers. Ovule primordia develop from parietal placentae which form along the carpel margins within each chamber of the ovary. In contrast to A. thaliana, the model angiosperm Medicago truncatula possesses a plicate, monocarpous gynoecium (Benlloch et al., 2003). At an early stage of M. truncatula flower development, the gynoecial primordium becomes crescent- shaped and its margins then fuse together to enclose the single chamber of the ovary. Ovule primordia in M. truncatula form from a parietal placenta that develops along the fused carpel margin. MATERIALS AND METHODS Carpel fusion in A. thaliana is regulated by a genetic module, generically termed here the NAM/miR164 module, which consists of a subset of NAC-family (NAC for NAM, ATAF and CUC; Aida et al., 1997) transcription factors and their post-transcriptional regulator miR164 (Mallory et al., 2004). In A. thaliana, the NAC genes involved in this module are CUP-SHAPED COTYLEDON1 (CUC1) and CUC2 (Aida et al., 1997), which are orthologs of the single gene NO APICAL MERISTEM (NAM) from Petunia hybrida (Souer et al., 1996). Loss of miR164 function through mutations to all three MIR164 paralogs in A. thaliana (Sieber et al., 2007), or genetic transformation of A. thaliana with a miR164-resistant version of CUC2 (CUC2g-m4; Nikovics et al., 2006), results in a breakdown of carpel fusion. Accordingly, in In Situ Hybridization Double-stranded cDNAs representing the full-length coding sequences of A. thaliana CUC1 and CUC2 and of M. truncatula MtNAM were generated by reverse-transcriptase PCR, incorporating a T7-RNA-Polymerase promoter sequence in the reverse primer. Digoxgenin-labeled riboprobes were prepared from these templates using T7 RNA-polymerase and these were then purified and used in in situ hybridizations to sections of fixed floral buds embedded in Paraplast Xtra (Leica- Surgipath), as described by Vialette-Guiraud et al. (2011b). Gene expression patterns were observed and photographed under bright field illumination using a Leica Axio Imager M2 inverted microscope fitted with a Leica AxioCam MRc digital camera. Monocarpy in Medicago truncatula Arose by Reversion from Syncarpy in a Common Ancestor Shared with Arabidopsis thaliana To elucidate transitions in carpel fusion in the angiosperms, with emphasis on the model eurosids M. truncatula and A. thaliana, we mapped this character state onto a cladogram (Figure 1) representing the consensus view of angiosperm phylogeny (Bremer et al., 2009). This analysis confirms the findings of earlier studies (Armbruster et al., 2002) which indicated that the MRCA of living angiosperms was apocarpous, and that syncarpy arose several times independently, including in Nymphaeaceae, monocots, Papaveraceae and a common ancestor of the rosids and asterids. Within the eurosids, our analysis indicates that monocarpy in Fabales (including M. truncatula), arose secondarily from syncarpy, which was present in a common ancestor shared with Brassicales (including A. thaliana), Celastrales and Malpighiales. By localizing transitions between apocarpy/monocarpy and syncarpy, this analysis provides a phylogenetic framework for the evolutionary interpretation of data on the molecular mechanisms involved in these processes in living angiosperms. Plant Cultivation Arabidopsis thaliana plants were grown from seed on peat- based compost in growth chambers at a daytime temperature of ∼21◦C and ∼55% relative humidity (RH). Plants were initially grown under 8/16 h day/night cycles generated using fluorescent lighting consisting of equal numbers of “cool daylight” (Osram Lumilux L36W/865) and “warm white” (Osram Lumilux L36W/830) lamps, giving a total photon flux at bench level of 170 µmol.m−2.s−1. To induce flowering, plants were transferred to long days (16/8 h day/night cycles) under otherwise similar conditions. January 2016 | Volume 6 | Article 1239 Frontiers in Plant Science | www.frontiersin.org 2 Evolution of Carpel Margin Fusion in Eurosids Vialette-Guiraud et al. Phenotypic Observations Flower buds were dissected, observed, and photographed using a Leica MZ12 dissecting microscope fitted with an AxioCam ICc5 digital camera. Carpel anatomy was revealed in transverse sections of fixed flower buds, prepared as for in situ hybridization and stained with 0.05% (w/v) Toluidine Blue-0 in 0.1 M sodium phosphate buffer (pH 6.8). Scanning electron microscopy was performed on unfixed material using a Hirox 3000 bench-top environmental scanning electron microscope (SEM). Medicago truncatula plants were grown from seed on peat- based compost in a greenhouse at a daytime temperature of ∼22.5◦C and 40–60% RH under natural daylight, extended to 16 h daylength using sodium lamps, as necessary. Plant Transformation Arabidopsis thaliana aux1-22 mutants (null mutants of AUX1; Bennett et al., 1996) were transformed by the “floral dip” method (Clough and Bent, 1998) using the CUC2g-wt and CUC2g-m4 constructs of Nikovics et al. (2006) in Agrobacterium tumefaciens strain GV3101 harboring the plasmids pMP90 (Koncz and Schell, 1986) and pSOUP (Hellens et al., 2000). Transformants were selected on plant agar containing 50 µg/mL kanamycin. MtNAMg-wt and MtNAMg-m4 constructs were introduced into A. tumefaciens GV3101, as described above, and used to transform M. truncatula R108 leaf disks by the protocol of Cosson et al. (2015), in which transgenic calli were selected on media containing 30 µg/mL hygromycin. Vector Construction MtNAM (MTR_2g078700; Cheng et al., 2012) was initially isolated by radioisotopic screening of an M. truncatula bacterial artificial chromosome (BAC) library (Nam et al., 1999). A 1.2-kb fragment containing the miR164-binding site of MtNAM was released from a sub-cloned BAC DNA fragment by cleavage with SstI and re-ligated into the pGEM T-Easy vector. The resulting plasmid was subjected to oligonucleotide-directed site-specific mutagenesis following the method of Kirsch and Joly (1998), using the sense- and antisense-strand oligonucleotides 5′- GAGCACGTGTCCTGTTTtagtACAACATCTACAACATC and 5′-GATGTTGTAGATGTTGTactaAAACAGGACACGTGCTC, respectively. These oligonucleotides generate the same four-base mismatch (shown above in lower case) present in the miR164- binding site of CUC2g-m4 (Nikovics et al., 2006). Mutagenised and wild-type versions of a MtNAM genomic sequence of 9883 bp, from an EcoRI site situated 6437 bp upstream of the MtNAM initiation codon to an NcoI site situated 2151 bp downstream of its termination codon, were then inserted by ligation between unique EcoR1 and Not1 sites situated between the Left and Right T-DNA borders of the pGREEN II-NosHyg plant transformation vector, thereby generating the plasmids MtNAMg-m4 and MtNAMg-wt, respectively. Character State Mapping A partial cladogram of angiosperm phylogeny was produced, based on the current consensus view of angiosperm phylogeny given by the Angiosperm Phylogeny Group III (APG III, http://www.mobot.org/MOBOT/research/APweb/; Bremer et al., 2009). Carpel fusion character states, obtained from the APG III website and from bibliographic searches, were mapped on this cladogram by maximum parsimony using MacClade4 software. The NAM/miR164 in Arabidopsis thaliana Plays a Role in Both Syncarpy and the Closure of Single Carpels As the NAM/miR164 developmental module is necessary for carpel fusion in wild-type, syncarpous A. thaliana (Nikovics et al., 2006; Sieber et al., 2007), we aimed to discover whether this mechanism could also contribute to the closure of single carpels in this species. To do this, we tested whether the introduction of a miR164-resistant version of CUC2 (CUC2g-m4) could cause a breakdown in the closure of the single carpels that are January 2016 | Volume 6 | Article 1239 Frontiers in Plant Science | www.frontiersin.org 3 Evolution of Carpel Margin Fusion in Eurosids Vialette-Guiraud et al. FIGURE 1 | A partial cladeogram of the angiosperms showing evolutionary transitions in carpel fusion. Monocarpy in Fabales (including Medicago trucatula) is revealed in this analysis to have reverted from syncarpy present in a common ancestor shared with Brassicales (including Arabidopsis thaliana), Celastrales and Malpighiales. FIGURE 1 | A partial cladeogram of the angiosperms showing evolutionary transitions in carpel fusion. Monocarpy in Fabales (including Medicago trucatula) is revealed in this analysis to have reverted from syncarpy present in a common ancestor shared with Brassicales (including Arabidopsis thaliana), Celastrales and Malpighiales. produced in A. thaliana aux1-22 mutants (Bennett et al., 1996), as compared to control plants transformed with a wild-type construct (CUC2g-wt). partial lack of carpel closure, remaining open over part or all of the valve margin. In these eight plants, carpel fusion/closure defects resulted in an almost complete loss of female fertility. Thus, disruption of the NAM/miR164 developmental module in monocarpous mutant gynoecia of A. thaliana causes the failure of developmental closure in these structures in a similar manner to the disruption of carpel fusion in syncarpous, wild-type gynoecia. Wild-type Col-0 gynoecia are syncarpous (Figure 2A), as are approximately 50% of gynoecia produced in aux1- 22 mutants (Figure 2B). The ovary wall in these gynoecia contains two valves, alternating with two abaxial repla. The monocarpous gynoecia, which are also produced in aux1-22 mutants (Figures 2C,D), develop as closed structures whose ovary wall consists of only one valve and one abaxial replum (Figure 2C). These monocarpous gynoecia are not divided by a septum, or adaxial replum. Transformation of aux1-22 mutants with CUC2g-wt produced no apparent change in the morphology of monocarpous gynoecia (Figure 2E). However, transformation of these mutants with CUC2g-m4 produced a high proportion of monocarpous gynoecia that remained open to maturity (Figures 2F–I). The NAM/miR164 in Arabidopsis thaliana Plays a Role in Both Syncarpy and the Closure of Single Carpels In eight of 20 T1 transformants analyzed, all flowers containing two carpels showed carpel fusion defects, while all monocarpous flowers showed a complete or Expression of NAM Orthologs is Absent or Reduced During Carpel Margin Fusion in Arabidopsis thaliana and Medicago truncatula The observation that the NAM/miR164 module regulates developmental closure events in the gynoecium in both syncarpous and monocarpous genotypes of A. thaliana led us to speculate that this molecular mechanism might be widely conserved within the angiosperms. We chose M. truncatula, Frontiers in Plant Science | www.frontiersin.org January 2016 | Volume 6 | Article 1239 Frontiers in Plant Science | www.frontiersin.org 4 Evolution of Carpel Margin Fusion in Eurosids Vialette-Guiraud et al. FIGURE 2 | Gynoecium morphology of A. thaliana aux1-22 mutants transformed with miR164-resistant (CUC2g-m4) or un-mutated control (CUC2g-wt) constructs. (A–C) Toluidine blue staining of transverse sections of Col-0 wild-type (A) and aux1-22 mutant gynoecia composed of two fused carpels (B), and one closed carpel (C), respectively. (D,E) An untransformed aux1-22 mutant (D) and an aux1-22 CUC2g-wt (control) transformant showing entirely closed monocarpous gynoecia. (F–I) aux1-22 CUC2g-m4 transformants showing a breakdown carpel closure in monocarpous gynoecia. (G is a magnification of the apex of the carpel shown in (F). I is a scanning electron microscope image) ar, abaxial replum; s, septum (or adaxial replum); v, valve. Bars = 200 µm in (A,B), 100 µm in (C), and 1 mm in (D–F,H,I). FIGURE 2 | Gynoecium morphology of A. thaliana aux1-22 mutants transformed with miR164-resistant (CUC2g-m4) or un-mutated control (CUC2g-wt) constructs. (A–C) Toluidine blue staining of transverse sections of Col-0 wild-type (A) and aux1-22 mutant gynoecia composed of two fused carpels (B), and one closed carpel (C), respectively. (D,E) An untransformed aux1-22 mutant (D) and an aux1-22 CUC2g-wt (control) transformant showing entirely closed monocarpo s g noecia (F I) a 1 22 CUC2g m4 transformants sho ing a breakdo n carpel clos re in monocarpo s g noecia (G is a magnification of the ape FIGURE 2 | Gynoecium morphology of A. thaliana aux1-22 mutants transformed with miR164-resistant (CUC2g-m4) or un-mutated control (CUC2g-wt) constructs. (A–C) Toluidine blue staining of transverse sections of Col-0 wild-type (A) and aux1-22 mutant gynoecia composed of two fused carpels (B), and one closed carpel (C), respectively. (D,E) An untransformed aux1-22 mutant (D) and an aux1-22 CUC2g-wt (control) transformant showing entirely closed monocarpous gynoecia. (F–I) aux1-22 CUC2g-m4 transformants showing a breakdown carpel closure in monocarpous gynoecia. (G is a magnification of the apex of the carpel shown in (F). I is a scanning electron microscope image) ar, abaxial replum; s, septum (or adaxial replum); v, valve. Expression of NAM Orthologs is Absent or Reduced During Carpel Margin Fusion in Arabidopsis thaliana and Medicago truncatula Bars = 200 µm in (A,B), 100 µm in (C), and 1 mm in (D–F,H,I). In situ hybridization in A. thaliana flowers at Stage 7 (Smyth et al., 1990), in which a central slot is beginning to form in the gynoecial cylinder, revealed the expression of CUC2 in the adaxial domain of the gynoecium and in the loculi of the developing anthers (Figure 3A). Recent studies (Galbiati et al., 2013) revealed similar results for CUC1. Thus, no expression of either CUC1 or CUC2 has been detected in regions of the ovary wall destined to become the abaxial repla, or the fusion zones between these tissues and the valves. At Stage 9–10, both CUC1 and CUC2 were expressed in the placentae and at presumptive tissue boundaries within the elongating ovule primordia (Figures 3B,C). At Stage 11, CUC1 was expressed at the base of the expanding ovule integuments (Figure 3D). which produces in its wild-type form a single carpel in each flower, as a candidate model species in which to test this hypothesis. The MRCA between M. truncatula and A. thaliana, which is also the MRCA of the living eurosid clade (comprising Fabidae, or eurosids I and Malvidae, or eurosids II), is estimated to have lived 114–113 million years ago (MYA; Wang et al., 2009). Prior to initiating functional experiments in M. truncatula, we used in situ hybridization to examine the conservation of expression of NAM orthologs in flower tissues between A. thaliana and M. truncatula and thereby ascertain the likelihood that the NAM/miR164 module might function in carpel closure in the latter species. January 2016 | Volume 6 | Article 1239 Frontiers in Plant Science | www.frontiersin.org 5 Evolution of Carpel Margin Fusion in Eurosids Vialette-Guiraud et al. FIGURE 3 | Expression of NAM orthologs in A. thaliana and Medicago truncatula flower buds. (A–D) A. thaliana flower buds hybridized to CUC1 and 2 probes. (A) A Stage-7 bud showing CUC2 expression in the abaxial domain of the gynoecium and anther locculi. (B,C) Buds at Stages 9–10 showing CUC1 (B) and CUC2 (C) expression in the placenta and within ovule primordia. (D) A bud at Stage 11 showing CUC1 expression at the base of the expanding ovule integuments. ad, adaxial zone of the gynoecium; al, anther loculus; ar, abaxial replum; g, gynoecium; oi, ovule integuments; op, ovule primordium. Bars = 50 µm. (E–I) M. truncatula flower buds hybridized to an MtNAM probe. Expression of NAM Orthologs is Absent or Reduced During Carpel Margin Fusion in Arabidopsis thaliana and Medicago truncatula (E) A bud at Stages 3–4 showing MtNAM expression between and within organ primordia. (F) A Stage-7 bud showing strong MtNAM expression in the unfused carpel margins. (G) A bud at Stage 8, in which MtNAM expression is reduced in the fused carpel margins. (H,I) Later stages of flower development in which MtNAM expression is absent in the carpel margins, but present first in ovule primordia (H) and then at the base of the expanding integuments (I). cm, carpel margin; cp, common primordium; g, gynoecium; gp, gynoecial primordium; oi, ovule integument; op, ovule primordium; pm, petal margin. Bars = 100 µm. FIGURE 3 | Expression of NAM orthologs in A. thaliana and Medicago truncatula flower buds. (A–D) A. thaliana flower buds hybridized to CUC1 and 2 probes. (A) A Stage-7 bud showing CUC2 expression in the abaxial domain of the gynoecium and anther locculi. (B,C) Buds at Stages 9–10 showing CUC1 (B) and CUC2 (C) expression in the placenta and within ovule primordia. (D) A bud at Stage 11 showing CUC1 expression at the base of the expanding ovule integuments. ad, adaxial zone of the gynoecium; al, anther loculus; ar, abaxial replum; g, gynoecium; oi, ovule integuments; op, ovule primordium. Bars = 50 µm. (E–I) M. truncatula flower buds hybridized to an MtNAM probe. (E) A bud at Stages 3–4 showing MtNAM expression between and within organ primordia. (F) A Stage-7 bud showing strong MtNAM expression in the unfused carpel margins. (G) A bud at Stage 8, in which MtNAM expression is reduced in the fused carpel margins. (H,I) Later stages of flower development in which MtNAM expression is absent in the carpel margins, but present first in ovule primordia (H) and then at the base of the expanding integuments (I). cm, carpel margin; cp, common primordium; g, gynoecium; gp, gynoecial primordium; oi, ovule integument; op, ovule primordium; pm, petal margin. Bars = 100 µm. In situ hybridization in M. truncatula at Stages 3–4 of flower development, following the time course defined by Benlloch et al. (2003), showed MtNAM expression between the gynoecium primordium and the surrounding common primordia that give rise to both stamens and petals (Figure 3E). Signals were also detected within these common primordia (Figure 3E), marking the boundary between the zones destined to produce petals and stamens. A Role of the NAM/miR164 Module in the Fusion of Carpel Margins has Been Conserved at Least Since the MRCA of the Eurosids In this study, we show that a previously characterized developmental module involving the post-transcriptional regulation of NAM orthologs by miR164 is involved not only in carpel fusion in syncarpous A. thaliana (Nikovics et al., 2006; Sieber et al., 2007), but also in the closure of the single carpels present in two species whose lineages diverged at the base of the eurosid clade, some 114–113 MYA. The two species concerned are A. thaliana itself, as aux1-22 mutants of A. thaliana produce single carpels, and M. truncatula, which is monocarpous in its wild-type form. We show that disruption of the NAM/miR164 module in both A. thaliana aux1-22 mutants (Figure 2) and a wild-type background of M. truncatula (Figure 4) produces single carpels that are no longer completely fused at their margins. Toluidene-blue staining was performed to highlight the ovule and the fused region of the carpel margins in the gynoecium of untransformed M. truncatula (Figure 4A). Transformation with MtNAMg-wt (Figures 4B–E) showed no effects on flower development compared to wild type M. truncatula. Accordingly, in MtNAMg-wt transformants, as in wild-type, five petals were produced, including two fused “keel” petals, two unfused “wing” petals, and a single “standard” petal (Figures 4B,C). As in the wild-type, all stamen filaments, with the exception of a single stamen positioned adjacent to the standard, were fused into a sheath surrounding the gynoecium (Figure 4D). The carpel margins of MtNAMg-wt transformants were also developmentally fused in the mature gynoecium, as in wild-type (Figure 4E). These data indicate that the NAM/miR164 module has conserved a role in developmental fusion events between carpel margins at least since the MRCA of the eurosids. The mapping of character states onto angiosperm phylogeny (Figure 1) indicates that the MRCA of the eurosids was syncarpous, and we may thus conclude that the NAM/miR164 module contributed to carpel fusion in that key ancestor, from which some 70 000 extant species are descended (Wang et al., 2009). By contrast, a range of mutant phenotypes were noted in flowers of plants transformed with the MtNAMg-m4 construct (Table 2; Figures 4F–Q). Two standard petals were produced in some flowers (Figure 4K), while in others, petals with altered morphology and fusion were produced, rendering difficult their identification as standard, wing or keel petals (Figures 4G,O). Unfused stamens were produced in some cases (Table 2; Figures 4H,L), while stamens were absent in others (Table 2; Figure 4Q). A Role of the NAM/miR164 Module in the Fusion of Carpel Margins has Been Conserved at Least Since the MRCA of the Eurosids The carpel margins remained unfused in many flowers (Table 2; Figures 4I,M), revealing the ovules within these, though a small proportion of flowers did show completely fused carpel margins (Table 2; Figure 4Q). Expression of NAM Orthologs is Absent or Reduced During Carpel Margin Fusion in Arabidopsis thaliana and Medicago truncatula At Stage 7, the gynoecium appeared crescent-shaped in transverse section and MtNAM was clearly expressed in the carpel margins, and at the margins of the developing free petals (Figure 3F). By early Stage 8, expression of MtNAM was observed to decline in the carpel margins (Figure 3G), which had, by this time, fused together to close the gynoecium. At later developmental stages, MtNAM expression is present in presumptive tissue boundaries in the elongating ovule primordia (Figure 3H) and, following this, at the base of the expanding integuments of the ovule (Figure 3I). Similar NAM-ortholog expression patterns in floral organ and ovule primordia were previously shown in another species of Fabaceae, Pisum sativum (Blein et al., 2008). These expression data reveal several underlying similarities in the expression of miR164-regulated NAM orthologs between A. thaliana and M. truncatula. These orthologs are highly expressed in both species at frontiers between and within floral organs, particularly during ovule development. These data do, however, reveal a difference in NAM expression in the carpel margins- no such expression was detected in the presumptive abaxial repla of the gynoecial tube at early stages of A. thaliana flower development, whereas NAM expression was detected in the carpel margins of the early M. truncatula gynoecium. This difference may relate to the contrasting modes of congenital and post-genital carpel margin fusion in A. thaliana and M. truncatula, respectively. Despite the differences observed, we concluded that the presence of MtNAM expression in M. truncatula carpel margins suggested that the NAM/miR164 module may be involved in the fusion of these structures, leading us to test this hypothesis experimentally. January 2016 | Volume 6 | Article 1239 Frontiers in Plant Science | www.frontiersin.org Frontiers in Plant Science | www.frontiersin.org 6 Evolution of Carpel Margin Fusion in Eurosids Vialette-Guiraud et al. Expression of a miR164-Resistant form of MtNAM Leads to a Breakdown in Carpel Margin Fusion and Other Developmental Fusion Events in Medicago truncatula Flowers particular interest to the current work, the elimination of post-transcriptional regulation of MtNAM in the gynoecium is shown to have a similar effect in M. truncatula to that shown on aux1-22 mutants of A. thaliana (Figure 2) by disrupting the fusion of carpel margins. To test the role of the NAM/miR164 developmental module on carpel closure in M. truncatula, we produced transgenic plants expressing genomic constructs of MtNAM (MtNAMg- m4 and MtNAMg-wt), respectively, with or without four point mutations in their predicted miR164-binding sites, identical to those present in the CUC2g-m4 construct (Nikovics et al., 2006). Three independent transgenic MtNAMg-m4 calli were generated, two of which were successfully regenerated into fertile adult plants, as was one transgenic callus containing an MtNAMg-wt construct (Table 1). Phenotypic observations were made on T2 progeny representative of one of each of these transformed lines, and on untransformed plants for comparison (Table 2; Figure 4). The NAM/miR164 Module Maintained its Role in Carpel Margin Fusion During a Transition from Syncarpy to Monocarpy in an Ancestor of Fabales Character-state mapping (Figure 1) further indicates that the monocarpy present in Fabales (including M. truncatula) is a derived condition that occurred by reversion from syncarpy, present in earlier eurosids. In the present work, we show that the role of the NAM/miR164 module in carpel margin fusion was conserved during this developmental transition. Thus, our study These data indicate a range of roles of the NAM/miR164 developmental module in fusion events in the corolla, androecium, and gynoecium of M. truncatula flowers. Of TABLE 1 | Results summary for transformation of Medicago truncatula. Constructs Number of transformation experiments performed Number of trangenic calli produced (combining all experiments) Number of plantlets regenerated Number of T1 plants surviving to reproductive phase MtNAMg-wt 2 7 (cal 1–7) 7 from cal 1 2 from cal 1 MtNAMg-m4 2 3 (cal 1–3) 18 from cal 1 1 from cal 2 1 from cal 3 12 from cal 1 1 from cal 2 Frontiers in Plant Science | www.frontiersin.org 7 January 2016 | Volume 6 | Article 1239 Frontiers in Plant Science | www.frontiersin.org Evolution of Carpel Margin Fusion in Eurosids Vialette-Guiraud et al. | p yp p g T1 parent T2 plant Total number of flowers dissected Number of flowers showing abnormalities in the corolla Number of flowers showing abnormalities in the androecium Number of flowers showing abnormalities in the gynoecium Unfused stamens Stamens absent Slight defects in carpel fusion Extensive defects in carpel fusion m4 cal 1 1_1 3 3 0 0 0 0 m4 cal 1 1_2 17 6 2 0 2 7 m4 cal 1 4_1 13 11 3 0 0 4 m4 cal 1 5_1 14 5 0 0 3 2 m4 cal 1 6_3 12 12 4 5 2 8 | f ti i strongly suggests that the NAM/miR164 module provides an underlying mechanism that is necessary for fusion events at the carpel margins of both syncarpous and monocarpous eurosids. strongly suggests that the NAM/miR164 module provides an underlying mechanism that is necessary for fusion events at the carpel margins of both syncarpous and monocarpous eurosids. It is interesting to note that the aux1-22 mutation in A. thaliana causes a transition from a congenitally fused gynoecium of two carpels to a closed, monocarpous gynoecium. Thus, a single loss-of-function mutation in a gene involved in auxin signaling can bring about, in A. The NAM/miR164 Module Maintained its Role in Carpel Margin Fusion During a Transition from Syncarpy to Monocarpy in an Ancestor of Fabales thaliana, a similar type of morphological transition to that which led to monocarpy in Fabales. The genetic simplicity of this transition suggests that reversions from syncarpy to monocarpy might occur frequently in natural populations. The general trend in the angiosperms, however, is for evolutionary transitions toward syncarpous gynoecia, which are believed to confer numerous selective advantages (Armbruster et al., 2002). Thus, while the loss of syncarpy may be a genetically “easy” transition to make, the fixation of this trait in populations by natural selection may occur much less frequently. A Possible Role for the NAM/miR164 Module in the Timing of Carpel Fusion (B–E) A typical flower of an MtNAMg-wt transformant showing (B) the intact flower, (C) petal morphology, (D) the sheath of anther filaments surrounding the gynoecium, and (E) the carpel margins. All structures in (B–E) appear identical to wild-type. (F–I), (J–M), and (N–Q) Three representative flowers from MtNAM-m4 transformants showing (F,J,N) the intact flower, (G,K,O) petal morphology, (H,L,P) after removal of the perianth, and (I,M,Q) the carpel margins. Defects in the corolla, androecium and gynoecium are apparent, including a marked breakdown in carpel margin fusion in most flower buds (e.g., I,M). cm, carpel margin; k, keel petal(s); o, ovule; ss, stamen sheath; st, standard petal; up, unidentified petal(s); us, unfused stamens; w, wing petal. Bars = 100 µm in (A), 1 mm in (B,C,F,G,J,K,N,O), and 0.5 mm in (D,E,H,I,L,M,P,Q). FIGURE 4 | Dissections of M. truncatula flowers transformed with miR164-resistant (MtNAMg-m4) or wild-type control (MtNAMg-wt) constructs. (A) Transverse section of wild-type M. truncatula gynoecium stained with toluidine blue. (B–E) A typical flower of an MtNAMg-wt transformant showing (B) the intact flower, (C) petal morphology, (D) the sheath of anther filaments surrounding the gynoecium, and (E) the carpel margins. All structures in (B–E) appear identical to wild-type. (F–I), (J–M), and (N–Q) Three representative flowers from MtNAM-m4 transformants showing (F,J,N) the intact flower, (G,K,O) petal morphology, (H,L,P) after removal of the perianth, and (I,M,Q) the carpel margins. Defects in the corolla, androecium and gynoecium are apparent, including a marked breakdown in carpel margin fusion in most flower buds (e.g., I,M). cm, carpel margin; k, keel petal(s); o, ovule; ss, stamen sheath; st, standard petal; up, unidentified petal(s); us, unfused stamens; w, wing petal. Bars = 100 µm in (A), 1 mm in (B,C,F,G,J,K,N,O), and 0.5 mm in (D,E,H,I,L,M,P,Q). A Possible Role for the NAM/miR164 Module in the Timing of Carpel Fusion g p In A. thaliana, the gynoecium forms as a radially symmetrical cylinder that later differentiates to show the positions of the carpel margins. By contrast, the single carpel of the M. truncatula gynoecium is plicate, and closes post-genitally by the fusion of preexisting carpel margins. In situ hybridization in this work (Figure 3) and other studies (Galbiati et al., 2013) failed to detect any expression of CUC1 or CUC2 in the carpel margins of A. thaliana. However, CUC2 is known to be highly expressed in the carpel margins of the unfused gynoecium at Stage 9 of flower development in mir164abc triple mutants (Sieber et al., 2007). Comparison of these data strongly suggests that the NAM/miR164 expression balance in A. thaliana lies heavily in favor of miR164 from the earliest stages of gynoecium development. By contrast, detectable levels of MtNAM were present in margin tissues at early stages of M. truncatula carpel development, and these levels were observed to decline at subsequent stages, as the margins fused (Figure 3). Thus, the different balances of NAM and miR164 expression observed at very early stages of A. thaliana and M. truncatula carpel development (Figure 3; Galbiati et al., 2013) correlate closely with the different timings of carpel closure observed in these species (Smyth et al., 1990; Benlloch et al., 2003). y Given the role of the NAM/miR164 module in carpel closure in both A. thaliana and M. truncatula (Figures 2 and 4), and the gene expression differences we have noted between the congenitally and post-genitally fused carpel margins of these two respective species, it would be interesting to compare the expression of NAM orthologs in a range of Fabales that show different spatial and temporal patterns of carpel closure. Candidate species for this analysis include Acacia celastrifolia and Inga bella (Paulino et al., 2014), in which the carpels include both congenitally fused (ascidiate) and later-fusing (plicate) zones, and Amberstia nobilis and Caesalpina spp. (Tucker and Kantz, 2001), in which the carpel margins remain unfused until after ovule initiation, much later than in most other Fabales. January 2016 | Volume 6 | Article 1239 8 Evolution of Carpel Margin Fusion in Eurosids Vialette-Guiraud et al. FIGURE 4 | Dissections of M. truncatula flowers transformed with miR164-resistant (MtNAMg-m4) or wild-type control (MtNAMg-wt) constructs. (A) Transverse section of wild-type M. truncatula gynoecium stained with toluidine blue. The Role of the NAM/miR164 Module in Carpel Evolution Such experiments, in quite closely related species showing marked differences in gynoecium anatomy, could provide strong correlative evidence of a role for the subtle modulation of gynoecium development by changes to the balance of the NAM/miR164 module. Notably, the NAM/miR164 expression balance at very early stages of carpel development may be important in determining whether carpel margins will fuse congenitally or postgenitally. As its genetic components are present in both gymnosperms and angiosperms (Axtell and Bartel, 2005; Larsson et al., 2012), the NAM/miR164 genetic module is clearly of ancient origin in seed plants. This module is involved in leaf, carpel, and ovule development in model angiosperms (Nikovics et al., 2006; Blein et al., 2008; Galbiati et al., 2013; Goncalves et al., 2015), January 2016 | Volume 6 | Article 1239 Frontiers in Plant Science | www.frontiersin.org Frontiers in Plant Science | www.frontiersin.org 9 Evolution of Carpel Margin Fusion in Eurosids Vialette-Guiraud et al. while expression studies in Amborella trichopoda, the only living representative of Amborellales (see Figure 1), and hence the likely sister to all other living angiosperms, suggest that its role in ovule development, at least, has been conserved from the earliest stages of angiosperm evolution (Vialette-Guiraud et al., 2011a). factor CRABS CLAW (crc-1 spt-2; Alvarez and Smyth, 1999). Like the NAM/miR164 module, it seems that SPT may have conserved its function in carpel development from the earliest stages of angiosperm evolution (Reymond et al., 2012). Thus, the establishment of negative regulation by SPT of a miR164- regulated NAM gene in a common ancestor of the angiosperms may have been a crucial step in the evolution of the closed carpel. Analysis of the pathway linking SPT, and its cofactors such as the HECATE transcription factors (Schuster et al., 2015), with the NAM/miR164 module in model angiosperms could provide insights into this possibility, and thus potentially indicate a molecular mechanism for the enclosure of the ovule with the carpel in the first angiosperms. Like most basally diverging angiosperms, A. trichopoda is apocarpous and has ascidiate carpels. Thus, in both A. trichopoda and A. thaliana, the carpel margins are congenitally fused from the earliest stages of gynoecium development, albeit in the different contexts of apocarpy and syncarpy, respectively. No expression of the NAM ortholog from A. trichopoda, AtrNAM, was observed in the early carpel wall (Vialette-Guiraud et al., 2011a), as is the case for CUC1 and CUC2 in A. FUNDING This work was supported by research grants ANR-13-BSV2-0009 “ORANGe” to CS and ANR-11-BSV2-0005 “Charmful” to PR. AV-G is funded through an ENS-Lyon research and teaching position. The Role of the NAM/miR164 Module in Carpel Evolution thaliana (Galbiati et al., 2013; Figure 3). Thus, it appears reasonable to postulate that the NAM/miR164 module operates in favor of the expression of miR164, and against that of NAM orthologs, from the earliest stages of gynoecium development in A. trichopoda, as it does in A. thaliana. ACKNOWLEDGMENTS Interestingly, it is known that in A. thaliana, the role of CUC2 in the closure of the gynoecium apex is under indirect negative control by the bHLH transcription factor SPATULA (SPT; Nahar et al., 2012). In addition, SPT is known to play a role in carpel fusion along the entire length of the gynoecium, which is revealed in double-mutant combinations with the YABBY transcription We thank Téva Vernoux for supplying aux1-22 seed and Patrick Laufs for making available the CUC2g-m4 and CUC2g- wt constructs. We are grateful to Patrick Laufs and Mike Frohlich for helpful discussions. AUTHOR CONTRIBUTIONS AV-G performed all of the work except Medicago transformation, prepared the figures and collaborated with CS to plan and write the paper. AC assisted with in situ hybridizations of Arabidopsis. JG-M assisted with in situ hybridizations of Medicago. AE performed Medicago transformations. PR supervised Medicago transformations. CS supervised all work except Medicago transformation and collaborated with AV-G to plan and write the paper. From the above observations, we hypothesize that the NAM/miR164 module may have played a role in the fusion of carpel margins in the MRCA of the living angiosperms, as it does in present-day model angiosperms. An important test of this hypothesis will depend on the development of plant transformation strategies in basally diverging angiosperms, which would allow, for example, the transformation of A. trichopoda with a miR164-resistant form of AtrNAM. Comparison of early diverging angiosperm lineages strongly suggests that the first flowering plants possessed ascidiate, rather than plicate carpels (Endress and Igersheim, 2000). Accordingly, we furthermore hypothesize, based on our gene expression analyses (Figure 3), that the origin of plicate carpels in various later-emerging angiosperm lineages may have depended on subtle modifications to the NAM/miR164 module that allowed a limited level of early expression of NAM orthologs in the carpel margins, as occurs in present-day M. truncatula. REFERENCES Axtell, M. J., and Bartel, D. P. (2005). Antiquity of microRNAs and their targets in land plants. Plant Cell 17, 1658–1673. doi: 10.1105/tpc.105.032185 Aida, M., Ishida, T., Fukaki, H., Fujisawa, H., and Tasaka, M. (1997). Genes involved in organ separation in Arabidopsis: an analysis of the cup-shaped cotyledon mutant. Plant Cell 9, 841–857. doi: 10.1105/tpc.9.6.841 Benlloch, R., Navarro, C., and Beltran, J. P. (2003). Floral developmentof the model legume Medicago truncatula: ontogeny studies as a tool to better characterize homeotic mutations. Sex. 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January 2016 | Volume 6 | Article 1239 REFERENCES doi: 10.1016/j.cub.2004.06.022 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. Nahar, M. A.-U., Ishida, T., Smyth, D. R., Tasaka, M., and Aida, M. (2012). Interactions of CUP-SHAPED COTYLEDON and SPATULA genes control carpel margin development in Arabidopsis thaliana. Plant Cell Physiol. 53, 1134–1143. doi: 10.1093/pcp/pcs057 Copyright © 2016 Vialette-Guiraud, Chauvet, Gutierrez-Mazariegos, Eschstruth, Ratet and Scutt. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Copyright © 2016 Vialette-Guiraud, Chauvet, Gutierrez-Mazariegos, Eschstruth, Ratet and Scutt. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Nam, Y.-W., Penmetsa, R. V., Endre, G., Uribe, P., Kim, D., and Cook, D. R. (1999). Construction of a bacterial artificial chromosome library of Medicago truncatula and identification of clones containing ethylene-response genes. Theor. Appl. Genet. 98, 638–646. doi: 10.1007/s001220051115 Nikovics, K., Blein, T., Peaucelle, A., Ishida, T., Morin, H., Aida, M., et al. (2006). The balance between the MIR164A and CUC2 genes controls leaf Frontiers in Plant Science | www.frontiersin.org January 2016 | Volume 6 | Article 1239 11

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SAT-Based Counterexample-Guided Inductive Synthesis of Distributed Controllers

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Received September 30, 2020, accepted October 29, 2020, date of publication November 16, 2020, date of current version November 27, 2020. Digital Object Identifier 10.1109/ACCESS.2020.3037780 Digital Object Identifier 10.1109/ACCESS.2020.3037780 KONSTANTIN CHUKHAREV 1,2, DMITRII SUVOROV1,2, DANIIL CHIVILIKHIN 1,2, AND VALERIY VYATKIN2,3,4, (Senior Member, IEEE) Funding: The reported study was funded by RFBR, project number 19-37-51066. ABSTRACT This article proposes a new method for automatic synthesis of distributed discrete-state controllers from given temporal specification and behavior examples. The proposed method develops known synthesis methods to the distributed case, which is a fundamental extension. This method can be applied for automatic generation of correct-by-design distributed control software for industrial automation. The pro- posed approach is based on reduction to the Boolean satisfiability problem (SAT) and has Counterexample- Guided Inductive Synthesis (CEGIS) at its core. We evaluate the proposed approach using the classical distributed alternating bit protocol. INDEX TERMS Control system synthesis, inference algorithms, Boolean satisfiability, counterexample- guided inductive synthesis, formal verification, model checking. INDEX TERMS Control system synthesis, inference algorithms, Boolean satisfiability, counterexample- guided inductive synthesis, formal verification, model checking. is work is licensed under a Creative Commons Attribution 4.0 License. For more information, see https://creativecommons.org/licenses/by/4.0/ Received September 30, 2020, accepted October 29, 2020, date of publication November 16, 2020, date of current version November 27, 2020. SAT-Based Counterexample-Guided Inductive Synthesis of Distributed Controllers KONSTANTIN CHUKHAREV 1,2, DMITRII SUVOROV1,2, DANIIL CHIVILIKHIN 1,2, AND VALERIY VYATKIN2,3,4, (Senior Member, IEEE) 1Sirius University of Science and Technology, 354340 Sochi, Russia 2Computer Technologies Laboratory, ITMO University, 197101 Saint Petersburg, Russia 3Department of Electrical Engineering and Automation, Aalto University, 02150 Espoo, Finland 4Department of Computer Science, Electrical and Space Engineering, Luleå University of Technology, 971 87 Luleå, Sweden Corresponding authors: Konstantin Chukharev ([email protected]) and Daniil Chivilikhin ([email protected]) Funding: The reported study was funded by RFBR, project number 19-37-51066. I. INTRODUCTION directly applied for controller model inference. However, these methods are mostly focused on inference of a mono- lithic controller, i.e. controller that is implemented as one sin- gle finite-state machine. In this work, we address the problem of synthesizing finite-state models for a set of independent controllers which communicate with each other via network. More specifically, we target systems implemented following the international standard for distributed automation systems development IEC 61499 [5], which defines control systems as networks of interacting function blocks (FBs), specified by their interfaces and implementations (control algorithms). Essentially, control algorithms are finite-state automata. The design of robust industrial automation systems is a com- plex problem. It is common to use deterministic finite-state models in order to reduce the complexity of engineering and benefit from automated software verification techniques [1]–[5]. Thus, controller logic is often designed as a set of interacting automata. Usually, finite-state models of controllers are designed by hand. This is a tedious and error-prone process, especially when long-term maintenance is required. Often there is no access to the controller logic source code, leading to the prob- lem of migrating to new automation standards, e.g. migra- tion from the IEC 61131-3 [6] standard for programmable logic controllers (PLCs) to the modern IEC 61499 [5] for distributed industrial automation systems. Alternatively, one could employ finite-state automata synthesis techniques and infer controller models from behavioral examples and/or tem- poral specification [7]–[14]. The main contribution of this article is a method for syn- thesis of finite-state models for a distributed controller (set of interconnected individual controllers) from behavior exam- ples and temporal specification. We implement the proposed method as an extension of the tool fbSAT [18], [19] and evaluate it using the Alternating Bit Protocol (ABP) [20] as an example. Automatic synthesis of finite-state models is a well-known problem [3], [4], [7], [8], [11], [15]–[17]. Over the past years, various methods have been proposed that can be The rest of this article is structured as follows. In Section II, we introduce the necessary definitions and notation. In Section III, we formally state the problem addressed in this article. Section IV contains a survey of related work. In Section V, we briefly describe the previous work done The associate editor coordinating the review of this manuscript and approving it for publication was Dong Shen . 207485 207485 K. A. FUNCTION BLOCKS AND AUTOMATA In this work we consider function blocks (FBs), defined in the IEC 61499 standard, as a target implementation for syn- thesized systems. A function block is defined by an interface and a control algorithm. An interface (see e.g. Fig. 1) speci- fies interaction between function block and environment, and consists of the sets of input and output events (denoted with I and O respectively), and the sets of input and output vari- ables (denoted with X and Z respectively). We only consider Boolean input/output variables. B. EXECUTION SCENARIOS FIGURE 1. A function block. An execution scenario is a sequence of scenario elements si. A scenario element is a pair of an input action and an output action: si = ⟨i[¯x], o[¯z]⟩. An automaton A satisfies a scenario s, if after processing a sequence of input actions in s it produces the exact same sequence of output actions as in s. A positive scenario is an execution scenario, that repre- sents a desired behavior of the automaton. Positive scenar- ios can either be obtained by running a simulation of some model or by accessing a real automation system. For example, in [22], execution scenarios are obtained directly from the legacy automation system: each legacy PLC is physically connected with a data collection PLC that observes and logs all input/output signals of the legacy PLC, thus producing a positive execution scenario. A separate problem addressed in [22] is denoising the hardware-derived execution scenarios that may exhibit various types of errors. In this work we assume that execution scenarios do not contain errors. FIGURE 1. A function block. A control algorithm is a Moore-type finite-state machine, called execution control chart (ECC). Later we will refer to such ECC simply as automaton. A rigorous description of FB semantics including a formal definition of ECC can be found in [21]. In this article we only consider canonical ECCs with logical inputs and outputs. Formally, an automaton A is a tuple (Q, qinit, I, O, X, Z, λ, ψ, ω), where: • Q is a set of states; I. INTRODUCTION Chukharev et al.: SAT-Based CEGIS of Distributed Controllers An automaton reads a sequence of input actions and trans- forms it into a sequence of output actions. An input action i[¯x] is a pair of an input event i ∈I and a vector of input variable values ¯x = ⟨x1, . . . , x|X |⟩, where xi ∈X. Similarly, an output action o[¯x] is pair of an output event o ∈O ∪{ε} and a vector of output variable values ¯z = ⟨z1, . . . , z|Z|⟩, where zi ∈Z. An empty output event ε is produced when automaton ignores an input action and does not make a transition. Later we will refer to ¯x and ¯z simply as input and output. The semantics of an execution step is as follows. An automaton receives an input action i[¯x] and changes its state to q′ = λ(q, i, ¯x), where q is the current state and ¯x are current input variable values. Next, it produces an output event o′ = ψ(q, i, ¯x) changes the output variable values to ¯z′ = ω(q, i, ¯x, ¯z), where ¯z are current output variable values. Note that the automaton may ignore some input actions, in that case the output event is an empty event: o′ = ε, and the state and the values of output variables do not change: q′ = q, ¯z′ = ¯z. on the synthesis of monolithic controllers [18]. Then, in Section VI, we describe the main contribution of this article: extension of monolithic controller synthesis for distributed systems. Section VII describes our experiments with ABP. The results are discussed in Section VIII, and Section IX concludes the paper. IV. RELATED WORK O k i i l i IV. RELATED WORK k i i l i Our work is mainly inspired by [16], where an approach for automatic synthesis of distributed protocols has been proposed. Distributed protocols are similar to distributed industrial control systems in the sense that they too can be modeled with a set of communicating finite-state machines. Correctness of protocols is also specified with safety and liveness temporal properties. The approach proposed in [16] uses the idea of program sketching [30], in which the devel- oper of the protocol specifies an incomplete implementation, and the synthesis tool automatically completes the manual implementation by means of search and verification of tem- poral properties. This approach of protocol completion deals with the known undecidability of the distributed synthesis problem [31]: limiting the sizes of the synthesized finite-state machines and their number of transitions makes the comple- tion problem decidable [16]. The approach of [16], similar to our work, is based on counterexample-guided inductive synthesis (CEGIS) [32]. However, it uses the following strong assumptions. Instead of developing an ad-hoc algorithm for a hard prob- lem at hand, in many cases one may develop a reduction of the problem to SAT: a function that, given a problem instance, produces a CNF formula that is satisfiable if and only if the original problem has a solution. The resulting CNF formula is then fed as input to a SAT solver tool. The outcome of SAT solving process is either a satisfying assignment, or UNSAT – an indication that the formula is unsatisfiable. 1) An incomplete protocol process (automaton or module) is defined as an automaton in which some transitions are missing (in comparison with a complete process). Thus, it is assumed that the protocol developer provides a partial implementation, in which some needed tran- sitions are missing, and, more importantly, all present transitions are correct. In this assumption, the synthesis (or rather, completion) algorithm only needs to add some missing transitions. This scenario does not appear to be very practical: an error in the manually prepared transitions will prevent the algorithm from finding the solution. C. FORMAL VERIFICATION WITH MODEL CHECKING • qinit ∈Q is the initial state; In this work we specify a system with a set of linear tem- poral logic (LTL) [23] formulas. An LTL formula is defined over execution paths and describes some temporal properties of these paths. LTL formulas include atomic propositions (some elementary statements about the system), propositional logic connectives (∧, ∨, ¬, →), and temporal operators (e.g., X – ‘‘next’’, U – ‘‘until’’, G – ‘‘always’’, F – ‘‘even- tually’’). With LTL formulas one can specify safety (‘‘some- thing bad never happens’’) and liveness (‘‘something good will eventually happen’’) properties of a given system. An example of a safety property is a formula G ¬P, which states that some predicate P is always false. An example of a liveness property is a formula G(P →F Q), which states that if a predicate P is true, then a predicate Q will eventually become true. • I, O are the sets of input/output events; • X, Z are the sets of input/output variables; • λ : Q×I ×B|X | 7→Q is the partial transition functio X p • ψ : Q × I × B|X | →(O ∪{ε}) is the output event function; p • ψ : Q × I × B|X | →(O ∪{ε}) is the output event function; • ω : Q × I × B|X | × B|Z| →B|Z| is the output function. • ω : Q × I × B|X | × B|Z| →B|Z| is the output function. The states of an automaton are marked with output events and algorithms. Algorithm is a function that changes the values of output variables Z. In this article we consider algorithms of form B|Z| →B|Z|, where each output variable only depends on its previous value. The transitions of an automaton are marked with input events and guard conditions – functions of a form B|X | →B which enables/disables a given transition for current input variables values. We label a transition with R & x to indicate it is marked with an input event R and a guard x. Model checking [24] is a technique that can be used to verify a given finite-state model w.r.t. a given specification 207486 VOLUME 8, 2020 VOLUME 8, 2020 K. Chukharev et al.: SAT-Based CEGIS of Distributed Controllers scenarios and LTL/specification. Denote the number of mod- ules with M, and let m ∈[1 .. C. FORMAL VERIFICATION WITH MODEL CHECKING M] be the index of a module in the rest of this article. Ultimately, the problem addressed in this work is to infer a set of automata {A(m)} for all modules, that comply with a given set of individual positive scenarios S+(m) for each module, and an LTL/specification L for the whole system. The module interfaces (i.e. sets X (m), Z(m), I(m), and O(m)) are known beforehand, and LTL formulas use variables from these sets as atomic propositions. (here, set of LTL formulas) and obtain a counterexample exe- cution path if this specification is violated. Counterexamples can further be translated to negative scenarios – execution scenarios representing undesired behavior of an automaton or a system of automata. This process is described in detail in Section V-A2. D. BOOLEAN SATISFIABILITY PROBLEM The SAT problem consists in determining whether a Boolean formula in Conjunctive Normal Form (CNF) is satisfiable. A Boolean variable has values from the set B = {0, 1}. Boolean variable x and its negation ¬x are called literals; lit- erals x and ¬x are called contrary. A clause is a disjunction of non-contrary literals, e.g., x1 ∨x2 ∨¬x3. A Boolean formula in CNF is a conjunction of clauses. A formula is called sat- isfiable if there exists an assignment of variable values such that all clauses evaluate to 1; such an assignment is called sat- isfying. If a satisfying assignment does not exist, the formula is called unsatisfiable. The problem is to determine if a given formula is satisfiable, and if it is, to find a satisfying assign- ment. SAT is the classical NP-complete problem [25], [26]. Extensive research in SAT theory and algorithms resulted in modern SAT solvers based on the conflict-driven clause learning (CDCL) algorithm [27], which are now considered to be a standard computational tool used in various applica- tion domains [28]. VOLUME 8, 2020 III. DISTRIBUTED CONTROLLER SYNTHESIS PROBLEM In this work we consider a setup of a distributed controller, in which multiple individual controllers, also called mod- ules, communicate with each other via some environment. We assume that the controllers are implemented (or could be implemented) with FBs, hence the communication is done by exchanging messages and setting Boolean variables. We do not formalize what an environment is, but instead require that it can be modeled with an input language of some model checker (in our experiments we use NuSMV [29]). By avoiding a more rigorous definition of an environment we gain more flexibility in the application of the proposed approach. For instance, we can model a synchronous setup, when controllers are attached to a common bus, like in [22], or we can model a true distributed system, in which modules communicate via unreliable channels. 2) Following from the first assumption, the input scenarios in [16] are assumed to cover all necessary states of each protocol. If this assumption is violated, the algorithm will fail to complete the distributed protocol. 3) Due to previous assumptions, the learner part of the CEGIS in [16], the one that is supposed to provide candidate completions for verification, is a simple enu- meration algorithm. Though the use of enumeration algorithms is quite a valid approach when the search space is small, in practice it quickly becomes infeasible. A better approach employed in our research is delegation of enumeration to a SAT solver, which performs it in a more intelligent and efficient way. We assume that we can observe the system and gather behavior examples, and that there is some temporal specifi- cation of the system. Our goal is to infer a set of FB mod- els, one for each module of the distributed controller, such that the distributed system complies with the given positive 207487 VOLUME 8, 2020 K. Chukharev et al.: SAT-Based CEGIS of Distributed Controllers FIGURE 2. Positive scenario tree. 4) Finally, the algorithm used in [16] to construct incom- plete automata from input scenarios heavily depends on so-called labels that annotate messages [33]. Labels affect the way that the scenario elements are merged, thus having an effect on the produced automata. In fact, labels mostly define the mapping of scenario elements to states of the incomplete process. III. DISTRIBUTED CONTROLLER SYNTHESIS PROBLEM In contrast to the above, in our approach we do not make any assumptions about the input scenarios (apart that they must define a deterministic system), do not require scenarios to cover all states of the distributed controller, use an intel- ligent SAT encoding to construct state machines from input scenarios and provide candidate distributed controllers for verification. FIGURE 2. Positive scenario tree. a scenario element. A scenario tree constructed from a set of positive scenarios is called a positive scenario tree and denoted with T +. Use of SAT solvers is a common approach in automatic passive synthesis of automata [7]–[9], [12], [17], [34], as well as in many other application domains [28]. A related paper that is based on the use SAT solvers is [22], where a modular controller is synthesized from one set of positive scenarios, not considering temporal properties. This is in contrast with the problem addressed in the present paper, where each part of the distributed controller (each automaton) is synthesized from a separate set of positive scenarios, and the functioning of the distributed system as a whole is bounded by temporal properties, producing a set of negative scenarios for the entire system. The following notation will be used throughout the paper. The set of all unique inputs in the scenario tree is denoted with U ⊆B|X |. The set of scenario tree nodes is denoted with V, ρ ∈V is the root of the tree. The following functions are used to describe the scenario tree: • tp(v) ∈V is the parent of node v ∈V, v ̸= ρ; • tp(v) ∈V is the parent of node v ∈V, v ̸= ρ; • tie(v) ∈I is the input event on the incoming edge of node v ∈V, v ̸= ρ; • toe(v) ∈O ∪{ε} is the output event in node v ∈V; • tin(v) ∈U is the input on the incoming edge of node v ∈V, v ̸= ρ; • tov(v, z) ∈B is the value of the output variable z ∈Z in node v ∈V. • tov(v, z) ∈B is the value of the output variable z ∈Z in node v ∈V. V. OVERVIEW OF MONOLITHIC CONTROLLER SYNTHESIS WITH fbSAT Tree nodes, other than the root, are divided into active V(act) and passive V(pass) nodes, those with a non-empty and empty output event respectively: In this section we briefly describe the fbSAT approach for the synthesis of a single monolithic finite-state func- tion block model from a set of positive scenarios and an LTL/specification, proposed in [18]. The method is based on a reduction to SAT. First, fbSAT builds an automaton that satisfies the positive scenarios, that are represented with a (positive) scenario tree. This is done with a SAT encoding that ensures correspondence of scenario elements with corre- sponding states of the automaton. • V(act) = {v ∈V \ {ρ}toe(v) ̸= ε}; • V(pass) = {v ∈V \ {ρ}toe(v) = ε}. ( ) • V(pass) = {v ∈V \ {ρ}toe(v) = ε}. ( ) • V(pass) = {v ∈V \ {ρ}toe(v) = ε}. The scenario tree for the following positive scenarios is shown in Fig. 2: [⟨R[00], ε[0]⟩; ⟨R[01], B[1]⟩; ⟨R[00], ε[1]⟩; ⟨R[01], B[0]⟩], [⟨R[00], ε[0]⟩; ⟨R[10], A[0]⟩; ⟨R[00], ε[0]⟩; ⟨R[01], B[1]⟩], [⟨R[00] ε[0]⟩; ⟨R[10] A[0]⟩; ⟨R[10] A[0]⟩] Second, the solution constructed from positive scenarios is refined by means of counterexample-guided inductive syn- thesis: on each iteration a model checker is used to ver- ify the compliance of the current automaton with the LTL/ specification; if counterexamples are generated, then SAT formula is appended with additional clauses that encode that the sought solution must not demonstrate the behavior repre- sented by the counterexample. [⟨R[00], ε[0]⟩; ⟨R[10], A[0]⟩; ⟨R[00], ε[0]⟩; ⟨R[01], B[1]⟩], [⟨R[00], ε[0]⟩; ⟨R[10], A[0]⟩; ⟨R[10], A[0]⟩]. 2) NEGATIVE SCENARIO TREE An automaton can be verified against a given LTL/ specification with a model checker tool, such as NuSMV [29]. A model checker finds a counterexample for each violated LTL property in the form of an execution state sequence. Note that execution states of NuSMV models should not be confused with states of automata. FIGURE 3. Example automaton with a looping behavior. the following counterexample (the notation [q1/0] indicates the execution state, in which the automaton is in the state q1, and the current value of z is 0): [q1/0] R[1] −−−→[q2/0] R[1] −−−→[q4/0] R[1] −−−→[q5/1]. This counterexample is translated into the following loopless negative scenario: [⟨R[1], C[0]⟩; ⟨R[1], C[0]⟩; ⟨R[1], C[1]⟩]. [⟨R[1], C[0]⟩; ⟨R[1], C[0]⟩; ⟨R[1], C[1]⟩]. A counterexample for a liveness property is an infinite sequence, which can be represented as a finite prefix followed by a loop. Consider a liveness property F z for the same system. A counterexample for it would be the following: Finally, to represent how the automaton changes states when processing the positive scenarios, variable λq,i,u ∈Q0 is introduced. It denotes the state to which the automaton switches after processing the input action i[u] in the state q. λq,i,u = q0 denotes the situation when the automaton ignores the input action i[u] and remains in the state q. To reduce the search space size, symmetry-breaking con- straints [10], [37] are declared. These constraints enforce that the states of the automaton are enumerated in the breadth-first search (BFS) traversal order. Variable τ bfs qi,qj ∈B (qi, qj ∈Q) encodes whether there is a transition from state qi to state qj. Variables τ bfs qi,qj ∈B are defined through τ in the following way: A negative execution scenario obtained from this coun- terexample contains a loopback and looks like this (the beginning of the loop is underlined): [⟨R[1], A⟩;⟨R[1],C[0]⟩; ⟨R[0],C[0]⟩; ⟨R[1],C[0]⟩]. A negative scenario tree T −is a prefix tree constructed from a set of negative scenarios. It contains auxiliary edges that represent loopbacks in negative scenarios. A set of tree nodes connected via a loopback edge with a nodebv is denoted with c tbe(bv) ⊆bV. The notation for negative trees is the same as for positive trees, but all symbols are marked with a hat: bv ∈bV, btp(v), bV (act), etc. τ bfs qi,qj ↔ _ k∈[1..K] (τqi,k = qj). Variable πbfs qj ∈{q1, . . . , qj−1} (j ∈[2 .. C]) denotes the parent of the state qj in the BFS traverse tree: (πbfs qj = qi) ↔τ bfs qi,qj ∧ ^ r<i (¬τ bfs qr,qj). B. FB MODEL INFERENCE USING SAT SOLVER Finally, the symmetry-breaking constraint requires that each state has a parent in the BFS traverse tree with a smaller number: The described approach is based on a reduction to SAT: a Boolean formula is constructed which is satisfiable if and only if there exists an automaton A of a predefined size that satisfies positive scenarios and does not satisfy negative scenarios. For non-Boolean variables with finite domains the standard pairwise [35] encoding is used. Tseytin transforma- tions [36] are employed to convert all constraints to CNF. The encoding consists of four parts which are covered in subsequent sections. (πbfs qj = qi) → ^ r<i (πbfs qj+1 ̸= qr). 1) POSITIVE SCENARIO TREE A scenario tree T is a prefix tree constructed from the set of execution scenarios. Each execution scenario is prepended with an empty element ε[⟨0 . . . 0⟩] so that they share a com- mon prefix. Each node of a scenario tree is marked with an output action, and each incoming edge is marked with an input action. A node with its incoming edge correspond to Counterexamples are translated into negative scenarios. A counterexample for a safety property is a finite sequence of execution states. Consider the system depicted in Fig. 3. Every state produces an output event C and it is omitted in the picture. A safety LTL property G ¬z is violated as shown by 207488 VOLUME 8, 2020 K. Chukharev et al.: SAT-Based CEGIS of Distributed Controllers FIGURE 3. Example automaton with a looping behavior. 1) AUTOMATON STRUCTURE ENCODING The first part of the encoding defines automaton states and transitions. Variable φq ∈O ∪{ε} denotes the output event associated with state q. Variable γq,z,b ∈B denotes the new value for the output variable z in dependence from its old value b and state q. Thus, variable γ encodes the algorithm associated with state q. The first part of the encoding defines automaton states and transitions. Variable φq ∈O ∪{ε} denotes the output event associated with state q. Variable γq,z,b ∈B denotes the new value for the output variable z in dependence from its old value b and state q. Thus, variable γ encodes the algorithm associated with state q. A transition is characterized by its destination state, input event, and guard condition. Variable τq,k ∈ Q0, where Q0 = Q∪{q0}, denotes the destination state of the k-th transi- tion from state q. Some states may have less than K transitions – to deal with it, an auxiliary state q0 is introduced: τq,k = q0 indicates the absence of the k-th transition from the state q. Variable ξq,k ∈I ∪{ε} denotes the input event associated with the k-th transition from the state q. To encode guard conditions the following variables are introduced. θq,k,u ∈B denotes the value of the guard condition associated with the k-th transition from the state q for the input u, and δq,k,i,u ∈B is true if and only if that guard condition fires for an input action i[u]. FIGURE 3. Example automaton with a looping behavior. 3) POSITIVE SCENARIO TREE MAPPING ENCODING wherebv ∈bV (pass),bp = btp(bv), q = ˆµbp, i = b tie(bv), u = b tin(bv). wherebv ∈bV (pass),bp = btp(bv), q = ˆµbp, i = b tie(bv), u = b tin(bv). In order to make the automaton A comply with a set of positive scenarios represented as scenario tree T +, a map- ping µ : V →Q is organized between the tree nodes and the automaton states. The variable µv ∈Q denotes the satisfying state (also called ‘‘mapped’’) in which the automaton finishes processing a sequence of input actions on the path from ρ to v ∈V. p Active nodes map to the state in which the automaton finishes processing the input action i[u]: ( ˆµbv = q′) ↔(ˆλq,i,u = q′) ∧( ˆφq′ = o) ∧ ^ z∈Z ( ˆγq′,z,b = b′), ( ˆµbv = q′) ↔(ˆλq,i,u = q′) ∧( ˆφq′ = o) ∧ ^ z∈Z ( ˆγq′,z,b = b′), where bv ∈ bV (act), bp = btp(bv), q = µbp, q′ ∈ Q, i = b tie(bv), u = b tin(bv), o = c toe(bv), z ∈Z, b = c tov(bp, z), b′ = c tov(bv, z). Note that this constraint, compared to positive mapping, uses equivalence (↔) instead of implication (→): a node maps to some state if and only if the automaton has matching behavior. This allows to declare the definitions of ˆµbv = q′ only for q′ ∈Q, without explicitly considering the case ˆµbv = q0 (which would require a large number of long clauses, which generally makes the SAT problem harder for a SAT solver [28]). When the automaton does not exhibit the behavior specified in the scenario tree node, this node remains unmapped (i.e. maps to q0). The root of the tree maps to the initial automaton state: µρ = qinit. A passive node in the scenario tree represents the case when the automaton ignores an input action and does not change state. Thus, passive nodes map to the same state as their parents: (µv = q) ∧(λq,i,u = q0), where v ∈V(pass), p = tp(v), q = µp, i = tie(v), u = tin(v). where v ∈V(pass), p = tp(v), q = µp, i = tie(v), u = tin(v). where v ∈V(pass), p = tp(v), q = µp, i = tie(v), u = tin(v). 4) NEGATIVE SCENARIO TREE MAPPING ENCODING b Similar to the positive tree mapping, a mapping ˆµ : bV →Q0 is organized between the negative tree nodes and the automa- ton states. Variable ˆµbv ∈Q0 denotes the satisfying state (or its absence) in which the automaton finishes processing a sequence of input actions on the path from bρ to ¬ ∈bV. Note that it is acceptable for the automaton not to behave in the way specified in the negative scenario tree, e.g., it might produce mismatching output actions – in such cases the corresponding nodes will be unsatisfied (also called unmapped), which is denoted by ˆµbv = q0, where q0 is an auxiliary automaton state. 3) POSITIVE SCENARIO TREE MAPPING ENCODING Active nodes, on the contrary, represent the situation when the automaton reacts to an input action i[u] and follows a tran- sition – exactly to the satisfying state, where the automaton must produce exactly the same output action as specified in the active node: If a negative tree node is unmapped, then all its descendants are unmapped as well: ( ˆµbtp(bv) = q0) →( ˆµbv = q0). (µv = q′) →(λq,i,u = q′) ∧(φq′ = o) ∧ ^ z∈Z (γq′,z,b = b′), where v ∈V(act), p = tp(v), q = µp, q′ ∈Q, i = tie(v), u = tin(v), o = toe(v), z ∈Z, b = tov(p, z), b′ = tov(v, z). (µv = q′) →(λq,i,u = q′) ∧(φq′ = o) ∧ ^ z∈Z (γq′,z,b = b′), Finally, for undesired behavior prohibition, it is required that the first and the last nodes of the loop in the negative tree either map to different states, or both are unmapped (i.e. map to q0): where v ∈V(act), p = tp(v), q = µp, q′ ∈Q, i = tie(v), u = tin(v), o = toe(v), z ∈Z, b = tov(p, z), b′ = tov(v, z). ^ bv′∈c tbe(bv)  ( ˆµbv ̸= ˆµbv′) ∨( ˆµbv = ˆµbv′ = q0)  . ^ ( ˆµbv ̸= ˆµbv′) ∨( ˆµbv = ˆµbv′ = q0)  . 2) GUARD CONDITIONS STRUCTURE ENCODING In the approach described so far, guard conditions are rep- resented as truth tables (via variable θ). This representation is inconvenient and is not human-readable. In [18], guard conditions are represented with parse trees of corresponding Boolean formulas and explicitly encoded in SAT. In this encoding, the parameter P is the guard condition parse tree size, and the parameter N is the upper bound for a total The following notation is used hereinafter. The number C = |Q| is the number of states in automaton A, each state has at most K ≤C outgoing transitions. It is also assumed that b ∈B = {0, 1}, q ∈Q, k ∈[1 .. K], i ∈I, o ∈O, u ∈U, v ∈V, unless stated otherwise. 207489 VOLUME 8, 2020 K. Chukharev et al.: SAT-Based CEGIS of Distributed Controllers where bv ∈bV (pass), q′ ∈Q, i = b tie(bv), u = b tin(bv). And in the second case, the automaton does the inverse – follows some transition: number of parse tree nodes in all guard conditions. We do not describe this encoding here, since it is not necessary for the understanding of the rest of the paper. ( ˆµbv = q0) →(ˆλq,i,u ̸= q0), C. COUNTEREXAMPLE-GUIDED INDUCTIVE SYNTHESIS The approach described so far accepts positive and neg- ative scenarios as input, without specifying the mecha- nism of producing the negative scenarios. This is done via counterexample-guided inductive synthesis [32]. A CEGIS iteration consists of three steps. First, some automaton A is inferred based on current positive and negative scenario trees. Second, the inferred automaton is checked against the specification L with a model checker, and, possibly, some counterexamples describing incorrect behavior are obtained. Finally, these counterexamples are translated into negative scenarios, which are added to the negative tree, and the proce- dure repeats until there are no more counterexamples, or there is no such automaton that satisfies positive scenarios and does not satisfy negative scenarios. In the first case the inferred automaton complies with the given temporal specification. A CEGIS loop is schematically shown in Fig. 4. The root bρ of the negative scenario tree T −maps to the initial state of the automaton: ˆµbρ = qinit. Passive nodes either (1) map to the same state as their parent, or (2) become unmapped (this happens when the automaton does not exhibit the passive behavior): ( ˆµbv = ˆµbp) ∨( ˆµbv = q0), where bv ∈bV (pass), bp = btp(bv). In the first case, the automa- ton ignores the input action and remains in the same state (i.e. does not follow any transition): OM MONOLITHIC TO DISTRIBUTED SYNTHESIS This article proposes an extension of the fbSAT approach [18] described in the previous section. The proposed extension allows for synthesis of distributed ( ˆµbv = q′) →(ˆλq′,i,u = q0), 207490 207490 VOLUME 8, 2020 VOLUME 8, 2020 K. Chukharev et al.: SAT-Based CEGIS of Distributed Controllers FIGURE 4. CEGIS loop. module of the distributed controller. Variable µ(m) v denotes a state in which the automaton for the m-th module finishes processing a sequence of inputs formed by following the positive tree from the root till the node v (since the tree is deterministic, for each node v there is only one path from the root). This variable represents a mapping µ(m) : V (m) →Q(m) between the nodes of T + m and the states of an automaton A(m), and the constraints on this mapping are the same to those described in Section V-B3. FIGURE 4. CEGIS loop. controllers, and is based on two ideas. The first one is to declare a reduction for monolithic case for each module independently. B. COMPOUND NEGATIVE SCENARIO TREE A compound negative scenario is a sequence of compound scenario elements ci, where ci is an M-tuple of scenario elements for each synthesized module: ci = (sm i ), where sm i = ⟨i[¯x], o[¯z]⟩. i Consider a system of two interconnected modules depicted in Fig. 5. The graph and the table in Fig. 5 show the execution state sequence of this system, illustrating the violation of the LTL property: GF ¬z. A compound negative scenario obtained from this execution sequence is shown below (the beginning of the loop is marked with an underline). [(⟨R[0], ε[0]⟩, ⟨R, C[1]⟩); (⟨R[1], C[1]⟩, ⟨R, C[0]⟩); (⟨R[0], ε[1]⟩, ⟨R, C[0]⟩); (⟨R[0], ε[1]⟩, ⟨R, C[1]⟩); (⟨R[1], C[1]⟩, ⟨R, C[0]⟩); (⟨R[0], C[1]⟩, ⟨R, C[0]⟩)] [(⟨R[0], ε[0]⟩, ⟨R, C[1]⟩); (⟨R[1], C[1]⟩, ⟨R, C[0]⟩); (⟨R[0], ε[1]⟩, ⟨R, C[0]⟩); (⟨R[0], ε[1]⟩, ⟨R, C[1]⟩); (⟨R[1], C[1]⟩, ⟨R, C[0]⟩); (⟨R[0], C[1]⟩, ⟨R, C[0]⟩)] The naïve approach would be to prohibit each negative scenario for all modules, but it would be incorrect to do so: a behavior that is incorrect for one of the modules may be completely normal for another one. Thus, this naïve approach would lead to unsatisfiability of corresponding Boolean for- mulas, and the sought distributed controller would not be synthesized. One important difference from the monolithic case is that in distributed synthesis we assume that the input event might be empty: i ∈I ∪{ε}. This corresponds to the case when one of the modules does not receive any input at some point in time. We also assume that in valid scenarios i = ε implies o = ε, i.e. if a module does not receive any input event, then it remains idle. Note that in negative traces derived from counterexamples obtained from the model checker, it might be the case that all synthesized modules do not receive an input event. It happens when all modules are idle, awaiting some input events from the environment. We filter out such elements, since they do not carry any information useful for the synthesis. The key idea to cope with this difficulty is to make sure that each negative scenario is prohibited for at least one module. This is described in Section VI-C. Finally, in Section VI-D we discuss the algorithm for minimization of inferred automata in terms of their parameters, e.g., number of states and total size of guard conditions. C. COUNTEREXAMPLE-GUIDED INDUCTIVE SYNTHESIS This is described in Section VI-A. The second idea has to do with the adaptation of CEGIS developed in [18] to make it applicable for distributed synthesis, and is the main idea of the paper. The difficulty of using CEGIS for distributed synthesis is that since the specification is written for the system as a whole, negative scenarios derived from counterexamples are also written for the system as a whole. Section VI-B describes the structure of these scenarios. The difficulty is that it is not known in advance, which modules behave incorrectly in some particular negative scenario, and thus we do not know, in which module do we need to prohibit the negative scenario. VOLUME 8, 2020 A. REDUCTION FOR DISTRIBUTED SYNTHESIS FROM POSITIVE SCENARIOS We declare the reduction described in Section V for each module independently, adding the module index to every variable. Thus, respective constraints remain the same, but are now quantified over all modules. For instance, variable τ (m) q,k ∈Q0 denotes the destination state of the k-th transition from state q in the automaton for the m-th module, and variable ξ(m) q,k ∈I∪{ε} denotes the input event associated with that transition. With the constraint (τ (m) q,k = q0) ↔(ξ(m) q,k = ε) we enforce that only transitions to q0 are marked with the ε input event. We also add module indices to the reduction parameters. For example, C(m) denotes the number of states in A(m) – the automaton for the m-th module. All other parts of the encoding from Section V are extended in a similar way, except for the negative mapping, which we discuss below. A compound negative scenario tree (or compound tree) CT −is a negative scenario tree built from a set of com- pound negative scenarios in a similar way as described in Section V-A. Each node of CT −and its incoming edge cor- respond to a compound scenario element. We denote the set of nodes of tree CT −with c CV and reuse the negative tree auxiliary functions (btp(bv), b tie(bv), etc.) for the compound tree. A projection of a compound scenario element to module m is simply its m-th element. A projection T − m of a compound tree CT −to module m is a tree isomorphic to CT −, each node of which is a projection of the scenario element in the respective node of CT −to module m. A compound negative scenario tree (or compound tree) CT −is a negative scenario tree built from a set of com- pound negative scenarios in a similar way as described in Section V-A. Each node of CT −and its incoming edge cor- respond to a compound scenario element. We denote the set of nodes of tree CT −with c CV and reuse the negative tree auxiliary functions (btp(bv), b tie(bv), etc.) for the compound tree. A projection of a compound scenario element to module m is simply its m-th element. C. COMPOUND NEGATIVE SCENARIO TREE MAPPING ( ) The main idea is to mark each state used/unused, which allows us to minimize the total number of used states by encoding it with the totalizer [38]. In order to enable the minimization of the synthesized automata in terms of total number of states we perform the following extensions of the reduction to SAT. To begin with, observe that parameters C(m) – maximum number of states in each module m ∈[1 .. M] – have to be known before the beginning of inference process, and the solution (if found) will have exactly the specified size (number of states). The simplest strategy for finding a satisfying solution with a minimal total number of states would be an iterative strategy The constraints for passive and active nodes are the same as in the reduction for monolithic synthesis. Each idle node maps to the same state as its parent. The only difference from passive nodes is that we do not need to encode that the automaton ignores an input action, since, in fact, it does not receive any: (bµ(m) bv = bµ(m) btp(bv)), wherebv ∈c CV(idle). – linear bottom-up search. However, in case of M > 1 (i.e. any non-monolithic synthesis) it is unclear how to per- form the iteration of parameters – efficiently, whereas main- taining the minimality – and this becomes a problem by itself. ( ) tp(v) Similar to the monolithic case, we ensure that if the node bv ∈c CV is unmapped (i.e. mapped to q0), then this property is propagated down the tree: (bµ(m) btp(bv) = q0) →(bµ(m) bv = q0). Finally, we need to prohibit the looping behavior described in counterexamples, but in such a way that each loop is prohibited in at least one module. This is done by adding a disjunction over m ∈[1 .. M] for each loop: ^ bv′∈c tbe(bv) _ m∈[1..M] h (bµ(m) bv ̸= bµ(m) bv′ ) ∨(bµ(m) bv = bµ(m) bv′ = q0) i . Thus, on each CEGIS iteration we accumulate clauses in the SAT formula to encode that this particular negative scenario is prohibited for at least one module. Essentially, this approach is an implicit search delegated to a SAT solver. Variable ζq ∈B denotes whether the state q is used by an automaton, i.e. is reachable from the initial state qinit. Clearly, the initial state is always used. C. COMPOUND NEGATIVE SCENARIO TREE MAPPING ( ) Variable bµ(m) bv denotes the satisfying state in automaton A(m) for nodebv ∈c CV, with bµ(m) bv = q0 in case when the automaton does not exhibit this particular behavior, and hence there is no satisfying state forbv (auxiliary state q0 is common for all modules). For each module, the root node of the compound negative tree maps to the initial state of the corresponding automaton: bµ(m) bρ = qinit(m). The projection of a compound tree may contain nodes with i = ε on an incoming edge – we call such nodes idle. Among non-idle nodes, we recognize passive (with c toe(bv) = ε) and active nodes (with c toe(bv) ̸= ε) with the same properties as for regular negative trees. We denote the sets of idle, passive and active nodes of the compound negative tree as c CV(idle), c CV(pass), and c CV(act) respectively. In order to enable the minimization of the synthesized automata in terms of total number of states we perform the following extensions of the reduction to SAT. To begin with, observe that parameters C(m) – maximum number of states in each module m ∈[1 .. M] – have to be known before the beginning of inference process, and the solution (if found) will have exactly the specified size (number of states). The simplest strategy for finding a satisfying solution with a minimal total number of states would be an iterative strategy – linear bottom-up search. However, in case of M > 1 (i.e. any non-monolithic synthesis) it is unclear how to per- form the iteration of parameters – efficiently, whereas main- taining the minimality – and this becomes a problem by itself. One possible way is to iterate all C(m) altogether until we find some solution (all ‘‘too small’’ values result in UNSAT), and then try to decrease some of C(m). However, the latter – inference with different C(m) values – requires restarts of the SAT solver, since the declared variables (those with index q ∈Q(m)) and constraints depend on the C(m) statically, i.e. this parameter cannot be easily changed while preserving the satisfiability. Hence, we need a dynamic way to gradually ‘‘disable’’ some states in the modules. This can be done using the ‘‘used states’’ approach proposed in [22]. C. COMPOUND NEGATIVE SCENARIO TREE MAPPING ( ) A (non-initial) state is used if and only if it has an incoming transition: A. REDUCTION FOR DISTRIBUTED SYNTHESIS FROM POSITIVE SCENARIOS A projection T − m of a compound tree CT −to module m is a tree isomorphic to CT −, each node of which is a projection of the scenario element in the respective node of CT −to module m. The difference with positive scenarios is that now we have a set of positive scenario trees {T + m }m∈[1..M], one for each 207491 207491 VOLUME 8, 2020 K. Chukharev et al.: SAT-Based CEGIS of Distributed Controllers FIGURE 5. Looping distributed system. FIGURE 5. Looping distributed system. FIGURE 5. Looping distributed system. the hardware. However, if we start minimizing (e.g., by adding additional constraints) the solution – set of modules {A(m)} – found by a CEGIS, the resulting minimal solution may (and, in practice, will) not comply with the specification L, though the already obtained negative scenarios will still not be satisfied, as expected. Therefore, the minimization step should be included into the CEGIS loop. Moreover, producing minimal models on each CEGIS iteration allows to perform model checking much faster, because smaller models have a smaller state space. Later we will refer to CEGIS with minimization included as CEGIS-min. label start // Estimate D for D ←1 to ∞do {A(m)} ←infer(∗∗∗) if {A(m)} ̸= null then break // Estimate D for D ←1 to ∞do {A(m)} ←infer(∗∗∗) if {A(m)} ̸= null then break while true do // Minimize Cused {A(m)} ←infer(∗∗∗, Cused < ∞) if {A(m)} = null then goto start while {A(m)} ̸= null do Cused min ←numberOfUsedStates({A(m)}) {A(m)} ←infer(∗∗∗, Cused < Cused min ) // Minimize N used {A(m)} ←infer(∗∗∗, Cused = Cused min , N used < ∞) while {A(m)} ̸= null do N used min ← numberOfParseTreeNodes({A(m)}) {A(m)} ←infer(∗∗∗, Cused = Cused min , N used < N used min ) {A(m)} ←infer(∗∗∗, Cused = Cused min , N used = N used min ) CS− new ←performModelChecking({A(m)}, L) if CS− new = ∅then // No counterexamples return {A(m)} CS−←CS−∪CS− new ¬ζqj ↔(πbfs-mod qj = q0). Variable τ bfs qi,qj ∈B and all other constraints – definitions of τ bfs and πbfs-mod, and the actual BFS constraint – do not require any additional changes. The main procedure implementing the CEGIS-min is shown in Algorithm 1. First, we estimate parameter D: the common upper bound for the number of states in each mod- ule. The estimation is done by a simple bottom-up linear search. On each iteration we call the function infer(∗∗∗), where ∗∗∗denotes the common arguments ({S+(m)}, CS−, {C(m) = D}, P). The infer procedure is responsible for: 1) building positive scenario trees for {S+(m)} and a nega- tive compound scenario tree for CS−; 2) reducing the inference problem to SAT by declaring the constraints described in this article; 3) delegating to a SAT solver to actually solve the SAT problem; 4) building a solution – set of automata (modules) {A(m)} satisfying given scenarios – from the satisfying assign- ment found by the SAT solver. they will be taken into account on the next iteration, else the CEGIS is done and {A(m)} is the satisfying solution. Next, we start an actual CEGIS loop. First, we minimize Cused using a top-down approach. Here, we call infer with an extra predicate/argument ‘‘Cused < UB’’, where UB denotes the upper bound for a total number of used (reach- able) states in all synthesized automata. This predicate rep- resents a cardinality constraint which is encoded using a totalizer technique [38]. Next, we minimize N used using the same approach. VII. CASE STUDY: ALTERNATING-BIT PROTOCOL We evaluate the proposed approach on the example of the classical Alternating-bit protocol (ABP) [20]. It is widely used to illustrate the concepts of formal verification and model checking, e.g. [16], [39], [40]. label start After that, we perform model check- ing of the synthesized distributed controller represented by automata {A(m)} for compliance with the given LTL/ specification L using NuSMV [29] model checker, and obtain a set of negative compound scenarios CS− new – if there are any, Input: set of positive scenario sets {S+(m)}m∈[1..M], p p { }m∈[1..M], LTL-specification L, parse tree size P. Output: automata {A(m)}m∈[1..M] complying with L. ¬ζqi →¬ζqi+1. In the context of distributed synthesis, this variable is declared for each module m ∈[1 .. M]: ζ (m) q . The total number of used states in all modules is denoted Cused and encoded using the totalizer [38]. In the context of distributed synthesis, this variable is declared for each module m ∈[1 .. M]: ζ (m) q . The total number of used states in all modules is denoted Cused and encoded using the totalizer [38]. // Compound negative scenarios CS−←∅ // Common arguments (for “infer”) ∗∗∗:= ({S+(m)}, CS−, {C(m) = D}, P) Symmetry-breaking constraints declared earlier (Section V-B1 require the states of the automaton to be enumerated in the BFS traversal order, i.e. to be included in the BFS traverse tree. This implies that all states (except the initial one) have an incoming transition, due to the definition of πbfs q variable – parent of the state q in the BFS traverse tree. Consequently, this implies that all states are used, by definition. In order to both reduce the search space by using the BFS constraints and allow the states to be unused, the former have to be modified in the following way. Modified variable πbfs-mod qj ∈{q0, q1, . . . , qj−1} (j ∈[2 .. C]) includes q0 in its domain, which represents the absence of a parent for the state qj ∈Q. Only unused states do not have a parent: label start label start D. FINDING A MINIMAL DISTRIBUTED CONTROLLER Ultimately, we want to infer the minimal distributed con- troller complying with the given specification L. Mini- mal models have more value in practice – they are much more comprehensible for humans and more efficient in ζqinit ∧ ^ q∈Q\{qinit}  ζq ↔ _ q∈Q k∈[1..K] (τq′,k = q)  . VOLUME 8, 2020 VOLUME 8, 2020 207492 K. Chukharev et al.: SAT-Based CEGIS of Distributed Controllers Algorithm 1: Distributed-CEGIS-min({S+(m)}, L, P) Input: set of positive scenario sets {S+(m)}m∈[1..M], LTL-specification L, parse tree size P. Output: automata {A(m)}m∈[1..M] complying with L. // Compound negative scenarios CS−←∅ // Common arguments (for “infer”) ∗∗∗:= ({S+(m)}, CS−, {C(m) = D}, P) label start // Estimate D for D ←1 to ∞do {A(m)} ←infer(∗∗∗) if {A(m)} ̸= null then break while true do // Minimize Cused {A(m)} ←infer(∗∗∗, Cused < ∞) if {A(m)} = null then goto start while {A(m)} ̸= null do Cused min ←numberOfUsedStates({A(m)}) {A(m)} ←infer(∗∗∗, Cused < Cused min ) // Minimize N used {A(m)} ←infer(∗∗∗, Cused = Cused min , N used < ∞) while {A(m)} ̸= null do N used min ← numberOfParseTreeNodes({A(m)}) {A(m)} ←infer(∗∗∗, Cused = Cused min , N used < N used min ) {A(m)} ←infer(∗∗∗, Cused = Cused min , N used = N used min ) CS− new ←performModelChecking({A(m)}, L) if CS− new = ∅then // No counterexamples return {A(m)} CS−←CS−∪CS− new they will be taken into account on the next iteration, else the CEGIS is done and {A(m)} is the satisfying solution. Algorithm 1: Distributed-CEGIS-min({S+(m)}, L, P) Input: set of positive scenario sets {S+(m)}m∈[1..M], LTL-specification L, parse tree size P. Output: automata {A(m)}m∈[1..M] complying with L. D. FINDING A MINIMAL DISTRIBUTED CONTROLLER // Compound negative scenarios CS−←∅ // Common arguments (for “infer”) ∗∗∗:= ({S+(m)}, CS−, {C(m) = D}, P) label start // Estimate D for D ←1 to ∞do {A(m)} ←infer(∗∗∗) if {A(m)} ̸= null then break while true do // Minimize Cused {A(m)} ←infer(∗∗∗, Cused < ∞) if {A(m)} = null then goto start while {A(m)} ̸= null do Cused min ←numberOfUsedStates({A(m)}) {A(m)} ←infer(∗∗∗, Cused < Cused min ) // Minimize N used {A(m)} ←infer(∗∗∗, Cused = Cused min , N used < ∞) while {A(m)} ̸= null do N used min ← numberOfParseTreeNodes({A(m)}) {A(m)} ←infer(∗∗∗, Cused = Cused min , N used < N used min ) {A(m)} ←infer(∗∗∗, Cused = Cused min , N used = N used min ) CS− new ←performModelChecking({A(m)}, L) if CS− new = ∅then // No counterexamples return {A(m)} CS−←CS−∪CS− new Additionally, the following constraint ensures that unused states have the largest indices: A. ABP DESCRIPTION The purpose of the ABP is to allow for a communication over an unreliable channel. The system shown in Fig. 6 con- sists of several modules, which interact with each other via message passing. The Sender and the Receiver modules 207493 VOLUME 8, 2020 K. Chukharev et al.: SAT-Based CEGIS of Distributed Controllers FIGURE 6. Alternating-bit protocol scheme. FIGURE 6. Alternating-bit protocol scheme. variables of this module have changed value. And it produces a CNF (confirmation) output event to notify the system that it has reacted on the request and modified values of its output variables accordingly. In fact, every event-oriented system can be transformed into a REQ/CNF-system, and the reverse transformation is also possible. Thus, Sender has the following interface: implement the protocol, and our goal is to infer finite-state FB models for them. There are also several environment mod- ules, which we model with NuSMV. Environment modules have the following semantics. • Forward Channel and Backward Channel transmit messages from Sender to Receiver and vice versa, respectively. Channels model unreliable transmission: messages can either be lost or duplicated. • Is = {REQ}; • Is = {REQ}; • Os = {CNF}; • Timer sends timeouts to Sender in order to force a mes- sage retransmission to cope with a lost message. • X s = {send, timeout, acknowledge, • X s = {send, timeout, acknowledge, • Sender Client and Receiver Client model protocol clients, which communicate with each other via the pro- tocol and expect reliable message transmission. input_bit}; • Zs = {done, packet, output_bit}. The Receiver module has the following interface: The Receiver module has the following interface: • Ir = {REQ}; More specifically, the modules communicate as follows. • Or = {CNF}; • Or = {CNF}; • Sender receives a SendMsg from Sender Client when- ever the latter wants to send a message, and replies with DoneMsg upon a successful message transmission. • X r = {packet, input_bit}; • Zr = {deliver, acknowledge, output_bit}. We transform safety and liveness monitors that are used in [16] to formally specify the ABP system into the following LTL properties. • Sender receives a TimeoutMsg from Timer and uses this information to retransmit messages over Forward Channel. First, we introduce four safety properties Lsafe which ensure the correct order between send and deliver, and between deliver and done. • Sender sends PacketMsg to Receiver via a Forward Channel. B. EXPERIMENTAL EVALUATION We apply the proposed Distributed-Cegis-min method to infer a minimal distributed controller complying with the given positive scenarios {S+(m)} and the LTL/specification L. For a model checker we used NuSMV [29]. For a SAT solver we used MiniSAT [41] through the native interface. Experiments were conducted on a computer with an Intel(R) Core(TM) i7-7700HQ CPU @ 3.40GHz and 32GB of RAM. It should be noted that the sizes of scenarios in Table 1 denote the number of execution states in the NuSMV simu- lation traces, which are generated randomly and may contain plenty of idle steps, resulting in effectively poorly informative scenarios. Since our designed liveness properties contain fairness assumptions, their verification – even for ‘‘small’’ mod- els – takes an excessive amount of time (typically, around 20 seconds, but sometimes it spans up to 3 minutes). This should be taken into an account, because the verification step is performed on each iteration of CEGIS. Hence, we split the specification and additionally perform the further described experiments using safety properties only. However, the results of such evaluations are less informative and can only be regarded as proof of CEGIS convergence. To sum up, the results in Table 1 indicate the effectiveness of the proposed Distributed-Cegis-min method on datasets of various sizes, especially if the given positive scenarios are sufficient to capture the desired behavior of the distributed system. y Models for Sender (left) and Receiver (right) modules of ABP synthesized from S10×50 with Lall using the proposed algorithm are shown in Fig. 7. By manually assessing the synthesized automata, we make sure that they indeed guar- antee the packet delivery. Obviously, by construction they comply with the scenarios and the LTL/specification. The automaton for the Sender module contains only three states, whereas manually constructed has four. Sender and Receiver have guard conditions of sizes N (s) = 13 and N (r) = 7, as opposed to 11 and 28 in the manually prepared automata. The synthesized automata are not easily understandable by human and operate in such a way that might seem unnatural. A. ABP DESCRIPTION This message is annotated with a single bit of information. 1) ¬(¬send U deliver): deliver can only happen after send. 1) ¬(¬send U deliver): deliver can only happen after send. • Receiver reports with DeliverMsg to Receiver Client upon a successful packet deliver. 2) G(send →X ¬(¬deliver U send)): two sends cannot occur in a row without a deliver in between. • Receiver sends AcknowledgeMsg to Sender via a Backward Channel. This message is annotated with a single bit of information and acknowledges the delivery of a PacketMsg annotated with the same bit. 3) ¬(¬deliver U done): done can only happen after deliver. 4) G(deliver → X ¬(¬done U deliver)): two deliver’s cannot occur in a row without a done in between. 4) G(deliver → X ¬(¬done U deliver)): two deliver’s cannot occur in a row without a done in between. For technical reasons we represent messages with input/output variables instead of direct representation with events. To store the bit annotating PacketMsg, we introduce variables input_bit and output_bit for both Sender and Receiver. We also introduce a special input event REQ and a special output event CNF (widely used in IEC 61499 [5]) with the following semantics. A module receives a REQ (request) event from the environment whenever some input Next, we augment the specification with three liveness prop- erties Llive. With P we denote the formula for fairness assumption under which we verify liveness properties: we do not take into account infinite execution paths with no timeout, and we ensure that the channels cannot lose 207494 VOLUME 8, 2020 K. Chukharev et al.: SAT-Based CEGIS of Distributed Controllers TABLE 1. Experimental results. TABLE 1. Experimental results. packets infinitely often. The first two properties guarantee the message delivery. The third one ensures that the Sender Client is not stale and at any moment in time it will eventually send a packet. 1) P →G(send →F deliver): any send is eventu- ally followed by deliver. 2) P →G(send →F done): any send is eventually followed by done. 3) P →GF send: send occurs infinitely often. Manually designed automata for Sender and Receiver com- plying with the described LTL/specification are shown in Fig. 6. B. EXPERIMENTAL EVALUATION Models for Sender (left) and Receiver (right) modules of ABP synthesized from S10×50 with Lall. scenarios, and remove its transitions one by one, each time running the completion tool to synthesize a single cor- rect complete solution. The results reported by the authors indicate that the initial incomplete protocol could be com- pleted in 65 iterations in 19 seconds. Further removal of transitions led to an increase of running time, and in the extreme case, when all 8 transitions from the incomplete protocol were removed, the tool execution was aborted after 4 hours. which ensure that the Receiver reacts with immediate AcknowledgeMsg to every PacketMsg. However, this is not a drawback of the proposed method, and the question of what should be considered a complete specification is beyond the scope of this article. B. EXPERIMENTAL EVALUATION For example, if Receiver receives a PacketMsg annotated with 0 in the state 3, it (1) produces an AcknowledgeMsg annotated with 1, and relies on the Sender, that it will handle this correctly (in fact it must handle this case, because mes- sages may duplicate); (2) changes its state to 1, and awaits for another PacketMsg from the Sender. Thus, Receiver behaves suboptimally. If optimal behavior is desired, the specification could be supplemented with the following LTL properties: A sufficient number of behavior examples allows inferring minimal models complying with the given LTL/specification in just several CEGIS iterations. In particular, 10 scenarios of length 25 or more are sufficient to cover all safety properties Lsafe so that the initial inference only from those scenarios produces satisfying automata – in Table 1 this corresponds to ‘‘#iter = 1’’. The liveness properties Llive are much harder to cover by positive scenarios alone, so the inference process requires multiple CEGIS iterations, but the size and number of scenarios still contribute a lot. The key observation is the following: if the initial automata constructed only from pos- itive scenarios have sizes equal (e.g., 3+4 within this exper- iment) to the sizes of the sought automata – which means that the given scenarios are sufficient to render the overall structure – then the CEGIS process will converge in several iterations. On the other hand, when the given positive sce- narios by themselves are insufficient (extreme case is when there are no positive scenarios at all) to capture the desired behavior, the CEGIS process resembles the enumerative LTL synthesis – in such cases, the proposed method is not very suitable. Example of such case is the evaluation on S1×50 – 1 scenario of length 50 – execution was aborted after 6 hours without a satisfying result. This confirms the clause in [16] that the problem of inference from scratch is quite challenging. G(pi →X ai), where p0 = packet ∧input_bit, p1 = packet ∧¬input_bit, a0 = acknowledge ∧output_bit, a1 = acknowledge ∧¬output_bit, G(pi →X ai), where p0 = packet ∧input_bit, p1 = packet ∧¬input_bit, a0 = acknowledge ∧output_bit, a1 = acknowledge ∧¬output_bit, 207495 VOLUME 8, 2020 K. Chukharev et al.: SAT-Based CEGIS of Distributed Controllers FIGURE 7. Models for Sender (left) and Receiver (right) modules of ABP synthesized from S10×50 with Lall. FIGURE 7. C. COMPARISON WITH THE PROTOCOL COMPLETION APPROACH Recall the protocol completion approach proposed by the authors of [16] for distributed synthesis. First, they use exe- cution scenarios to build incomplete processes (automata), which comprise the distributed protocol. Here, they assume that the scenarios are sufficient to cover all process states. Next, they try to complete those incomplete processes by adding some missing transitions, basically using an enumer- ative CEGIS approach. The main downside of this approach is that the initially derived states and transitions are fixed throughout the CEGIS process, which may (and should) lead to unsatisfiability results in practice. We can perform a set of experiments resembling the second experiment set and compare the outcomes. However note that since the target models of our research and of [16] are quite different, the following cannot be considered a one-to-one comparison. We will consider two sets of execution scenarios: S10×50 (10 scenarios, each of length 50) will play a role of ‘‘suffi- cient’’ set of scenarios, and S1×50 (1 scenario of length 50) will be considered ‘‘insufficient set of scenarios’’. Presum- ably, the larger scenario set size is, the more automaton state it will cover. Hence, using sufficient set of scenar- ios will result in a more efficient inferring with CEGIS. Inference of ABP only from ‘‘sufficient’’ scenarios results in the Sender with 3 states and the Receiver with 4 states. On the contrary, inference only from ‘‘insufficient’’ scenarios results in the Sender with only 2 states and the Receiver with only 3 states. Note that in both cases the resulting automata do not comply with the LTL/specification Lall, yet. As already shown in Table 1, the application of the proposed Distributed-Cegis-min method to the scenarios S10×50 allows inferring the satisfying ABP complying with Lall in just 2 iterations in 8 seconds. Ultimately, the goal of experiments reported in [16] is to synthesize ABP from scratch, by only specifying the number of states of the Sender and Receiver, i.e. by completing an incomplete protocol that has no defined transitions at all. Striving to reach this goal, authors of [16] performed two interesting sets of experiments. In the first set of experiments they start with the manually designed ABP, and remove transitions one by one (though, not all), consecutively asking the completion tool to synthe- size all possible completions. REFERENCES [1] B. Cornelissen, A. Zaidman, A. van Deursen, L. Moonen, and R. Koschke, ‘‘A systematic survey of program comprehension through dynamic analy- sis,’’ IEEE Trans. Softw. Eng., vol. 35, no. 5, pp. 684–702, Sep. 2009. [2] M. Shahbaz and R. Groz, ‘‘Analysis and testing of black-box component- based systems by inferring partial models,’’ Softw. Test., Verification Rel., vol. 24, no. 4, pp. 253–288, Jun. 2014. Observe that in [16] the process completion strictly requires a set of execution scenarios that cover all states of protocol processes. The approach for distributed synthesis proposed in the present paper, on the other hand, requires some representative behavior examples: ideally, covering all states as in [16], but not necessarily. [3] N. Walkinshaw and K. Bogdanov, ‘‘Inferring finite-state models with tem- poral constraints,’’ in Proc. 23rd IEEE/ACM Int. Conf. Automated Softw. Eng., Sep. 2008, pp. 248–257. [4] N. Walkinshaw, R. Taylor, and J. Derrick, ‘‘Inferring extended finite state machine models from software executions,’’ Empirical Softw. Eng., vol. 21, no. 3, pp. 811–853, Jun. 2016. [5] V. Vyatkin, ‘‘IEC 61499 as enabler of distributed and intelligent automa- tion: State-of-the-Art review,’’ IEEE Trans. Ind. Informat., vol. 7, no. 4, pp. 768–781, Nov. 2011. Also note that in our experiments we observed that model checking is taking 90-95 % of run time in each CEGIS itera- tion. This can be partially explained by the fact that we used a symbolic model checker NuSMV in our implementation. Research (e.g. [42]) indicates that in many cases explicit- /state model checking (e.g., using SPIN1) is much faster than symbolic model checking. Thus, if we switch to explicit-state model checking, the verification time may decrease, leading to better overall performance of our algorithm. Also note that in our experiments we observed that model checking is taking 90-95 % of run time in each CEGIS itera- tion. This can be partially explained by the fact that we used a symbolic model checker NuSMV in our implementation. [6] Programmable Controllers—Part 1: General Information, document IEC 61131-1:2003, 2003. [Online]. Available: https://webstore.iec.ch/ publication/4550 [7] M. J. H. Heule and S. Verwer, ‘‘Exact DFA identification using SAT solvers,’’ in Grammatical Inference: Theoretical Results and Applications. Berlin, Germany: Springer, 2010, pp. 66–79. [8] P. Faymonville, B. Finkbeiner, and L. Tentrup, ‘‘BoSy: An experimentation framework for bounded synthesis,’’ in Computer Aided Verification. Cham, Switzerland: Springer, 2017, pp. 325–332. [9] V. Ulyantsev, I. Buzhinsky, and A. REFERENCES Shalyto, ‘‘Exact finite-state machine identification from scenarios and temporal properties,’’ Int. J. Softw. Tools Technol. Transf., vol. 20, no. 1, pp. 35–55, Feb. 2018. The presented experimental evaluation was conducted on rather small examples, however, we observe a relatively short execution time (especially the synthesis time compared to the verification time), which indicates that the proposed approach would scale well on larger data. [10] V. Ulyantsev, I. Zakirzyanov, and A. Shalyto, ‘‘BFS-based symme- try breaking predicates for DFA identification,’’ in Language and Automata Theory and Applications. Cham, Switzerland: Springer, 2015, pp. 611–622. [11] G. Giantamidis and S. Tripakis, ‘‘Learning Moore machines from input– output traces,’’ in Formal Methods. Cham, Switzerland: Springer, 2019, pp. 291–309. 1http://spinroot.com/ C. COMPARISON WITH THE PROTOCOL COMPLETION APPROACH In the second set of experiments they start with the incomplete protocol built from a sufficient set of execution VOLUME 8, 2020 VOLUME 8, 2020 207496 207496 K. Chukharev et al.: SAT-Based CEGIS of Distributed Controllers The execution of the proposed method on S1×50 was aborted after several hours without a result. However, since we know the parameters (number of states C(m) and parse trees nodes N (m)) of the final ABP, we can specify them to the proposed method, which greatly reduces the search space. By initially specifying C(m) = 3 + 4 and N (m) = 13 + 7 we obtained ABP satisfying S1×50 and complying with Lall in 622 iterations in 27867 seconds. Note that most of this time was spend on model checking, not on SAT solving – verifica- tion time ranged from 10 to 200 seconds. A similar run, but only with safety properties Lsafe, completed in 565 iterations in 1231 seconds. approach generates a set of finite-state machines implement- ing the distributed controller, one for each designated ele- ment (module) of the distributed control system. Thought the proposed method itself is quite general, our imple- mentation reported in this article is specifically tailored to IEC 61499 function blocks, allowing direct application to synthesis of distributed controllers in an industrial format and their use in real industrial automation systems. Future research may include speeding up CEGIS by exten- sively employing the iterative SAT solving, e.g., by elim- inating the solver restarts after the minimization on each CEGIS iteration. Also, LTL properties during the CEGIS can be taken into account gradually, one by one, in the order of their complexity, which might reduce the total execution time – due to not spending time on the model checking on early iterations. Another research direction is the support of non-canonical ECCs – those with multiple output events in automata states. ACKNOWLEDGMENT ACKNOWLEDGMENT An extreme case of the problem is to infer automata from LTL/specification only, which makes CEGIS practi- cally infeasible, because it is much harder to describe the automaton with a set of negative scenarios only, than with a set of positive scenarios. In other words, positive scenar- ios describe what a system should do, while negative sce- narios describe what it should not do. Naturally, the state space of undesired behaviors is much larger than that of desired ones. (Konstantin Chukharev and Dmitrii Suvorov contributed equally to this work.) VIII. DISCUSSION The experiments described in the previous section show that the synthesis works most effectively when supplied with a sufficient set of positive scenarios (e.g. S10×50), resulting in a single CEGIS iteration for Lsafe and a few more for Lall. The solving time increases as we decrease the total size of scenarios, resulting in a timeout for S1×50. IX. CONCLUSION AND FUTURE WORK van Maaren, and T. Walsh, Eds., Handbook of Sat- isfiability (Frontiers in Artificial Intelligence and Applications). vol. 185. Amsterdam, The Netherlands: IOS Press, 2009, p. 980 DANIIL CHIVILIKHIN received the bachelor’s and master’s degrees in applied mathematics and informatics and the Ph.D. degree in technical sci- ences (mathematics and software for computing systems) from ITMO University, Saint Petersburg, Russia, in 2011, 2013, and 2015, respectively. He is currently an Associate Professor with the Computer Technologies Laboratory, ITMO Uni- versity. His research interests include program synthesis and verification, industrial informatics, evolutionary algorithms, and SAT solver applications. DANIIL CHIVILIKHIN received the bachelor’s and master’s degrees in applied mathematics and informatics and the Ph.D. degree in technical sci- ences (mathematics and software for computing systems) from ITMO University, Saint Petersburg, Russia, in 2011, 2013, and 2015, respectively. DANIIL CHIVILIKHIN received the bachelor’s and master’s degrees in applied mathematics and informatics and the Ph.D. degree in technical sci- ences (mathematics and software for computing systems) from ITMO University, Saint Petersburg, Russia, in 2011, 2013, and 2015, respectively. [29] A. Cimatti, E. Clarke, F. Giunchiglia, and M. Roveri, ‘‘NUSMV: A new symbolic model checker,’’ Int. J. Softw. Tools for Technol. Transf. (STTT), vol. 2, no. 4, pp. 410–425, Mar. 2000. [30] A. Solar-Lezama, ‘‘Program sketching,’’ Int. J. Softw. Tools Technol. Trans., vol. 15, no. 5, pp. 475–495, Oct. 2013. He is currently an Associate Professor with the Computer Technologies Laboratory, ITMO Uni- versity. His research interests include program synthesis and verification, industrial informatics, evolutionary algorithms, and SAT solver applications. pp [31] A. Pneuli and R. Rosner, ‘‘Distributed reactive systems are hard to synthe- size,’’ in Proc. 31st Annu. Symp. Found. Comput. Sci., vol. 2, Oct. 1990, pp. 746–757. pp [32] A. Solar-Lezama, L. Tancau, R. Bodik, S. Seshia, and V. Saraswat, ‘‘Com- binatorial sketching for finite programs,’’ ACM SIGARCH Comput. Archit. News, vol. 34, no. 5, pp. 404–415, Oct. 2006. [33] R. Alur, M. Martin, M. Raghothaman, C. Stergiou, S. Tripakis, and A. Udupa, ‘‘Synthesizing finite-state protocols from scenarios and require- ments,’’ in Hardware and Software: Verification and Testing. Cham, Switzerland: Springer, 2014, pp. 75–91. VALERIY VYATKIN (Senior Member, IEEE) received the Ph.D. and Dr.Sc. degrees in applied computer science from the Taganrog Radio Engi- neering Institute, Russia, in 1992 and 1999, respectively, the Dr.Eng. degree from the Nagoya Institute of Technology, Japan, in 1999, and the Habilitation degree in Germany, in 2002. [34] A. IX. CONCLUSION AND FUTURE WORK g y p [21] V. Dubinin and V. Vyatkin, ‘‘Towards a formal semantic model of IEC [21] V. Dubinin and V. Vyatkin, ‘‘Towards a formal semantic model of IEC 61499 function blocks,’’ in Proc. IEEE Int. Conf. Ind. Informat., Aug. 2006, pp. 6–11. [21] V. Dubinin and V. Vyatkin, ‘‘Towards a formal semantic model of IEC 61499 function blocks,’’ in Proc. IEEE Int. Conf. Ind. Informat., Aug. 2006, y 61499 function blocks,’’ in Proc. IEEE Int. Conf. Ind. Informat., Aug. 2006, pp. 6–11. [22] D. Chivilikhin, S. Patil, K. Chukharev, A. Cordonnier, and V. Vyatkin, ‘‘Automatic state machine reconstruction from legacy PLC using data collection and SAT solver,’’ IEEE Trans. Ind. Informat., vol. 16, no. 12, pp. 7821–7831, Dec. 2020. DMITRII SUVOROV received the bachelor’s and master’s degrees in applied mathematics and infor- matics from ITMO University, Saint Petersburg, Russia, in 2016 and 2018, respectively, where he is currently pursuing the Ph.D. degree with the Computer Technologies Laboratory. He has worked as a Software Engineer. He is currently a Junior Research Associate with the Computer Technologies Laboratory, ITMO Uni- versity. He also teaches discrete mathematics. His research interests include programming languages, formal verification, and distributed systems. DMITRII SUVOROV received the bachelor’s and master’s degrees in applied mathematics and infor- matics from ITMO University, Saint Petersburg, Russia, in 2016 and 2018, respectively, where he is currently pursuing the Ph.D. degree with the Computer Technologies Laboratory. [23] Z. Manna and A. Pnueli, Temporal Verification of Reactive Systems: Safety. Berlin, Germany: Springer-Verlag, 1995, p. 512. y p g g p [24] E. M. Clarke, O. Grumberg, and D. Peled, Model Checking. Cambridge, MA, USA: MIT Press, 1999, p. 330. He has worked as a Software Engineer. He is currently a Junior Research Associate with the Computer Technologies Laboratory, ITMO Uni- versity. He also teaches discrete mathematics. His research interests include programming languages, formal verification, and distributed systems. , , , p [25] S. A. Cook, ‘‘The complexity of theorem-proving procedures,’’ in Proc. 3rd Annu. ACM Symp. Theory Comput. (STOC), 1971, pp. 151–158. [26] L. A. Levin, ‘‘Universal sequential search problems,’’ Problems Inf. Trans- miss., vol. 9, no. 3, pp. 265–266, 1973. [27] J. Marques-Silva, I. Lynce, and S. Malik, ‘‘Conflict-driven clause learning SAT solvers,’’ in Handbook of Satisfiability, (Frontiers in Artificial Intel- ligence and Applications), vol. 185. Amsterdam, The Netherlands: IOS Press, 2009, pp. 131–153. [28] A. Biere, M. Heule, H. IX. CONCLUSION AND FUTURE WORK [12] F. Avellaneda and A. Petrenko, ‘‘FSM inference from long traces,’’ in Formal Methods. Cham, Switzerland: Springer, 2018, pp. 93–109. We have developed a complete SAT-based counterexample- guided approach for the synthesis of a distributed finite-state controller from given behavior examples of a legacy system and formal specification in the form of LTL properties. The [13] C.-H. Cheng, C.-H. Huang, H. Ruess, and S. Stattelmann, ‘‘G4LTL-ST: Automatic generation of PLC programs,’’ in Computer Aided Verification. Cham, Switzerland: Springer, 2014, pp. 541–549. [14] R. Smetsers, P. Fiterău-Broştean, and F. Vaandrager, ‘‘Model learning as a satisfiability modulo theories problem,’’ in Language and Automata The- ory and Applications. Cham, Switzerland: Springer, 2018, pp. 182–194. VOLUME 8, 2020 207497 K. Chukharev et al.: SAT-Based CEGIS of Distributed Controllers KONSTANTIN CHUKHAREV received the bache- lor’s degree in control systems and informatics and the master’s degree in applied mathematics and informatics from ITMO University, Saint Peters- burg, Russia, in 2018 and 2020, respectively, where he is currently pursuing the Ph.D. degree with the Computer Technologies Laboratory. He finished the one-year program ‘‘Algorithmic Bioinformatics’’ at the Bioinformatics Institute, Saint Petersburg, in 2017. He is currently a Junior Research Associate with the Computer Technologies Laboratory, ITMO University. He studies formal methods, software engineering, SAT and phy- logenetics, while teaching students discrete mathematics and working on his Ph.D. degree. [15] E. M. Gold, ‘‘Complexity of automaton identification from given data,’’ Inf. Control, vol. 37, no. 3, pp. 302–320, Jun. 1978. KONSTANTIN CHUKHAREV received the bache- lor’s degree in control systems and informatics and the master’s degree in applied mathematics and informatics from ITMO University, Saint Peters- burg, Russia, in 2018 and 2020, respectively, where he is currently pursuing the Ph.D. degree with the Computer Technologies Laboratory. pp [16] R. Alur and S. Tripakis, ‘‘Automatic synthesis of distributed protocols,’’ ACM SIGACT News, vol. 48, no. 1, pp. 55–90, Mar. 2017. [17] I. Buzhinsky and V. Vyatkin, ‘‘Automatic inference of finite-state plant models from traces and temporal properties,’’ IEEE Trans. Ind. Informat., vol. 13, no. 4, pp. 1521–1530, Aug. 2017. pp g [18] K. Chukharev and D. Chivilikhin, ‘‘FbSAT: Automatic inference of min- imal finite-state models of function blocks using SAT solver,’’ 2019, arXiv:1907.03285. [Online]. Available: http://arxiv.org/abs/1907.03285 p g [19] fbSAT. Accessed: Nov. 12, 2020. [Online]. Available: http://www.github. com/ctlab/fbSAT [20] J. Kurose and K. Ross, Computer Networking: A Top-down Approach, 5th ed. Reading, MA, USA: Addison-Wesley, 2009, p. 862. IX. CONCLUSION AND FUTURE WORK Petrenko, F. Avellaneda, R. Groz, and C. Oriat, ‘‘FSM inference and checking sequence construction are two sides of the same coin,’’ Softw. Qual. J., vol. 27, no. 2, pp. 651–674, Jun. 2019. pp [35] T. Walsh, ‘‘SAT υ CSP,’’ in Proc. 6th Int. Conf. Princ. Pract. Constraint Program. Berlin, Germany: Springer, 2000, pp. 441–456. [36] G. S. Tseytin, ‘‘On the complexity of derivation in propositional calculus,’’ in Automation of Reasoning: 2: Classical Papers on Computational Logic 1967–1970. Berlin, Germany: Springer, 1983, pp. 466–483. He was a Visiting Scholar with Cambridge Uni- versity, U.K., and had permanent appointments with the University of Auckland, New Zealand; and Martin Luther University, Germany, as well as in Japan and Russia. He is currently on joint appointment as the Chair of Dependable Compu- tations and Communications, Luleå University of Technology, Sweden, and a Professor of information technology in automation with Aalto University, Finland. He is also the Co-Director of the International Research Laboratory ‘‘Computer Technologies,’’ ITMO University, Saint Petersburg, Russia. His research interests include dependable distributed automation and industrial informatics; software engineering for industrial automation systems; artifi- cial intelligence; distributed architectures; and multi-agent systems in vari- ous industries: smart grid, material handling, building management systems, datacenteres, and reconfigurable manufacturing. [37] I. Zakirzyanov, A. Morgado, A. Ignatiev, V. Ulyantsev, and J. Marques-Silva, ‘‘Efficient symmetry breaking for SAT-based minimum DFA inference,’’ in Language and Automata Theory and Applications. Springer, 2019, pp. 159–173. [38] O. Bailleux and Y. Boufkhad, ‘‘Efficient CNF encoding of Boolean cardi- nality constraints,’’ in Principles and Practice of Constraint Programming. Berlin, Germany: Springer, 2003, pp. 108–122. [39] G. Holzmann, The SPIN Model Checker: Primer Reference Manual. Reading, MA, USA: Addison-Wesley, 2004, p. 608. g y p [40] N. Lynch, Distributed Algorithms. San Mateo, CA, USA: Morgan Kaufmann, 1996. g [41] N. Eén and N. Sörensson, ‘‘An extensible SAT-solver,’’ in Theory and Applications of Satisfiability Testing. Berlin, Germany: Springer, 2003, pp. 502–518. Dr. Vyatkin received the Andrew P. Sage Award for the Best IEEE TRANSACTIONS paper in 2012. He is the Chair of IEEE IES Technical Com- mittee on Industrial Informatics. pp [42] I. Buzhinsky, A. Pakonen, and V. Vyatkin, ‘‘Explicit-state and symbolic model checking of nuclear I&C systems: A comparison,’’ in Proc. 43rd Annu. Conf. IEEE Ind. Electron. Soc., Oct./Nov. 2017, pp. 5439–5446. VOLUME 8, 2020 207498

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⁶⁸Ga-DOTATOC PET/CT to detect immune checkpoint inhibitor-related myocarditis

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ABSTRACT To cite: Boughdad S, Latifyan S, Fenwick C, et al. 68Ga-­DOTATOC PET/CT to detect immune checkpoint inhibitor-­related myocarditis. Journal for ImmunoTherapy of Cancer 2021;9:e003594. doi:10.1136/ jitc-2021-003594 assess the full extent of the disease. In contrast, four patients with CMR imaging had negative findings despite having elevated serum cTnI levels (range 20.5–5896.1 ng/ mL), thus defining possible myocarditis. Newly identified immune correlates could provide specific biomarkers for the diagnosis of ICI-­related myocarditis. Most tested patients (six of seven patients) had serum increases in the inflammatory cytokine interleukin (IL)-­6 and in the chemokines CXCL9, CXCL10, and CXCL13, and the mass cytometry phenotypes of immune cell populations in the blood also showed correlations with myocardial inflammation. Four of five patients with myocarditis exhibited a Th1/Th2 imbalance favoring a pronounced inflammatory Th1, Th1/Th17, and Th17 CD4 memory T-­cell response. The high proportion of non-­classical monocytes and significantly reduced levels of CD31 in four to five patients was also consistent with an inflammatory disease. Conclusion  The use of 68Ga-­DOTATOC PET/CT along with immune correlates is a highly sensitive method to detect ICI-­related myocarditis especially in the early stage of myocardial inflammation, as patients with elevated cTnI may present normal CMR imaging results. 68Ga-­DOTATOC PET/CT is also useful for detecting concomitant myositis. These results need to be confirmed in a larger population of patients and validated against a histological gold standard if available. Background  Immune checkpoint inhibitor (ICI)-­related myocarditis is a rare but potentially fatal adverse event that can occur following ICI exposure. Early diagnosis and treatment are key to improve patient outcomes. Somatostatin receptor-­based positron emission tomography–CT (PET/CT) showed promising results for the assessment of myocardial inflammation, yet information regarding its value for the diagnosis of ICI-­related myocarditis, especially at the early stage, is limited. Thus, we investigated the value of 68Ga-­DOTA(0)-­Phe(1)-­Tyr(3)-­ octreotide (68Ga-­DOTATOC) PET/CT for the early detection and diagnosis of ICI-­related myocarditis. ►Additional supplemental material is published online only. To view, please visit the journal online (http://​dx.​doi.​org/​10.​ 1136/​jitc-​2021-​003594). Methods  Consecutive patients with clinically suspected ICI-­related myocarditis from July 2018 to February 2021 were retrospectively evaluated in this single-­center study. All patients underwent imaging for the detection of ICI-­related myocarditis using either cardiac magnetic resonance (CMR) imaging or 68Ga-­DOTATOC PET/CT. Original research Original research 68Ga-­DOTATOC PET/CT to detect immune checkpoint inhibitor-­related myocarditis Sarah Boughdad  ‍ ‍ ,1 Sofiya Latifyan,2 Craig Fenwick,3 Hasna Bouchaab,2 Madeleine Suffiotti,3 Javid J Moslehi,4 Joe-­Elie Salem  ‍ ‍ ,5 Niklaus Schaefer  ‍ ‍ ,1 Marie Nicod-­Lalonde  ‍ ‍ ,1 Julien Costes  ‍ ‍ ,6 Matthieu Perreau,3 Olivier Michielin,2 Solange Peters,2 John O Prior  ‍ ‍ ,1 Michel Obeid  ‍ ‍ 3 ABSTRACT PET/ CT images were acquired 90 min after the injection of 2 MBq/kg 68Ga-­DOTATOC with pathological myocardial uptake in the left ventricle (LV) suggestive of myocarditis defined using a myocardium-­to-­background ratio of peak standard uptake value to mean intracavitary LV standard uptake (MBRpeak) value above 1.6. Patients had a full cardiological work-­up including ECG, echocardiography, serum cardiac troponin I (cTnI), cardiac troponin T and creatine kinase (CK), CK-­MB. Endomyocardial biopsy and inflammatory cytokine markers were also analyzed. The detection rate of ICI-­related myocarditis using 68Ga-­ DOTATOC PET/CT and CMR was assessed. ►Additional supplemental material is published online only. To view, please visit the journal online (http://​dx.​doi.​org/​10.​ 1136/​jitc-​2021-​003594). To cite: Boughdad S, Latifyan S, Fenwick C, et al. 68Ga-­DOTATOC PET/CT to detect immune checkpoint inhibitor-­related myocarditis. Journal for ImmunoTherapy of Cancer 2021;9:e003594. doi:10.1136/ jitc-2021-003594 Accepted 27 September 2021 Open access cannot exclude the progression toward major cardiac complications.18 findings suggest that the uncontrolled activation of auto- reactive T cells may occur in patients with ICI-­related myocarditis. Interestingly, cardiomyocyte PD-­L1 expres- sion is upregulated in cardiac injury and inflammation, suggesting that PD-­L1 signaling might have a cardio- protective immunomodulatory effect.7 Early detection, timely intervention, and the initiation of adequate immu- nosuppressive (IS) treatment, can reduce mortality and improve the prognosis of ICI-­related myocarditis. The measurement of troponin I serum levels has also yielded promising results for the early diagnosis of ICI-­associated myocarditis, with a specificity and predictive value of 95% and 62%, respectively, for cardiac involvement in patients with myositis.8–10 p Molecular imaging could be a valuable addition when the diagnosis of ICI-­related myocarditis remains possible but not definite myocarditis after a complete cardiac work-­ up, as we previously reported in a patient with refractory, severe ICI-­associated myocarditis.2 Bonaca et al proposed a definition of myocarditis for application in clinical trials in the setting of cancer therapeutics, and they included 18F-­fluorodeoxyglucose (FDG) positron emission tomog- raphy–CT (PET/CT) imaging as a potential tool to define probable myocarditis.19 However, it is also known that somatostatin receptor is highly expressed by inflam- matory cells.20 Somatostatin receptor-­based PET/CT such as the 68Ga-­labeled somatostatin analog 68Ga-­DOTA(0)-­ Phe(1)-­Tyr(3)-­octreotide (68Ga-­DOTATOC) PET/CT also showed interesting results for the assessment of myocardium inflammation with a close spatial correlation with CMR in a study by Lapa et al.20 In this context, we present our results showing that imaging with 68Ga-­DO- TATOC PET/CT, and the correlation with some specific cytokine correlates might be beneficial in the diagnostic work-­up of ICI-­related myocarditis. y The diagnosis of myocarditis is complex and is based on the clinical picture and the results of ECG, transthoracic echocardiography, cardiac biomarkers, and exclusion of acute coronary syndrome using coronary angiography. Open access The gold standard for definite myocarditis remains the endomyocardial biopsy (EMB) but the value of cardiac magnetic resonance (CMR) for a non-­invasive diagnosis of myocarditis and monitoring disease progression is now recognized.11 The CMR features indicating myocar- ditis are the combination of myocardial edema (using T2-­weighted imaging), myocardial hyperemia, and myocardial necrosis/fibrosis following a non-­ischemic distribution (using late-­gadolinium enhancement (LGE) imaging according to the initial Lake Louise criteria.12 Tissue characterization using parametric imaging (T1 mapping, extracellular volume calculation (ECV), and T2 mapping) allowed further improvement of the diag- nostic accuracy of CMR by combining the parameters of myocardial edema (T2-­weighted imaging or T2 mapping) and non-­ischemic injury (LGE, ECV, or T1 mapping).13 14 Imaging also plays an important role in the detection of ICI-­related myocarditis, although currently supported by less evidence.15 Interestingly, in a series of 103 patients with ICI-­related myocarditis assessed with CMR, LGE was observed in only 48%, and T2-­weighted imaging was positive in only 28%, indicating a lower than expected sensitivity of CMR, especially when CMR was performed early (<4 days) after admission. Furthermore, neither LGE nor T2-­weighted imaging was associated with major adverse cardiac events (MACEs).16 On the other hand, using modern parametric imaging in a cohort of 136 patients with ICI-­related myocarditis, Thavendiranathan et al reported that the criteria of non-­ischemic injury was present in 95% and the criteria of myocardial edema in 53% of the patients. Increased T1 values alone were found in 78% of myocarditis cases and had an excel- lent discriminatory value for subsequent major cardiac events.17 Myocardial tissue characterization therefore appears as a critical step in the assessment of ICI-­related myocarditis: in a series of 35 patients with ICI-­related myocarditis, 46% of the patients experienced MACE in the follow-­up and the majority of them had normal left ventricle (LV) ejection fraction at admission, high- lighting the fact that an initial normal LV function alone The diagnosis of myocarditis is complex and is based on the clinical picture and the results of ECG, transthoracic echocardiography, cardiac biomarkers, and exclusion of acute coronary syndrome using coronary angiography. Patient population and diagnostic work-up h l ll p p g p Consecutive patients with clinically suspected ICI-­ associated myocarditis from July 2018 to February 2021 at our institution were included in this study. All patients underwent a cardiological work-­up including cardiac biomarkers (serum cardiac troponin I (cTnI), cardiac troponin T, creatine kinase (CK), CK-­MB and inflam- matory cytokine markers when available), 12-­lead ECG, transthoracic echocardiography (TTE). Most patients underwent CMR imaging for the detection of ICI-­ associated myocarditis. The results of EMB and coronary angiography were also part of the diagnostic work-­up, when available. Based on this assessment, the probability of ICI-­related myocarditis was defined according to the recommendations of Bonaca et al as definite, probable or possible. Imaging also plays an important role in the detection of ICI-­related myocarditis, although currently supported by less evidence.15 Interestingly, in a series of 103 patients with ICI-­related myocarditis assessed with CMR, LGE was observed in only 48%, and T2-­weighted imaging was positive in only 28%, indicating a lower than expected sensitivity of CMR, especially when CMR was performed early (<4 days) after admission. Furthermore, neither LGE nor T2-­weighted imaging was associated with major adverse cardiac events (MACEs).16 On the other hand, using modern parametric imaging in a cohort of 136 patients with ICI-­related myocarditis, Thavendiranathan et al reported that the criteria of non-­ischemic injury was present in 95% and the criteria of myocardial edema in 53% of the patients. Increased T1 values alone were found in 78% of myocarditis cases and had an excel- lent discriminatory value for subsequent major cardiac events.17 Myocardial tissue characterization therefore appears as a critical step in the assessment of ICI-­related myocarditis: in a series of 35 patients with ICI-­related myocarditis, 46% of the patients experienced MACE in the follow-­up and the majority of them had normal left ventricle (LV) ejection fraction at admission, high- lighting the fact that an initial normal LV function alone INTRODUCTION Immune checkpoint inhibitors (ICIs), PD-­1, PD-­L1 and CTLA-­4 have been approved in a wide variety of cancers; hence, the optimal and timely management of immune-­related adverse events (irAEs), especially life-­ threatening toxicities, is required.1–3 ICI-­ related myocarditis is a rare but potentially lethal irAE with an incidence of 0.5% to 1.0% but a high mortality rate of up to 60%.4 5 The pathophysiology of ICI-­related myocarditis has not been clearly elicited, but postmortem assessments of fatal cases have confirmed myocarditis with substantial inflammatory cell infiltrate characterized by the presence of both CD4-­positive and CD8-­positive T cells and macrophages but not B cells.6 These Results  A total of 11 patients had clinically suspected ICI-­related myocarditis; 9 underwent 68Ga -DOTATOC PET/ CT. All nine (100%) patients with 68Ga-­DOTATOC PET/CT presented with pathological myocardial uptake in the LV that was suggestive of myocarditis (MBRpeak of 3.2±0.8, range 2.2–4.4). Eight patients had CMR imaging and 3/8 (38%) patients had lesions evocative of myocarditis. All PET-­positive patients were previously treated with a high dose of steroids and intravenous immunoglobulin prior to PET/CT had elevated serum cTnI except for one patient for whom PET/CT was delayed several days. Interestingly, in 5/6 (83%) patients who presented with concomitant myositis, pathological uptake was seen on whole-­body 68Ga-­DOTATOC PET/CT images in the skeletal muscles, suggesting an additional advantage of this method to Correspondence to Dr Michel Obeid; ​michel.​obeid@​chuv.​ch 1 Boughdad S, et al. J Immunother Cancer 2021;9:e003594. doi:10.1136/jitc-2021-003594 Open access Open access The gold standard for definite myocarditis remains the endomyocardial biopsy (EMB) but the value of cardiac magnetic resonance (CMR) for a non-­invasive diagnosis of myocarditis and monitoring disease progression is now recognized.11 The CMR features indicating myocar- ditis are the combination of myocardial edema (using T2-­weighted imaging), myocardial hyperemia, and myocardial necrosis/fibrosis following a non-­ischemic distribution (using late-­gadolinium enhancement (LGE) imaging according to the initial Lake Louise criteria.12 Tissue characterization using parametric imaging (T1 mapping, extracellular volume calculation (ECV), and T2 mapping) allowed further improvement of the diag- nostic accuracy of CMR by combining the parameters of myocardial edema (T2-­weighted imaging or T2 mapping) and non-­ischemic injury (LGE, ECV, or T1 mapping).13 14 Echocardiographic and CMR imaging protocol Echocardiographic and CMR imaging protocol Complete echocardiographic imaging was performed using either a Vivid E9 (GE Healthcare) or an Epic V.7 (Philips Healthcare) according to the current guide- lines.21 Biplane Simpson method was used to calculate LV volumes, and the threshold for normal LV ejection frac- tion was ≥52% in men and ≥54% in women. The presence of regional wall motion abnormalities was specifically assessed by experienced board-­certified cardiologists. ECG-­gated CMR was performed on a 1.5 T MAGNETOM Sola scanner (Siemens Healthcare) with a 32-­channel phased-­array surface receiver coil. Imaging protocol for suspected myocarditis included cine images acquired with breath-­hold steady-­state free precession sequences in short-­axis orientation from base to apex (slice thickness Boughdad S, et al. J Immunother Cancer 2021;9:e003594. doi:10.1136/jitc-2021-003594 2 Open access 8 mm, no gap) and in three long axis orientation (two-­ chamber, three-­chamber and four-­chamber orientations). Edema detection was performed using T2 mapping acquisitions in short-­axis orientation from the base to the apex of the LV. Normal values for T2 relaxation time were defined on a segment basis in our center and edema was considered present when the myocardial T2 relaxation time was superior to 2 SDs above the local normal values. T1 mapping sequences were performed both before and after contrast injection, but they were acquired only on a single basal short-­axis slice orientation. Ten minutes after the administration of a 0.2 mmol/kg intravenous bolus of Gadobutrol (Gadovist, Bayer Healthcare), LGE images were acquired using a two-­dimensional breath-­hold phase-­sensitive segmented inversion–recovery gradient echo pulse sequence in the same orientations as the cine images. Experienced (European Association of Cardio- vascular Imaging level 3) cardiologists visually assessed the presence and distribution of myocardial lesions on LGE images. (IFN)-α, IFN-γ, and tumor necrosis factor (TNF)-α), chemokines (eg, CCL2 (MCP-­1), CCL3 (MIP-­1α), CCL4 (MIP-­1β), CCL5 (RANTES), CCL11 (Eotaxin), CXCL1 (GRO-α/KC), CXCL8 (IL-­8), CXCL9 (MIG), CXCL10 (IP-­10), CXCL12 (SDF-­1α), CXCL13 (BLC), and TNF-­beta), and growth factors (eg, NGF-­beta, BDNF, EGF, FGF-­2, HGF, LIF, PDGF-­BB, PlGF-­1, SCF, VEGF-­A, VEGF-­D, BAFF, GM-­CSF, and G-­CSF) were determined by multiplex bead assays as previously described.23 The reference values of these 49 serum markers were defined on the basis of the results obtained from 450 healthy subjects. The references are age-­adjusted values and vali- dated by the Centre Hospitalier Universitaire Vaudois (CHUV) Immunology and Allergy diagnostics platforms for clinical routine practice. Mass cytometry (CyTOF) Mass cytometry (CyTOF) As described previously,24 25 fresh blood samples were mixed with 1× red blood cell lysis buffer (eBiosci- ences) to remove erythrocytes according to the manu- facturer’s protocol. Pelleted cells were stained using metal-­conjugated antibodies according to the CyTOF manufacturer’s instructions (Fluidigm, San Francisco, California, USA). Through direct staining in whole blood, patient immune cell distributions will be compared with age-­adjusted values for 450 normal healthy donors and longitudinal changes were monitored over the course of the ICI therapy. The CyTOF panel will consist of 28 markers for cell surface staining and up to 12 intracellular markers to evaluate the accumulation of intracellular cytokines and cytotoxic molecules. The panel included conjugated antibodies against CD45 141 Pr, CD3 154 Sm, CD4 115 In, CD8 115 In, CD19 142 Nd, CD27 155 Gd, CCR7 159 Tb, CD14 160 Gd, CD11c 162 Dy, CXCR3 163 Dy, CD38 167 Er, CD25 169 Tm, HLA-­DR 174 Yb, PD-­1 175 Lu, and CD16 209 Bi (Fluidigm. Cells were subsequently washed with cell staining medium and phosphate-­buffered saline (PBS), fixed with 2.4% formaldehyde (Thermo Fisher) in PBS for 5 min and then resuspended in DNA interca- lation solution (PBS, 1 µM Ir-­Intercalator, 1% formalde- hyde, 0.3% saponin) and stored at 4°C until analysis. For CyTOF analysis, cells were washed three times with Milli-­Q water and resuspended at 0.5×10E6 cells/mL in 0.1% EQ Four Element Calibration Beads solution (Fluidigm). Samples were acquired on a Helios instrument at a flow rate of 30 µL/min. Flow Cytometry Standard files were normalized for the EQ bead intensities using MATLAB Echocardiographic and CMR imaging protocol ROC curves were generated to evaluate the discriminative ability of each cytokine to separate patients with ICI-­related myocarditis from controls. The areas under the ROC curves (AUC values), sensitivity, specificity, positive predictive value (ppv), negative predictive value (npv) for cytokines of interest were calculated. In addition, principal component anal- ysis (PCA) was performed to evaluate the variability and to determine which markers mostly contribute to the separation of control and ICI-­related myocarditis groups. All calculations were performed using R software V.3.6.3 and prcomp function for PCA analysis. 8 mm, no gap) and in three long axis orientation (two-­ chamber, three-­chamber and four-­chamber orientations). Edema detection was performed using T2 mapping acquisitions in short-­axis orientation from the base to the apex of the LV. Normal values for T2 relaxation time were defined on a segment basis in our center and edema was considered present when the myocardial T2 relaxation time was superior to 2 SDs above the local normal values. T1 mapping sequences were performed both before and after contrast injection, but they were acquired only on a single basal short-­axis slice orientation. Ten minutes after the administration of a 0.2 mmol/kg intravenous bolus of Gadobutrol (Gadovist, Bayer Healthcare), LGE images were acquired using a two-­dimensional breath-­hold phase-­sensitive segmented inversion–recovery gradient echo pulse sequence in the same orientations as the cine images. Experienced (European Association of Cardio- vascular Imaging level 3) cardiologists visually assessed the presence and distribution of myocardial lesions on LGE images. Metabolic imaging with 68Ga -DOTATOC PET/CT 68 g g 68Ga-­DOTATOC PET/CT was obtained from Biograph Vision Siemens (Erlangen, Germany). Thirty-­nine patients with neuroendocrine tumors that underwent 68Ga-­DOTATOC PET/CT in a diagnosis or follow-­up setting without pathological uptake on visual analysis were used as a control group. Pathological uptake in the myocardium of different regions of the LV, namely, the free wall, septum and apex, was calculated and measured with a previously defined threshold of 1.6 for the ratio between myocardium standardized uptake value (SUV) and the average left ventricular SUV (myocardium-­to-­ background ratio or myocardium-­to-­background ratio of peak standard uptake value to mean intracavitary LV stan- dard uptake (MBRpeak)), in agreement with the literature and our previous work in the field.22 On visual analysis, we described a pathological uptake of 68Ga-­DOTATOC seen on the three regions of the myocardium (apex, septum, and free wall) as ‘patchy diffuse’, whereas a less extensive pathological uptake was described as ‘heterogeneous’. We compared ratio values calculated on whole body 68Ga-­DO- TATOC PET/CT acquisitions between patients with clinically suspected ICI-­associated myocarditis and the control group using non-­parametric Mann-­Withney test and performed a receiver operating characteristic (ROC) analysis for determining the presence of a myocarditis.19 Patient characteristics Eleven consecutive patients (1 woman and 10 men) with various cancers treated with ICIs were admitted to the hospital for suspected ICI-­related myocarditis. Nine of them had a 68Ga-­DOTATOC PET/CT performed in the diagnostic work-­up and were included in the study (online supplemental figure S1A). The clinical characteristics of the patients are presented in table 1. The mean age of the patients (n=11) who developed ICI-­associated myocarditis was 71 years±11 (table  1). Before myocarditis, the pre-­ ICI left ventricular ejection fraction (LVEF) was normal, ≥52% in men and ≥54% in women in 10 out of the 11 patients with a baseline measurement. Six patients out of 11 (45%) received ICIs in the setting of melanoma, and 2 patients were on their second line of ICIs when ICI-­related myocarditis was diagnosed. Six patients (54%) received a combination of the ICIs ipilimumab–nivolumab. Overall, myocarditis occurred more commonly (54%) in patients receiving a combination of ICIs at the time of presenta- tion. All patients had experienced at least another ICI-­ related side effect: myositis (80%, 8 of 11), including 2 with ocular myositis without myasthenia gravis, hepatitis (18%) and colitis (18%) (table  1). For all the patients included in this study, ICI treatment was interrupted once the diagnosis of ICI-­associated myocarditis was suspected, and appropriate treatment with steroids and additional IS drugs was initiated in the case of corticosteroid (CS)-­ refractory myocarditis. g 68Ga-­DOTATOC PET/CT was performed on an average of 5.4 days (1–13 days) after the onset of clinical symptoms, with pathological uptake detected in 100% of patients with elevated troponin T, 100% of patients with elevated NT-­proBNP, 100% of patients with reduced LVEF and 100% of patients with abnormal ECG. CMR was performed an average of 3.0 days±3.1 days after the onset of clinical symptoms, with suggestive myocardial lesions seen in only 20% of the patients with elevated troponin T, 50% of patients with elevated N-­terminal pro-­brain natriuretic peptide (NT-­proBNP), none of patients with reduced LVEF and 50% of patients with abnormal ECG (table  2). According to the recommendations for the definition from Bonaca et al (15), we defined 3 patients as definite, 1 as probable and seven as possible in our population (table 2). Patient characteristics Looking at PET results, among the 5 patients with patchy diffuse uptake one had a definite myocarditis according to Bonaca et al whereas for patient with a probable myocarditis the uptake on 68Ga-­DO- TATOC PET/CT was patchy (table 2). We did not look into a possible association between Bonaca et al criteria and MBRpeak as among the 9 patients who had a PET/ Cytokine panel To define the specific serum cytokine signature associ- ated with ICI-­related myocarditis, the concentration of serum markers in different patients was evaluated by a multiplex bead assay (Thermo Fisher) at different time points before and after tocilizumab (TCZ) treatment. Briefly, serum concentrations of cytokines and soluble cytokine receptors (eg, IL-­1alpha, IL-­1RA, IL-­1beta, IL-­2, IL-­4, IL-­5, IL-­6, IL-­7, IL-­9, IL-­10, IL-­12p70, IL-­13, IL-­15, IL-­17A, IL-­18, IL-­21, IL −22, IL-­23, IL-­27, IL-­31, interferon 3 Boughdad S, et al. J Immunother Cancer 2021;9:e003594. doi:10.1136/jitc-2021-003594 Open access normalizer software to limit interanalysis staining intensi- ties. Data were processed and analyzed with the Cytobank software package. The references are age-­adjusted values and validated by the CHUV Immunology and Allergy diagnostics platforms for clinical routine practice. Myocarditis imaging normalizer software to limit interanalysis staining intensi- ties. Data were processed and analyzed with the Cytobank software package. The references are age-­adjusted values and validated by the CHUV Immunology and Allergy diagnostics platforms for clinical routine practice. Among those patients, nine underwent 68Ga-­DOTATOC PET/CT for the diagnosis of ICI-­associated myocarditis, whereas eight underwent CMR imaging. Seven patients had both PET/CT and CMR imaging. All patients except one patient had measurements of hs troponin I serum levels. All patients who underwent PET/CT imaging had pathological uptake in the myocardium compared with the intraventricular chamber, and the most frequent pattern of uptake seen was patchy diffuse uptake in the myocardium (figure 1). For patients who underwent both imaging PET/CT and CRM imaging, the rest of the six patients out of the eight were discordant (table 2). Inter- estingly, for five patients with negative CMR imaging results but positive PET/CT results, hs troponin I serum levels were elevated (table  2 and online supplemental figure S2). A total of 37 patients (18 women and 19 men) with 39 consecutive scans were included in the control group, with a mean age of 57.4 years (19.1–82.8). Amidst this control population, 17 out of 33 patients (clinical history was not available for 4 patients) had no clinical history of cardiovascular disease or risk factors; 1 patient had a history of myocardial infarction; 3 had a history of cardiovascular disease; and 12 had at least one risk factor. Looking at the nine patients with clinical suspicion of irAEs—myocarditis, one patient had a history of myocar- dial infarction; two had a history of cardiovascular disease; and five had at least one risk factor. There were significant differences for all the ratio values calculated on different regions of the LV myocardium (free wall, septum, and apex) between the patients with clinically suspected ICI-­ associated myocarditis and the control group (p value <0.001) with good diagnostic performance on ROC anal- ysis using a value of 2 for the ratio on the free wall (AUC 0.954, 94% sensitivity, and 87% specificity; online supple- mental figure S3). 68 Myocarditis presentation The clinical and biological characteristics of the patients are shown in table 2 (online supplemental figure S1B). The median time to the onset of myocarditis from ICI initiation (or the first day of second-­line ICI treatment for three patients) was 63 days (IQR 11–124 days), with 91% presenting within 3 months of therapy initiation. Eight patients presented with cardiac symptoms (mainly chest pain) at diagnosis, and early investigations showed new alterations on ECG in 4 out of 10 patients (4 atrioventric- ular block and 1 right branch block), whereas no alter- ations in the left ventricular function were seen on TTE. Indeed only one patient had an altered left ventricular function at diagnosis (18% EF) with a similar ECG to a previous one done 6 months before. All myocarditis (11 out of 11) patients had a high-­sensitivity (hs) troponin T elevation (100%), though it was subnormal in one patient, and nearly all patients had an hs troponin I (hs troponin I) elevation (80%, 8 out of 10). Besides, one patient had a cardiac biopsy. Boughdad S, et al. J Immunother Cancer 2021;9:e003594. Myocarditis presentation doi:10.1136/jitc-2021-003594 4 Open access Open acc Table 1  Clinical characteristics of the patients Patient ID Age/ gender Cancer Previous chemotherapy TNM cancer staging ICI (number of cycles) irAEs (grade) Treatments Clinical resolution Patient 1 77/F Merkel carcinoma No Stade IIA Pembrolizumab (six cycles) Myocarditis G2 Pericarditis None Yes Patient 2 79.6/M Melanoma No Stage IV First line: pembrolizumab (two cycles) Second line: ipilimumab and nivolumab (three cycles) Myocarditis G3 Ocular myositis CS +infliximab Yes Patient 3 64.9/M Melanoma No Stage IIIC Pembrolizumab (two cycles) Myocarditis (G3) Hepatitis (G3) Myositis (G2) CS +MMF+infliximab Yes Patient 4 57.7/M Small cell neuro-­endocrine carcinoma Cisplatine– etoposide Stage IIIB Ipilimumab and nivolumab (two cycles) Myocarditis (G3–G4) Ocular myositis CS +TCZ Yes Patient 5 66.3/M Prostate adenocarcinoma Taxotere and cabazitaxel Stage IV Nivolumab (one cycle) Myocarditis (G3–G4) Myositis CS +MMF+TCZ Yes Patient 6 64.9/M Squamous cell anal carcinoma Capecitabine– mytomycin Recurrence (LN Pembrolizumab (two cycles) Myocarditis (G3) Hepatitis (G2) Myositis (G3) CS Yes Patient 7 84.6/M Melanoma No Stage IV Nivolumab (four cycles) Myocarditis (G2) Myositis CS Yes Patient 8 87.7/M Melanoma No Stage IV First line: pembrolizumab (two cycles) Second line: ipilimumab and nivolumab (two cycles) Myocarditis (G3–G4) Myositis CS +TCZ Yes Patient 9 74.9/M Hepatocellular carcinoma No Stage IV Atezolizumab (four cycles) Myocarditis (G2) Myositis CS Yes Patient 10 56/M Melanoma No Stage IV Ipilimumab and nivolumab (four cycles) Myocarditis (G1) Colitis (G3) Acute interstitial nephritis CS +infliximab+TCZ Yes Patient 11 64.6/M Melanoma No Stage IIIC First line: nivolumab (16 cycles) Second line: ipilimumab and nivolumab (four cycles) Myocarditis (G1) Colitis (G3) Thyroiditis Hypophysitis CS +infliximab Yes CS, corticosteroid; F, female; ICI, immune checkpoint inhibitor; irAE, immune-­related adverse event; LN, lymph node; M, male; MMF, mycophenolate mofetil; TCZ, tocilizumab; TNM, tumour, node, metastasis. 5 Boughdad S, et al. J Immunother Cancer 2021;9:e003594. Myocarditis presentation Patient ID 68Ga-­DOTATOC PET/CT CMR imaging hs troponine I values (n >25 ng/L) Other investigations Classification bonaca et al Uptake pattern Highest ratio SUVpeak_M/SUVmean_IC * Days from symptom onset and PET/CT Patient 10 Heterogenous 3.5 1 † Absence of edema or intramyocardial scars of inflammatory type 28 TTE=no alteration of LV function or kinetics, minimal pericardial effusion/ECG=sinusal rythm/ troponin T=40 ng/L/CK=235 U/L Possible Patient 11 Heterogenous 4.4 13 ND 110 TTE=low LV function (18%) and alterations of segmental kinetics/ ECG=sinusal rythm/troponin T=150 ng/L/CK=40 U/L Probable Troponin I normal values <26 ng/L; troponin T normal values <14 ng/L; CK normal values <190 ng/L. *MBRpeak: SUVpeak_M to left ventricular intracavitary background SUVmean ratio. †Two patients had immunosuppressive treatment initiated on the same day as the PET/CT. AVB, atrioventricular block; CK, creatine kinase; 68Ga-­DOTATOC, 68Ga-­DOTA(0)-­Phe(1)-­Tyr(3)-­octreotide; hs, high-­sensitivity; LAHB, left anterior hemiblock; LV, left ventricle; MBRpeak, myocardium-­to-­background ratio of myocardium SUVpeak to LV intracavitary SUVmean; ND, not done; NF, not found; PET, positron emission tomography; SUVpeak_M, myocardial SUVpeak; TTE, transthoracic echocardiogram sive treatment initiated on the same day as the PET/CT. atine kinase; 68Ga-­DOTATOC, 68Ga-­DOTA(0)-­Phe(1)-­Tyr(3)-­octreotide; hs, high-­sensitivity; LAHB, left anterior hemiblock; LV, left ventricle; MBRpeak, f myocardium SUVpeak to LV intracavitary SUVmean; ND, not done; NF, not found; PET, positron emission tomography; SUVpeak_M, myocardial SUVpeak; TTE, g p Additionally, in five out six patients who presented with concomitant myositis, pathological uptake was seen in the muscles, especially the paravertebral and inter- costal musculature, as well as the diaphragm on PET/ CT imaging (online supplemental figure S4A). CK levels were also elevated in four of the patients with significant uptake muscle uptake on 68Ga-­DOTATOC PET/CT. One patient presented with ocular myositis, which was difficult to detect on PET/CT imaging. Significant uptake in the mediastinal and hilar lymph nodes was also seen in several patients, with one patient having very intense uptake, suggesting a highly intense locoregional inflammatory response (online supplemental figure S4B). Additionally, one patient with acute biological autoimmune pancre- atitis presented with high and diffuse pancreatic uptake on 68Ga-­DOTATOC PET/CT (online supplemental figure S4B). Interestingly, some patients with tumorous lesions seen on the 68Ga-­DOTATOC PET/CT showed moderate uptake, but this was not explored in this study. Conversely, only three out of the eight patients who underwent CMR showed lesions suggestive of ICI-­related myocarditis, and no edema or intramyocardial scars of the inflammatory type were seen for the other five patients. Myocarditis presentation doi:10.1136/jitc-2021-003594 Open access g g g p ent ID 68Ga-­DOTATOC PET/CT CMR imaging hs troponine I values (n >25 ng/L) Other investigations Classification bonaca et al Uptake pattern Highest ratio SUVpeak_M/SUVmean_IC * Days from symptom onset and PET/CT ent 1 ND ND ND Intramyocardial lesions, edema and pericarditis ND TTE =no alteration of LV function/ ECG=no kinetic alterations/hs troponin T=229 ng/L (n <14 ng/L)/ creatinin kinase=140 CK U/L (N 25–190) U/L Definite ent 2 ND ND ND Minimal subepicardial lesions, no edema 18.8 TTE=no alteration of LV function/ ECG=no kinetic alteration/ troponin T=387 ng/L/CK=178 U/L Definite ent 3 Heterogenous 2.2 7 Absence of edema or intramyocardial scars of inflammatory type 88.7 TTE=ND/ECG=AVB I and LAHB/ troponin T=1231 ng/L/CK=6249 U/L Possible ent 4 Patchy diffuse 2.3 3 ND 981.9 TTE=no alteration of LV function/ ECG=new AVB/troponin T=1045 ng/L (<14) /CK=2234 U/L (<190) Possible ent 5 Patchy diffuse 3.1 4 ND 169.9 TTE=no alteration of LV function or kinetics/ECG=new intermittent high-­grade AVB/troponin T=2389 ng/L/CK=524 U/L Possible ent 6 Patchy diffuse 4.4 10 † Absence of edema or intramyocardial scars of inflammatory type. 1604.7 TTE=no alteration of LV function or kinetics/ECG=diffuse alterations of the repolarization with submillimetre undershift and flattened T wave/troponin T=1281 ng/L/CK=4752 U/L Possible ent 7 Patchy diffuse 2.9 3 Absence of edema or intramyocardial scars of inflammatory type 212.6 TTE=no alteration of LV function or kinetics/ECG=sinusal rythm, no modification/troponin T=936 ng/L/ CK=844 U/L Possible ent 8 Patchy diffuse 3.3 5 Late-­gadolinium enhancement (patchy pattern) and edema 5896.1 TTE=no alteration of LV function or kinetics/ECG=new bundle branch block/troponin T=966 ng/L/CK=127 U/L Definite ent 9 Heterogenous 2.4 3 Absence of edema or intramyocardial scars of inflammatory type 20.5 TTE=NF/ECG=sinusal rythm/ troponin T=1210 ng/L/CK=280 U/L Possible Table 2  Imaging and biological characteristics of the patients 68Ga DOTATOC PET/CT Patient 9 Patient 8 Boughdad S, et al. J Immunother Cancer 2021;9:e003594. doi:10.1136/jitc-2021-003594 6 Open access CT, 7 were defined as possible myocarditis, 1 with definite and the other with probable, thus not allowing a compar- ison between groups. CT, 7 were defined as possible myocarditis, 1 with definite and the other with probable, thus not allowing a compar- ison between groups. Treatments Of the 11 ICI-­related myocarditis cases, only patient one did not have IS treatments. Among patients that had 68Ga-­DOTATOC PET/CT, seven out of nine patients had IS treatment several days prior to the scan with an average delay of 4.2 days (0–15 days). Two patients had IS treatment initiated on the same day as the PET/CT (table 2). Only three patients (6, 7, and 9) had a satisfac- tory response to CS without the need for additional IS treatment. Patients 3 and 5 received a third IS regimen with infliximab because of refractoriness to progression with mycophenolate mofetil (MMF) and CS. Two MMF-­ refractory patients with myocarditis (patients 3 and 5) were treated with infliximab and TCZ, respectively. One patient with infliximab-­refractory myocarditis was treated with TCZ. Four patients with CS-­refractory myocarditis were treated with infliximab (patients 2 and 11) or TCZ (patients 4 and 8, table 1). All in all, the clinical course for all ICI-­related myocarditis were favorable despite CS-­refractory cases (table  1) and corroborating with a persistent drop in troponin I following different treat- ments (online supplemental figure S1B). Myocarditis presentation CMR did not provide an alternative diagnosis in these patients, particu- larly ischemic events. Two patients had additional angiog- raphy to exclude an ischemic origin, and one patient had a cardiac biopsy that did not show any signs of inflamma- tion in the LV. Immune correlates Most patients had serum increases in the inflamma- tory cytokine IL-­6 (six of eight patients) and in the 7 Boughdad S, et al. J Immunother Cancer 2021;9:e003594. doi:10.1136/jitc-2021-003594 Open access Figure 1  Fused 68Ga -DOTATOC PET/CT images in the axial plane centered on the long axis of the LV (A–I): patients 3–11 with pathological uptake in the myocardium; pathological uptake in the paravertebral and intercostal muscle is also seen in some patients (A,B,D,E,G); SUV scale 0–2 g/mL. 68Ga -DOTATOC, 68Ga-­DOTA(0)-­Phe(1)-­Tyr(3)-­octreotide; LV, left ventricle. Figure 1  Fused 68Ga -DOTATOC PET/CT images in the axial plane centered on the long axis of the LV (A–I): patients 3–11 with pathological uptake in the myocardium; pathological uptake in the paravertebral and intercostal muscle is also seen in some patients (A,B,D,E,G); SUV scale 0–2 g/mL. 68Ga -DOTATOC, 68Ga-­DOTA(0)-­Phe(1)-­Tyr(3)-­octreotide; LV, left ventricle. chemokines CXCL9, CXCL10 and CXCL13 (five to six of eight patients) (figure 2A). As expected and reported previously, the serum level of IL-­6 increased during TCZ treatment.24 26 Furthermore, elevated levels of the growth factors hepatocyte growth factor (HGF) and/or vascular endothelial growth factor (VEGF-­A) were observed in four of eight patients (figure 2A). Major immune cell popula- tions in blood samples from five patients (patients 6–10) were profiled using a mass cytometry panel consisting of 44 different metal isotope-­labeled antibodies targeting immune-­related markers for cell lineage, memory subsets, chemokine receptors, and activation markers. Of note, T cells and monocyte immune cell subsets in patient samples showed features that were distinct from those of healthy donor samples. All five patients tested displayed significant increases in the proportion of Th1, Th1/Th17, and Th17 helper memory CD4 T-­cell populations that expressed elevated levels of the CXCR3, CXCR3/CCR6, and CCR6/ CCR4 chemokine receptors, respectively, compared with a panel of 146 healthy donor controls. Patients also had a corresponding reduction in levels of Th2 helper cells, as defined by CCR4+/CXCR3−/CCR6− expression. Patient chemokines CXCL9, CXCL10 and CXCL13 (five to six of eight patients) (figure 2A). As expected and reported previously, the serum level of IL-­6 increased during TCZ treatment.24 26 Furthermore, elevated levels of the growth factors hepatocyte growth factor (HGF) and/or vascular endothelial growth factor (VEGF-­A) were observed in four of eight patients (figure 2A). Immune correlates Major immune cell popula- tions in blood samples from five patients (patients 6–10) were profiled using a mass cytometry panel consisting of 44 different metal isotope-­labeled antibodies targeting immune-­related markers for cell lineage, memory subsets, chemokine receptors, and activation markers. Of note, T cells and monocyte immune cell subsets in patient samples showed features that were distinct from those of healthy donor samples. All five patients tested displayed significant increases in the proportion of Th1, Th1/Th17, and Th17 helper memory CD4 T-­cell populations that expressed elevated levels of the CXCR3, CXCR3/CCR6, and CCR6/ CCR4 chemokine receptors, respectively, compared with a panel of 146 healthy donor controls. Patients also had a corresponding reduction in levels of Th2 helper cells, as defined by CCR4+/CXCR3−/CCR6− expression. Patient memory CD8 T cells also exhibited elevated expression of CXCR3, either with or without the expression of the CD38 activation marker. With regard to the monocyte cell population, the proportion of non-­classical monocytes relative to classical and intermediate monocyte popula- tions also increased in four of five patients. Non-­classical monocytes from patients also showed significantly lower levels of CD45RA, CD31, and CD1c phenotypical markers than those from healthy donor controls (figure 2B). y g ROC curves were constructed showing the abilities of serum cytokines to distinguish patients with ICI-­related myocarditis from healthy controls. The diagnostic values of each cytokine, including AUC, cut-­off value (value that maximizes sensitivity and minimize false positive rate, sensitivity, specificity, positive predictive value, and negative predictive value are shown in online supple- mental figure S5). The cytokines that provided accept- able discrimination for use in the diagnostic test (ie, AUC >0.7) included IL-­6, CXCL9, CXCL10, and CXCL13. Among the cytokines with high diagnostic values, IL-­6, CXCL9, CXCL10, and CXCL13 had hs (~100 %), while IL-­6 and CXCL13 had the lowest specificity (71% and Boughdad S, et al. J Immunother Cancer 2021;9:e003594. doi:10.1136/jitc-2021-003594 8 Open access Figure 2  Heatmap of cytokines, chemokines and growth factors identified in patients diagnosed with ICI-­related myocarditis. Values are displayed as colors ranging from white to dark red according to deviation to the reference. Day 0 corresponds to the day of clinical presentation of the myocarditis, and the numbers correspond to the delay in days between clinical presentation of the myocarditis and the analysis of the chemokines and cytokines in the serum. ICI, immune checkpoint inhibitor; IFN, tumor necrosis factor; IL, interleukin; TNF, tumor necrosis factor. Immune correlates Figure 2  Heatmap of cytokines, chemokines and growth factors identified in patients diagnosed with ICI-­related myocarditis. Values are displayed as colors ranging from white to dark red according to deviation to the reference. Day 0 corresponds to the day of clinical presentation of the myocarditis, and the numbers correspond to the delay in days between clinical presentation of the myocarditis and the analysis of the chemokines and cytokines in the serum. ICI, immune checkpoint inhibitor; IFN, tumor necrosis factor; IL, interleukin; TNF, tumor necrosis factor. defined a threshold of 1.6 for the MBRpeak for the diag- nosis of myocarditis on 68Ga-­DOTATOC PET/CT in a population of 21 patients referred for suspected myocar- ditis, of whom 14 had pathological uptake in comparison to a control group of 44 patients with neuroendocrine tumors and no clinical history of cardiac inflammation.22 However, in routine practice, we tend to favor a higher threshold of 2 for the MBRpeak in order to increase spec- ificity and which would have led to similar results in this study (table 2). CMR imaging was negative for six of the eight patients who underwent both imaging modalities, though it should be mentioned that for one patient with altered renal function, CMR was performed without gadolinium contrast, limiting its sensitivity. Moreover, in our opinion, selection bias might have existed, as patients recommended for PET/CT already had a high suspi- cion of ICI-­related myocarditis, with high blood levels of hs troponin I. Indeed, except for one patient, there was a good concordance between a positive PET/CT and elevated blood levels of hs troponin I. The patient who had normal hs troponin I serum levels at the time of PET/CT was treated with a high dose of steroids and intravenous immunoglobulin for several days prior to PET/CT. Additionally, ICI treatment was interrupted, 85%, respectively) in contrast to 100% for CXCL9 and CXCL10 (online supplemental figure S5A). The color distribution of patients with ICI-­related myocarditis differed from that of controls. The first principal compo- nent (dim 1) accounts for 47.2% of the variability (sepa- ration) of control and ICI-­related myocarditis groups (online supplemental figure S5B). Boughdad S, et al. J Immunother Cancer 2021;9:e003594. doi:10.1136/jitc-2021-003594 DISCUSSION These results concord with the recent data in the literature, even though the lack of sensitivity of CMR imaging for the detection of acute myocarditis has been reported.27 Interestingly, soma- tostatin receptors (SSTR)-­targeted radiotracers for PET/ CT imaging do not present physiological uptake.28 The higher sensitivity of 68Ga-­DOTATOC PET/CT might be related to an infiltration of the myocardium by lympho- cytic cells expressing somatostatin receptor 2 (STTR2),28 which can be detected before significant myocardial damage detectable by CMR occur. Indeed, SSTR-­based PET/CT directly detects myocardium inflammation in vivo, as activated inflammatory cells have been known to overexpress SSTRs (SSTR1 and 2).28 29 Borchert et al also showed that polarized macrophages, T cells/natural killer (NK) cells and B cells had a higher uptake of 68Ga-­DOTATATE than neutrophils.30 These results are interesting, as myocardial histological findings showed predominantly lymphocytic cells (CD8  + and CD4+ T cells) with macrophaparge/histiocyte/giant cell (mainly CD68 + cells) cellular infiltration in most cases.27 Thus, it could facilitate the detection of myocarditis at an early stage, especially in patients with a diagnosis of possible myocarditis after complete cardiological work-­up and for the subsequent therapeutic monitoring.20 22 There were no comparisons between 68Ga-­DOTATOC findings and pathology results, as only one patient (patient 4) had a myocardial biopsy that did not show signs of inflammation despite having highly elevated levels of serum troponin I and inflammatory cytokine markers and a diffuse but heterogeneous pathological uptake in the myocardium on 68Ga -DOTATOC PET/CT. EMB is still being consid- ered the gold standard for the definitive diagnosis of myocarditis, but it is technically challenging, which could explain the limited number of patients with pathology data.29 31 32 p g It is quite interesting to note that serum IFN-γ was not increased especially that IFN-γ-induced chemokines such as CXCL9 and CXCL10 and IFN-γ-inducing cytokine such as IL-­18 are elevated. Furthermore, CXCL9 production is induced specifically by IFN-γ, but CXCL10 and CXCL11 can also be induced by IFN-α, IFN-β, and TNF-α. In our case series, IFN-α and TNF-α were not increased. DISCUSSION To the best of our knowledge, this is the first article reporting the usefulnesss of 68Ga-­DOTATOC for the diag- nosis of ICI-­related myocarditis at an early stage. Patho- logical uptake was seen in the myocardium of all the patients with suspected ICI-­related myocarditis. In this study, it showed hs for detecting pathological uptake in the myocardium of the LV in patients with clinical symp- toms and an increase in troponin levels and inflamma- tory cytokines that were suggestive of myocarditis. This was confirmed by the comparison of myocardium uptake on whole-­body acquisition between our group of patients with clinical suspicion of ICI-­related myocarditis and a population of 37 consecutive patients (39 scans) with neuroendocrine tumors that underwent 68Ga-­DOTATOC PET/CT (online supplemental figure S6). We previously 9 Boughdad S, et al. J Immunother Cancer 2021;9:e003594. doi:10.1136/jitc-2021-003594 Open access Immune profiles, as determined by serum levels of cytokines/chemokines and phenotypes of immune cell populations in the blood, also show a correlation with myocardial inflammation. Elevated levels of IL-­6 were detected in five of eight patients and are indica- tive of ongoing inflammation, while increased levels of the CXCL9 and/or CXCL10 chemoattractants in six of eight patients could be involved in the recruitment of CXCR3-­expressing immune cells, including cytotoxic lymphocytes, NK cells, and macrophages, to a site of inflammation. Augmented CXCL9 and CXCL10 expres- sion has been shown to be correlated with the develop- ment of heart failure, left ventricular dysfunction, and the development of adverse cardiac remodeling.33 34 and IS treatment was initiated, at the onset of clinical symptoms, which may have led to a false-­negative finding for troponin levels. In addition, this patient underwent a neurological evaluation that confirmed the presence of a motor deficiency in relation to myositis, which was reflected by electromyographic abnormalities. and IS treatment was initiated, at the onset of clinical symptoms, which may have led to a false-­negative finding for troponin levels. In addition, this patient underwent a neurological evaluation that confirmed the presence of a motor deficiency in relation to myositis, which was reflected by electromyographic abnormalities. In contrast, among the eight patients with a strong clin- ical suspicion of ICI-­related myocarditis who underwent CMR imaging, its sensitivity for detecting lesions associ- ated with myocarditis was moderate, with a positive result seen in only three patients. Open access It is important to note that CS-­refractory, infliximab-­ refractory, or MMF-­refractory myocarditis responded very well to anti-­IL6R therapy, which could further support the role of this therapy in the treatment of refractory myocarditis. The few published case series of myocar- ditis with EMB have described T-­cell infiltration, mostly CD8 + T cells intermixed with CD4 + T cells, and CD68 + macrophages.6 36 ICI-­related myocarditis is histologically characterized by a more lymphohistiocytic inflammatory infiltrate with an increased CD68:CD3 ratio and increased PD-­L1  + macrophages and myocytes,36 and by the tran- scriptional involvement of CD8 and the IFN-γ pathway.38 Our patients displayed significant blood increases in the proportion of Th1, Th1/Th17, and Th17 helper memory CD4 T-­cell populations, suggesting a possible pathogenic correlation between the predominant blood immune T-­cell activation and the T-­cell immune infil- trates reported on EMB. These immune-­related pheno- typical and functional determinations need to be further sequentially explored in order to identify robust predic- tive biomarkers specific to ICI-­related myocarditis the clinical presentation with chest pain and elevated troponins at a mean age above 70 years. It might be explained by the poor health status of those patients with metastatic disease and the strong suspicion of ICI-­related myocarditis as the median time for the onset of myocar- ditis from ICI initiation was 2 months, in agreement with the current literature. In addition, all patients had a favorable evolution after initiation of an IS treatment. Similarly, CMR was not performed in all patients, and the imaging protocol to detect myocarditis in our institution was based on T2 mapping and LGE, whereas T1 mapping of the whole myocardium was not systematic. Performing a systematic T1 mapping might have resulted in a higher rate of abnormal CMR as suggested by the recent study of Thavendiranathan et al. Finally, the use of a control group with NET patients without abnormal uptake of the myocardium on visual analysis might not have been the ideal control, but it was in our opinion the best option. Indeed, except for the detection of myocarditis, the current recruitment for 68Ga-­DOTATOC in our institu- tion is patients with NET tumors, and those patients are not usually treated with ICI, which limits the possibility of a comparison to a group of patient undergoing ICI treat- ments and without clinical suspicion of myocarditis. p y Interestingly, our longitudinal profiling of the immune cells by global immune cell CyTOF panel with 40 markers reported for the first time a possible role of Th17 in ICI-­related myocarditis, especially in patients 8 and 9, who had refractory myocarditis but responded to IL6R blockade therapy. Importantly, IL-­6 induces the develop- ment of Th17 cells from naïve CD4 + T cells.39 The key role that this pathogenic cytokine plays in the differentia- tion of Th17 cells could suggest that there is a causal link between the increase in IL-­6 and the involvement of Th17 cells. This observation is very important in the context of CS-­refractory irAEs because a number of reports have postulated that Th17 cells may play a role in CS-­refrac- tory irAEs in patients who respond to anti-­IL6 therapies without providing direct evidence. Thus, the possibility that Th17 could be a potential predictive biomarker of irAEs and resistance or low response to CS is plausible but should be confirmed in a large population with different types of CS-­refractory irAEs. This case series of ICI-­related myocarditis supports the use of anti-­IL-­6 therapy in CS-­re- fractory myocarditis, as we have reported before.2 p y Nonetheless, our data indicate a high sensitivity of 68Ga-­DOTATOC PET/CT for the detection of ICI-­related myocarditis, though its specificity should be further explored in future studies. Indeed, using a control popu- lation of patients with cancer treated with ICI without clinical suspicion of ICI-­related myocarditis or a direct comparison of EMB guided by the pathological uptake seen on 68Ga-­DOTATOC PET/CT would help to validate its diagnostic accuracy. Furthermore, the main advan- tages of 68Ga-­DOTATOC PET/CT for the detection of ICI-­related myocarditis are the absence of physiological uptake of the myocardium and that no special several days’ preparation regimen is needed by contrast to 18F-­ FDG PET/CT well suited for emergency setting.20 Boughdad S, et al. J Immunother Cancer 2021;9:e003594. doi:10.1136/jitc-2021-003594 DISCUSSION Inter- estingly, it is was reported in both murine models and patients undergoing ICI that macrophages were the predominant source of CXCL9, and their depletion abrogated CD8+ T-­cell infiltration and the therapeutic efficacy of ICI underlining the fundamental importance of macrophage-­derived CXCR3 ligands for therapeutic efficiency of ICI.35 This might also account for macro- phages infiltrating the myocardium in case of an ICI-­ related myocarditis because it showed a higher density of CD68  + PD-­L1+ macrophages in high-­grade ICI-­related myocarditis.36 Unfortunately, we did not assess the pres- ence of macrophages in myocardial tissue (since there was only one patient biopsied, which was negative). This dissociation between IFN-γ and IFN-γ-induced chemok- ines was described in other diseases such as patients of polymyalgia rheumatica showed that serum levels CXCL9 and CXCL10 were elevated although the levels of IFN-γ and TNF-α were not increased.37 Therefore, it is not well established whether changes in serum chemokines reflect infiltration of immune cells in the myocardium and what is the exact part of macrophage versus CD8+ T-­cell involve- ment in the production of the serum CXCL9 and 10. p In line with the cytokine/chemokine profile, four of five patients with myocarditis evaluated by mass cytom- etry exhibited a Th1/Th2 imbalance that favored a pronounced inflammatory Th1, Th1/Th17 and Th17 CD4 memory T-­cell response. The PCA analysis clearly indicates that patients with ICI-­related myocarditis have a distinct cytokine profile compared with healthy donors. Indeed, patients with ICI-­related myocarditis cluster homogeneously close together and separated from healthy donors. We have already shown that the activation of the IL-­6/Th1 axis is an indicator of severe CS-­refractory irAEs, such as ICI-­related myocarditis,2 hemophagocytic lymphohistiocytosis25 and cholangiohepatitis,24 which could suggest that the IL-­6/Th1 axis may be a possible common blood biomarker for CS-­refractory irAEs. In addition, with 68Ga-­DOTATOC PET/CT imaging, it is possible to perform whole-­body acquisition after a single injection of the radiotracer. In the control group of patients with neuroendocrine tumors, there was no uptake in the muscle (online supplemental figure S6). This allows the detection of concomitant irAEs such as myositis, which is frequently associated with ICI-­associated myocarditis or, in one patient, with acute pancreatitis. Another advantage of 68Ga-­DOTATOC PET/CT imaging is that it is well suited for an emergency setting, as it does not require an extensive carbohydrate-­free diet, in contrast to 18F-­FDG PET/CT. 10 Boughdad S, et al. J Immunother Cancer 2021;9:e003594. doi:10.1136/jitc-2021-003594 Open access Contributors  SB, JP and MO conceived the study idea and supervised the project. SB and MO collected the data and wrote and prepared the manuscript. All authors participated in discussions and critically read the manuscript. Contributors  SB, JP and MO conceived the study idea and supervised the project. SB and MO collected the data and wrote and prepared the manuscript. All authors participated in discussions and critically read the manuscript. Funding  The authors have not declared a specific grant for this research from any funding agency in the public, commercial or not-­for-­profit sectors. Funding  The authors have not declared a specific grant for this research from any funding agency in the public, commercial or not-­for-­profit sectors. Funding  The authors have not declared a specific grant for this research from any funding agency in the public, commercial or not-­for-­profit sectors. 12 Friedrich MG, Sechtem U, Schulz-­Menger J, et al. Cardiovascular magnetic resonance in myocarditis: a JACC white paper. J Am Coll Cardiol 2009;53:1475–87. Competing interests  None declared. Competing interests  None declared. Competing interests  None declared. 13 Ferreira VM, Schulz-­Menger J, Holmvang G, et al. Cardiovascular Magnetic Resonance in Nonischemic Myocardial Inflammation: Expert Recommendations. J Am Coll Cardiol 2018;72:3158–76. Patient consent for publication  Consent obtained directly from patient(s) Patient consent for publication  Consent obtained directly from patient(s) Ethics approval  Institutional approval was obtained from the local ethics committee. 14 Lurz P, Luecke C, Eitel I, et al. Comprehensive cardiac magnetic resonance imaging in patients with suspected myocarditis: the MyoRacer-­trial. J Am Coll Cardiol 2016;67:1800–11. Provenance and peer review  Not commissioned; externally peer reviewed. 15 Pradhan R, Nautiyal A, Singh S. Diagnosis of immune checkpoint inhibitor-­associated myocarditis: a systematic review. Int J Cardiol 2019;296:113–21. Data availability statement  Data are available upon reasonable request. Supplemental material  This content has been supplied by the author(s). It has not been vetted by BMJ Publishing Group Limited (BMJ) and may not have been peer-­reviewed. Any opinions or recommendations discussed are solely those of the author(s) and are not endorsed by BMJ. BMJ disclaims all liability and responsibility arising from any reliance placed on the content. Where the content includes any translated material, BMJ does not warrant the accuracy and reliability of the translations (including but not limited to local regulations, clinical guidelines, terminology, drug names and drug dosages), and is not responsible for any error and/or omissions arising from translation and adaptation or otherwise. ORCID iDs 21 Lang RM, Badano LP, Mor-­Avi V, et al. Recommendations for cardiac chamber quantification by echocardiography in adults: an update from the American Society of echocardiography and the European association of cardiovascular imaging. Eur Heart J Cardiovasc Imaging 2015;16:233–71. Sarah Boughdad http://orcid.org/0000-0003-1900-758X Joe-­Elie Salem http://orcid.org/0000-0002-0331-3307 Niklaus Schaefer http://orcid.org/0000-0002-9732-7696 Marie Nicod-­Lalonde http://orcid.org/0000-0002-4886-0611 Julien Costes http://orcid.org/0000-0002-4528-7362 John O Prior http://orcid.org/0000-0003-1429-1374 Michel Obeid http://orcid.org/0000-0003-2095-2677 g g 22 Dalm VASH, van Hagen PM, van Koetsveld PM, et al. Expression of somatostatin, cortistatin, and somatostatin receptors in human monocytes, macrophages, and dendritic cells. Am J Physiol Endocrinol Metab 2003;285:E344–53. 23 Amelio P, Portevin D, Hella J, et al. Hiv infection functionally impairs Mycobacterium tuberculosis-­specific CD4 and CD8 T-­cell responses. J Virol 2019;93. doi:10.1128/JVI.01728-18. [Epub ahead of print: 01 03 2019]. Open access 16 Zhang L, Awadalla M, Mahmood SS, et al. Cardiovascular magnetic resonance in immune checkpoint inhibitor-­associated myocarditis. Eur Heart J 2020;41:1733–43. 17 Thavendiranathan P, Zhang L, Zafar A, et al. Myocardial T1 and T2 Mapping by Magnetic Resonance in Patients With Immune Checkpoint Inhibitor-­Associated Myocarditis. J Am Coll Cardiol 2021;77:1503–16. 18 Mahmood SS, Fradley MG, Cohen JV, et al. Myocarditis in patients treated with immune checkpoint inhibitors. J Am Coll Cardiol 2018;71:1755–64. Open access  This is an open access article distributed in accordance with the Creative Commons Attribution 4.0 Unported (CC BY 4.0) license, which permits others to copy, redistribute, remix, transform and build upon this work for any purpose, provided the original work is properly cited, a link to the licence is given, and indication of whether changes were made. See https://creativecommons.org/​ licenses/by/4.0/. 19 Bonaca MP, Olenchock BA, Salem J-­E, et al. Myocarditis in the setting of cancer therapeutics: proposed case definitions for emerging clinical syndromes in cardio-­oncology. Circulation 2019;140:80–91. 20 Lapa C, Reiter T, Li X, et al. Imaging of myocardial inflammation with somatostatin receptor based PET/CT - A comparison to cardiac MRI. Int J Cardiol 2015;194:44–9. Boughdad S, et al. J Immunother Cancer 2021;9:e003594. doi:10.1136/jitc-2021-003594 Open access Open access Author affiliations 1Nuclear Medicine and Molecular Imaging, Lausanne University Hospital, Lausanne, Switzerland 2Department of Medical Oncology, Lausanne University Hospital, Lausanne, Switzerland 3Immunology and allergy division, Department of Medicine, Lausanne University Hospital, Lausanne, Switzerland 4Section of Cardio-­Oncology & Immunology, Division of Cardiology and the Cardiovascular Research Institute, University of California San Francisco, San Francisco, California, USA 5Cardio-­Oncology Program, Pitié-Salpétrière Hospital, Paris, France, AP-­HP, Pitié- Salpétrière Hospital,Sorbonne, INSERM, CIC-­1901, Paris, France, Paris, France, France 6Radiopharmacy Unit, Department of Pharmacy, Lausanne University Hospital, Lausanne, Switzerland observational, retrospective, pharmacovigilance study. Lancet Oncol 2018;19:1579–89. observational, retrospective, pharmacovigilance study. Lancet Oncol 2018;19:1579–89. i 1Nuclear Medicine and Molecular Imaging, Lausanne University Hospital, Lausanne, Switzerland ; 5 Lyon AR, Yousaf N, Battisti NML, et al. Immune checkpoint inhibitors and cardiovascular toxicity. Lancet Oncol 2018;19:e447–58. 2Department of Medical Oncology, Lausanne University Hospital, Lausanne, Switzerland 6 Johnson DB, Balko JM, Compton ML, et al. Fulminant myocarditis with combination immune checkpoint blockade. N Engl J Med 2016;375:1749–55. 3Immunology and allergy division, Department of Medicine, Lausanne University Hospital, Lausanne, Switzerland 7 Müller OJ, Spehlmann ME, Frey N. Cardio-­toxicity of checkpoint inhibitors. J Thorac Dis 2018;10:S4400–4. 8 Lilleker JB, Diederichsen ACP, Jacobsen S, et al. Using serum troponins to screen for cardiac involvement and assess disease activity in the idiopathic inflammatory myopathies. Rheumatology 2018;57:1041–6. 5Cardio-­Oncology Program, Pitié-Salpétrière Hospital, Paris, France, AP-­HP, Pitié- Salpétrière Hospital,Sorbonne, INSERM, CIC-­1901, Paris, France, Paris, France, France 9 Bodor GS, Porterfield D, Voss EM, et al. Cardiac troponin-­I is not expressed in fetal and healthy or diseased adult human skeletal muscle tissue. Clin Chem 1995;41:1710–5. 6Radiopharmacy Unit, Department of Pharmacy, Lausanne University Hospital, Lausanne, Switzerland 6Radiopharmacy Unit, Department of Pharmacy, Lausanne University Hospital, Lausanne, Switzerland 10 Hughes M, Lilleker JB, Herrick AL, et al. Cardiac troponin testing in idiopathic inflammatory myopathies and systemic sclerosis-­spectrum disorders: biomarkers to distinguish between primary cardiac involvement and low-­grade skeletal muscle disease activity. Ann Rheum Dis 2015;74:795–8. Correction notice  This article has been corrected since it was first published to remove the acknowledgements statement. Correction notice  This article has been corrected since it was first published to remove the acknowledgements statement. 11 Caforio ALP, Pankuweit S, Arbustini E, et al. Current state of knowledge on aetiology, diagnosis, management, and therapy of myocarditis: a position statement of the European Society of cardiology Working group on myocardial and pericardial diseases. Eur Heart J 2013;34:2636–48. CONCLUSION 68 68Ga-­DOTATOC PET/CT showed promising results as an imaging modality for the diagnosis of ICI-­related myocarditis. Characterized by hs, it might be of value at the early stage of the disease in patients with suggestive clinical symptoms who may not yet present myocardial damage on CMR imaging. Its value is supported by a good concordance with serum levels of hs troponin I, the inflammatory IL-­6/Th1 cytokine markers and immune correlates, including Th17. 68Ga-­DOTATOC PET/CT was also useful for detecting concomitant myositis. These findings should be considered in light of certain limita- tions, including the smaller number of patients and the retrospective study design. Thus, the diagnostic value of 68Ga-­DOTATOC PET/CT for the detection of ICI-­ related myocarditis should be confirmed in future studies including a larger population and histological documen- tation of myocarditis. y y p The high proportion of non-­classical monocytes in four or five patients was also consistent with an inflammatory disease, and the significantly reduced levels of CD31, a checkpoint receptor for monocyte FcγR-­mediated phago- cytic activity, have been associated with acute coronary syndromes. These immune correlates provide increased confidence in the diagnosis of myocardial disease; however, 68Ga-­DOTATOC was the only method that had a 100% sensitivity for the identification of ICI-­related myocarditis. We should also acknowledge some limitations in this original and retrospective pilot study. First, the number of patients included in the study was limited. Histolog- ical confirmation was available in only one patient and was negative, and only two patients had coronary angi- ography to exclude acute coronary syndrome despite 11 Boughdad S, et al. J Immunother Cancer 2021;9:e003594. doi:10.1136/jitc-2021-003594 REFERENCES 24 Moi L, Bouchaab H, Mederos N, et al. Personalized Cytokine-­ Directed therapy with tocilizumab for refractory immune checkpoint Inhibitor-­Related cholangiohepatitis. J Thorac Oncol 2021;16:318–26. 1 Martins F, Sofiya L, Sykiotis GP, et al. Adverse effects of immune-­ checkpoint inhibitors: epidemiology, management and surveillance. Nat Rev Clin Oncol 2019;16:563–80. 1 Martins F, Sofiya L, Sykiotis GP, et al. Adverse effects of immune-­ checkpoint inhibitors: epidemiology, management and surveillance. Nat Rev Clin Oncol 2019;16:563–80. 2 Doms J, Prior JO, Peters S, et al. Tocilizumab for refractory severe immune checkpoint inhibitor-­associated myocarditis. Ann Oncol 2020;31:1273–5. 25 Özdemir BC, Latifyan S, Perreau M, et al. Cytokine-­directed therapy with tocilizumab for immune checkpoint inhibitor-­related hemophagocytic lymphohistiocytosis. Ann Oncol 2020;31:1775–8. 3 Haanen J, Ernstoff M, Wang Y, et al. Rechallenge patients with immune checkpoint inhibitors following severe immune-­related adverse events: review of the literature and suggested prophylactic strategy. J Immunother Cancer 2020;8:e000604. 26 Nishimoto N, Terao K, Mima T, et al. Mechanisms and pathologic significances in increase in serum interleukin-­6 (IL-­6) and soluble IL-­6 receptor after administration of an anti-­IL-­6 receptor antibody, tocilizumab, in patients with rheumatoid arthritis and Castleman disease. Blood 2008;112:3959–64. gy 4 Salem J-­E, Manouchehri A, Moey M, et al. Cardiovascular toxicities associated with immune checkpoint inhibitors: an 12 Boughdad S, et al. J Immunother Cancer 2021;9:e003594. doi:10.1136/jitc-2021-003594 Open access 27 Mahmood SS, Fradley MG, Cohen JV, et al. Myocarditis in patients treated with immune checkpoint inhibitors. J Am Coll Cardiol 2018;71:1755–64. 34 Altara R, Manca M, Hessel MH, et al. Cxcl10 is a circulating inflammatory marker in patients with advanced heart failure: a pilot study. J Cardiovasc Transl Res 2016;9:302–14. 28 Boughdad S, Meyer M, Pruvot E. Prior J: reference values for abnormal uptake of 68Ga-­DOTATOC in patients with myocardium inflammation. Europ J Nuc Med Molec Imag 2019;46:S163. 35 House IG, Savas P, Lai J, et al. Macrophage-­Derived CXCL9 and CXCL10 are required for antitumor immune responses following immune checkpoint blockade. Clin Cancer Res 2020;26:487–504. l 29 Cooper LT, Baughman KL, Feldman AM, et al. The role of endomyocardial biopsy in the management of cardiovascular disease. Europ Heart J 2007;28:3076–93. 36 Champion SN, Stone JR. Immune checkpoint inhibitor associated myocarditis occurs in both high-­grade and low-­grade forms. Mod Pathol 2020;33:99–108. 30 Borchert T, Beitar L, Langer LBN, et al. Dissecting the target leukocyte subpopulations of clinically relevant inflammation radiopharmaceuticals. J Nucl Cardiol 2021;28:1636–45. Boughdad S, et al. J Immunother Cancer 2021;9:e003594. doi:10.1136/jitc-2021-003594 REFERENCES 37 Han JH, Suh C-­H, Jung J-­Y, et al. Elevated circulating levels of the interferon-γ-induced chemokines are associated with disease activity and cutaneous manifestations in adult-­onset still's disease. Sci Rep 2017;7:46652. 31 Kasner M, Sinning D, Escher F, et al. The utility of speckle tracking imaging in the diagnostic of acute myocarditis, as proven by endomyocardial biopsy. Int J Cardiol 2013;168:3023–4. 38 Finke D, Heckmann MB, Salatzki J, et al. Comparative transcriptomics of immune checkpoint inhibitor myocarditis identifies guanylate binding protein 5 and 6 dysregulation. Cancers 2021;13 doi:10.3390/cancers13102498 32 Palaskas NL, Segura A, Lelenwa L. Immune checkpoint inhibitor myocarditis: elucidating the spectrum of disease through endomyocardial biopsy. Eur J Heart Fail 2021 doi:10.1002/ejhf.2265 33 Altara R, Gu Y-­M, Struijker-­Boudier HAJ, et al. Left ventricular dysfunction and CXCR3 ligands in hypertension: from animal experiments to a population-­based pilot study. PLoS One 2015;10:e0141394. 39 Bettelli E, Carrier Y, Gao W, et al. Reciprocal developmental pathways for the generation of pathogenic effector Th17 and regulatory T cells. Nature 2006;441:235–8. 39 Bettelli E, Carrier Y, Gao W, et al. Reciprocal developmental pathways for the generation of pathogenic effector Th17 and regulatory T cells. Nature 2006;441:235–8. 13 Boughdad S, et al. J Immunother Cancer 2021;9:e003594. doi:10.1136/jitc-2021-003594

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Editor in Chief Volker Gast, Jena AssiStant Editor Ann Kelly, Jena Editorial Board R. Harald Baayen, Tübingen Sonia Colina, Tucson, AZ Östen Dahl, Stockholm N. J. Enfield, Sydney Ricardo Etxepare, Bayonne Jennifer Hay, Christchurch Beth Levin, Stanford, CA Yen-hui Audrey Li, Los Angeles, CA Caroline Rowland, Nijmegen 2023 · Volume 61 · ISSUE 6 Linguistics an Interdisciplinary Journal of the Language Sciences 2023 · Volume 61 · ISSUE 6 AssiStant Editor Ann Kelly, Jena Board of consulting editors Patrícia Amaral, Bloomington, IN Matthew Baerman, Guildford Heike Behrens, Basel Delia Bentley, Manchester Ruth Berman, Tel Aviv Walter Bisang, Mainz Denis Creissels, Lyon William Croft, Albuquerque, NM Stuart Davis, Bloomington, IN Henrik De Smet, Leuven Stephen M. 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Stückle Druck und Verlag e.K., Ettenheim Linguistics 2023 | Volume 61 | Issue 6 Contents Special Issue: Re-assessing the explanatory potential of the alienability contrast Guest Editors: An Van linden and Françoise Rose Françoise Rose and An Van linden Introduction: the limits of the explanatory potential of the alienability contrast 1341 Natalia Chousou-Polydouri, David Inman, Thomas C. Huber and Balthasar Bickel Multi-variate coding for possession: methodology and preliminary results 1365 Isabelle Bril Categorizing possession in Zuanga-Yuanga and other Kanak languages (New Caledonia): a typological perspective 1403 Jorge Emilio Rosés Labrada Beyond alienability: factors determining possessive classes in Piaroa 1447 Françoise Rose Questioning the relevance of alienability in Arawak linguistics: an innovative analysis of possession in Mojeño Trinitario 1491 An Van linden When the alienability contrast fails to surface in adnominal possession: bound nouns in Harakmbut 1533 Natalia Aralova and Brigitte Pakendorf Non-canonical possessive constructions in Negidal and other Tungusic languages: a new analysis of the so-called “alienable possession” suffix 1563 Denis Creissels Coding splits in the adnominal possessive construction and alienability: the case of Mandinka (West Mande) 1593 Sonia Cristofaro Explaining alienability splits in the use of overt and zero possessive marking: a source oriented approach 1613

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ISSN 1021-0296 REVISTA NICARAGUENSE DE ENTOMOLOGIA N° 290 Noviembre 2022 LYGAEIDAE (HEMIPTERA: HETEROPTERA: LYGAEOIDEA) DE VENEZUELA, CON NUEVE NUEVOS REGISTROS Dalmiro Cazorla, Maritza Alarcón & Pedro Morales PUBLICACIÓN DEL MUSEO ENTOMOLÓGICO LEÓN - - - NICARAGUA Revista Nicaragüense de Entomología. Número 290. 2022. La Revista Nicaragüense de Entomología (ISSN 1021-0296) es una publicación reconocida en la Red de Revistas Científicas de América Latina y el Caribe, España y Portugal (Red ALyC). Todos los artículos que en ella se publican son sometidos a un sistema de doble arbitraje por especialistas en el tema. The Revista Nicaragüense de Entomología (ISSN 1021-0296) is a journal listed in the Latin-American Index of Scientific Journals. Two independent specialists referee all published papers. Consejo Editorial Jean Michel Maes Editor General Museo Entomológico Nicaragua Fernando Hernández-Baz Editor Asociado Universidad Veracruzana México José Clavijo Albertos Universidad Central de Venezuela Silvia A. Mazzucconi Universidad de Buenos Aires Argentina Weston Opitz Kansas Wesleyan University United States of America Don Windsor Smithsonian Tropical Research Institute, Panama Fernando Fernández Universidad Nacional de Colombia Jack Schuster Universidad del Valle de Guatemala Julieta Ledezma Museo de Historia Natural “Noel Kempf” Bolivia Olaf Hermann Hendrik Mielke Universidade Federal do Paraná, Brasil _______________ Foto de la portada: Ejemplar macho de Melacorhyphus circumlitus (Stål, 1862). Capturado en La Parroquia Osuna Rodríguez, Mérida, municipio Libertador, estado Mérida, Venezuela (Foto: Eduardo Alarcón Mendoza y Elisabeth Alarcón). Página 2 Revista Nicaragüense de Entomología. Número 290. 2022. LYGAEIDAE (HEMIPTERA: HETEROPTERA: LYGAEOIDEA) DE VENEZUELA, CON NUEVE NUEVOS REGISTROS Dalmiro Cazorla1,*, Maritza Alarcón2 & Pedro Morales1 RESUMEN En el presente estudio, se muestra un listado revisado y actualizado de la fauna venezolana de Lygaeidae (chinches de las semillas) (Hemiptera: Heteroptera: Lygaeoidea). En total, esta revisión reveló la presencia de 49 especies, agrupadas en 3 subfamilias (Ischnorhynchinae, Lygaeinae, Orsillinae), 3 Tribus (Metrargini, Nysiini, Orsillini) y 19 géneros. Se reporta por primera vez la presencia de la especie de Orsillinae (Orsillini) Neortholomus jamaicensis (Dallas, 1852) (nuevo registro) en el estado Mérida (región andina); así como también de ocho especies de Lygaeinae, incluyendo Acroleucus phoenix Brailovsky, 1980 (estado Mérida, región andina; nuevo registro en Venezuela), Lygaeus argutus Brailovsky, 1982, Oncopeltus (Erythrischius) longirostris Stål, 1874 y Spilostethus pandurus (Scopoli, 1763) (nuevos registros en el estado Anzoátegui, región nor-oriental), Oncopeltus (Oncopeltus) femoralis Stål, 1874 [nuevo registro en los estados Anzoátegui (región nor-oriental) y Falcón (región nor-occidental)], Oncopeltus (Erythrischius) sandarachatus (Say, 1831) [nuevo registro en los estados Anzoátegui (región nor-oriental) y Mérida (región andina)], Oncopeltus (Erythrischius) unifasciatellus Slater, 1964 y Melanopleurus bistriangularis (Say, 1831) (nuevos registros en el estado Mérida, región andina). Adicionalmente, se provee información sobre distribución geográfica, plantas hospedadoras y/o microorganismos patógenos que se vehiculizan. PALABRAS CLAVE: Chinches de las semillas, listado, taxonomía, Venezuela. DOI: 10.5281/zenodo.7315546 1 Universidad Nacional Experimental “Francisco de Miranda”, Decanato de Investigaciones, Centro de Investigaciones Biomédicas (CIB), Laboratorio de Entomología, Parasitología y Medicina Tropical (LEPAMET), Coro-Falcón, Venezuela * E-mail: [email protected] / [email protected]; ORCID ID: https://orcid.org/00000001-7199- 6325 2 Laboratorio de Parasitología Experimental (LAPEX), Departamento de Biología, Facultad de Ciencias, Universidad de Los Andes, Mérida, Estado Mérida, Venezuela. E-mail: [email protected] / [email protected]; ORCID ID: https://orcid.org/0000-00019035- 0933 Página 3 Revista Nicaragüense de Entomología. Número 290. 2022. ABSTRACT LYGAEIDAE (HEMIPTERA: HETEROPTERA: LYGAEOIDEA) OF VENEZUELA, WITH NINE NEW RECORDS In the present study, a revised and updated checklist of the Venezuelan fauna of Lygaeidae (seed bugs) (Hemiptera: Heteroptera: Lygaeoidea) is shown. In total, this revision revealed the presence of 49 seed bug species, grouped into 3 subfamilies (Ischnorhynchinae, Lygaeinae, Orsillinae), 3 Tribes (Metrargini, Nysiini, Orsillini) and 19 genera. A record is made of the presence for the first time in Venezuela of the Orsillinae (Orsillini) species Neortholomus jamaicensis (Dallas, 1852) (new record) in Merida state (Andes region); as well as of eight Lygaeinae species, including Acroleucus phoenix Brailovsky, 1980 (Merida state, Andes region; new record in Venezuela), Lygaeus argutus Brailovsky, 1982, Oncopeltus (Erythrischius) longirostris Stål, 1874 and Spilostethus pandurus (Scopoli, 1763) (new records in the Anzoategui state, north-east region), Oncopeltus (Oncopeltus) femoralis Stål, 1874 [new record in the Anzoategui (north-east region) and Falcon (north-western region) states], Oncopeltus (Erythrischius) sandarachatus (Say, 1831) [new record in the Anzoategui (north-east region) and Merida (Andes region) states], Oncopeltus (Erythrischius) unifasciatellus Slater, 1964 and Melanopleurus bistriangularis (Say, 1831) (new records in the Merida state, Andes region). In addition, geographical distribution, host or associated plants plants and/or vehiculized pathogens data is provided. KEY WORDS: Seed bugs, list, taxonomy, Venezuela. INTRODUCCIÓN Lygaeidae (sensu stricto) constituye una de las 16 familias que integran a la superfamilia Lygaeoidea (Hemiptera: Heteroptera: Pentatomomorpha), y se les denomina comúnmente como “chinches de las semillas”, esto debido a que muchas especies se alimentan de semillas maduras de una amplia variedad de plantas; sin embargo, se debe aclarar que este nombre de “seed bugs” se queda corto, esto debido a que otras especies se nutren de savia y unas pocas son predadoras (entomófagas) (Slater & Baranowski 1990, Burdfield-Steel & Shuker 2014, Henry et al. 2015, Larson y Scudder 2018, Dellapé y Henry 2022). Lygaeidae posee una amplia distribución en el globo terráqueo, y se encuentra integrada por alrededor de 102 géneros y 970 especies (Henry et al. 2015, Dellapé y Henry 2022). El sistema de clasificación actual mayormente aceptado de Lygaeidae, se basa en el análisis filogenético de tres subfamilias (Ischnorhynchinae, Lygaeinae, Orsillinae) dado por Henry (1997). Página 4 Revista Nicaragüense de Entomología. Número 290. 2022. Para la región Neotropical, en la subfamilia Ischnorhynchinae se conocen 4 géneros y 9 especies; mientras que en Lygaeinae se han señalado 22 géneros y alrededor de 175 especies, y en Orsillinae: 9 géneros y alrededor de 46 especies (Henry et al. 2015, Dellapé y Henry 2022). Los integrantes de la familia Lygaeidae poseen un rango de tallas diverso (pequeños, mediano y relativamente largo); y en cuanto a la coloración, similarmente es amplia y diversa en la familia: desde una coloración opaca, críptica pardo-mate a pardo-rojizo (Ischnorhynchinae) y pardo-amarillento (Orsillinae), hasta una coloración aposemática llamativa rojo-naranja-amarilla (Lygaeinae) (Burdfield-Steel & Shuker 2014, Dellapé 2014, Henry et al. 2015). Los taxones de la familia Lygaeidae poseen un aparato bucal picador-chupador y un ciclo de desarrollo hemimetábolo, siendo la mayoría fitófagos y algunas especies representan gran relevancia económica al constituir plagas de cultivos agrícolas de importancia económica [P. ej., Spilostethus pandurus (Scopoli, 1763), Lygaeus equestris (L.), Nysius raphanus Howard, 1872, Nysius cymoides (Spinola, 1837), Nysius simulans Stål, 1859] (Burdfield-Steel & Shuker 2014, Dellapé 2014, Dughetti et al. 2015, Henry et al. 2015, Yazic 2022). Es importante resaltar que algunos taxones de Lygaeidae poseen importancia sanitaria [P. ej., Oncopeltus fasciatus (Dallas, 1852), Nysius Dallas, 1852], al ocasionar, por ejemplo, picaduras adventicias en el humano (Cazorla 2020). En sus extensos “Catálogos de Lygaeidae del Mundo” (Catalogue of the Lygaeidae of the World), Slater (1964) y Slater & O´Donnell (1995) reunieron información relacionada con la fauna de “seed bugs” de Venezuela, junto con las de todas las regiones del globo terráqueo. Y actualmente, Dellapé y Henry (2022) en su sitio WEB “Lygaeoidea Species File” (Lygaeoidea SpeciesFile.org) realizaron los listados de las taxa de Lygaeoidea del Mundo, incluyendo los de la familia Lygaeidae. Varias de las contribuciones sobre Lygaeidae de Venezuela, se han realizado por investigadores extranjeros [P.ej., Brailovsky (1977,1978, 1979a, b, 1982,1984)] y nacionales [P. ej., Cazorla-Perfetti & Morales-Moreno (2019a,b,c), Cazorla Perfetti et al. (2019), Alarcón & Cazorla (2022)], de una manera, si se quiere, aislada o fragmentada; por lo que hasta el presente no existe un “Listado” o “Catálogo” particular o específico sobre la fauna de Lygaeidae para el territorio nacional. A la luz de lo expuesto, en el presente trabajo se muestra el primer intento por reunir globalmente la información de las fuentes bibliográficas, además de capturas puntuales de ejemplares en algunas entidades federales de Venezuela, y crear así el “Listado de Lygaeidae de Venezuela”. Página 5 Revista Nicaragüense de Entomología. Número 290. 2022. MATERIAL Y MÉTODOS Para la elaboración del presente listado de Lygaeidae de Venezuela, se realizó, en primera instancia, una minuciosa y exhaustiva revisión de la literatura científica del grupo taxonómico. En este sentido, sirvieron como base o punto de partida los catálogos, listados o revisiones mencionados anteriormente de Slater (1964), Slater & O´Donnell (1995) y Dellapé y Henry (2022). Figura 1: Mapa de Venezuela mostrando la demarcación de las entidades federales. Página 6 Revista Nicaragüense de Entomología. Número 290. 2022. Asimismo, se presentan datos de ejemplares adultos capturados en varias localidades de Venezuela. Las capturas se hicieron entre julio-diciembre 2022, de forma manual durante horas diurnas (8:00 AM-12:00 M) mientras se posaban y/o alimentaban sobre plantas [Amaranthus viridis L. (bledo), Amaranthaceae (Figuras 96-97), Allamanda cathartica L. (jazmín de Cuba) (Figuras 61-63), Calotropis procera (Ait.) Ait. (algodón de seda), Apocynaceae; Origanum vulgare, L. (orégano) (Figuras 64-65), Clerodendrum L. Figuras 93-95), Lamiaceae] o deambulaban en superficies de objetos inanimados, en tres localidades de Venezuela, incluyendo Anaco (09°26’00’’N, 64°28’00’’O; 220 m), municipio Anaco (estado Anzoátegui, región nor-oriental), Coro (11°24’N, 69°40’O, 20 m), municipio Miranda (estado Falcón, región nor-occidental), y La Parroquia Osuna Rodríguez (08°34’11”N, 71°11’52”O; 1323 m), Mérida, municipio Libertador (estado Mérida, región andina); dichas localidades se encuentran ubicadas en zonas bioclimáticas que corresponden a Bosque Seco Tropical (bs-T), Monte Espinoso Tropical (MET) y Bosque Muy Húmedo Tropical (bmh-T), respectivamente (Ewel et al. 1976). Los insectos se transportaron a los Laboratorios de Entomología, Parasitología y Medicina Tropical (LEPAMET), del Área Ciencias de la Salud de Universidad Nacional Experimental “Francisco de Miranda” (UNEFM), Coro, Estado Falcón, y de Parasitología Experimental (LAPEX), Facultad de Ciencias, Universidad de Los Andes (ULA), Mérida, estado Mérida; se sacrificaron con vapores de cloroformo y se revisaron bajo estereoscopio binocular (Carl Zeiss Stemi DRC), y se encuentran depositados en la colecciones de artrópodos de dichos laboratorios. Los heterópteros Lygaeidae se identificaron siguiendo trabajos de Ojeda (1973), Hamilton (1983), Slater (1992), Dellapé Montemayor (2012), Henry et al. (2015), Faúndez & Rocca (2017), Cazorla Perfetti & Morales Moreno (2019a), Cazorla Perfetti et al. (2019) y en datos “nivel identificación” de la plataforma iNaturalist [P. ej., iNaturalist (2022). iNaturalist Research grade Observations. iNaturalist.org. Occurrence dataset https://doi.org/ 10.15468/ab3s5x; 28/08/2022)]. En cada uno de los taxones, se dan en la medida que existan referencias o datos, rangos de distribución geográfica en Venezuela por cada entidad federal (Figuras 1, 2); asimismo, se aporta información sobre las plantas hospedadoras o asociadas y/o microorganismos patógenos que se vehiculizan reportados a nivel nacional y/o mundial. El esquema de clasificación de tres subfamilias (Ischnorhynchinae, Lygaeinae, Orsillinae) y sus respectivos taxones (tribus, géneros), y la terminología y nomenclatura taxonómica se basa en Henry (1997), el cual implementaron Henry et al. (2015) y Dellapé y Henry (2022). Las subfamilias, tribus, géneros y especies se encuentran ordenadas alfabéticamente. En los mapas (Figuras 1, 2), se muestran las entidades federales (Figura 1) y los relieves (Figura 2) de las diversas regiones de Venezuela. Página 7 Revista Nicaragüense de Entomología. Número 290. 2022. Figura 2: Mapa de Venezuela mostrando los relieves de las diferentes regiones geográficas. RESULTADOS LISTADO DE LAS ESPECIES DE LYGAEIDAE DE VENEZUELA Familia LYGAEIDAE Schilling, 1829 Subfamilia ISCHNORHYNCHINAE STÅL, 1872 Género Neokleidocerys Scudder, 1962 1. Neokleidocerys godmani (Distant, 1893) Plantas hospedantes o asociadas. Desconocidas. Fuentes bibliográficas: Slater & Brailovsky (1989), Dellapé y Henry (2022). Página 8 Revista Nicaragüense de Entomología. Número 290. 2022. Distribución en Venezuela. Sin información disponible de la (s) localidad (es). Género Polychisme Kirkaldy, 1904 2. Polychisme ferruginosus (Stål, 1874) Plantas hospedantes o asociadas. Desconocidas. Fuentes bibliográficas: Slater & Brailovsky (1986), Dellapé y Henry (2022), Faúndez & Muñoz-Cepeda (2022). Distribución en Venezuela. Estado Aragua: Estación Biológica “Rancho Grande”: Parque Nacional “Henri Pittier” (10°22’48”N, 67°37’08”O; 1800 m); estado Mérida: El Valle (2400 m) (08°41’21”N, 71°06’00”O), municipio Libertador; El Teleférico (3500 m); estado Táchira: La Grita (2300 m) (08°08’00”N, 71°59’00”O), municipio Jáuregui; estado Trujillo: Páramo de Guaramacal (3200 m) (09°12’22”N, 70°11’37”O), Boconó (09°15’07”N, 70°15’01”O), municipio Boconó (Slater & Brailovsky 1986). Subfamilia LYGAEINAE Schilling, 1829 Género Achlyosomus Slater Alex, 1992 3. Achlyosomus campestris (Brailovsky, 1979) Plantas hospedantes o asociadas. Orquidaceae: especie no identificada interceptada en New Jersey, EUA. Fuentes bibliográficas: Brailovsky (1979a), Slater (1992), Dellapé y Henry (2022). Distribución en Venezuela. Localidad no especificada (Brailovsky (1979a). Género Acroleucus Stål, 1874 4. Acroleucus caicaraensis Brailovsky, 1984 Plantas hospedantes o asociadas. Desconocidas. Fuentes bibliográficas: Brailovsky (1984), Dellapé y Henry (2022). Distribución en Venezuela. Estado Bolívar: 150 Km al sur de Caicara (07°36’41”N, 66°08’52”O), río Suapure (06°30’25”N, 66°39’24”O) (Brailovsky 1984). Página 9 Revista Nicaragüense de Entomología. Número 290. 2022. 5. Acroleucus conchatus Brailovsky, 1984 Plantas hospedantes o asociadas. Desconocidas. Fuentes bibliográficas: Brailovsky (1984), Dellapé y Henry (2022). Distribución en Venezuela. Estado Lara: El Blanquito (1350 m), Parque Nacional Yacambú (09°42’28”N, 69°34’41”O); estado Mérida: Cacute (2200 m) (08°40’43”N, 71°00’52”O), municipio Rangel (Brailovsky 1984). 6. Acroleucus haemopterus Stål, 1874 Plantas hospedantes o asociadas. Desconocidas. Fuentes bibliográficas: Brailovsky & Barrera (2015), Dellapé y Henry (2022). Distribución en Venezuela. Estado Mérida: La Mucuy (08°37’13”N, 71°04’07”O; 2400, 2700 m), municipio Santos Marquina (Brailovsky & Barrera 2015). 7. Acroleucus municeps Brailovsky & Barrera, 1984 Plantas hospedantes o asociadas. Desconocidas. Fuentes bibliográficas: Brailovsky & Barrera (1984), Dellapé y Henry (2022). Distribución en Venezuela. Estado Falcón: Curimagua (11°10’31”N, 69°40’20”O; 1150 m), municipio Petit (Brailovsky & Barrera 1984). 8. Acroleucus neomaurus Slater, 1964 Plantas hospedantes o asociadas. Desconocidas. Fuentes bibliográficas: Slater (1964), Dellapé y Henry (2022). Distribución en Venezuela. Sin información disponible de la (s) localidad (es). Página 10 Revista Nicaragüense de Entomología. Número 290. 2022. 9. Acroleucus nigrovittatus Stål, 1874 Plantas hospedantes o asociadas. Desconocidas. Fuentes bibliográficas: Slater (1964), Dellapé y Henry (2022). Distribución en Venezuela. Sin información disponible de la (s) localidad (es). 10. Acroleucus orinocoensis Brailovsky, 1984 Plantas hospedantes o asociadas. Desconocidas. Fuentes bibliográficas: Brailovsky (1984), Dellapé y Henry (2022). Distribución en Venezuela. Estado Trujillo: La Mesa (09°02’39”N, 70°42’23”O; 1768 m), municipio Urdaneta (Brailovsky 1984). 11. Acroleucus phoenix Brailovsky, 1980 (Figuras 12-21) Plantas hospedantes o asociadas. Apocynaceae: Allamanda cathartica L. (jazmín de Cuba) (Nuevo registro) (Figuras 61-63); Anacardiaceae: Mangifera L. (mango). Fuentes bibliográficas: Brailovsky (1980), Slater (1992), Dellapé y Henry (2022), iNaturalist org. Distribución en Venezuela. Estado Mérida: La Parroquia Osuna Rodríguez (08°34’11”N, 71°11’52”O; 1323 m), Mérida, municipio Libertador (Nuevo registro, presente estudio). Comentarios. El presente aparece como el primer registro de la especie para Venezuela, la cual se encuentra también distribuida en Perú, Ecuador y Colombia (Brailovsky 1980, iNaturalist org). Los reportes de las plantas hospedantes o asociadas requieren de estudios más detallados para confirmarse. 12. Acroleucus scitulus Brailovsky & Barrera, 1984 Plantas hospedantes o asociadas. Desconocidas. Fuentes bibliográficas: Brailovsky & Barrera (1984), Dellapé y Henry (2022). Página 11 Revista Nicaragüense de Entomología. Número 290. 2022. Distribución en Venezuela. Estado Aragua: Portachuelo (Rancho Grande) (10°12’26”N, 67°14’26”O; 960 m), municipio José R. Revenga (Brailovsky & Barrera 1984). 13. Acroleucus tullus (Stål, 1862) Plantas hospedantes o asociadas. Solanaceae: Solanum nigrum L., Solanum nudum H.B. Fuentes bibliográficas: Brailovsky & Cervantes (2008), Dellapé y Henry (2022). Distribución en Venezuela. Sin información disponible de la (s) localidad (es). 14. Acroleucus vittaticeps Stål, 1874 Plantas hospedantes o asociadas. Desconocidas. Fuentes bibliográficas: Slater (1964), Dellapé y Henry (2022). Distribución en Venezuela. Sin información disponible de la (s) localidad (es). 15. Acroleucus vulturnus Brailovsky, 1984 Plantas hospedantes o asociadas. Desconocidas. Fuentes bibliográficas: Brailovsky (1984), Dellapé y Henry (2022). Distribución en Venezuela. Estado Trujillo: Carretera (2200 m) Trujillo (09°25’01”N, 70°30’00”O), municipio Trujillo-Boconó (09°15’07”N, 70°15’01”O), municipio Boconó; estado Mérida: Timotes (08°59’14”N, 70°44’14”O, 2025 m), municipio Miranda; localidad no especificada (Brailovsky 1984). 16. Acroleucus yacambuensis Brailovsky, 1984 Plantas hospedantes o asociadas. Desconocidas. Fuentes bibliográficas: Brailovsky (1984), Dellapé y Henry (2022). Página 12 Revista Nicaragüense de Entomología. Número 290. 2022. Distribución en Venezuela. Estado Lara: El Blanquito (1350 m), Parque Nacional Yacambú (09°42’28”N, 69°34’41”O) (Brailovsky 1984). Género Anochrostomus Slater Alex, 1992 17. Anochrostomus formosus (Blanchard, 1840) Plantas hospedantes o asociadas. Asparagaceae: Agave L.; Cannaceae: Canna L., Canna indica L.; Convolvulaceae: Convolvulus equitans Benth., Ipomoea L., Merremia quinquefolia (L.) Hallier F., Merremia umbellata (L.) Hallier F., Rivea corymbosa (L.) Raf.; Fabaceae: Lysiloma Benth.; Fagaceae: Quercus L.; Poaceae: Zea mays L.; Rutaceae: Citrus L., Citrus x sinensis Osbeck. Fuentes bibliográficas: Brailovsky y Barrera (1984), Slater & Baranowsky (1990), Maes (1998), Dellapé y Henry (2022). Distribución en Venezuela. Distrito Capital: El Valle (Caracas) (10°28’02”N, 66°54’26”O, 900-1400 m) (Brailovsky y Barrera 1984). Género Craspeduchus Stål, 1874 18. Craspeduchus attrahens Brailovsky & Barrera, 1984 Plantas hospedantes o asociadas. Desconocidas. Fuentes bibliográficas: Brailovsky & Barrera (1984), Dellapé y Henry (2022). Distribución en Venezuela. Estado Aragua: Cata (10°27’45”N, 67°44’02”O; 30 m), municipio Ocumare de La Costa de Oro; estado Carabobo: Montalbán (10°15’08”N, 68°18’07”O; 670 m), municipio Montalbán; estado Falcón: Cerro Galicia (1500 m) (11°10’39”N, 69°42’11”O), municipio Petit; estado Mérida: Las González (08°29’60”N, 71°19’14”O; 1800 m), municipio Campo Elías (Brailovsky & Barrera 1984). 19. Craspeduchus circumseptus Stål, 1867 Plantas hospedantes o asociadas. Desconocidas. Fuentes bibliográficas: Brailovsky (1979a), Slater (1964), Dellapé y Henry (2022). Página 13 Revista Nicaragüense de Entomología. Número 290. 2022. Distribución en Venezuela. Estado Aragua: Estación Biológica “Rancho Grande”: Parque Nacional “Henri Pittier” (10°22’48”N, 67°37’08”O; 1800 m) (Brailovsky 1979a). 20. Craspeduchus variegatus (DeGeer, 1773) Plantas hospedantes o asociadas. Desconocidas. Fuentes bibliográficas: Slater (1964), Dellapé y Henry (2022). Distribución en Venezuela. Sin información disponible de la (s) localidad (es). Género Dalmochrimnus Brailovsky, 1982 21. Dalmochrimnus ayalai (Brailovsky, 1984) Plantas hospedantes o asociadas. Desconocidas. Fuentes bibliográficas: Slater & O´Donnell (1995), Dellapé y Henry (2022). Distribución en Venezuela. Sin información disponible de la (s) localidad (es). 22. Dalmochrimnus guatemalanus (Distant, 1893) Plantas hospedantes o asociadas. Desconocidas. Fuentes bibliográficas: Slater (1964), Dellapé y Henry (2022). Distribución en Venezuela. Sin información disponible de la (s) localidad (es). Género Latochrimnus Brailovsky, 1982 23. Latochrimnus aduncus Brailovsky, 1984 Plantas hospedantes o asociadas. Desconocidas. Fuentes bibliográficas: Slater & O´Donnell (1995), Dellapé y Henry (2022). Distribución en Venezuela. Sin información disponible de la (s) localidad (es). Página 14 Revista Nicaragüense de Entomología. Número 290. 2022. 24. Latochrimnus funereus Brailovsky, 1984 Plantas hospedantes o asociadas. Desconocidas. Fuentes bibliográficas: Slater & O´Donnell (1995), Dellapé y Henry (2022). Distribución en Venezuela. Sin información disponible de la (s) localidad (es). Género Lygaeus Fabricius, 1794 25. Lygaeus argutus Brailovsky, 1982 (Figuras 24-26) Plantas hospedantes o asociadas. Apocynaceae: Asclepias L., Calotropis procera (Ait.) Ait. Fuentes bibliográficas: Brailovsky (1978, 1982), Cazorla-Perfetti y MoralesMoreno (2019a), Dellapé y Henry (2022). Distribución en Venezuela. Estado Falcón: Coro (11°24’N, 69°40’O; 20 m), municipio Miranda (CazorlaPerfetti y Morales-Moreno 2019a); estado Anzoátegui: Anaco (09°26’00’’N, 64°28’00’’O; 220 m), municipio Anaco (Nuevo registro, presente estudio). Comentarios. En un previo artículo (Cazorla-Perfetti y Morales-Moreno 2019a), hemos documentado la presencia de L. argutus para Venezuela, y adicionalmente se describió la hembra de la misma. Por lo tanto, el presente aparece como el segundo registro para el país de la especie, la cual fue capturada similarmente sobre C. procera. En la plataforma iNaturalist (https://inaturalist.ca/ observations/45485224; https://inaturalist.ca/observations/19218798), se le ha capturado sobre plantas no identificadas de Cactaceae y Poaceae en México y Honduras. 26. Lygaeus inaequalis Walker, 1972 Plantas hospedantes o asociadas. Desconocidas. Fuentes bibliográficas: Brailovsky (1978), Dellapé y Henry (2022). Distribución en Venezuela. Estado Mérida: Mérida (08°35’00”N, 71°08’00”O; 1630 m de altitud media), municipio Libertador (Brailovsky 1978). Página 15 Revista Nicaragüense de Entomología. Número 290. 2022. 27. Lygaeus truculentus Stål, 1862 Plantas hospedantes o asociadas. Desconocidas. Fuentes bibliográficas: Brailovsky (1978), Dellapé y Henry (2022). Distribución en Venezuela. Localidad (es) no especificada(s). Género Melacoryphus Slater Alex, 1988 28. Melacorhyphus circumlitus (Stål, 1862) Plantas hospedantes o asociadas. Apocynaceae: Asclepias angustifolia Schweigg.; Asparagaceae: Yucca L. Fuentes bibliográficas: Brailovsky (1979a), Behrstock (2021), Alarcón y Cazorla (2022), Dellapé y Henry (2022). Distribución en Venezuela. Distrito Capital: Caracas (10°30’00”N, 66°56’00”O, 900-1400 m); estado Aragua: Maracay (10°14’49”N, 67°35’45”O; 491 m), municipio Girardot; El Limón (10°18’01”N, 67°38’01”O; 483 m), municipio Mario Briceño Iragorry; estado Mérida: La Parroquia Osuna Rodríguez (08°34’11”N, 71°11’52”O; 1323 m) en el municipio Libertador de la ciudad de Mérida (Brailovsky 1979a, Alarcón y Cazorla 2022). 29. Melacoryphus nigrinervis (Stål, 1874) Plantas hospedantes o asociadas. Arecaceae: Brahea dulcis (Kunth) Marth; Asteraceae: Brickellia Elliot 1823, Brickellia glomerata Fernald. Fuentes bibliográficas: Brailovsky (1977), Osuna (2000), Cervantes-Peredo y Báez-Santa Cruz (2015), Dellapé y Henry (2022). Distribución en Venezuela. Estado Aragua: Parque Nacional “Henri Pittier” (10°22’48”N, 67°37’08”O; 1100 m); estado La Guaira: La Guaira (10°36’00”N, 66°55”59”; altitud media: 4 m), municipio Vargas (Distant 1880/1893, Osuna 2000). Página 16 Revista Nicaragüense de Entomología. Número 290. 2022. Género Melanopleurus Stål, 1874 30. Melanopleurus bistriangularis (Say, 1831) (Figuras 45-60) Plantas hospedantes o asociadas. Desconocidas. Fuentes bibliográficas: Distant (1880/1893), Henry et al. (2015), Dellapé y Henry (2022). Distribución en Venezuela. Estado La Guaira: La Guaira (10°36’00”N, 66°55”59”; altitud media: 4 m), municipio Vargas (Distant 1880/1893); estado Mérida: La Parroquia Osuna Rodríguez (08°34’11”N, 71°11’52”O; 1323 m), Mérida, municipio Libertador (Nuevo registro, presente estudio). Comentario. Usualmente, en Melanopleurus bistriangularis se han reconocido dos subespecies [M. bistriangularis bistriangularis (Say, 1831) y M. bistriangularis marginellus (Dallas, 1852)], las cuales fueron documentadas para el país por Distant (1880/1893); sin embargo, acá empleamos el criterio de Henry et al. (2015) de solo incluir el nivel de especie. Género Neacoryphus Scudder, 1965 31. Neacoryphus bicrucis (Say, 1825) Plantas hospedantes o asociadas. Asteraceae: Cacalia atriplicifolia L., Erechtites Raf., Erechtites hieraciifolia (L.) Raf. ex DC, Parasenecio W.W. SM. & Small, Senecio L., Senecio glabelus Poir., Senecio tomentosus Michx. Fuentes bibliográficas: Distant (1880/1893), Brailovsky (1977), Slater & Baranowsky (1990), Maes (1998), Dellapé y Henry (2022). Distribución en Venezuela. Estado La Guaira: La Guaira (10°36’00”N, 66°55”59”; altitud media: 4 m), municipio Vargas (Distant 1880/1893). Género Ochrimnus Stål, 1874 32. Ochrimnus (Phaeochrimnus) limbatipennis (Stål, 1858) Plantas hospedantes o asociadas. Desconocidas. Fuentes bibliográficas: Brailovsky y Barrera (1984), Dellapé y Henry (2022). Página 17 Revista Nicaragüense de Entomología. Número 290. 2022. Distribución en Venezuela. Estado Bolívar: Santa Rosalía (Caicara) (07°29’04”N, 65°39’17”O; 77 m), municipio Cedeño (Brailovsky y Barrera 1984). 33. Ochrimnus (Aglaochrimnus) nigrosteolaris Brailovsky, 2021 Plantas hospedantes o asociadas. Desconocidas. Fuentes bibliográficas: Brailovsky (2021), Dellapé y Henry (2022). Distribución en Venezuela. Estado Mérida: Km 20 Mérida-El Morro (2400 m) (08°26’36”N, 71°11’18”O), municipio Libertador; Valle Grande (08°40’22”N, 71°07’27”O; 2359 m), municipio Libertador; El Valle (2400 m) (08°37’54”N, 71°07’16”O), municipio Libertador; La Carbonera (2000 m) (08°38’36”N, 71°21’46”O), municipio Campo Elías; La Mucuy (08°37’13”N, 71°04’07”O), municipio Santos Marquina; La Azulita (2000 m) (08°42’52”N, 71°26’42”O), municipio Andrés Bello (Brailovsky 2021). Género Ochrostomus Stål, 1874 34. Ochrostomus pulchellus (Fabricius, 1794) Plantas hospedantes o asociadas. Anacardiaceae: Mangifera indica L.; Arecaceae: Cocos nucifera L.; Apocynaceae: Asclepias curassavica L.; Convolvulaceae: Ipomoea batatas (L.) Lam; Rivea corymbosa (L.) Raf.; Malvaceae: Corchorus hirsutus L., Corchorus siliquosus L., Malachra fasciata Jacq.; Musaceae: Musa paradisiaca L.; Poaceae: Digitaria eriantha Steud, especies no identificadas (zacate), Zea mays L.; Solanaceae: Capsicum L., Capsicum annuum L., Solanum lycopersicum L. Fuentes bibliográficas: Brailovsky (1979b), Slater & Baranowsky (1990), Maes (1998), Delancy y Landry (2017) Dellapé y Henry (2022). Distribución en Venezuela. Chaper (sic) (Brailovsky 1979b). Comentario. En su revisión sobre el taxón (como perteneciente al género Craspeduchus Stål, 1874), Brailovsky (1979) reporta la presencia para Venezuela de O. pulchellus en la localidad de “Chaper”; lo que a todas luces aparece como un error (¿lapsus calami?), ya que esta localidad no existe en la geografía venezolana. Página 18 Revista Nicaragüense de Entomología. Número 290. 2022. Por otra parte, Dellapé y Henry (2022) en su “Catálogo on line” sobre Lygaeoidea no incluyen a Venezuela dentro del rango de distribución O. pulchellus. Por lo tanto, el registro de la especie para el territorio nacional requiere ser confirmado. 35. Ochrostomus ulheri (Stål, 1874) Plantas hospedantes o asociadas. Apiaceae: Donnellsmithia hintonii Mathias y Constance; Apocynaceae: Asclepias angustifolia Schweigg., Calotropis procera (Ait.) Ait.; Asteraceae: Baccharis L.; Convolvulaceae: Ipomoea batatas (L.) Lam; Fabaceae: Phaseolus vulgaris L.; Musaceae: Musa paradisiaca L.; Rutaceae: Citrus L. (cítricos); Solanaceae: Solanum lycopersicum L. Fuentes bibliográficas: Brailovsky (1979b), Maes (1998), Cazorla-Perfetti y Morales-Moreno (2019b), Behrstock (2021), Dellapé y Henry (2022). Distribución en Venezuela. Estado Aragua: El Limón, (10°18’01”N, 67°38’01”O; 483 m), municipio Mario Briceño Iragorry; estado Falcón: Coro (11°24’N, 69°40’O; 20 m), municipio Miranda (Brailovsky 1979b, Cazorla-Perfetti y Morales-Moreno 2019b). Género Oncopeltus Stål, 1868 36. Oncopeltus (Erythrischius) cingulifer Stål, 1874 Plantas hospedantes o asociadas. Apocynaceae: Asclepias L., Asclepias curassavica L., Calotropis procera (Ait.) Ait.; Cucurbitaceae: Cucurbita L. Fuentes bibliográficas: Martorell (1939), O’Rourke (1976, 1979), Root y Chaplin (1976), Slater & Baranowsky (1990), Maes (1998), Dellapé y Henry (2022). Distribución en Venezuela. Distrito Capital: Caracas (10°30’00”N, 66°56’00”O, 900-1400 m); estado Aragua: Estación Biológica “Rancho Grande”; La Providencia (10°13’60”N, 67°31’60”O; 447 m), municipio Santiago Mariño; Turmero (10°13’42”N; 67°28’31”O; altitud media: 446 m), municipio Santiago Mariño; Maracay (10°14’49”N, 67°35’45”O; 491 m), municipio Girardot; estado Monagas: Caripito (10°06’40”N, 63°06’17”O; 17-48 m), municipio Bolívar; Maturín (09°44’44”N, 63°11’00”O; 67 m de altitud media), municipio Maturín (Martorell 1939, O’Rourke 1979). Página 19 Revista Nicaragüense de Entomología. Número 290. 2022. Figuras 3-4: Oncopeltus (Oncopeltus) femoralis Stål, 1874. Hembra, capturada en Coro, estado Falcón. 3. Habitus, vista dorsal. 4. Habitus, vista ventral. Página 20 Revista Nicaragüense de Entomología. Número 290. 2022. Figuras 5-7: Oncopeltus (Oncopeltus) femoralis Stål, 1874. Hembra, capturada en Coro, estado Falcón. 5. Habitus, vista lateral. 6. Vista frontal ampliada de cabeza y pronoto. 7. Vista ventral ampliada de cabeza. Página 21 Revista Nicaragüense de Entomología. Número 290. 2022. Figuras 8-10: Oncopeltus (Oncopeltus) femoralis Stål, 1874. Hembra, capturada en Coro, estado Falcón. 8. Vista dorsal ampliada de cabeza, pronoto, escutelo y región anterior de hemélitro. 9. Vista ventral ampliada de cabeza y tórax. 10. Vista ventral ampliada de región torácica. Página 22 Revista Nicaragüense de Entomología. Número 290. 2022. Figura 11: Oncopeltus (Oncopeltus) femoralis Stål, 1874. Hembra, capturada en Anaco, estado Anzoátegui. Habitus, vista dorsal. Página 23 Revista Nicaragüense de Entomología. Número 290. 2022. Figuras 12: Acroleucus phoenix Brailovsky, 1980. Macho. 12. Habitus, vista dorsal. Página 24 Revista Nicaragüense de Entomología. Número 290. 2022. Figuras 13-16: Acroleucus phoenix Brailovsky, 1980. Macho. 13. Vista dorsal de cabeza y pronoto. 14. Vista ampliada de antenas. 15. Vista ampliada de escutelo. 16. Vista dorsal ampliada de región terminal de hemélitros. Página 25 Revista Nicaragüense de Entomología. Número 290. 2022. Figuras 17-18: Acroleucus phoenix Brailovsky, 1980. Macho. 17. Habitus, vista lateral. 18. Vista ampliada de peritrema ostiolar de glándula odorífera metatorácica (círculo). Página 26 Revista Nicaragüense de Entomología. Número 290. 2022. Figuras 19-21: Acroleucus phoenix Brailovsky, 1980. Macho. 19. Habitus, vista ventral. 20. Vista ventral ampliada de esternitos terminales. 21. Vista posterior de pigóforo. Página 27 Revista Nicaragüense de Entomología. Número 290. 2022. Figuras 22-23: Spilostethus pandurus (Scopoli, 1763). Hembras. 22,23. Habitus, vista dorsal. Página 28 Revista Nicaragüense de Entomología. Número 290. 2022. Figuras 24-26: Lygaeus argutus Brailovsky, 1982. Macho. 24. Habitus, vista dorsal. 25. Vista dorsal ampliada de cabeza y pronoto. 26. Vista ampliada de región terminal de hemélitros. Escala: 1 mm. Página 29 Revista Nicaragüense de Entomología. Número 290. 2022. 37. Oncopeltus (Erythrischius) fasciatus (Dallas, 1852) Plantas hospedantes o asociadas. Apocynaceae: Asclepias angustifolia Schweigg., Asclepias curassavica L.; Calotropis procera (Ait.) Ait.; Nerium oleander L.; Asteraceae: Baccharis neglecta Britton. Transmisor de Microrganismos patógenos. Protozoa: Phytomonas elmassiani (Migone, 1916) Wenyon, 1926 (Kinetoplastea: Trypanosomatidae). Fuentes bibliográficas: Slater & Baranowsky (1990), Urtiaga (2007), Behrstock (2021), Dellapé y Henry (2022). Distribución en Venezuela. Estado Lara: Barquisimeto (10°04’04”N, 69°20’48”O; altitud media: 640 m), municipios Iribarren y Palavecino; Siquisique (10°39’09”N, 69°42’20”O; altitud media: 290 m), municipio Urdaneta (Urtiaga 2007). 38. Oncopeltus (Oncopeltus) femoralis Stål, 1874 (Figuras 3-11) Plantas hospedantes o asociadas. Apocynaceae: Calotropis procera (Ait.) Ait. (Nuevo registro). Fuentes bibliográficas: Slater (1964, 1992), Dellapé y Henry (2022). Distribución en Venezuela. Estado Anzoátegui: Anaco (09°26’00’’N, 64°28’00’’O; 220 m), municipio Anaco; estado Falcón: Coro (11°24’N, 69°40’O; 20 m), municipio Miranda (ambas localidades en el presente estudio; Nuevos registros). Comentario. El presente aparece como el primer registro documentado en revistas especializadas de localidades específicas para O. (Oncopeltus) femoralis en Venezuela, así como también de su asociación con una planta (C. procera), sobre la cual se capturaron hasta 8 ejemplares adultos alimentándose sobre semillas y/o frutos. En la plataforma iNaturalist (https://spain.inaturalist.org/ observations/ 116144117; https://spain.inaturalist.org/observations/ 120148097; https://spain.inaturalist.org/observations/129201346; https:// spain.inaturalist.org/observations/128992850) aparecen fotos de ejemplares adultos de O. (Oncopeltus) femoralis posándose sobre plantas no identificadas de Verbenaceae, Poaceae y Fabaceae, y sobre C. procera (Apocynaceae) en Cumaná (10°27’00”N, 64°10’00”O; altitud media: 43 m), municipio Sucre (estado Sucre; región nor-oriental). Página 30 Revista Nicaragüense de Entomología. Número 290. 2022. Figuras 27-28: Oncopeltus (Erythrischius) sandarachatus (Say, 1831). Hembra. 27. Habitus, vista dorsal. 28. Vista dorsal ampliada de cabeza y pronoto (los círculos señalan manchas rojizas en márgenes laterales del lóbulo anterior del pronoto). Página 31 Revista Nicaragüense de Entomología. Número 290. 2022. Figuras 29-30: Oncopeltus (Erythrischius) sandarachatus (Say, 1831). Hembra. 29. Habitus, vista ventral. 30. Vista ventral ampliada de esternitos terminales. Página 32 Revista Nicaragüense de Entomología. Número 290. 2022. Figuras 31-32: Oncopeltus (Erythrischius) longirostris Stål, 1874. Ejemplar 1. Hembra. 31. Habitus, vista dorsal. 32. Vista dorsal ampliada de cabeza y pronoto. Página 33 Revista Nicaragüense de Entomología. Número 290. 2022. Figura 33: Oncopeltus (Erythrischius) longirostris Stål, 1874. Ejemplar 1. Hembra. 33. Habitus, vista ventral (II, III: segundo y tercer segmentos rostrales). Página 34 Revista Nicaragüense de Entomología. Número 290. 2022. Figuras 34-35: Oncopeltus (Erythrischius) longirostris Stål, 1874. Ejemplar 2. Hembra. 34. Habitus, vista dorsal. 35. Habitus, vista lateral (II, III: segundo y tercer segmentos rostrales). Página 35 Revista Nicaragüense de Entomología. Número 290. 2022. Figuras 36-37: Oncopeltus (Erythrischius) longirostris Stål, 1874. Ejemplar 3. Macho. 36. Habitus, vista ventral. 37. Vista ventral ampliada de tórax y región abdominal (II, III: segundo y tercer segmentos rostrales). Página 36 Revista Nicaragüense de Entomología. Número 290. 2022. Figuras 38-39: Oncopeltus (Erythrischius) longirostris Stål, 1874. Ejemplar 4. Macho. 38. Habitus, vista ventral. 39. Vista ventral ampliada de tórax y región abdominal (II, III: segundo y tercer segmentos rostrales). Página 37 Revista Nicaragüense de Entomología. Número 290. 2022. Figuras 40-41: Oncopeltus (Erythrischius) longirostris Stål, 1874. Ejemplar 5. Hembra. 40. Habitus, vista ventral. 41. Vista ventral ampliada de tórax y región abdominal (II, III: segundo y tercer segmentos rostrales). Página 38 Revista Nicaragüense de Entomología. Número 290. 2022. Figuras 42-44: Oncopeltus (Erythrischius) longirostris Stål, 1874. Ejemplar 6. Hembra. 42. Habitus, vista dorsal. 43. Habitus, vista ventral. 44. Vista ventral ampliada de tórax y región abdominal (II, III: segundo y tercer segmentos rostrales). Página 39 Revista Nicaragüense de Entomología. Número 290. 2022. Figuras 45-47: Melanopleurus bistriangularis (Say, 1831). Macho. 45. Habitus, vista dorsal. 46. Vista dorsal ampliada de cabeza y pronoto. 47. Vista ampliada de antena. Página 40 Revista Nicaragüense de Entomología. Número 290. 2022. Figuras 48-52: Melanopleurus bistriangularis (Say, 1831). Macho. 48. Vista ampliada de cabeza y pronoto. 49. Vista frontal ampliada de cabeza. 50. Vista parcial ampliada de hemélitro. 51. Vista ampliada pronoto y escutelo. 52. Vista ampliada de parte terminal de hemélitros. Página 41 Revista Nicaragüense de Entomología. Número 290. 2022. Figuras 53-55: Melanopleurus bistriangularis (Say, 1831). Macho. 53. Habitus, vista ventral. 54. Vista postero-ventral de región abdominal terminal (círculo señala el pigóforo). 55. Vista dorsal de pigóforo (círculo). Página 42 Revista Nicaragüense de Entomología. Número 290. 2022. Figuras 56-57: Melanopleurus bistriangularis (Say, 1831). Macho. 56. Habitus, vista lateral. 57. Vista lateral ampliada de cabeza y región torácica. Página 43 Revista Nicaragüense de Entomología. Número 290. 2022. Figuras 58-60: Melanopleurus bistriangularis (Say, 1831). Macho. 58. Habitus, vista lateral. 59, 60. Vista lateral ampliada de parte de tórax y región abdominal. Página 44 Revista Nicaragüense de Entomología. Número 290. 2022. Similarmente en iNaturalist (https://spain.inaturalist.org/observations/ 78393053; https://spain.inaturalist.org/observations/58511165) se presentan fotos de ejemplares de esta especie de Lygaeinae sobre planta no identificada en Barcelona (10°08’00’’N, 64°41’00’’O; 20 m de altitud media), municipio Simón Bolívar (estado Anzoátegui), y sobre C. procera en la Vela Coro (11°27’33”N, 69°34’04”O; 20 m), municipio Colina (estado Falcón). 39. Oncopeltus (Erythrischius) longirostris Stål, 1874 (Figuras 31-44) Plantas hospedantes o asociadas. Desconocidas. Fuentes bibliográficas: Slater (1964, 1992), O’Rourke (1979), Osuna (2000), Dellapé y Henry (2022). Distribución en Venezuela. Estado Anzoátegui: Anaco (09°26’00’’N, 64°28’00’’O; 220 m), municipio Anaco (Nuevo registro, presente estudio); estado Aragua: Parque Nacional “Henri Pittier” (10°22’48”N, 67°37’08”O; 1100 m) (Osuna 2000). Comentarios. El único registro documentado de O. (Erythrischius) longirostris para Venezuela se debe a Osuna (2000), quien en su libro “Entomología del Parque Nacional Henri Pittier, Aragua, Venezuela” la menciona como parte integrante de la fauna insectil de dicho parque nacional en la región nor-central. Por lo tanto, el presente aparece como el segundo registro para el país de esta especie de Lygaeinae. De acuerdo a Slater (1992), la longitud de los segmentos rostrales II y III constituye uno de los caracteres morfológicos diagnósticos más importantes para separar entre los adultos de O. (Erythrischius) longirostris y los de Oncopeltus (Erythrischius) semilimbatus (Stål, 1874), la especie que le es más afín morfológicamente. En este sentido, en cuanto a sus longitudes ambos segmentos rostrales son subiguales en O. (Erythrischius) semilimbatus; mientras que, por contraste, en los adultos de O. (Erythrischius) longirostris el segmento II es mucho más corto que el III, tal como se observó en los ejemplares capturados en Anaco, estado Anzoátegui. Sin embargo, en algunos especímenes se observó que dichas longitudes del rostrum no se presentaron tan conspicuas, lo que sugiere un grado de hibridación e introgresión, tal como se presenta en otras especies de Oncopeltus (O’Rourke 1979). Página 45 Revista Nicaragüense de Entomología. Número 290. 2022. 40. Oncopeltus (Oncopeltus) luctuosus (Stål, 1867) Plantas hospedantes o asociadas. Apocynaceae: Calotropis procera (Ait.) Ait., otra especie no identificada. Fuentes bibliográficas: O’Rourke (1976), Ojeda (1973), Cazorla-Perfetti y Morales-Moreno (2019c), Dellapé y Henry (2022). Distribución en Venezuela. Distrito Capital: El Valle (Caracas) (10°28’02”N, 66°54’26”O, 900-1400 m); estado Falcón: Coro (11°24’N, 69°40’O; 20 m), municipio Miranda (O’Rourke 1976, Cazorla-Perfetti y Morales-Moreno 2019c). 41. Oncopeltus (Erythrischius) sandarachatus (Say, 1831) (Figuras 27-30, 81-95) Plantas hospedantes o asociadas. Apocynaceae: Calotropis procera (Ait.) Ait. (Nuevo registro, estado Anzoátegui); Clerodendrum L. (Lamiaceae) (Nuevo registro, estado Mérida) (Figuras 93-95). Fuentes bibliográficas: Martorell (1939), Slater (1964,1992), Dellapé y Henry (2022). Distribución en Venezuela. Estado Aragua: Turmero (10°13’42”N; 67°28’31”O; altitud media: 446 m), municipio Santiago Mariño (Martorell 1939); estado Anzoátegui: Anaco (09°26’00’’N, 64°28’00’’O; 220 m), municipio Anaco (Nuevo registro, presente estudio); estado Mérida: La Parroquia Osuna Rodríguez (08°34’11”N, 71°11’52”O; 1323 m), Mérida, municipio Libertador (Nuevo registro, presente estudio). Comentarios. El presente aparece como el segundo registro de la especie para el territorio nacional, cuya primera documentación se hizo hace más de 80 años; además, las capturas de ejemplares de esta especie representan el primer registro de la misma en las regiones nor-oriental y andina de Venezuela. En la plataforma iNaturalist (https://inaturalist.ca/observations/13030112; https://inaturalist. ca/observations/19003277; https://inaturalist.ca/observations/31546926; https://inaturalist.ca/observations/39827289), se muestran fotos de adultos de O. (Erythrischius) sandarachatus capturados en Ecuador y México sobre plantas de Asclepias curassavica L. y Calotropis procera (Ait.) Ait. (Apocynaceae); y en la Guayana Francesa, Ecuador, Costa Rica y México sobre plantas no identificadas de Poaceae y de otras familias, similarmente no identificadas (https://inaturalist.ca/observations/70452305; https:// inaturalist.ca/taxa/261476-Oncopeltussandarachatus/browse_photos; Página 46 Revista Nicaragüense de Entomología. Número 290. 2022. https://inaturalist.ca/observations/36328984; https://inaturalist.ca/ observations/71192646). Las observaciones acerca de las plantas asociadas/hospedantes tanto la del presente estudio (que representan nuevos registros) como las de iNaturalist, requieren de estudios más detallados y amplios para llegar a conclusiones más certeras. Aparece importante resaltar la presencia de anomalías en pata media izquierda (oligomeria, por ausencia de fémur, tibia, tarsos) y hemélitro (abultamiento) en ejemplar capturado en Mérida (Figuras 81-92). 42. Oncopeltus (Erythrischius) semilimbatus (Stål, 1874) Plantas hospedantes o asociadas. Apocynaceae: Asclepias curassavica L., Gomphocarpus fruticosus (L.), C. procera; Euphorbiaceae: Euphorbia L., Euphorbia hirta L., Euphorbia hypericifolia L. Transmisor de Microrganismos patógenos. Protozoa: Phytomonas Donovan, 1909 (Kinetoplastea: Trypanosomatidae). Fuentes bibliográficas: Barreto (1982), Uzcátegui et al. (2017), SegarraCarmona et al. (2020), Dellapé y Henry (2022). Distribución en Venezuela. Estado Mérida: Cerro La Morita a 12 Km N de la ciudad de Mérida (08°36’30”N, 71°12’00”O; 1935 m), municipio Libertador; estado Sucre: Güiria (10°34’26”N, 62°17’54”O; 10 m de altitud media), municipio Valdez (Península de Paria); Irapa (10°34’14”N, 62°34’56”O; 3 m de altitud media), municipio Mariño (Península de Paria); Yaguaraparo (10°34’05”N, 62°49’39”O), municipio Cagigal (Península de Paria) (Barreto 1982, Uzcátegui et al. 2017). 43. Oncopeltus (Erythrischius) unifasciatellus Slater, 1964 (Figuras 96106). Plantas hospedantes o asociadas. Amaranthaceae: Amaranthus viridis L. (bledo) Nuevo registro, estado Mérida) (Figuras 96-97); Apocynaceae: Asclepias curassavica L. Fuentes bibliográficas: Distant (1880/1893), Slater (1964), Root y Chaplin (1976), Dellapé y Henry (2022). Distribución en Venezuela. Localidad (es) no especificada (s) (Distant 1880/1893); estado Mérida: La Parroquia Osuna Rodríguez (08°34’11”N, 71°11’52”O; 1323 m), Mérida, municipio Libertador (Nuevo registro, presente estudio). Página 47 Revista Nicaragüense de Entomología. Número 290. 2022. Comentario. El presente aparece como el primer registro de una localidad específica para la especie en Venezuela, por lo que se confirma su presencia en el territorio nacional. 44. Oncopeltus (Oncopeltus) varicolor stalii Distant, 1882 Plantas hospedantes o asociadas. Desconocidas. Fuentes bibliográficas: Distant (1880/1893), Dellapé y Henry (2022). Distribución en Venezuela. Localidad (es) no especificada (s) (Distant 1880/1893). 45. Oncopeltus (Oncopeltus) varicolor varicolor (Fabricius, 1794) Plantas hospedantes o asociadas. Desconocidas. Fuentes bibliográficas: Distant (1880/1893), Dellapé y Henry (2022). Distribución en Venezuela. Localidad (es) no especificada (s) (Distant 1880/1893). Género Spilostethus Stål, 1868 46. Spilostethus pandurus (Scopoli, 1763) (Figuras 22-23) Plantas hospedantes o asociadas. Apocynaceae: Asclepias sinaica (Boiss.), Calotropis procera (Ait.) Ait., Calotropis gigantea (L.) W.T. Aiton, Nerium oleander L.; Anacardiaceae: Mangifera indica L., Pistacia vera L.; Asteraceae: Cyathillium cinereum (L.) H. Rob., Helianthus annuus L.; Betulaceae: Corylus avellana L.; Brassicaceae: Brassica oleracea var. capitata L. 1753; Combretaceae: Conocarpus erectus L.; Convolvulaceae: Ipomoea batatas (L.) Lam, Convolvulus arvensis L.; Cucurbitaceae: Citrullus lanatus (Thunb.) Matsum. & Nakai, 1916, Cucurbita maxima Duchesne in Lam., 1786, Cucumis melo L., Luffa acutangula (Roxb.); Fabaceae: Arachis hypogaea L., Cajanus cajan (L.) Huth, Cicer arietinum L., Cyamopsis tetragonoloba L., Medicago sativa L., Phaseolus vulgaris L., Trifolium alexandrium L., Vicia faba L., Vigna inguiculata (L.) Walp., Vigna mungo (L.) Hepper; Juglandaceae: Carya illinoinensis (Wangenh.) K. Koch, 1869; Lamiaceae: Plectranthus ornatus Codd., Stachys affinis Bunge; Malvaceae: Abelmoschus esculentus (L.) Moench, 1794, Gossypium hirsutum L., Hibiscus sabdariffa L.; Myrtaceae: Psidium guajava L., Syzygium cumini (L.) Skeels Página 48 Revista Nicaragüense de Entomología. Número 290. 2022. (= Eugenia jambolana Lam); Pedaliaceae: Sesamum indicum L.; Poaceae: Eleusine coracana Gaertn., Pennisetum glaucum (L.) R.Br., Saccharum officinarum L., Sorghum bicolor (L.) Moench var. curra, Sorghum vulgare L., Triticum aestivum L.; Rosaceae: Prunus dulcis (Mill.) D. A. Webb., Prunus armeniaca L., Prunus persica Batsch; Rutaceae: Citrus L.; Sapindaceae: Litchi chinensis Sonn., Sapindus mukorossi Gaertn.; Solanaceae: Datura stramonium L., Nicotiana L., Solanum lycopersicum L., Solanum melongena L.; Vitaceae: Vitis vinifera L. Transportador de microorganismos patógenos. Hongos: Eremothecium coryli Kurtzman, 1995 (= Nematospora coryli) (Ascomycota), causante de estigmatomicosis (mancha amarilla). Fuentes bibliográficas: Thangavelu (1979), Pericart (1998), Sweet (2000), Rengifo-Correa y González-Obando (2011), Burdfield-Steel y Shuker (2014), Cazorla-Perfetti et al. (2019), Ibrahim y Elshewy (2020), Bastardo & PérezGelabert (2021). Distribución en Venezuela. Estado Anzoátegui: Anaco (09°26’00’’N, 64°28’00’’O; 220 m), municipio Anaco (el presente estudio, Nuevo registro); estado Falcón: Coro (11°24’N, 69°40’O; 20 m), municipio Miranda (Cazorla-Perfetti et al. 2019). Comentario. Spilostethus pandurus es una especie polífaga de Lygaeidae de distribución primariamente transpaleártica y paleotropical, y representa una plaga importante de cultivos (30-40 especies de 15-16 familias de plantas). Sin embargo, desde la primera década del presente siglo se ha reportado su presencia como una “especie invasora” (aliens) en la región Neotropical (Colombia, República Dominicana, Venezuela) (Rengifo-Correa y GonzálezObando 2011, Cazorla-Perfetti et al. 2019, Bastardo & Pérez-Gelabert 2021). El presente aparece como el segundo registro de la especie en Venezuela. Subfamilia ORSILLINAE Distant, 1880 Tribu Metrargini Kirkaldy, 1902 Género Xyonysius Ashlock & Lattin, 1963 47. Xyonysius cf. californicus (Stål, 1859) Plantas hospedantes o asociadas. Asparagaceae: Yucca L.; Asteraceae: Erigeron quercifolius Lam., Flaveria Juss.; Flaveria linearis Lag.; Bromeliaceae: Tillandsia L.; Fabaceae: Medicago L.; Medicago lupulina L.; Malvaceae: Gossypium L.; Poaceae: Saccharum L. Página 49 Revista Nicaragüense de Entomología. Número 290. 2022. Fuentes bibliográficas: Slater y Baranowsky (1990), Maes (1998), Schaefer (1998), Alarcón y Cazorla (2022), Dellapé y Henry (2022). Distribución en Venezuela. Estado Mérida: La Parroquia Osuna Rodríguez (08°34’11”N, 71°11’52”O; 1323 m), Mérida, municipio Libertador (Alarcón y Cazorla 2022). Comentario. La presencia de Xyonysius para el territorio nacional fue documentado muy recientemente (Alarcón y Cazorla 2022); sin embargo, el estatus de la especie (Xyonysius californicus) aún requiere revisarse y establecerse con exactitud. Tribu Nysiini Uhler, 1876 Género Nysius Dallas, 1852 48. Nysius ericae (Schilling, 1829) Plantas hospedadoras o asociadas. Poaceae: Saccharum officinarum L. Fuentes bibliográficas: Slater (1964), Urtiaga (2007), Dellapé y Henry (2022). Distribución en Venezuela. Estado Lara: Valle del Turbio (44°15’00”N- 72°00’00”O, 44°30’00”N72°20’00”O), extenso valle a ambos lados del río Turbio, entre municipios Iribarren y Palavecino, y el municipio Peña del estado Yaracuy (Urtiaga 2007). Comentario. Nysius ericae se encuentra compuesta por dos subespecies: N. ericae alticola Hutchinson, 1934 (Distribución: India) y N. ericae ericae (Schilling, 1829) (Distribución: Sudáfrica, Egipto, Senegal, Astrakán, Turkestán, EUA, México, Panamá, Puerto Rico, Austria, Bélgica, República Checa, Inglaterra, Francia, Alemania, Hungría, Polonia, Portugal, Rumania, Rusia, España, Suecia, Suiza, Yugoslavia, Argelia, Arabia, Libia, China, Mongolia, Turquía, Bulgaria, Croacia, Grecia, Eslovaquia, Ucrania, Marruecos, Túnez, Armenia, Irán, Irak, Taiwán, Yemen) (Dellapé y Henry 2022). Página 50 Revista Nicaragüense de Entomología. Número 290. 2022. Figuras 61-63: Acroleucus phoenix Brailovsky, 1980. Planta hospedante/asociada. Allamanda cathartica L. en conjunto residencial. Página 51 Revista Nicaragüense de Entomología. Número 290. 2022. Figuras 64-65: Neortholomus jamaicensis (Dallas, 1852). Planta hospedante/asociada. Origanum vulgare L. en peridomicilio. Página 52 Revista Nicaragüense de Entomología. Número 290. 2022. Figuras 66-68: Neortholomus jamaicensis (Dallas, 1852). Hembra. 66. Habitus, vista dorsal. 67. Vista ampliada de antena. 68. Habitus, vista ventral. Página 53 Revista Nicaragüense de Entomología. Número 290. 2022. Figuras 69-71: Neortholomus jamaicensis (Dallas, 1852). Hembra. 69. Habitus, vista lateral. 70. Vista ventral ampliada de cabeza y tórax. 71. Vista ventral ampliada de región abdominal. Página 54 Revista Nicaragüense de Entomología. Número 290. 2022. Figuras 72-74: Neortholomus jamaicensis (Dallas, 1852). Macho. 72. Habitus, vista dorsal. 73. Habitus, vista ventral. 74. Vista dorsal ampliada de parte de cabeza, tórax escutelo y hemélitros. Página 55 Revista Nicaragüense de Entomología. Número 290. 2022. Figuras 75-77: Neortholomus jamaicensis (Dallas, 1852). Macho. 75. Habitus, vista lateral. 76. Vista lateral ampliada de región torácica (el círculo señala peritrema ostiolar de las glándulas odoríferas metatorácicas). 77. Vista ampliada de pata delantera. Página 56 Revista Nicaragüense de Entomología. Número 290. 2022. Figuras 78-80: Neortholomus jamaicensis (Dallas, 1852). Macho. 78. Vista ampliada de pronoto, escutelo y parte de tergitos. 79. Vista ampliada de escutelo y tergitos. 80. Vista ampliada de ala. Página 57 Revista Nicaragüense de Entomología. Número 290. 2022. Figuras 81-84: Oncopeltus (Erythrischius) sandarachatus (Say, 1831). Macho (estado Mérida). 81. Habitus, vista dorsal. 82. Vista dorsal ampliada de cabeza y pronoto (los círculos señalan manchas rojizas en márgenes laterales del lóbulo anterior del pronoto). 83. Vista dorsal ampliada de parte de pronoto, escutelo y hemélitros. 84. Vista ampliada de parte terminal de hemélitros. Las flechas señalan abultamiento anómalo en hemélitros. Página 58 Revista Nicaragüense de Entomología. Número 290. 2022. Figuras 85-87: Oncopeltus (Erythrischius) sandarachatus (Say, 1831). Macho (estado Mérida). 85. Habitus, vista ventral. 86. Vista ventral ampliada de cabeza y tórax. 87. Vista ampliada de región torácica. Las flechas señalan anomalía (oligomeria) en pata media izquierda. Página 59 Revista Nicaragüense de Entomología. Número 290. 2022. Figuras 88-92: Oncopeltus (Erythrischius) sandarachatus (Say, 1831). Macho (estado Mérida). 88. Vista lateral ampliada de cabeza y región torácica. 89. Vista lateral ampliada de región abdominal. 90. Vista ventral ampliada de cabeza. 91. Vista ampliada de esternitos terminales. 92. Vista posterior ampliada de terminalia. Los círculos señalan el pigóforo. Página 60 Revista Nicaragüense de Entomología. Número 290. 2022. Figuras 93-95: Oncopeltus (Erythrischius) sandarachatus (Say, 1831), estado Mérida. Planta hospedante/asociada. 93,94, 95. Clerodendrum L. (Lamiaceae). Página 61 Revista Nicaragüense de Entomología. Número 290. 2022. Figuras 96-97: Oncopeltus (Erythrischius) unifasciatellus Slater, 1964. Planta hospedante/asociada. 96,97. Amaranthus viridis L. (Amaranthaceae). Página 62 Revista Nicaragüense de Entomología. Número 290. 2022. Figuras 98-100: Oncopeltus (Erythrischius) unifasciatellus Slater, 1964. Hembra. 98. Habitus, vista dorsal. 99. Vista dorsal ampliada de cabeza y pronoto. 100. Vista dorsal ampliada de pronoto, escutelo y parte de hemélitros. Página 63 Revista Nicaragüense de Entomología. Número 290. 2022. Figuras 101-103: Oncopeltus (Erythrischius) unifasciatellus Slater, 1964. Hembra. 101. Habitus, vista ventral. 102. Vista ventral ampliada de cabeza y región torácica. 103. Vista ampliada de esternitos terminales. Página 64 Revista Nicaragüense de Entomología. Número 290. 2022. Figuras 104-106: Oncopeltus (Erythrischius) unifasciatellus Slater, 1964. Hembra. 104. Habitus, vista lateral. 105. Vista lateral ampliada de región torácica. 106. Vista ampliada de esternitos terminales. Página 65 Revista Nicaragüense de Entomología. Número 290. 2022. Tribu Orsillini Ashlock & Slater 1988 Género Neortholomus Hamilton, 1983 49. Neortholomus jamaicensis (Dallas, 1852) (Figuras 64-80) Plantas hospedadoras o asociadas. Asteraceae: Baccharis L.; Ageratina adenophora (Spreng.) R. M. King & H. Rob. (= Eupatorium adenophorum); Cleomaceae: Cleome spinosa Jacq.; Cucurbitaceae: Cucurbita moschata (Duchesne ex Lam.) Duchesne ex Poir. 1786; Grossulariaceae: Ribes californicum Hook & Arn. var. hesperium (McClatchie) Jeps. (=hesperium); Lamiaceae: Origanum vulgare L. (orégano) (Nuevo record; presente estudio) (Figuras 64-65); Ocimum gratissimum (L.) (= Ocimum suave); Hyptis Jacq.; Vitex pyramidata B. L. Robinson; Salvia xalapensis Benth.; Salvia splendens Sellow ex Schult., 1822; Stachys L.; Pedaliaceae: Sesamum indicum L; Solanaceae: Solanum tuberosum L. (papa); Verbenaceae: Verbena bracteata Cav. Ex Lag. & Rodr. (= prostrata Savi); Lippia L. Fuentes bibliográficas: Martorell (1939), Hamilton (1983), Slater y Baranowsky (1990), Maes (1998), Narváez (2003), Dellapé & Montemayor (2012), Henry et al. (2015), Dellapé y Henry (2022).Distribución en Venezuela. Estado Monagas: Caripito (10°06’40”N, 63°06’17”O; 17-48 m), municipio Bolívar (Martorell, 1939); estado Mérida: La Parroquia Juan Rodríguez Suárez (08°33’32,84”N, 71°11’59,38”O; 1269 m), Mérida, municipio Libertador (Nuevo registro, presente estudio). Comentarios. El presente aparece como el segundo registro de la especie para el territorio nacional, cuya primera documentación fue realizada hace más de 80 años (Martorell 1939). Hamilton (1983) y Dellapé & Montemayor (2012) indican que es muy difícil la diferenciación morfológica entre Neortholomus jamaicensis y N. rubricatus (Berg, 1874), la especie que le es más afín morfológicamente, especialmente si se toma en consideración que ambas especies varían ampliamente en talla y coloración; de allí que los últimos autores señalados consideran que aún se requiere realizar más estudios para encontrar caracteres más robustos y determinantes para la diferenciación entre ambas especies. Aparece importante comentar que Hamilton (1983) señaló que Neortholomus scolopax (Say, 1831) posee la conducta de esconderse dentro de los capítulos o conglomerados de flores (flower head), y al parecer “esto pudiera ser característico de todas las especies de Neortholomus”. En el presente trabajo, se observaron varios ejemplares de N. jamaicensis apareándose y alimentándose sobre las flores de O. vulgare (Lamiaceae). Página 66 Revista Nicaragüense de Entomología. Número 290. 2022. DISCUSIÓN Desde 2021, hemos venido documentando actualizaciones y revisiones de varias familias de Hemiptera-Heteroptera de Venezuela, incluyendo Miridae (Cazorla 2021a), Coreidae (Cazorla 2021b), Pentatomidae (Cazorla 2021c), Pyrrhocoridae (Cazorla-Perfetti et al. 2021), Tingidae (Cazorla & Knudson 2021) y Rhopalidae (Cazorla & Alarcón 2022). La extensa y detallada revisión bibliográfica más las capturas e identificación de ejemplares en algunas entidades federales de Venezuela, reveló que hasta el presente en el territorio nacional se ha documentado la presencia de 49 especies y 19 géneros de Lygaeidae. Estos guarismos representarían alrededor del 54% de los 35 géneros y el 21,30% de las 230 especies, respectivamente, reportados para la región Neotropical (Henry et al. 2015). Por ello, aún se requiere estudiar y muestrear extensas áreas inexploradas, y revisar las colecciones entomológicas presentes en el país. Asimismo, se hace necesario revisar los estatus taxonómicos y la presencia de varias especies para el territorio nacional. AGRADECIMIENTOS A Gabriel Eduardo Alarcón Mendoza y Elisabeth Alarcón por su valiosa ayuda en captura y fotografiado de los insectos capturados en Mérida, estado Mérida, Venezuela. Dres. Eduardo Faúndez (Laboratorio de Entomología, Instituto de la Patagonia, Universidad de Magallanes, Punta Arenas, Chile) y María Cecilia Melo (División de Entomología, Museo de La Plata, U.N.L.P., La Plata, Buenos Aires, Argentina), por sus consejos y orientaciones. A Dr. Harry Brailovsky (Instituto de Biología UNAM, Depto. de Zoología, Ciudad de México, México), por su apoyo bibliográfico. REFERENCIAS BIBLIOGRÁFICAS ALARCÓN M. & CAZORLA D. (2022) Registro de Melacorhyphus circumlitus (Stål, 1862) (Lygaeinae) y Xyonysius cf. californicus (Stål, 1859) (Orsillinae: Metrargini) (Hemiptera-Heteroptera: Lygaeidae) en la región andina de Venezuela. Revista Nicaragüense de Entomología, 271: 1-26. BASTARDO R. & PÉREZ-GELABERT D. (2021) Spilostethus pandurus (Scopoli, 1763) (Hemiptera: Heteroptera: Lygaeidae), nuevo registro para República Dominicana y el Caribe insular. Novitates Caribaea, 17: 179-183. BARRETO J. M. (1982) Marchitez del cocotero asociada con protozoarios flagelados (Phytomonas sp.) en la península de Paria – Estado Sucre. Agronomía Tropical, 32(1-6): 291–302. Página 67 Revista Nicaragüense de Entomología. Número 290. 2022. BEHRSTOCK R. (2021) New records of pollinators and other insects associated with Arizona milkweed, Asclepias angustifolia, at four sites in Southeastern Arizona. Journal of Pollination Ecology, 27(1): 1-24. BRAILOVSKY H. (1977) Contribución al estudio de los Hemiptera- Heteroptera de México. XII. El género Neacoryphus Scudder (Lygaeidae-Lygaeinae) y descripción de tres nuevas especies. Anales del Instituto de Biología de la Universidad Nacional Autónoma de México, Serie Zoología 48(1): 97-122. BRAILOVSKY H. (1978) Estudio del género Lygaeus Fabricius 1794, del Nuevo Mundo, con descripción de cinco nuevas especies. Anales del Instituto de Biología de la Universidad Nacional Autónoma de México, Serie Zoología, 49(1): 123-166. BRAILOVSKY H. (1979a) El género Neocoryphus Scudder en Sudamérica y descripción de tres nuevas especies (Hemiptera – Heteroptera – Lygaeidae – Lygaeinae). Anales del Instituto de Biología de la Universidad Nacional Autónoma de México, Ser. Zoología, 50(1):227-239. BRAILOVSKY H. (1979b) Revisión del género Craspeduchus Stål, con descripción de dos nuevas especies (Hemiptera – Heteroptera – Lygaeidae – Lygaeinae). Anales del Instituto de Biología de la Universidad Nacional Autónoma de México, Ser. Zoología, 50(1):205-226. BRAILOVSKY H. (1980) Revisión del género Acroleucus Stål (HemipteraHeteroptera-Lygaeidae-Lygaeinae). Folia Entomológica Mexicana, 44: 39-120. BRAILOVSKY H. (1982) Un nuevo arreglo nomenclatorial y descripción de tres nuevos géneros y dos nuevas especies americanas de la subfamilia Lygaeinae (Hemiptera-Heteroptera-Lygaeidae). Anales del Instituto de Biología de la Universidad Nacional Autónoma de México, Serie Zoología, 52(1): 259-276. BRAILOVSKY H. (1984) Nuevas adiciones al género Acroleucus Stål (Hemiptera – Heteroptera – Lygaeidae – Lygaeinae). Anales del Instituto de Biología de la Universidad Nacional Autónoma de México, Ser. Zoología, 54(1):35-52. BRAILOVSKY H. & BARRERA E. (1984) Cinco especies nuevas, nuevos datos distribucionales y notas biológicas acerca de Lygaeinae americanos del género Acroleucus (Lygaeidae: Hemiptera: Heteroptera). Anales del Instituto de Biología de la Universidad Nacional Autónoma de México, Serie Zoología, 55(2): 95-110. BRAILOVSKY H. & CERVANTES L. (2008) Two new species and distribution records of the genus Acroleucus in Mexico (Hemiptera: Heteroptera: Lygaeidea: Lygaeinae). Florida Entomologist, 91(1): 49-54. Página 68 Revista Nicaragüense de Entomología. Número 290. 2022. BRAILOVSKY H. & BARRERA E. (2015) Dos especies nuevas del género Acroleucus (Hemiptera: Heteroptera: Lygaeoidea: Lygaeinae) de Colombia y Ecuador. Revista Mexicana de Biodiversidad, 86: 634-637. BRAILOVSKY H. (2021) New taxa of neotropical Lygaeinae (Hemiptera: Heteroptera: Lygaeoidea: Lygaeidae). Dugesiana, 28(2): 139-146. BURDFIELD-STEEL E. & SHUKER D. (2014) The evolutionary ecology of the Lygaeidae. Ecology and Evolution, 4(11): 2278-2301. CAZORLA PERFETTI D. & MORALES MORENO P. (2019a) Primer registro en Venezuela de Lygaeus argutus Brailovsky, 1982 (Heteroptera: Lygaeidae) con la descripción de la hembra. Revista Nicaragüense de Entomología, 184: 1-18. CAZORLA-PERFETTI D. & MORALES-MORENO P. (2019b) Registro de Ochrostomus ulheri (Stål) (Heteroptera: Lygaeidae) asociada con Calotropis procera (Ait.) Ait. (Apocynaceae) en el semiárido urbano del estado Falcón, Venezuela. 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Número 290. 2022. OSUNA E. (2000) Entomología del Parque Nacional Henri Pittier, Aragua, Venezuela. 1a edición. Fundación Polar: Museo del Instituto de Zoología Agrícola Francisco Fernández Yépez, Caracas, Venezuela 199 pp. PERICART J. (1998) Faune de France 84a. Hémiptères Lygaeidae euroméditerranéens. Vol. 1. Généralités Systématique: première partie. Féderation Française des Sociétés de Sciences Naturelles, Paris, Francia 475 pp. RENGIFO-CORREA L. & GONZÁLEZ-OBANDO R. (2011) Lygaeoidea (Hemiptera: Heteroptera) de Parques Nacionales Naturales (PNN) con nuevos registros para Colombia. Revista Colombiana de Entomología, 37(1): 331-340. ROOT R. & CHAPLIN S. (1976) The life-styles of tropical milkweed bugs, Oncopeltus (Hemiptera: Lygaeidae) utilizing the same hosts. Ecology, 57: 132140. SCHAEFER C. (1998) The taxonomic status of Xyonysius major (Berg) (Hemiptera: Lygaeidae), an occasional pest of sunflower in Brazil. Annal of The Entomological Society of Brasil, 27(1): 55-58. 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Lygaeidae of Florida (Hemiptera: Heteroptera). Florida Department of Agriculture and Consumer Services. Vol. 14. Contribution N° 725, Florida, USA 211 pp. SLATER A. (1992) A genus level revision of Western Hemisphere Lygaeinae (Heteroptera: Lygaeidae) with keys to species. The University of Kansas Science Bulletin, 55(1): 1-56. Página 72 Revista Nicaragüense de Entomología. Número 290. 2022. SLATER J. A. & O’DONNEL J. E. (1995) A catalogue of the Lygaeidae of the World (1960-1964). New York, USA 410 pp. SWEET II M. H. (2000) Seed and chinch bugs (Lygaeoidea). Pp. 143-264. In: Schaefer C. W. & Panizzi A. R. (eds.). Heteroptera of economic importance. CRC Press, Boca Raton, Florida, EUA. THANGAVELU K. (1979) The pest status and biology of Spilostethus pandurus (Scopoli) (Lygaeidae: Heteroptera). Entomon, 4(2): 137-141. URTIAGA R. (2007) Catálogo de los insectos de la región central. Agronomía Mesoamericana. https://revistas.ucr.ac.cr/docs/ AgronomiaMesoamericana/ catalogo-de-los-insectos-de-la-region-central.pdf (Accesado abril 2022). UZCÁTEGUI D., ÁVILA-NÚÑEZ J. & ALONSO-AMELOT M. (2017) Rapid adaptation of neotropical Oncopeltus semilimbatus (Hemiptera; Lygaeidae) to Gomphocarpus fruticosus, (Apocynaceae, Asclepiadoideae), an introduced African host, in the Northern Andes, Venezuela. Tropical Ecology, 58(1): 127137. YAZIC G. (2022) New Hosts and Spread Areas of Invasive Species Nysius cymoides (Spinola, 1837) (Hemiptera: Heteroptera: Lygaeidae) in Crop Plants in Turkey. KSU Journal of Agriculture & Nature, 25 (2): 267-273. Página 73 Revista Nicaragüense de Entomología. Número 290. 2022. La Revista Nicaragüense de Entomología (ISSN 1021-0296) es una publicación del Museo Entomológico de León, aperiódica, con numeración consecutiva. Publica trabajos de investigación originales e inéditos, síntesis o ensayos, notas científicas y revisiones de libros que traten sobre cualquier aspecto de la Entomología, Acarología y Aracnología en América, aunque también se aceptan trabajos comparativos con la fauna de otras partes del mundo. No tiene límites de extensión de páginas y puede incluir cuantas ilustraciones sean necesarias para el entendimiento más fácil del trabajo. The Revista Nicaragüense de Entomología (ISSN 1021-0296) is a journal published by the Entomological Museum of Leon, in consecutive numeration, but not periodical. RNE publishes original research, monographs, and taxonomic revisions, of any length. RNE publishes original scientific research, review articles, brief communications, and book reviews on all matters of Entomology, Acarology and Arachnology in the Americas. Comparative faunistic works with fauna from other parts of the world are also considered. Color illustrations are welcome as a better way to understand the publication. Todo manuscrito para RNE debe enviarse en versión electrónica a: (Manuscripts must be submitted in electronic version to RNE editor): Dr. Jean Michel Maes (Editor General, RNE) Museo Entomológico de León Apartado Postal 527, 21000 León, NICARAGUA Teléfono (505) 2319-9327 / (505) 7791-2686 [email protected] [email protected] Costos de publicación y sobretiros. La publicación de un artículo es completamente gratis. Los autores recibirán una versión pdf de su publicación para distribución. Página 74 Revista Nicaragüense de Entomología. Número 290. 2022. Ejemplar adulto de Spilostethus pandurus (Scopoli, 1763) succionando savia sobre fruto de Calotropis procera Ait. (Ait.) (Apocynaceae). Capturado en Coro, municipio Miranda, estado Falcón, Venezuela (Foto: Dalmiro Cazorla) Página 75 Revista Nicaragüense de Entomología. Número 290. 2022. Ejemplar macho de Lygaeus argutus Brailovsky, 1982. 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Applicability of Time Domain Transform Methods for Frequency Dependent Dynamic Stiffness

Frontiers in built environment

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ORIGINAL RESEARCH published: 04 April 2022 doi: 10.3389/fbuil.2022.854838 ORIGINAL RESEARCH published: 04 April 2022 doi: 10.3389/fbuil.2022.854838 Applicability of Time Domain Transform Methods for Frequency Dependent Dynamic Stiffness Naohiro Nakamura 1*, Kunihiko Nabeshima 1, Yoshihiro Mogi 2 and Akira Ota 3 1Graduate School of Advanced Science and Engineering, Hiroshima University, Higashi-hiroshima, Japan, 2Design Division, Taisei Corporation, Tokyo, Japan, 3Nuclear Facilities Division, Taisei Corporation, Tokyo, Japan Some types of dynamic stiffness, such as the dynamic ground stiffness used in soil- structure interaction analyses and the viscoelastic body used in vibration control systems, have strong frequency dependency. To perform seismic response analysis considering this frequency dependence and the nonlinearity of the model, the dynamic stiffness in the frequency domain must be transformed into the time domain, and a time-history nonlinear response analysis is required. Therefore, many studies on these time-domain transforms have been conducted. One of the present authors has already studied and proposed transform methods for this purpose, and some of their results were used to design new types of damping models. In the present study, the outline and characteristics of the proposed methods (A to C) for this transform are described first. Next, typical problems with strong frequency dependency (i.e., the dynamic soil stiffness, Maxwell element, viscoelastic body, Biot model, and causal hysteretic damping) were transformed into the time domain using these transform methods. The applicability of the transform methods was examined. Subsequently, the characteristics of each problem in the frequency domain and the characteristics of the obtained impulse response in the time domain were analyzed. Finally, it was confirmed that the proposed methods were applicable to all studied problems. These studies are important to understand the physical meaning of these problems, which have strong frequency dependency. Edited by: Edited by: Arturo Tena-Colunga, Autonomous Metropolitan University, Mexico Reviewed by: Shehata E. Abdel Raheem, Assiut University, Egypt Farhad Behnamfar, Isfahan University of Technology, Iran *Correspondence: Naohiro Nakamura [email protected] Specialty section: This article was submitted to Earthquake Engineering, a section of the journal Frontiers in Built Environment Received: 14 January 2022 Accepted: 07 February 2022 Published: 04 April 2022 Citation: Nakamura N, Nabeshima K, Mogi Y and Ota A (2022) Applicability of Time Domain Transform Methods for Frequency Dependent Dynamic Stiffness. Front. Built Environ. 8:854838. doi: 10.3389/fbuil.2022.854838 Specialty section: This article was submitted to Earthquake Engineering, a section of the journal Frontiers in Built Environment Keywords: dynamic stiffness, frequency dependency, time domain, impulse response, time history response analysis INTRODUCTION The dynamic ground stiffness used in soil-structure interaction analyses and the viscoelastic body used in vibration control systems may show strong frequency dependence. To perform a seismic response analysis considering this frequency dependence and the nonlinearity of the ground and buildings during strong earthquakes, the dynamic stiffness in the frequency domain must be transformed to the impulse response in the time domain. For this purpose, a time-history response analysis is required. Therefore, many studies on these time-domain transformations have been conducted since the 1980s (i.e., Wolf and Obernhuber, 1985; Wolf and Motosaka, 1989; Hayashi and Katukura, 1990; Meek, 1990; Motosaka and Nagano, 1992). Received: 14 January 2022 Accepted: 07 February 2022 Published: 04 April 2022 Method A (Basic Method) Let D(ω) be a complex function with frequency dependence and consider its time-domain transform. For D(ω), it is assumed that N complex numbers D(ω1), D(ω2), ..D(ωN) (hereinafter, data points) are given. Where 0 < ω1 < ω2 < ..ωN. The time-domain transform of D(ω) can be considered to be equivalent to passing through all data points, smoothly interpolating between the data points and approximating to a causal (=satisfying causal law) function. These methods have the following features: 1) Transforming calculation is simpler and easier. Transformation can be done by solving simultaneous equations only. 2) The number of dynamic stiffness data needed for the transformation is small. Consequently, the obtained impulse response is also simple, and the calculation of nonlinear response analysis is not heavy. The most basic method is Eq. 1. HA(ω) is an approximated complex function. yA(ω) and x(ω) are the output and input values in the frequency domain. tj  Δtpj(j  0 to N −1). hj, hj are the real undetermined constants and can be determined by solving simultaneous linear equations. These are the impulse responses in the time domain required. Details are described in Determination of Unknowns Using Simultaneous Equations. 3) The causality is automatically satisfied. Even a noncausal stiffness in the frequency domain can be transformed approximately and used for nonlinear response analysis. As Δt, the reciprocal of the maximum frequency of the data point (fmax  ωN/2π) or a value close to it is recommended. Δt does not have to match the time step (ΔT) of the time-history response analysis, and in many cases, ΔT < Δt. For example, if ΔT  0.01 s and ΔT  0.1 s, the past displacements and velocities used to calculate the time-delay terms will be the values of every 10th-time-history analysis result. 4) Matrix stiffness can be transformed in the same way. The applicability of the time-domain transform method and time-history response analysis of ground impedance calculated using a finite element model when a building is embedded in heterogeneous soil was shown in Nakamura (2008a). Moreover, the applicability of the time-domain transform method to the viscoelastic damper nonlinear problem, which has strong frequency and strain amplitude dependence, was studied (Nakamura, 2008b). In the time domain, Eq. 1 is expressed by Eq. 2. Citation: Moreover, the viscoelastic body used in vibration control systems may have nonlinear characteristics, such as strain amplitude, temperature, and frequency dependences. To evaluate the nonlinear dynamic response considering these characteristics, many studies have been conducted April 2022 | Volume 8 | Article 854838 1 Frontiers in Built Environment | www.frontiersin.org Nakamura et al. Time Domain Transform Methods to express the complex stiffness in the frequency domain using a dynamic model in the time domain (i.e., Makris and Constantinou, 1991; Kirekawa et al., 1992; Xue et al., 1992; Kasai et al., 2004). to express the complex stiffness in the frequency domain using a dynamic model in the time domain (i.e., Makris and Constantinou, 1991; Kirekawa et al., 1992; Xue et al., 1992; Kasai et al., 2004). However, in these studies, each dynamic stiffness characteristics and how the characteristics are expressed as an impulse response in the time domain were not clear. In this study, we collected the representative examples of various frequency-dependent functions. We divided them into several groups based on the transformation’s obtained impulse response. Then, we examined them and analyzed the applicability of the transform methods. However, these methods are difficult to apply in actual problems because some have low-accuracy results. Moreover, some require a heavy calculation load for transformation and response analyses, and others need to estimate the property of ω →∞of the complex stiffness appropriately, but this estimation is very difficult. Further, all these methods have difficulty transforming when the stiffness is not “causal.” Causal refers to a state that satisfies the temporal order that the “result” must happen after the “cause.” First, we listed the outline and features of the above time- domain transform methods. Next, using them, typical frequency- dependent problems were transformed. Then, these problems were classified, and the applicability of the transform method was evaluated. This approach will scrutinize the applicability of these transform methods to new problems in the future. Conversely, noncausal implies a state in which this temporal order is broken, and a part of the result occurs before the cause. Even in such a state in the frequency domain, it is possible to analyze the response. However, in the real world of the time domain, that state never happens and cannot be analyzed. OUTLINE OF THE PROPOSED TRANSFORM METHOD Given these issues, a method of transforming the dynamic stiffness of a rigid foundation on layered soil into the time domain and performing time-history response analysis was proposed by Nakamura (2006a). However, the complex stiffness in the frequency domain sometimes has noncausal properties. Further, methods that enable approximate time-domain transform were proposed even in noncausal cases. These transform methods were organized as Methods A–C (Nakamura, 2006b). Moreover, the theoretical explanation of these methods by comparing with the Duhamel integral was studied (Nakamura, 2012). The outline of the proposed time-domain transform method and its characteristics are shown below. Because the notation method of the formula differs among studies, it is organized based on the most common notation for complex functions (Nakamura, 2012). Frontiers in Built Environment | www.frontiersin.org Method B (Method to Consider Virtual Mass) Where, In Nakamura (2006b), the improvement of Method A was examined. First, to improve the accuracy of the transform, the 2nd-order component simultaneous term 2h0 was added to make HB (ω) and yB (t) (see Eqs 3, 4). The 2h0 is a value related to the input second derivative value of the current time. This value corresponds to the virtual mass in the transform of the ground impedance. This method is called Method B. Δ2h0  V3n′ −V2V4 V1n′ −V2 2 , Δ0h0  V2n3 −V1V4 V1n′ −V2 2 (8) V1   n′ i1 ω4 i , V3   n′ i1 ω2 i ReHB′(ωi) −Re(D(ωi)) V2   n′ i1 ω2 i , V4   n′ i1 ReHB′(ωi) −Re(D(ωi)) ⎫ ⎪ ⎪ ⎪ ⎪ ⎪ ⎬ ⎪ ⎪ ⎪ ⎪ ⎪ ⎭ (9) (8) (9) yB(ω)  HB(ω) · x(ω) HB(ω)  −ω2·2h0 + iω·1h0+0h0 + ⎛ ⎝iω ·  N−2 j1 1hj · e−iωt +  N−1 j1 0hj · e−iωt⎞⎠ ⎫ ⎪ ⎪ ⎬ ⎪ ⎪ ⎭ (3) yB(t)2h0 · €x(t)+1h0 · _x(t)+0h0 · x(t) + ⎧ ⎨ ⎩ N−2 j1 1hj · _xt −tj +  N−2 j1 0hj · xt −tj ⎫ ⎬ ⎭ (4) yB(ω)  HB(ω) · x(ω) HB(ω)  −ω2·2h0 + iω·1h0+0h0 + ⎛ ⎝iω ·  N−2 j1 1hj · e−iωt +  N−1 j1 0hj · e−iωt⎞⎠ ⎫ ⎪ ⎪ ⎬ ⎪ ⎪ ⎭ (3) Similarly, 1h0, a simultaneous term of the imaginary part, is modified to obtain Eq. 10 (Waas, 1972). Here, Δ1h0 is the correction value obtained using Eqs 11, 12 via the least- squares method. This method is called Method C. yB(ω)  HB(ω) · x(ω) yB(ω)  HB(ω) · x(ω) ImHC′(ω)  ImHB′(ω) + ω · Δ1h0 (10) Δ1h0  −V5 V2 (11) ImHC′(ω)  ImHB′(ω) + ω · Δ1h0 (10) Δ1h0  −V5 V2 (11) V5   n′ i1 ωiImHB′(ωi) −ImD(ωi) (12) (10) yB(t)2h0 · €x(t)+1h0 · _x(t)+0h0 · x(t) + ⎧ ⎨ ⎩ N−2 j1 1hj · _xt −tj (11) ⎩ j +  N−2 j1 0hj · xt −tj ⎫ ⎬ ⎭ (4) (4) V5   n′ i1 ωiImHB′(ωi) −ImD(ωi) (12) (12) In many cases, the time-delay term tends to be smaller as j becomes larger. This is equivalent to the property that the input state quantity in the near past has a considerable effect. Method B (Method to Consider Virtual Mass) The state quantity in the distant past has a minor effect on the current output value. It can be understood generally (with exceptions, of course). Suppose the first n’ time-delay terms are significant values among the calculated time-delay terms; the values after n’ are negligible. In that case, it is possible to reduce the upper limit of Σ in Eqs 1–4 from N−1 and N−2 to n’. Applying this idea to Eqs 3, 4 yields Eqs 5, 6, respectively. Here, n’ < N−1. However, in this case, H’B (ω) passes near the original data point but does not pass on the data point. When the transform target is dynamic stiffness obtained from the analysis in the frequency domain using complex damping, a larger damping ratio results in a more noncausal function; from the above transform methods, Method B is recommended when the data points are almost causal. Method C is recommended when noncausality is strong. Method A (Basic Method) yA(t), x(t), _x(t) are the output value, input value, and first derivative of the input value (hereinafter, input first derivative value) at time t (hereinafter, current time), respectively. Eq. 2 shows that the output value of the current time depends on the current time and past input state values (input value and its first derivative value). Furthermore, using these methods, the imaginary unit i in the frequency domain was transformed and approximated as a causal function in the time domain. This function was called the causal unit imaginary function. Using this function, new damping models with small frequency dependency were proposed (Nakamura, 2007; Nakamura, 2016). This method is hereinafter referred to as Method A. Moreover, 0hj is called the 0th-order component, and 1hj is April 2022 | Volume 8 | Article 854838 Frontiers in Built Environment | www.frontiersin.org 2 Nakamura et al. Time Domain Transform Methods YB′(t)2h0 · €x(t)+1h0 · _x(t)+0h0 · x(t) + ⎧ ⎨ ⎩ n′ j1 1hj · _xt −tj +  n′ j1 0hj · xt −tj ⎫ ⎬ ⎭ (6) called the 1st-order component. It can be said that the former is multiplied by displacement and has the dimension of the stiffness. The latter is multiplied by velocity and has the dimension of the damping coefficient. 0hj and 1hj, 0h0 and 1h0 related to the current time state quantity are called simultaneous terms, and 0h1 to 0hN−1, 1h1 to 1hN−1 related to the past quantity are called time-delay terms. (6) yA(ω)  HA(ω) · x(ω) HA(ω)  iω · 1h0 + 0h0 + ⎛ ⎝iω ·  N−1 j1 1hj · e−iωtj +  N−1 j1 0hj · e−iωtj⎞⎠ ⎫ ⎪ ⎪ ⎬ ⎪ ⎪ ⎭ (1) yA(t)  h0 · _x(t)+h0 ·x(t)+⎛ ⎝ N−1 j1 1hj · _xt−tj+  N−1 j1 0hj ·xt−tj⎞⎠ (2) Method C (Correction Method to Correct When Noncausality is Strong) yA(ω)  HA(ω) · x(ω) When Noncausality is Strong) Strictly speaking, the time-domain transform is impossible if D(ω) is not causal. However, it may be possible approximately. When the noncausal data points are approximated using Eq. 5, there is a tendency to differentiate between the real and imaginary parts. Therefore, Δ0h0 and Δ2h00, simultaneous terms of the real part, are modified to obtain Eq. 7. Here, Δ0h0 and Δ2h0 are the correction values obtained using Eqs 8, 9 via the least-squares method. (2) ReHC′(ω)  ReHB′(ω) −ω2 · Δ2h0 + Δ0h0 (7) (7) 4) Does not require simultaneous term separation. {S(ωi)}   SR(ωi) SI(ωi)   ⎧ ⎪ ⎪ ⎪ ⎪ ⎪ ⎨ ⎪ ⎪ ⎪ ⎪ ⎪ ⎩  N−1 j0 cos θij·0hj + ωi  N−2 j0 sin θij·1hj −ω2 i h0 − N−1 j0 sin θij·0hj +  N−2 j0 cos θij·1hj ⎫ ⎪ ⎪ ⎪ ⎪ ⎪ ⎬ ⎪ ⎪ ⎪ ⎪ ⎪ ⎭ (13) ⎧ ⎪ ⎪ ⎨ ⎪ ⎪ ⎩ {D(ω1)} .. . {D(ωN)} ⎫ ⎪ ⎪ ⎬ ⎪ ⎪ ⎭  C0 C1 C2 · ⎧ ⎪ ⎨ ⎪ ⎩ {G0} {G1} G2 ⎫ ⎪ ⎬ ⎪ ⎭ (14) When θij  ωi · tj, tj  Δt · j {D(ωi)}  Re[D(ωi)] Im[D(ωi)], {G0}  ⎧ ⎪ ⎪ ⎪ ⎪ ⎨ ⎪ ⎪ ⎪ ⎪ ⎩ 0h0 0h1 ... 0hN−1 ⎫ ⎪ ⎪ ⎪ ⎪ ⎬ ⎪ ⎪ ⎪ ⎪ ⎭ , {G1}  ⎧ ⎪ ⎪ ⎪ ⎪ ⎨ ⎪ ⎪ ⎪ ⎪ ⎩ 1h0 1h1 .. . 1hN−2 ⎫ ⎪ ⎪ ⎪ ⎪ ⎬ ⎪ ⎪ ⎪ ⎪ ⎭ , G2  2h0 C0  ⎡⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎣ c01,0 / c01,N−1 .. . 1 .. . c0N,0 / c0N,N−1 ⎤⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎦, C1  ⎡⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎣ c11,0 / c11,N−2 .. . 1 .. . c1N,0 / c1N,N−2 ⎤⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎦,C2  ⎧ ⎪ ⎪ ⎨ ⎪ ⎪ ⎩ c21 .. . c2N ⎫ ⎪ ⎪ ⎬ ⎪ ⎪ ⎭ c0i,j   cosθij −sinθij , c1i,j   ωi · sinθij ωi · cosθij  c2i  −ω2 i 0  (15) Characteristics of the Transform Methods The characteristics of the proposed transform method can be summarized as follows. {S(ωi)}   SR(ωi) SI(ωi)   ⎧ ⎪ ⎪ ⎪ ⎪ ⎪ ⎨ ⎪ ⎪ ⎪ ⎪ ⎪ ⎩  N−1 j0 cos θij·0hj + ωi  N−2 j0 sin θij·1hj −ω2 i h0 − N−1 j0 sin θij·0hj +  N−2 j0 cos θij·1hj ⎫ ⎪ ⎪ ⎪ ⎪ ⎪ ⎬ ⎪ ⎪ ⎪ ⎪ ⎪ ⎭ (13) ⎧ ⎪ ⎪ ⎨ ⎪ ⎪ ⎩ {D(ω1)} .. . {D(ωN)} ⎫ ⎪ ⎪ ⎬ ⎪ ⎪ ⎭  C0 C1 C2 · ⎧ ⎪ ⎨ ⎪ ⎩ {G0} {G1} G2 ⎫ ⎪ ⎬ ⎪ ⎭ (14) When θij  ωi · tj, tj  Δt · j With the inverse Fourier transform methods, the complex stiffness needs to be integrable with −∞< ω < ∞. 5) Noncausal functions can be converted approximately. 5) Noncausal functions can be converted approximately. When θij  ωi · tj, tj  Δt · j {D(ωi)}  Re[D(ωi)] Im[D(ωi)], {G0}  ⎧ ⎪ ⎪ ⎪ ⎪ ⎨ ⎪ ⎪ ⎪ ⎪ ⎩ 0h0 0h1 ... 0hN−1 ⎫ ⎪ ⎪ ⎪ ⎪ ⎬ ⎪ ⎪ ⎪ ⎪ ⎭ , {G1}  ⎧ ⎪ ⎪ ⎪ ⎪ ⎨ ⎪ ⎪ ⎪ ⎪ ⎩ 1h0 1h1 .. . 1hN−2 ⎫ ⎪ ⎪ ⎪ ⎪ ⎬ ⎪ ⎪ ⎪ ⎪ ⎭ , G2  2h0 C0  ⎡⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎣ c01,0 / c01,N−1 .. . 1 .. . c0N,0 / c0N,N−1 ⎤⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎦, C1  ⎡⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎣ c11,0 / c11,N−2 .. . 1 .. . c1N,0 / c1N,N−2 ⎤⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎦,C2  ⎧ ⎪ ⎪ ⎨ ⎪ ⎪ ⎩ c21 .. . c2N ⎫ ⎪ ⎪ ⎬ ⎪ ⎪ ⎭ c0i,j   cosθij −sinθij , c1i,j   ωi · sinθij ωi · cosθij  c2i  −ω2 i 0  (15) Even if the complex stiffness is noncausal, it can be transformed into an approximate causal impulse response. In this sense, the proposed methods are capable of the causal approximation of noncausal functions. However, if the noncausality is extremely strong, the deviation of the obtained causal function from the original function is considerable and the result may not be satisfactory. 6) The number of significant components in obtained impulse responses is small. 6) The number of significant components in obtained impulse responses is small. Characteristics of the Transform Methods The characteristics of the proposed transform method can be summarized as follows. The number of the components of the two-series impulse responses obtained via the transform of the proposed methods is N or N−1, which is almost the same as the number of the data points (N) of complex stiffness. However, because there are few small and negligible components in the obtained impulse responses, the number of significant components n’ is relatively small (10 or less in each series in many cases). Therefore, the calculation load in the time-history response analysis is relatively small. The time step (Δt) of the impulse response obtained using these methods is usually larger than the time step (ΔT) of the time-history response analysis (in many cases, ΔT = 0.005–0.01 s and Δt = 0.05–0.1 s). This implies that the past displacements and velocities used in the time-history response analysis are the values of the skipping ΔT. Determination of Unknowns Using Simultaneous Equations In any of the Methods A–C, the impulse response components (simultaneous terms 0h0, 1h0, 2h0 and time-delay terms 0h 1 ‥, 1h 1 ‥) are unknown and can be obtained by solving simultaneous equations using N known complex data points D (ω1), D (ω2), ‥ D (ωΝ). The case of Method B is shown below as an example. The case of Method C is also common, but in Method A, 2h0 = 0. HB′(ω)  −ω2·2h0 +iω·1h0+0h0 + ⎧ ⎨ ⎩iω· n′ j1 1hj ·e−iωtj + n′ j1 0hj ·e−iωtj⎫ ⎬ ⎭ (5) April 2022 | Volume 8 | Article 854838 Frontiers in Built Environment | www.frontiersin.org 3 Time Domain Transform Methods Nakamura et al. 1) Using two series of an impulse response. This point is different from many transform methods such as the Duhamel integral. It can be said to be the greatest characteristic of these methods. This is particularly effective for the vibration amplitude increase in proportion to ω (i.e., problem No.1 in Classification and Organization of Typical Dynamic Stiffness). 2) Can be transformed using discrete data within a finite frequency range. The proposed methods target complex data that are given discretely within a finite frequency range (0 to ωN). In some transform methods, it may be necessary to predict the properties of complex stiffness ω →∞; however, the proposed methods do not require it. Moreover, the number of data used for the transform is relatively small (in many cases, less than 20). 7) High conversion accuracy. 3) Causality is automatically satisfied. Eq. 3 of Method B can be expressed using Eq. 13 separately for the real and imaginary parts. Eqs 14, 15 are the matrix representations of this relationship for N data of complex stiffness (D (ω1) to D (ωN)). By solving the simultaneous Eq. 14 having a coefficient matrix of 2N × 2N, the value of the unknown impulse response (0h 0 to 0h N−1, 1h 0–1h N−2 and 2h 0) can be obtained. Notice that data D (0) should never be used. If D (0) is used, the coefficients on the right side of the imaginary part SI(ω) in Eq.13 become all 0 and Eq. 14 becomes singular and cannot be calculated. The causality may be impaired with methods such as the inverse Fourier transform. To avoid this, there is a method of calculating other parts using the Hilbert transform from the real or imaginary part of complex stiffness. However, it distorts the original function. In the proposed methods, the impulse response components are considered in the range of t ≥0, so the causality is always satisfied. 5) Noncausal functions can be converted approximately. For example, 10 skipping values are used, when ΔT = 0.01 s and Δt = 0.1 s. The characteristics of the proposed transform method can be summarized as follows. 4) Does not require simultaneous term separation. Therefore, removing the singular components (corresponding to the simultaneous terms in the proposed methods) that do not satisfy this condition before transform is necessary. However, for this purpose, it is necessary to estimate the property of ω →∞ of complex stiffness. This method requires neither separation of simultaneous terms nor property prediction in the infinite range because transform is possible with simultaneous terms included. (13) ⎧ ⎪ ⎪ ⎨ ⎪ ⎪ ⎩ {D(ω1)} .. . {D(ωN)} ⎫ ⎪ ⎪ ⎬ ⎪ ⎪ ⎭  C0 C1 C2 · ⎧ ⎪ ⎨ ⎪ ⎩ {G0} {G1} G2 ⎫ ⎪ ⎬ ⎪ ⎭ (14) 5) Noncausal functions can be converted approximately. 7) High conversion accuracy. In many cases, the original complex stiffness could be well approximated with 10 or fewer impulse Classification and organization of typical dynamic stiffness. April 2022 | Volume 8 | Article 854838 Frontiers in Built Environment | www.frontiersin.org 4 Time Domain Transform Methods Nakamura et al. RE 1 | Typical transform examples for time-domain transform. * The dimension of lateral axis is (Hz) for stiffness and (s) for the impulse response (0th- and 1 components). In the impulse response, the value corresponds to time = 0 is the simultaneous term and the others are time-delay terms. The dimension o stiffness (N/m) for Nos. 1–7 and dimensionless for No. 8. FIGURE 1 | Typical transform examples for time-domain transform. * The dimension of lateral axis is (Hz) for stiffness and (s) for the impulse response (0th- and 1st- order components). In the impulse response, the value corresponds to time = 0 is the simultaneous term and the others are time-delay terms. The dimension of vertica axis is stiffness (N/m) for Nos. 1–7 and dimensionless for No. 8. FIGURE 1 | Typical transform examples for time-domain transform. * The dimension of lateral axis is (Hz) for stiffness and (s) for the impulse response (0th- and 1st- order components). In the impulse response, the value corresponds to time = 0 is the simultaneous term and the others are time-delay terms. The dimension of ver axis is stiffness (N/m) for Nos. 1–7 and dimensionless for No. 8. April 2022 | Volume 8 | Article 854838 Frontiers in Built Environment | www.frontiersin.org Nakamura et al. Time Domain Transform Methods which is the main cause of frequency dependency; thus, periodic vibration appears clearly. FIGURE 2 | Soil properties for No. 1. As for No. 1, in the impulse response obtained using the time- domain transform, pulses are generated at 0.1 s for the 0th- and 1st- order components. This time-delay can be considered as follows. A wave generated by the forced vibration of the rigid base at the soil’s surface propagates underground. If it is uniform soil, it will dissipate as it is. However, in layered soil, the wave is reflected at the boundary of the bottom layer and comes back. A reaction force is generated to shake the rigid base again. This appears as a time-delay term of the impulse response, and the time-delay corresponds to the round-trip time of the wave. CLASSIFICATION AND ORGANIZATION OF TYPICAL DYNAMIC STIFFNESS Figure 1 shows eight transform examples of typical dynamic stiffness studied in the literature to be shown later as the problems with frequency dependency. This figure shows the following; 1) The physical image of each problem. 2) Dynamic stiffness in the frequency domain. The dimension of the lateral axis is the frequency (Hz). 2) Dynamic stiffness in the frequency domain. The dimension of the lateral axis is the frequency (Hz). 3) Obtained impulse response in the time domain (both 0th- and 1st-order components). The dimension of the lateral axis is the time (s)—the value when t = 0 is the simultaneous term, and the others are time-delay terms. Another common characteristic of Nos. 1 and 2 is that the effect of the time-delay terms of the 1st-order compnents cannot be ignored. For such problems, the proposed method using two types of impulse responses, the 0th- and 1st-order components, is considered particularly useful. For other impedances (Nos. 3–8), the time-delay terms of 1st-order component are small. y 4) Recovered dynamic stiffness from obtained impulse response using time-delay terms only by Eqs 5, 7. For all figures, the dimension of the vertical axis is stiffness (N/ m) for Nos. 1–7 and dimensionless for No. 8. These eight problems can be classified into the following three types according to the impulse response characteristics. Below, we analyze and organize each problem. Ground Impedance With Cut-Off Frequency (Nos. 3 and 4) No. 3 is the dynamic stiffness at the top of a semi-infinite material whose cross-section increases exponentially (Wolf and Motosaka, 1985) and has a cut-off frequency. It is a typical example of the problem that the radiation damping is almost 0 below a certain frequency. In this problem, the theoretical value of the impulse response and impedance are obtained. Impedance S(ω) and impulse response G(t) are shown using Eqs 16, 17, respectively. (1) Horizontal ground impedance (dynamic stiffness) of layered soil (Nos. 1 and 2). (1) Horizontal ground impedance (dynamic stiffness) of layered soil (Nos. 1 and 2). (2) Ground impedance with cut-off frequency (Nos. 3 and 4). (3) Maxwell element, viscoelastic body, and similar impedance (Nos. 5–8) S(ω)  E · A0 2x0 1 + !!!!!! 1 −4a2 0 " # (16) G(t)  E · A0 x0 $1 2 δ(t) + x0 C0 · d(δ(t)) dt + 1 2tJ1$C0 x0 · t%% a0  ω · x0 C0 , C0  !! E ρ & (17) (16) 7) High conversion accuracy. In this ground model, the shear wave velocity Vs of the surface layer is 400 m/s. The surface layer thickness is 20 m, so the wave trips 40 m, and the time delay is 0.1 s. FIGURE 2 | Soil properties for No. 1. y In No. 2, it is considered that the wave generated by the vibration at the midpoint position propagates in the ground, reaches the ground surface position, and corresponds to the reaction force that tries to shake the point. The time delay of 0.1 s in this model is considered the time required for wave propagation from the midpoint to the ground surface. The Vs of the surface layer is 200 m/s, and the surface layer thickness is 20 m for this problem. Because the reaction force does not occur simultaneously as the vibration, the simultaneous terms do not appear (although a minute value appears, it is considered a numerical cause). Consequently, the impedance becomes a property close to the periodic harmonic wave. Periodic vibration corresponding to the time mentioned above delay is often seen in the off-diagonal components of the impedance matrix. Ground Impedance of Layered Soil (Nos. 1 and 2) No. 1 is the horizontal ground impedance of the rigid base on the two-layered soil. Figure 2 shows its soil properties. No. 2 is the off- diagonal component of the ground impedance matrix of the building embedded in the two-layered soil. That component corresponds to the reaction force at the ground point position when forced vibration is performed on the rigid base at the bottom of the structure. Both impedances are greatly wavy. The rightmost column of the figure shows the impedance recovered using only the time-delayed terms, (17) Where, Here, E denotes the Young’s modulus; ρ denotes the mass density; and J1 denotes the first-order Bessel function. The first April 2022 | Volume 8 | Article 854838 Frontiers in Built Environment | www.frontiersin.org 6 Time Domain Transform Methods Nakamura et al. and second terms of Eq. 17 correspond to the simultaneous terms of the proposed methods 0h0 and 1h0, respectively. The third term corresponds to the time-delay terms of the 0th-order component. The theoretical value of the impulse response in Eq. 17 is given as a continuous quantity concerning time t, whereas the impulse response obtained the proposed methods is discrete values for each Δt. Therefore, the theoretical value was converted using Eq. 18, and it was confirmed that the impulse response obtained using the proposed method corresponds well to this. and second terms of Eq. 17 correspond to the simultaneous terms of the proposed methods 0h0 and 1h0, respectively. The third term corresponds to the time-delay terms of the 0th-order component. to 0 while oscillating as the time delay increases. The 1st- component has almost simultaneous terms only. Even in the impedance recovered using only the time-delay terms, Nos. 3 and 4 have almost the same shape. The theoretical value of the impulse response in Eq. 17 is given as a continuous quantity concerning time t, whereas the impulse response obtained the proposed methods is discrete values for each Δt. Therefore, the theoretical value was converted using Eq. 18, and it was confirmed that the impulse response obtained using the proposed method corresponds well to this. Maxwell Element, Viscoelastic Body, and Similar Impedance (Nos 5–8) p ( ) The Maxwell element of No. 5 is a spring (K0) and a dashpot (C0) connected in series and its characteristics are expressed using the relaxation, shown in Figure 3 time τr = C0/K0. Further, the impedance is expressed using Eq. 21. When ω = 0, the real part and the imaginary part become 0, and when ω →∞, the real part becomes K0, and the imaginary part gradually approaches K0/(τrω). Eq. 22 provides the theoretical impulse response, and the simultaneous and time-delay terms are only in the 0th- component. Similar to No. 3, the theoretical impulse response value was converted to a discrete value using Eq. 18. It was confirmed that the impulse response obtained using the proposed method corresponds well to this. G′tj  ' tj+Δt 2 tj−Δt 2 G(t)dt tj  Δt · j (18) (18) No. 4 is the dynamic stiffness of the side of the massless rigid cylinder embedded in the viscoelastic surface layer on the rigid soil and is known as a problem having a cut-off frequency similar to No. 3. Tajimi (1969) performed a rocking vibration analysis of rigid foundations and calculated theoretical solutions. Harada et al. (1983) improved this and expressed it as a spring per unit layer thickness in the underground sidewall and showed that this distribution became almost constant in the depth direction. S(ω)  K0⎛ ⎝1 − 1 τr iω + 1 τr ⎞⎠ (21) G(t)  K0$δ(t) −e−t τr τr % (22) (21) Eq. 19 shows the impedance of the Harada’s spring. Here, H denotes the surface layer thickness; Vs denotes the surface shear wave velocity; r0 denotes the equivalent radius of the foundation; μ denotes the shear modulus of the ground; h denotes the damping ratio of the layer; ] denotes the Poisson’s ratio; and Kn (ω) denotes the nth order of the modified Bessel function. (22) No. 6 is a constitution rule identified by Kasai et al. (2004) from the experimental results of acrylic materials. The storage stiffness G’(ω) and loss coefficient η(ω) are expressed using Eqs 23, 24, respectively. The parameters of this material are obtained as a = 5.60 × 10–5, b = 2.10, μ = 3.92 × 102 N/m2, and α = 0.558. KH(ω)  8r2 0μ H2 ·  N n ξ2 n · Ωn n3 · (−1) n−1 2 (n  1, 3, 5 . . . CONCLUSION extended Rayleigh damping model (Nakamura, 2016). The applicability of these models to practical problems was examined (Nakamura, 2019; Mogi et al., 2021; Ota et al., 2021). The impulse responses of Nos. 5–8 obtained via transform have the following characteristics. extended Rayleigh damping model (Nakamura, 2016). The applicability of these models to practical problems was examined (Nakamura, 2019; Mogi et al., 2021; Ota et al., 2021). Herein, the following studies were conducted. First, the outline of the transform Methods A to C proposed so far was shown in more general expressions and their characteristics were analyzed and organized. When the noncausality of the dynamic stiffness in the frequency domain is weak, Method B is recommended. When the hysteretic damping of the dynamic stiffness is large, the stiffness has a strong and considerable noncausality; for this case, Method C is recommended. g The impulse responses of Nos. 5–8 obtained via transform have the following characteristics. 1) For the 0th-order component, the time-delay term becomes a significant value, which occurs on the negative side of the figure and converges to 0 as the time delay increases. The simultaneous term is a positive and significant value in Nos. 5–7, but 0 in No. 8. Next, the above time-domain transform methods were applied to eight typical problems with frequency dependence and their properties were examined. All dynamic stiffnesses could be transformed well, and the effectiveness of the transform method was confirmed. For all problems, it was found that in the time- delay terms related to frequency dependence, the 0th-order component was essential. Moreover, they were roughly classified into three types, and the characteristics of them are as follows: 2) In the 1st-order component, the time-delay term is almost 0. 2) In the 1st-order component, the time-delay term is almost 0. Consequently, the impedances of Nos. 5–8 recovered only using the time-delay term have similar shapes. It is considered that these frequency dependences are of the same type. The viscoelastic damper of No. 6 is often approximated using the generalized Maxwell element of No. 8, consistent with this result. 1) The ground impedance of layered soil (Nos. 1 and 2) 1) The ground impedance of layered soil (Nos. 1 and 2) The time-domain transform could perform with generally good accuracy in any problem in this section. Table 1 summarizes the properties of the impulse responses transformed for each problem. 3) Maxwell element, viscoelastic body, and similar impedance (Nos. 5–8). Frontiers in Built Environment | www.frontiersin.org Maxwell Element, Viscoelastic Body, and Similar Impedance (Nos 5–8) aX: the value is significant; Blank: the value is small or nearly 0. CONCLUSION X in the figure indicates that each component plays an important role in the impulse response. The time delay corresponds to the wave propagation time, so the physical meaning of the time delay is clear. Because the vibration amplitude tends to increase as the frequency increases, the time-delay terms of the 1st-order component are important to represent it. Regarding the time-delay term related to frequency dependency, the 0th-order component was indispensable in all the problems examined while the 1st-order component was not necessary in many cases. However, it was shown that the 1st- order component is also important in the impedance of the foundation of layered soil (Nos. 1 and 2). It is considered that this is because their dynamic stiffness tends to increase the amplitude of vibration as the frequency increases, and the 1st- order component is required to express this property. Maxwell Element, Viscoelastic Body, and Similar Impedance (Nos 5–8) N) (19) (19) Where, G′(ω)  μ 1 + abω2α + (a + b)ωα cos(απ/2) 1 + a2ω2α2 + aωα cos(απ/2) (23) η(ω)  (−a + b)ωα sin(απ/2) 1 + abω2α + (a + b)ωα cos(απ/2) (24) Ωn  4K1γRK1βR + βRK1γRK0βR + γnK0γRK1βR (γRK0γR + K1γR).(βRK0βR + K1βR) −K1γRK1βR βR  βnr0, γR  γnr0 βn  π 2H · 1 !!!!!!! 1 + 2i · h √ · ξn, γn  π 2H · !!!!!!! 1 −2] 2(1 + ]) & · ξn ξn  !!!!!!!!!!!!!!!!!!!! (1 + 2i · h)n2 −ω*ωg 2 + , ωg  πVs 2H ⎫ ⎪ ⎪ ⎪ ⎪ ⎪ ⎪ ⎪ ⎪ ⎪ ⎪ ⎪ ⎪ ⎪ ⎬ ⎪ ⎪ ⎪ ⎪ ⎪ ⎪ ⎪ ⎪ ⎪ ⎪ ⎪ ⎪ ⎪ ⎭ (20) (23) (24) No. 7 is the Biot model (Biot, 1958), the limit of the generalized Maxwell element. The 0th-order component of the impulse response is similar to Nos. 5 and 6. No. 8 is a time-domain approximation of the unit imaginary function obtained using the transform of the imaginary unit i from the frequency to time domain. This is the main component of the damping models with small frequency dependency as the causal history damping model (Nakamura, 2007) and the (20) The impulse responses of Nos. 3 and 4 show almost the same properties. The 0th-component of the impulse response has simultaneous term and time-delay terms. The latter converges FIGURE 3 | Examples of the generalized Maxwell elements. Frontiers in Built Environment | www.frontiersin.org April 2022 | Volume 8 | Article 854838 7 FIGURE 3 | Examples of the generalized Maxwell elements. FIGURE 3 | Examples of the generalized Maxwell elements. April 2022 | Volume 8 | Article 854838 Frontiers in Built Environment | www.frontiersin.org 7 Time Domain Transform Methods Nakamura et al. TABLE 1 | Components of each case in the time domain. No. of dynamic stiffness Simultaneous term Time-delay term 0th-Order component (0h0) 1st-Order component (1h0) 0th-Order component (0h 1 ~) 1st-Order component (1h 1 ~) 1 X X X X 2 X X 3 X X X 4 X X X 5 X X 6 X X X 7 X X X 8 X X aX: the value is significant; Blank: the value is small or nearly 0. TABLE 1 | Components of each case in the time domain. REFERENCES Nakamura, N. (2008a). Seismic Response Analysis of Deeply Embedded Nuclear Reactor Buildings Considering Frequency-dependent Soil Impedance in Time Domain. Nucl. Eng. Des. 238, 1845–1854. doi:10.1016/j.nucengdes.2007.12.006 Biot, M. A., (1958), Linear Thermodynamics and the Mechanics of the Solid. In Proc. 3rd US National Congress of Applied Mechanics, ASCE, 1–18. de Barros, F. C. P., and Luco, J. E. (1990). Discrete Models for Vertical Vibrations of Ota, A., Nakamura, N., and Mogi, Y. (2021). Examination of Applicability of Causality-Based Damping Model to Dynamic Explicit Method. Nihon Kenchiku Gakkai Kozokei Ronbunshu 86, 1168–1179. doi:10.3130/aijs.86.1168 de Barros, F. C. P., and Luco, J. E. (1990). Discrete Models for Vertical Vibrations of Surface and Embedded Foundations. Earthquake Engng. Struct. Dyn. 19, 289–303. doi:10.1002/eqe.4290190211 Surface and Embedded Foundations. Earthquake Engng. Struct. Dyn. 19, 289–303. doi:10.1002/eqe.4290190211 Harada, T., Kubo, K., Katayama, T., and Hirose, T. (1983). Dynamic Stiffness of Embedded Cylindrical Rigid Foundation. Proc. Jpn. Soc. Civil Eng. 1983, 79–88. doi:10.2208/jscej1969.1983.339_79 Saitoh, M. (2007). Simple Model of Frequency-dependent Impedance Functions in Soil-Structure Interaction Using Frequency-independent Elements. J. Eng. Mech. 133, 1101–1114. doi:10.1061/(asce)0733- 9399(2007)133:10(1101) Hayashi, Y., and Katukura, H. (1990). Effective Time-Domain Soil-Structure Interaction Analysis Based on FFT Algorithm with Causality Condition. Earthquake Engng. Struct. Dyn. 19, 693–708. doi:10.1002/eqe.4290190506 Tajimi, H., (1969), Dynamic Analysis of Structure Embedded in an Elastic Stratum, Proc. 4th World Conference on Earthquake Engineering, A-6, 53–69. Hsu, H. P. (1967). Fourier Analysis. New York, U.S.A: Simon & Schuster. Wolf, J. P., and Motosaka, M. (1989). Recursive Evaluation of Interaction Forces of Unbounded Soil in the Time Domain. Earthquake Engng. Struct. Dyn. 18, 345–363. doi:10.1002/eqe.4290180304 Kasai, K., Ooki, Y., Tokoro, K., Amemiya, K., and Kimura, K. (2004). JSSI Manual for Building Passive Control Technology, Time-History Analysis Model for Viscoelastic Dampers, 13th World Conference on Earthquake Engineering. Vancouver, Canada: IAEE. part 9. Wolf, J. P., and Obernhuber, P. (1985). Non-linear Soil-Structure-Interaction Analysis Using Dynamic Stiffness or Flexibility of Soil in the Time Domain. Earthquake Engng. Struct. Dyn. 13, 195–212. doi:10.1002/eqe. 4290130205 Kirekawa, A., Ito, Y., and Asano, K., (1992). A Study of Structural Control Using Viscoelastic Material, 10th World Conference of Earthquake Engineering, 2047-2054. Wolf, J. P. (1997). Spring-Dashpot-Mass Models for Foundation Vibrations. Earthquake Engng. Struct. Dyn. 26, 931–949. doi:10.1002/(sici)1096- 9845(199709)26:9<931::aid-eqe686>3.0.co;2-m Makris, N., and Constantinou, M. C. (1991). Fractional-Derivative Maxwell Model for Viscous Dampers. J. Struct. Eng. 117, 2708–2724. doi:10.1061/(asce)0733- 9445(1991)117:9(2708) Meek, J. W. (1990). Recursive Evaluation of Dynamic Phenomenon in Civil Engineering. Bautechnik 67, 205–210. DATA AVAILABILITY STATEMENT It is considered these findings useful for understanding the properties and physical meanings of dynamic stiffnesses with frequency dependence in the time domain. Through these studies, if the frequency dependency of the dynamic stiffness is stable, it is considered that there is no major problem with this transform method. However, if the frequency dependency change largely due to strain or temperature dependency, it is necessary to interpolate and use the transformed results according to the strain and temperature changes. However, it requires a considerable calculation effort; therefore, it is necessary to improve practical methods for such problems. The raw data supporting the conclusion of this article will be made available by the authors, without undue reservation. 2) Ground impedance with cut-off frequency (Nos. 3 and 4) The 0th-order component has simultaneous and time-delayed terms. The latter converges to 0 while oscillating as the time delay increases. The 1st-order component has almost simultaneous terms only. Even in the impedance recovered using only the time- delay term, Nos. 3 and 4 have almost the same shape. Furthermore, most problems show either the 0th- or 1st- order component for simultaneous terms. This indicates that many dynamic stiffnesses are related to the current displacement or velocity. The exception is No. 2. This corresponds to the fact that the reaction force does not occur simultaneously. This soil stiffness corresponds to the reaction force from where the forced vibration is applied at different places. Therefore, it needs some time that the effect propagates to a different place. 3) Maxwell element, viscoelastic body, and similar impedance (Nos. 5–8). 3) Maxwell element, viscoelastic body, and similar impedance (Nos. 5–8). For the 0th-order component, the time-delay terms become significant values, which occur on the negative side of the figure and converge to 0 as the time delay increases. For the 1st-order component, the time-delay term is small. April 2022 | Volume 8 | Article 854838 8 Nakamura et al. Time Domain Transform Methods AUTHOR CONTRIBUTIONS NN contributed to conception, design of the study, and wrote the first draft of the manuscript. KN, YM, and AO checked and advised to improved the manuscript. All authors contributed to manuscript revision, read, and approved the submitted version. Nakamura, N. (2007). Practical Causal Hysteretic Damping. Earthquake Engng Struct. Dyn. 36, 597–617. doi:10.1002/eqe.644 REFERENCES Wu, W.-H., and Lee, W.-H. (2002). Systematic Lumped-Parameter Models for Foundations Based on Polynomial-Fraction Approximation. Earthquake Engng. Struct. Dyn. 31, 1383–1412. doi:10.1002/eqe.168 Mogi, Y., Nakamura, N., and Ota, A. (2021). Application of Extended Rayleigh Damping Model to 3D Frame Analysis. Nihon Kenchiku Gakkai Kozokei Ronbunshu 86, 738–748. doi:10.3130/aijs.86.738 Xue, S. t., Tobita, J., Hanzawa, T., and Izumi, M. (1992). Wave Attenuation in Viscoelastic Continuum with Fading Memory. J. Eng. Mech. 118, 1597–1611. doi:10.1061/(asce)0733-9399(1992)118:8(1597) Motosaka, M., and Nagano, M. (1992). Recursive Evaluation of Convolution Integral in Nonlinear Soil-Structure Interaction Analysis and its Applications. J. Struct. Construction Eng. 436, 71–80. doi:10.3130/aijsx.436.0_71 Conflict of Interest: Authors YM and AO are employed by Taisei Corporation. Conflict of Interest: Authors YM and AO are employed by Taisei Corporation. Nakamura, N. (2019). Application of Causal Hysteretic Damping Model to Nonlinear Seismic Response Analysis of Super High-Rise Building. J. Struct. Construction Eng. (Transactions AIJ) 759, 627–637. doi:10.3130/aijs.84.597 The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest Nakamura, N. (2016), Extended Rayleigh Damping Model, Front. Built Environ. 2, doi:10.3389/fbuil.2016.00014 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. Nakamura, N. (2006a). A Practical Method to Transform Frequency Dependent Impedance to Time Domain. Earthquake Engng Struct. Dyn. 35, 217–231. doi:10.1002/eqe.520 Nakamura, N. (2012). Basic Study on the Transform Method of Frequency- dependent Functions into Time Domain: Relation to Duhamel’s Integral and Time-Domain-Transfer Function. J. Eng. Mech. 138, 276–285. doi:10. 1061/(asce)em.1943-7889.0000330 Copyright © 2022 Nakamura, Nabeshima, Mogi and Ota. 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. Nakamura, N. (2006b). Improved Methods to Transform Frequency-dependent Complex Stiffness to Time Domain. Earthquake Engng Struct. Dyn. 35, 1037–1050. doi:10.1002/eqe.570 Nakamura, N. (2008b). Nonlinear Response Analysis Considering Dynamic Stiffness with Both Frequency and Strain Dependencies. J. Eng. Mech. 134, 530–541. doi:10.1061/(asce)0733-9399(2008)134:7(530) April 2022 | Volume 8 | Article 854838 Frontiers in Built Environment | www.frontiersin.org 9

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PULMONARY FINDINGS ON HIGH RESOLUTION COMPUTED TOMOGRAPHY IN PATIENTS WITH JUVENILE DERMATOMYOSITIS: RETROSPECTIVE STUDY

Blucher Medical Proceedings

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PULMONARY FINDINGS ON HIGH RESOLUTION COMPUTED TOMOGRAPHY IN PATIENTS WITH JUVENILE DERMATOMYOSITIS: RETROSPECTIVE STUDY CARLA HELENA CAPPELLO (UNICAMP, CAMPINAS, SP, Brasil), EDUARDO BRONZATTO (UNICAMP, CAMPINAS, SP, Brasil), ROBERTO MARINI (UNICAMP, CAMPINAS, SP, Brasil), SERGIO DERTKIGIL (UNICAMP, CAMPINAS, SP, Brasil), SIMONE APPENZELLER (UNICAMP, CAMPINAS, SP, Brasil) PULMONARY FINDINGS ON HIGH RESOLUTION COMPUTED TOMOGRAPHY IN PATIENTS WITH JUVENILE DERMATOMYOSITIS: RETROSPECTIVE STUDY CARLA HELENA CAPPELLO (UNICAMP, CAMPINAS, SP, Brasil), EDUARDO BRONZATTO (UNICAMP, CAMPINAS, SP, Brasil), ROBERTO MARINI (UNICAMP, CAMPINAS, SP, Brasil), SERGIO DERTKIGIL (UNICAMP, CAMPINAS, SP, Brasil), SIMONE APPENZELLER (UNICAMP, CAMPINAS, SP, Brasil) PULMONARY FINDINGS ON HIGH RESOLUTION COMPUTED TOMOGRAPHY IN PATIENTS WITH JUVENILE DERMATOMYOSITIS: RETROSPECTIVE STUDY CARLA HELENA CAPPELLO (UNICAMP, CAMPINAS, SP, Brasil), EDUARDO BRONZATTO (UNICAMP, CAMPINAS, SP, Brasil), ROBERTO MARINI (UNICAMP, CAMPINAS, SP, Brasil), SERGIO DERTKIGIL (UNICAMP, CAMPINAS, SP, Brasil), SIMONE APPENZELLER (UNICAMP, CAMPINAS, SP, Brasil) BACKGROUND Juvenile Dermatomyositis (JDM), are complex, heterogeneous, autoimmune diseases that affect skeletal muscles and the skin. The interstitial lung disease (ILD) is a complication that has been observed in 20- 78% of patients and can be a determining prognosis, causing greater morbidity and mortality. Objective: to evaluate the finds pulmonary by high-resolution Computerized Tomography (HRCT) in accompanying patients in outpatient pediatric rheumatology unit. RESULTS HRCT was retrieved for analysis in 62 of patients. We observed interstitial pneumopatia in 9 (14.51%), showed; nodules/micronodules in 20 (32.25%), opacity in ground glass in 21 (33.87%), fibroatelectasias in 13 (20.96%), septal thickening in 7 (11.29%), bronchial wall thickening in 10 (16.12%) and pneumomediastium in 1 (1.61%). Findings were observed during the first 2 years of disease In 62 patients. MATERIALS AND METHODS Retrospective study of 62 patients diagnosed with JDM, which are submitted to the examination of image for clinical monitoring of the disease. TCAR tests were observed in approximately 10 years, including repeated CT scans assessed: interstitial pneumopatia; pneumomediastium; the presence of nodules; opacity in ground glass; fibros elastic stretch marks; septal thickening; bronquica wall thickening. Two patients were excluded because they had overlap with systemic lupus erythematosus. CONCLUSION We conclude that pulmonary involvement in the diseases presented above has been more evident and that high resolution computed tomography provides additional information to clinical evaluation.

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Low-Cost, Accurate, Effective Treatment of Hypertensive Cerebral Hemorrhage With Three-Dimensional Printing Technology

Frontiers in neurology

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ORIGINAL RESEARCH published: 25 February 2021 doi: 10.3389/fneur.2021.608403 ORIGINAL RESEARCH published: 25 February 2021 doi: 10.3389/fneur.2021.608403 Ke Li 1, Xiangqian Ding 2, Qingbo Wang 1, Gangxian Fan 1, Wei Guo 3, Chenglong Li 1, Meng Li 1 and Zefu Li 1* 1 Department of Neurosurgery, Binzhou Medical University Hospital, Binzhou, China, 2 Department of Neurosurgery, First Affiliated Hospital of Kunming Medical University, Kunming, China, 3 Department of Neurology, Binzhou Medical University Hospital, Binzhou, China Background: Hypertensive intracerebral hemorrhage (HICH) is an acute, severe neurosurgical disease. Puncture drainage of the hematoma has gradually been accepted as a surgical treatment for HICH because of its minimally invasive nature. The precision of the puncture is extremely high because of particular physiological functions. This study was performed to explore the effect of a navigation mold created by three-dimensional printing (3DP) technology in the surgical treatment of HICH. Edited by: Simona Sacco, University of L’Aquila, Italy Reviewed by: Xiaolei Chen, Chinese PLA General Hospital, China Craig S. Anderson, University of New South Wales, Australia *Correspondence: Zefu Li [email protected] Edited by: Simona Sacco, University of L’Aquila, Italy Reviewed by: Xiaolei Chen, Chinese PLA General Hospital, China Craig S. Anderson, University of New South Wales, Australia Material and methods: We conducted a retrospective analysis of all consecutive patients with ICH treated with minimally invasive surgery using 3DP navigation or craniotomy to remove the hematoma through a small bone window at the Binzhou Medical University Hospital from June 2017 to March 2019. In total, 61 patients were treated with minimally invasive surgery using 3DP navigation (3DP group), and 67 patients were treated with craniotomy to remove the hematoma through a small bone window (craniotomy group). A comparative study of the two groups was conducted to assess the preoperative and postoperative conditions. Specialty section: This article was submitted to Stroke, a section of the journal Frontiers in Neurology Results: The duration of the surgery was significantly longer in the craniotomy group than in the 3DP group (3.27 ± 1.14 h vs. 1.52 ± 0.23 h). Postoperative complication rates were significantly lower in the 3DP group than in the craniotomy group (18.0 vs. 34.3%). Moreover, the rate of patients with a Glasgow Outcome Scale score ≥4 points was not statistically significantly different in the two groups. Received: 20 September 2020 Accepted: 19 January 2021 Published: 25 February 2021 ORIGINAL RESEARCH published: 25 February 2021 doi: 10.3389/fneur.2021.608403 Low-Cost, Accurate, Effective Treatment of Hypertensive Cerebral Hemorrhage With Three-Dimensional Printing Technology Ke Li 1, Xiangqian Ding 2, Qingbo Wang 1, Gangxian Fan 1, Wei Guo 3, Chenglong Li 1, Meng Li 1 and Zefu Li 1* Keywords: hypertensive intracerebral hemorrhage, minimally invasive, three dimensional printing, individualization, accuracy Abbreviations: 3DP, three-dimensional printing; CT, computed tomography; GCS, Glasgow Coma Scale; GOS, Glasgow Outcome Scale; HICH, hypertensive intracerebral hemorrhage. INTRODUCTION (3) absence of a cerebral hernia, cerebrovascular malformation aneurysm, or stroke; (4) first-onset lesion; and (5) treatment with minimally invasive surgery using 3DP navigation or craniotomy to remove the hematoma through a small bone window. In total, 61 patients were treated with minimally invasive surgery using 3DP navigation (3DP group), and 67 patients were treated with craniotomy to remove the hematoma through a small bone window (craniotomy group). A comparative study of the two groups was conducted to assess the preoperative and postoperative conditions. Hypertensive intracerebral hemorrhage (HICH) is a common critical neurosurgical illness with high mortality and disability rates (1–3). At present, surgical treatment for HICH mainly involves removing the hematoma by craniotomy or minimally invasive surgery (4–6). Although craniotomy removal quickly relieves the compression of the hematoma on brain tissue and effectively stops bleeding, the operation is lengthy, which causes great trauma to the patient; it is also expensive (7). Minimally invasive surgery is widely performed as surgical treatment of HICH because it is a short procedure that causes little trauma and is low cost (8–10). Patients We conducted a retrospective analysis of all consecutive patients with ICH treated at the Binzhou Medical University Hospital from June 2017 to March 2019. The inclusion criteria were (1) a diagnosis of HICH based on the Guidelines for the Diagnosis and Treatment of Cerebral Hemorrhage in China formulated by the Chinese Medical Association in 2014 and confirmed by brain computed tomography (CT); (2) location of the bleeding site at the basal ganglia (i.e., the hematoma did not enter the ventricle); Citation: Li K, Ding X, Wang Q, Fan G, Guo W, Li C, Li M and Li Z (2021) Low-Cost, Accurate, Effective Treatment of Hypertensive Cerebral Hemorrhage With Three-Dimensional Printing Technology. Front. Neurol. 12:608403. doi: 10.3389/fneur.2021.608403 Conclusion: Minimally invasive surgery assisted by 3DP navigation to treat patients with HICH appears to be safe and effective. The 3DP technique may improve the individualization and accuracy of the surgery. Keywords: hypertensive intracerebral hemorrhage, minimally invasive, three dimensional printing, individualization, accuracy February 2021 | Volume 12 | Article 608403 1 Frontiers in Neurology | www.frontiersin.org 3D-Printing Assisted Treatment of HICH Li et al. Li et al. Surgical Procedure The diagnosis of HICH was confirmed by preoperative brain CT (Figures 4a1–c1). After appropriately positioning the patient, we placed the 3D individualized model close to his or her face, ensuring that the bridge of the nose and zygomatic arch were accurately positioned. Next, along the puncture channel, we placed a marker at the patient’s frontal puncture point. The surgical area was then routinely disinfected. Lidocaine 2% was used for local infiltration anesthesia. After confirmation of successful anesthesia, a 0.5-cm transverse incision was made at the center of the puncture point on the forehead. The model was then positioned on the patient’s face and kept as close to the patient’s skin as possible. A cranial drill was then introduced into a bone hole to penetrate the dura mater, moving in the direction of the puncture channel. A drainage tube was slowly inserted in the same direction until the desired depth was reached (guide- channel length plus intracranial length), and advancement of the tube was stopped when dark red liquid began to flow. Adjuvant Therapy Brain CT was performed 6 h postoperatively (Figures 4a2–c2). Urokinase was injected through the drainage tube for 2–3 days (once or twice a day) after the surgery. Strict sterility was observed during the operational procedures. Brain CT was performed to check the intracranial condition after the injection of urokinase (Figures 4a3–c3). MATERIALS AND METHODS All patients provided written informed consent for the publication of all images and data, including any potentially identifiable images or data included in this article. The Medical Science Ethics Committee and Chinese Clinical Trial Registry approved this study (ChiCTR1800014543, 20/01/2018). The participating patients or their relatives or legal guardians also provided written informed consent for performance of the operation. All methods of this study were performed in accordance with the Guidelines for the Diagnosis and Treatment of Cerebral Hemorrhage in China in 2014. Minimally Invasive Surgery Production of 3DP Guide Plate With the advances in medical technology in recent years, navigation and stereotactic, robotic, and other technologies have been introduced to the field of neurosurgery, which have greatly improved the accuracy of minimally invasive surgery and significantly benefitted patients. However, the role of this type of surgery has been limited because the equipment required is expensive and thus difficult to promote and popularize in developing countries. To create the 3DP guide plate, Digital Imaging and Communications in Medicine format data obtained from CT scans were imported into Mimics 17.0 software embedded in the computer system to construct the skull, brain tissue, and hematoma in a 1:1 ratio via threshold selection (Figure 1). Bone markers such as the nasal root and bilateral eyebrow arches were reconstructed to fix on the patient’s face (Figure 2). The puncture point was generally 3–5 cm from the eyebrow arch. The puncture channel avoided important structures, such as the frontal sinus. The guide plate for surgery was designed by a Boolean logic operation and then printed by the 3D printer (Figure 3). Three-dimensional printing (3DP) technology has recently become popular in biomedical studies. It is a rapid, prototyping technology based on digital model files that can be used to print a solid object with complex geometry through layered processing. It has been gradually applied to the medical field because of its speed and accuracy (11–13). We have used 3DP technology to construct navigation molds that assist in intracranial hematoma puncture, hoping to achieve more individualized and precise treatment that retains the therapeutic effects of stereotactic, navigational, and other equipment. Surgical Hematoma Removal via Small Bone Window To remove the hematoma with conventional surgery, a 5-cm straight skin incision was made in the patient’s temporal region. One or two bone holes were then drilled, and a milling cutter was used to create a bone window with a diameter of about February 2021 | Volume 12 | Article 608403 Frontiers in Neurology | www.frontiersin.org 2 Li et al. 3D-Printing Assisted Treatment of HICH FIGURE 1 | Three-dimensional reconstruction model of the hematoma and cranium, designed for optimal puncture passage. The end of the puncture passage points to the hematoma center and avoids the frontal sinus and important blood vessels. FIGURE 1 | Three-dimensional reconstruction model of the hematoma and cranium, designed for optimal puncture passage. The end of the puncture passage points to the hematoma center and avoids the frontal sinus and important blood vessels. FIGURE 1 | Three-dimensional reconstruction model of the hematoma and cranium, designed for optimal puncture passage. The end of the puncture passage points to the hematoma center and avoids the frontal sinus and important blood vessels. FIGURE 1 | Three-dimensional reconstruction model of the hematoma and cranium, designed for optimal puncture passage. The to the hematoma center and avoids the frontal sinus and important blood vessels. struction model of the hematoma and cranium, designed for optimal puncture passage. The end of the puncture passage points he frontal sinus and important blood vessels. (conscious but disabled and requiring assistance in daily life), (4) mild disability (able to live independently and work under protection), and (5) good recovery (a return to normal life despite minor defects). 3 cm. A cortical ostomy was performed in the superior or middle temporal gyrus, and the hematoma was removed under microscopic guidance. A drainage tube was placed inside the hematoma, and the bone flap was repositioned. Evaluation of Surgical Methods IBM SPSS 21.0 statistical software (IBM Corp., Armonk, NY, USA) was used for statistical analysis of data, which are expressed as mean ± standard deviation. We performed Student’s t-test to analyze the differences between the two groups. Count data were compared between the two groups using the χ2 test, with P < 0.05 indicating a statistically significant difference. The safety and feasibility of minimally invasive surgery with 3DP individualized guidance was evaluated based on the efficiency and treatment outcomes (compared with those of craniotomy). The efficiency of the surgery was judged according to the duration of the surgery. We evaluated the treatment outcome based on the mortality rate, number of postoperative complications (i.e., pulmonary infections, rebleeding, and intracranial infection), Glasgow Outcome Scale (GOS) score at 30 days postoperatively, and duration of postoperative hospitalization. Frontiers in Neurology | www.frontiersin.org DISCUSSION HICH is a critical illness commonly seen by neurosurgeons. Early release of the compression of the hematoma on the brain tissue is a key factor in improving a patient’s prognosis (14, 15). At present, surgical treatments for HICH mainly include removal of the hematoma via craniotomy through a small bone window, large-bone craniotomy, and drainage of the hematoma via minimally invasive puncture drainage. Large bone craniotomy is the traditional surgical procedure. Although this technique provides good visualization of the surgical field, it is not the preferred procedure because of its resultant trauma and postoperative complications (16). FIGURE 2 | Construction of the navigation mold. The puncture guide plate was applied as closely as possible to the patient’s face. FIGURE 2 | Construction of the navigation mold. The puncture guide plate was applied as closely as possible to the patient’s face. FIGURE 3 | Surgical procedure for the three-dimensional printer (3DP) group. With the continuing development of microsurgical techniques, removal of a hematoma via a craniotomy through small bone window has made significant progress and has offered obvious surgical benefits to patients. With the development of modern imaging technology and the introduction of stereotactic, navigation, robotic, and other procedures, minimally invasive surgery to accomplish puncture drainage is increasingly being applied in the clinical setting. However, this procedure shows no significant difference in surgical benefits compared with small bone window surgery (17). Minimally invasive puncture takes only a short time, causes little trauma, and has a low postoperative complication rate and a short recovery time. However, because stereotactic, navigation, and robotic devices are expensive, promotion of their use in developing countries has been difficult. The accuracy of empirical puncture is not high and has become the main factor affecting patients’ prognoses (16). FIGURE 3 | Surgical procedure for the three-dimensional printer (3DP) group. Three-dimensional printing technology has been increasingly applied in aerospace, medicine, automotive development, electronics, and other fields since the mid-1990’s. Continuous improvements in 3DP technology have increased its clinical value because of the ability to deliver individualized, precise medicine. Thus, it is being widely applied clinically (18, 19). We introduced 3DP technology to the clinical diagnosis and treatment of neurosurgical patients and created a navigation mold for puncturing and draining hemorrhagic hematomas. This technique can improve the precision of puncture surgery and plays a role in the functions of stereotactic, navigation, robotic, and other equipment. DISCUSSION The cost of a 3D printer is only about 700 USD, and the cost of printing a navigation mold is <100 RMB (14 USD). Because of the low cost of the 3DP equipment and the associated consumables, this technology is easy to promote in developing countries, thereby bringing precise treatment to most of these patients. Because our technique does not have strict posture requirements, it can be performed under local anesthesia, (GCS) score was 12 ± 2 on admission. Among the 67 patients in the craniotomy group (42 men, 25 women; mean age, 55.5 ± 9.6 years; range, 38–73 years), the mean systolic blood pressure was 178 ± 24 mmHg, the mean hematoma volume was 36.7 ± 6.6 ml, and the mean GCS score was 11 ± 3 on admission. No significant differences in sex, age, systolic blood pressure, hematoma volume, or GCS score were observed between the two groups (Table 1). RESULTS Comparison of Preoperative Data Among the 61 patients in the 3DP group (37 men, 24 women; mean age, 58.4 ± 9.5 years; range, 44–79 years), the mean systolic blood pressure was 174 ± 22 mmHg, the mean hematoma volume was 36.3 ± 6.6 ml, and the mean Glasgow Coma Scale Follow-up methods included telephone assessments and scheduled outpatient head CT examinations. Clinical efficacy evaluation referenced the GOS, which included the following: (1) death, (2) vegetative survival (i.e., minimal response, no sleep or wake cycle, if the eyes open on command), (3) severe disability February 2021 | Volume 12 | Article 608403 Frontiers in Neurology | www.frontiersin.org 3 3D-Printing Assisted Treatment of HICH Li et al. FIGURE 2 | Construction of the navigation mold. The puncture guide plate was applied as closely as possible to the patient’s face. FIGURE 3 | Surgical procedure for the three-dimensional printer (3DP) group. of patients with a GOS score of ≥4 points between the 3DP and craniotomy groups. The mean operation time, mean postoperative hospitalization duration, and complication rate were significantly different between the two groups (P < 0.05). However, the proportion of patients with a GOS score of ≥4 points was not significantly different between the two groups. FIGURE 2 | Construction of the navigation mold. The puncture guide plate was applied as closely as possible to the patient’s face. FIGURE 2 | Construction of the navigation mold. The puncture guide plate was applied as closely as possible to the patient’s face. Comparison of Postoperative Data The patients in the 3DP group underwent surgical drainage of their hematomas under local infiltration anesthesia. The puncture channel accurately located the position of the hematoma. All patients in the craniotomy group underwent complete surgical drainage under general intravenous anesthesia. Table 2 compares the mean operation time, mean postoperative hospitalization duration, complication rate, and proportion February 2021 | Volume 12 | Article 608403 Frontiers in Neurology | www.frontiersin.org 4 Li et al. 3D-Printing Assisted Treatment of HICH FIGURE 4 | Comparison of preoperative and postoperative computed tomography (CT) results. (a1–c1) Preoperative CT. (a2–c2) Postoperative CT, 6 h after surgery. (a3–c3) Postoperative CT, 3 days after surgery. FIGURE 4 | Comparison of preoperative and postoperative computed tomography (CT) results. (a1–c1) Preoperative CT. (a2–c2) Postoperative CT, 6 h after surgery. (a3–c3) Postoperative CT, 3 days after surgery. TABLE 2 | Comparison of postoperative data in two groups of patients. 3DP group Craniotomy group t/χ2 value P-value (n = 61) (n = 67) The preparation time of 3D model (3D modeling and printing), h 1.03 ± 0.09 The total operation duration, h 1.52 ± 0.23 3.27 ± 1.14 t = −11.68 <0.001* Postoperative hospitalization, days 20.54 ± 7.08 23.86 ± 10.72 t = −2.04 0.044* Overall complication rate 11 (18.0%) 23 (34.3%) χ2 = 4.35 0.037* Complication rate of pulmonary infection 8 (13.1%) 17 (25.4%) χ2 = 3.05 0.081 Complication rate of Intracranial infection 2 (3.3%) 3 (4.5%) Rebleeding rate 1 (1.6%) 3 (4.5%) Mortality rate 0 2 (3.3%) Rate of patients with a GOS ≥4 points 31 (50.8%) 38 (56.7%) χ2 = 0.45 0.503 TABLE 2 | Comparison of postoperative data in two groups of patients. TABLE 1 | Comparison of preoperative data in two groups of patients. TABLE 1 | Comparison of preoperative data in two groups of patients. Factors 3DP group Craniotomy group t/χ2 value (n = 61) (n = 67) Sex (male %) 37 (60.7) 42 (62.7) χ2 = 0.056 Age, years 58.4 ± 9.5 55.5 ± 9.6 t = 1.72 Systolic pressure, mmHg 174 ± 22 178 ± 24 t = −1.01 Hematoma volume, ml 36.3 ± 6.6 36.7 ± 6.6 t = −0.34 GCS score 12 ± 2 11 ± 3 t = 1.59 There was no statistically significant difference between the two groups. ere was no statistically significant difference between the two groups. which reduces its cost. Comparison of Postoperative Data The average cost of surgery in the 3DP group was half that in the craniotomy group. The present study revealed no significant difference in the prognosis of patients who underwent 3DP navigation mold- assisted puncture surgery vs. small bone window surgery to remove hematomas. The 3DP-guided surgery could be completed under local infiltration anesthesia to shorten the whole operation time and reduce the risks associated with anesthesia. Craniotomy must be performed under general anesthesia, which increase the operation time; additionally, the risk of general anesthesia is high. Because of the short operation time and minimal trauma in the 3DP group, the incidence of complications was significantly lower, and the mean hospitalization time was significantly shorter than those in the craniotomy group. Frontiers in Neurology | www.frontiersin.org ETHICS STATEMENT The studies involving human participants were reviewed and approved by the ethics committee of Binzhou Medical University Hospital. The patients/participants provided their written informed consent to participate in this study. Written informed consent was obtained from the individuals for the publication of any potentially identifiable images or data included in this article. AUTHOR CONTRIBUTIONS effective. This procedure is more advantageous than craniotomy because of its minimal invasiveness, superior efficacy, low anesthesia risk, and low complication rate. It is more accurate than normal minimally invasive surgery without navigation, which reduces the number of punctures and trauma of most puncture wounds. The 3DP technique may improve the individualization and accuracy of minimally invasive surgery. QW and XD drafted the manuscript. QW, GF, WG, CL, and ML completed the operation. XD, GF, and WG performed the data collection and data analysis. KL is responsible for the revision and data processing of the article. ZL participated in the design of this study and helped to check the manuscript. All authors read and approved the final manuscript. ACKNOWLEDGMENTS We thank Nancy Schatken BS, MT (ASCP), from Liwen Bianji, Edanz Group China (www.liwenbianji.cn/ac), for editing the English text of a draft of this manuscript. FUNDING The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author/s. This study was financially supported by Natural Science Foundation of Shandong (No. ZR2018LH007) and research projects of Binzhou Medical University Hospital (No. BY2018KJ02). This study was financially supported by Natural Science Foundation of Shandong (No. ZR2018LH007) and research projects of Binzhou Medical University Hospital (No. BY2018KJ02). CONCLUSION Using individualized 3DP guidance, minimally invasive surgery to treat patients with HICH appears to be technically feasible and February 2021 | Volume 12 | Article 608403 5 3D-Printing Assisted Treatment of HICH Li et al. Li et al. REFERENCES 12. Ernoult C, Bouletreau P, Meyer C, Aubry S, Breton P, Bachelet JT. Reconstruction assisted by 3D printing in maxillofacial surgery. Revue de stomatologie, de chirurgie maxillo-faciale et de chirurgie orale. (2015) 116:10. doi: 10.1016/j.revsto.2015.01.010 1. Chiquete E, Ruiz-Sandoval MC, Álvarez-Palazuelos LE, Padilla- Martínez JJ, González-Cornejo S, Ruiz-Sandoval JL. 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Hostettler IC, Seiffge DJ, Werring DJ. Intracerebral hemorrhage: an update on diagnosis and treatment. Expert Rev Neurotherapeut. (2019) 19:679– 94. doi: 10.1080/14737175.2019.1623671 3. Zhang Y, Yi B, Ma J, Zhang L, Zhang H, Yang Y, et al. Quercetin promotes neuronal and behavioral recovery by suppressing inflammatory response and apoptosis in a rat model of intracerebral hemorrhage. Neurochemical Res. (2015) 40:195–203. doi: 10.1007/s11064-014-1457-1 15. Cappellari M, Zivelonghi C, Moretto G, Micheletti N, Carletti M, Tomelleri G, et al. The etiologic subtype of intracerebral hemorrhage may influence the risk of significant hematoma expansion. J Neurol Sci. (2015) 359:293– 7. doi: 10.1016/j.jns.2015.11.024 4. Fiorella D, Zuckerman SL, Khan IS, Kumar NG, Mocco J. Intracerebral Hemorrhage: a common and devastating disease in need of better treatment. World Neurosurg. (2015) 84:1136. doi: 10.1016/j.wneu.2015.05.063 16. Xia L, Han Q, Ni XY, Chen B, Yang X, Chen Q, et al. Different techniques of minimally invasive craniopuncture for the treatment of hypertensive intracerebral hemorrhage. World Neurosurg. (2019) 126:e888– 94. doi: 10.1016/j.wneu.2019.03.006 5. Li ZY, Luo YN, Jin MS, Chen DW, Shi PQ, Xu XG. Clinical therapeutic effect of different surgical approaches on the hypertensive intracerebral. J Dalian Medical University. (2012) 34:60–63. 17. Gui C, Gao Y, Hu D, Yang X. Neuroendoscopic minimally invasive surgery and small bone window craniotomy hematoma clearance in the treatment of hypertensive cerebral hemorrhage. Pakistan J Med Sci. (2019) 35:377. doi: 10.12669/pjms.35.2.463 6. REFERENCES Wang W, Zhou N, Wang C. Minimally invasive surgery for hypertensive intracerebral hemorrhage patients with large hematoma volume: a retrospective study. World Neurosurg. (2017) 105:158. doi: 10.1016/j.wneu.2017.05.158 6. Wang W, Zhou N, Wang C. Minimally invasive surgery for hypertensive intracerebral hemorrhage patients with large hematoma volume: a retrospective study. World Neurosurg. (2017) 105:158. doi: 10.1016/j.wneu.2017.05.158 18. Choy WJ, Parr WC, Phan K, Walsh WR, Mobbs RJ. 3-dimensional printing for anterior cervical surgery: a review. J Spine Surg. (2018) 4:757. doi: 10.21037/jss.2018.12.01 7. Mendelow AD, Gregson BA, Rowan EN, Murray GD, Gholkar A, Mitchell PM, et al. Early surgery versus initial conservative treatment in patients with spontaneous supratentorial lobar intracerebral haematomas (STICH II): a randomised trial. Lancet. (2013) 382:397–408. doi: 10.1016/S0140-6736(13)60986-1 19. Awad A, Trenfield SJ, Gaisford S, Basit AW. 3D printed medicines: a new branch of digital healthcare. Int J Pharmaceut. (2018) 548:586– 96. doi: 10.1016/j.ijpharm.2018.07.024 8. Wang W. Minimally invasive surgical treatment of acute epidural hematoma: case series. BioMed Res Int. (2016) 20:1–8. doi: 10.1155/2016/6507350 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. 9. Mould WA, Carhuapoma JR, Muschelli J, Lane K, Morgan TC, McBee NA, et al. Minimally invasive surgery plus recombinant tissue-type plasminogen activator for intracerebral hemorrhage evacuation decreases perihematomal edema. Stroke. (2013) 44:627. doi: 10.1161/STROKEAHA.111.000411 Copyright © 2021 Li, Ding, Wang, Fan, Guo, Li, Li and Li. 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. 10. Rennert RC, Signorelli JW, Abraham P, Pannell JS, Khalessi AA. Minimally invasive treatment of intracerebral hemorrhage. Expert Rev Neurotherapeut. (2015) 15:919–33. doi: 10.1161/STROKEAHA.118.020688 11. Jakus AE, Rutz AL, Shah RN. Advancing the field of 3D biomaterial printing. Biomedical Mater. (2016) 11:014102. doi: 10.1088/1748-6041/11/1/0 14102 February 2021 | Volume 12 | Article 608403 Frontiers in Neurology | www.frontiersin.org 6

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Review of Biofuel Technologies in WtL and WtE

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Review of Biofuel Technologies in WtL and WtE Bruno B. Garcia, Gonçalo Lourinho, Paulo Brito and Pedro Romano Abstract Processing of biomass feedstocks to produce energy, fuels, and chemicals via a combination of different applied technologies is considered a promising pathway to achieve sustainable waste management, with many environmental and economic benefits. In this chapter, we review the current state of the main processes associ- ated with energy recovery and biofuel production under the concept of waste biorefineries. The reviewed technologies are classified into thermochemical, bio- logical, and chemical, including combustion, gasification, steam explosion, pyroly- sis, hydrothermal liquefaction, and torrefaction; anaerobic digestion, fermentation, enzymatic treatment, and microbial electrolysis; and hydrolysis, solvent extraction, transesterification, and supercritical conversion. Their brief history, current status, and future developments are discussed within a perspective of valorization and managing of current waste streams with no solution. Keywords: biorefineries, biofuel production, energy recovery, waste-to-fuels, waste-to-energy, circular economy 1. Introduction Waste can be defined as any substance or object that has no further use and is intended to be discarded [1]. In this sense, waste production is inevitable in a society based on consumption, making waste management a huge challenge taking into account the enormous quantities of residues that are produced globally. In fact, about 2.6 billion Mg of waste were produced in the European Union (EU) during 2014, from which 41% was discarded in landfills, 36% was recycled, 10% used in earthmoving operations, 7% treated in wastewater treatment plants, and 6% incinerated either for energy production or for destruction. Based on this, in recent decades, humanity is shifting their focus of traditional waste management from the concept of “collection and disposal” in favor of pyramid-based management of the waste hierarchy in order to increase sustainability [2]. However, even when environmentally-friendly practices such as recycling and reuse are accomplished, much of the operations are performed “downcycling,” meaning that the recycled product has an economic value below its original purpose. As such, the linear economy model based on the pyramidal hierarchy of wastes that we tend to use nowadays also has limitations. Actually, there are still opportunities for efficiency gains in many industrial processes, but these gains will probably be increasingly marginal and undifferentiated. 1 Elements of Bioeconomy Elements of Bioeconomy The future adoption of the concept of circular economy is, therefore, a necessary change of paradigm, in contrast to the current linear model. This new concept is increasingly viewed as a source of innovation in products, processes, and busi- ness models, opening excellent opportunities that should be seen by companies and organizations as competitive advantages in a dynamic and global market [3]. Specifically, with a circular flux in the consumption of resources, every waste generated is a potential raw material for another process, introducing novel ways of valorization and development of second and third generation products. The ben- efits are clear as few wastes would be generated and disposed of without treatment, potentially reducing environmental pollution. Updated knowledge of current technologies is a crucial factor in determining the most suitable processes to valorize different types of wastes in future biorefineries. These waste biorefineries are facilities that integrate the necessary technologies in order to convert biomass feedstocks and other wastes into usable products, ensuring that circular economy transitions from theory to the real world. 1. Introduction The available waste streams can either be transformed by technologies producing biofuels (waste-to-liquids, WtL) or energy (waste-to-energy, WtE) with both categories expected to be a key element in future waste management. Based on this, in this chapter, we briefly review the current state of main WtL and WtE technologies within a perspective of their use as tools for managing post-process residues and by-products. The review ends with a brief discus- sion on future developments regarding mentioned technological options. 2. WtL and WtE technologies: historical and technological overview Biorefineries are a way to achieve sustainable waste management with many environmental and economic benefits. However, waste streams are often very selective in terms of the technological option most suitable for their valorization. As such, a complete understanding of each technology is a fundamental resource to determine if the different wastes available can be viewed as a raw material for valu- able products. Tables 1–3 summarize the different thermochemical, biological, and chemical processes discussed. A brief description of each technology follows. 2.1 Incineration/combustion Combustion is the most common waste energy recovery technology used in the production of heat, steam, and electricity. Historically, this technology is consid- ered one of the most “dirty” and polluting processes in waste management and dis- posal; however, advances in the treatment of emissions in the late 1980s and early 1990s, along with the development of command and control technologies and the pretreatment of waste, have led to combustion once again attracting the attention of researchers and investors around the world. In general, a modern incineration facility consists of pretreatment and/or sorting line from where the wastes are con- tinuously and uniformly fed to a furnace (Figure 1). The furnace operates at very high temperatures to ensure complete combustion. The combustion parameters are continuously controlled, and emissions are treated in a set of filters to ensure the removal of the toxic pollutants. As a WtE technology, combustion is very mature with the most recent studies focused on the recovery of energy and ashes resulting from the co-combustion or co-incineration of different wastes in nonspecialized equipment [4]. In fact, this process is widely used for thermal energy recovery of waste forms with good calorific value [5]. In 2016, for example, 28% of municipal solid waste (MSW) generated in the EU-28 was incinerated [6]. Furthermore, about 13.1% of hazardous waste was incinerated with and without energy recovery [7]. 2 Products and by-products Applications TRL/CRI/demonstration proj ublic opinion production, air Heat for boilers and furnaces. Potential metal recovery from slag Heating, electricity TRL9/CRI4 gh sensibility bility, risk of tars production H2, CO-rich syngas Biochar for soil remediation Heating, electricity, transportation, fuels and high-value chemicals TRL9/CRI3 Energos, Norway; Vaskiluodon Voima, Finland compounds, partial Sugars, digestible products Heating, electricity, transportation, fuels, and high-value chemicals TRL9/CRI2 enance and h bio-oil viscosity Bio-oils, biochar, syngas Additives, high-value chemicals, transportation, heating, and electricity TRL8/CRI2 ABRITech, Canada; Ensyn Several, Canada; Metso, Finland; Rise, Sweden ciency (20–60%), ipment and higher Heavy oil, intermediate value chemicals Additives, high-value chemicals, transportation, heating, and electricity TRL7/CRI1 Steeper Energy, Denmark; PNN USA; Genifuel, USA; PilotABP, Spain; TERAX, New Zealand , high ash quantity Torrefied biomass Heating, electricity TRL7/CRI1 Torplant, Switzerland; ECN, Netherlands; Norris Thermal Technologies, Vega Biofuels, USA –10]. opinion uction nsibilit risk of roduct ounds e and oil visc y (20–6 nt and ash qu Limitations Products Applications TRL/CRI/ demonstration projects Need to treat and clean the biogas; unstable system; large facilities are unattractive Biogas, bio-digestate Heating, electricity, transportation, fuels, and high-value chemicals TRL9/CRI3 Limited to sugar, starch, or cellulose-rich feedstocks Liquids and CO2 Additives, high-value chemicals, transportation, heating, and electricity TRL9/CRI2-3 Low efficiency, inhibited in the presence of oxygen Hydrogen, carbon dioxide, organic acids Additives, high-value chemicals, transportation, heating, and electricity TRL6/CRI1 Low theoretical limit, immature technology Hydrogen, acetic acid Additives, high-value chemicals, transportation, heating, and electricity TRL5/CRI1 DiSAA Milan, Italy; LanzaTech, New Zealand High cost of the enzymes, slow reactions, necessity of high purity, limited in temperature and pH range Ethanol, amino acids High-value chemicals, transportation, heating, and electricity TRL9/CRI2 PROESA™, Italy; Kalundborg Bioethanol, Denmark High internal resistance, high capital cost, production greatly affected by substrate composition Hydrogen, methane, acetate, formic acid Wastewater treatment, high-value chemicals, transportation, heating, and electricity TRL6/CRI1 ]. Limitations Products Applications TRL/CRI/ demonstration projects es Slow and inefficient, high alkalinity or acidity, formation of inhibitory salts Cellulose, hemicellulose, and lignite Additives, high-value chemicals TRL9/CRI5 nts, Intermediate products, solvent saturation Primary and secondary metabolites Additives, high-value chemicals TRL9/CRI3 on, CO2 Weak supply chain, high viscosity, high cost, odor FAME Transportation, electricity TRL9/CRI5 s; better High pressures required, supercritical state, difficult to maintain, complex maintenance and cleaning Chemicals Wastewater treatment, high- value chemicals, transportation, heating, and electricity TRL7/CRI-1 Thar Technology, USA; Integrated Plantrose Complex, USA; New Oil Resources, USA dex. ologies [8–10]. Limitations Products Applications TRL/CRI/ demonstration projects Slow and inefficient, high alkalinity or acidity, formation of inhibitory salts Cellulose, hemicellulose, and lignite Additives, high-value chemicals TRL9/CRI5 Intermediate products, solvent saturation Primary and secondary metabolites Additives, high-value chemicals TRL9/CRI3 Weak supply chain, high viscosity, high cost, odor FAME Transportation, electricity TRL9/CRI5 High pressures required, supercritical state, difficult to maintain, complex maintenance and cleaning Chemicals Wastewater treatment, high- value chemicals, transportation, heating, and electricity TRL7/CRI-1 Thar Technology, USA; Integrated Plantrose Complex, USA; New Oil Resources, USA [8–10]. ent, high ty, form ducts, so in, high quired, e, difficu ex maint Elements of Bioeconomy Figure 1. Example scheme of incineration/combustion technology. Figure 1. Example scheme of incineration/combustion technology. g Example scheme of incineration/combustion technology. 2.2 Gasification Gasification is a thermochemical decomposition process which occurs without the presence of sufficient oxygen for a complete combustion and allows the transforma- tion of waste feedstocks into a combustible gas known as syngas, a fuel with many potential applications (Figure 2). As a technology, gasification has a several-centuries- old history with progress made by advances and stalls. Widely used during industrial revolution in the 1850s to illuminate factories, streets, and houses, this technology fell into disuse during the twentieth century and only recently gained a continuous sup- port for its development due to energy security threats and climate change. Among WtE processes, gasification is one of the most promising with some specific barriers explaining its lack of penetration in the domestic and commercial sectors [11]. An extensive review on technology progress identified 50 companies offering “commercial” gasification plants in Europe, the USA, and Canada, mostly downdraft and fluidized bed systems (75 and 20%, respectively) [12]. Moreover, in 2013 there were more than 272 operating gasification plants worldwide with more under construction and planned until 2019 [13]. p Supply chain development, waste pretreatment (drying/grinding/pelletization), and the potential need for treatment of syngas are usually pointed out as the main barriers to be overcome. Conventional drying systems are known to be expensive and energy intensive. In addition, complete drying of the biomass represents a decrease in Figure 2. Example scheme of gasification technology. Figure 2. Figure 2. Example scheme of gasification technology. gu e . Example scheme of gasification technology. 6 6 6 Review of Biofuel Technologies in WtL and WtE DOI: http://dx.doi.org/10.5772/intechopen.84833 the amount of hydrogen that is potentially producible during gasification. Solar dry- ing, though inefficient, is cheap and should be studied and viewed as an alternative. The potential presence of tars, particulate emissions, SOx, NOx, and NH3 in the syn- gas also limits its range of use. Filtration of the syngas is important to obtain a syngas free of contaminants but requires constant cleaning of the filters as a way to prevent blockage and pressure drops. Tars are seen as the most complicated contaminant. In addition to filtration, it is also possible to resort to thermal decomposition and cata- lytic cracking as a form of treatment [14]. Thermal decomposition leads to melting of the ashes, which can also result in mechanical problems. 2.2 Gasification Catalytic treatment is seen as the most effective for dealing with tars but ineffective against particles and other toxic gases. The combination of various forms of treatment is the best solution [15]. g Pretreatment of the waste and biomass to be gasified, as well as reactor design and optimization of operational conditions, has been proven to be of great importance to maximize conversion efficiency, viability, and profitability [16]. In this regard, proce- dures such as sorting, grinding, and sifting are simple but essential. Fluidized bed reac- tors are considered the most suitable for a good and efficient process. Fluid bed material consisting of natural rocks such as dolomite and olivine is usually the best option due to reasonable prices. As for optimized conditions, mathematical models using 2D computational fluid dynamics (CFD) confirmed that gasification temperature has a key influence on the calorific value of the syngas produced [17]. Co-gasification of several wastes has been reported with promising results [18–20]. Inorganic additives such as calcium oxide (CaO) have been observed to decrease CO2 and increase the quality of the syngas [21]. Integrating gasification and co-gasification into solid oxide fuel cells (SOFC) or internal combustion engine (ICE) cogeneration systems is a very promising option and is already considered economically viable [22–24]. 2.3 Explosive steam decompression Explosive decompression is a thermochemical pretreatment process which disrupts the rigid structure of lignocellulosic materials using steam and high pres- sures. Patented in 1931 by Mason [25], this process consists in heating the waste in hot steam at 285°C and at a pressure of 3.5 MPa for 2 min, before increasing the pressure once again, this time to 7 MPa for 5 s [26]. Naturally, time and temperature are a major influence in the disruption of the fibers composing the biomass, with the pretreatment process potentially resulting in just some grooves in the wood or in the total conversion into pulp. The main application of this technology is as pre- treatment of lignocellulosic materials (Figure 3) which is essential for making the Figure 3. Example scheme of explosive steam decompression technology. Figure 3. Figure 3. Figure 3. Example scheme of explosive steam decompression technology. gu 3. Example scheme of explosive steam decompression technology. 7 7 Elements of Bioeconomy Elements of Bioeconomy biopolymers accessible for further treatment via other processes such as fermenta- tion, hydrolysis, anaerobic digestion, and densification. The production of biogas by anaerobic digestion using lignocellulosic wastes, for example, is considered a huge challenge due to its recalcitrant nature (non-biodegradability) [27]. In this regard, the use of explosive steam decompression as a form of pretreatment has been proven to enhance the production of biogas. Moreover, ethanol production and syngas production using lignocellulosic feedstocks have also been reported to proceed with higher calorific value and lower temperatures, respectively, when precluded with steam explosion [28, 29]. A promising solution for continu- ous steam explosion has been presented by a research team from South China University of Technology [30] allowing for process scale-up and its potential integration in second-generation biorefineries. 2.4 Pyrolysis Pyrolysis is a thermochemical decomposition process that occurs in the total absence of oxygen and at relatively low temperatures (500–800°C) when com- pared to gasification (800–1000°C). There are different types of pyrolysis, each favoring the production of three different products: pyrogas, pyrolysis oil, and char (Figure 4). The relative proportions of each product depend on the applied pyrolysis method, the type of feedstock, and temperature. Archeological evidence suggests that during the Middle Paleolithic, Neanderthals resorted to pyrolysis to produce a kind of tar which they would use as glue. The use of this process in the production of all types of products was widespread throughout the world until the beginning of the twentieth century. Nowadays, pyrolysis is once again being viewed as one interesting solution to produce energy, fuels, and chemicals using local wastes. The major advantage of pyrolysis in waste recovery may be in being able to convert low-energy-density materials into high-energy-density products. As an example, pyrolysis has been adopted as an alternative to the treatment of plastic wastes to produce plastic-derived oil (PDO) [31] and pyrogas [32]. PDO has been reported to be similar to diesel (C13–C20) [33]; however, additional pro- cessing is needed to deal with aromatic compounds. The use of calcium carbon- ate (CaCO3) in the pyrolysis of horse manure allows for lower temperatures due to the catalytic effects of CaCO3 as a possible source of CO2 [34]. Co-pyrolysis of different plastic mixtures [35], as well as the use of catalysts [36–39], has also yield interesting results concerning the productivity and quality of the PDO components. Figure 4. Example scheme of pyrolysis technology. Figure 4. Figure 4. Figure 4. Example scheme of pyrolysis technology. g 4 Example scheme of pyrolysis technology. 8 Review of Biofuel Technologies in WtL and WtE DOI: http://dx.doi.org/10.5772/intechopen.84833 2.5 Hydrothermal liquefaction (thermal depolymerization) Hydrothermal liquefaction or thermal depolymerization is the thermochemical conversion of solid waste into a liquid using moderate temperatures (250–375°C) and high pressures (4–22 MPa). Similar to pyrolysis but occurring with the waste immersed in water at high pressures and temperatures, the process leads to the break of long carbon chains, resulting in a bio-oil with a good calorific value. As a technological option, the process does not need catalysts, but research has indicated that the use of alkaline catalysts allows the formation of high-value chemicals. Hydrothermal liquefaction is attractive because efficiencies greater than 80% are common when converting biomass into fuels and other high-value chemicals [40]. This technology has enormous potential, particularly to produce biofuels and raw materials for further chemical processing. y g Hydrothermal liquefaction is attractive because efficiencies greater than 80% are common when converting biomass into fuels and other high-value chemicals [40]. This technology has enormous potential, particularly to produce biofuels and raw materials for further chemical processing. p g The concept of hydrothermal liquefaction was first explored in the 1920s and was further developed in the 1950s by H. Heinemann. However, only after the oil crisis in the 1970s did the first efforts to exploit this technology finally emerged, being the concept finally proved at pilot scale with the construction of Biomass Liquefaction Experimental Facility in Oregon, USA [41]. Recently, research regard- ing this technology has focused on finding new catalysts and developing novel ways of converting the produced bio-oils into high-value products. In practice, hydrothermal liquefaction is valued because it provides rapid conversion of waste biomass into bio-oil, avoiding the high energy cost of drying [42]. Most studies have shown that temperatures between 250 and 370°C are optimal for the production of bio-oil, with no general conclusion given about the effects of reaction time and moisture content [43]. Hydrothermal co-liquefaction is an interesting pathway and should be explored in future studies [44, 45]. Both the addition of potassium carbonate (K2CO3) [46] and the reuse of the liquid were reported to increase calo- rific value and productivity. The addition of solvents was also observed to enhance the process [47], while the addition of metallic catalysts led to deoxygenation and desulphurization of the bio-oil [48]. 2.6 Torrefaction Torrefaction is a form of thermal treatment which takes place between 200 and 500°C in the absence of oxygen. As temperature rises, moisture and superfluous volatiles are gradually released, and biopolymers such as hemicellulose, cellulose, and lignite are partially decomposed, depending on process conditions [49]. At mild temperatures (235–275°C), for example, the degradation of hemicellulose is acceler- ated, and the release of the volatiles is intensified, while cellulose is only consumed to some degree. On average, the process results in mass losses and decreases in calorific value (20% and 10%, respectively) but yields a more homogeneous waste composition and leads to higher energy densities. Some biomasses have characteris- tics that hinder their utilization as energy feedstocks; using this process as pretreat- ment allows the use of a broad spectrum of wastes in other WtE technologies. The main product of torrefaction is, therefore, a waste with improved characteristics regarding its energy use. More than 150 torrefaction installations worldwide with powers from 50 to 700 MWe have successfully tested the co-combustion of torre- fied biomass, reducing greenhouse gas emissions and dependence on fossil fuels. It is expected that torrefied biomass could represent 5–10% of industrial applications in Europe [49]. However, the market for torrefied waste products is still very recent, and there is not enough data available about the real use of technology, its imple- mentation, and its evolution. 9 Elements of Bioeconomy Among researchers, torrefaction has been viewed as an excellent pretreatment for improving the energy recovery features of several wastes creating products with low oxygen to carbon ratios and high calorific values for co-gasification and co- combustion applications [50]. As an example, the torrefaction of several pomaces [51] and prunings [52] led to very promising results with calorific values increased to near lignite levels. Other interesting results have been reported by research- ers [50] dealing with the very heterogeneous nature of MSW which along with high moisture contents make them challenging for application in WTE and WTL processes. Most studies reported a positive correlation between the calorific value and torrefaction temperature. 2.7 Anaerobic digestion Anaerobic digestion (AD) consists in the conversion of biodegradable organic matter in the absence of oxygen in which a biogas rich in methane is produced [53]. Typically, the resulting biogas is composed of 50–75% CH4, 25–50% CO2, and 1–15% of other gases such as H2O, NH3, and H2S. Another by-product of anaerobic digestion is the digestate, an excellent organic biofertilizer. Virtually all types of organic matter have the potential to be digested anaerobically to produce biogas. The most common organic wastes used in AD are agricultural, livestock industry, agroindustry, and municipal solid wastes and wastewater. Woody materials are less suitable because they contain a high proportion of lignite, making it very difficult to decompose biologically. p g y As a technology, AD is already mature and well developed. Since 2009, the num- ber of biogas plants has greatly increased in Europe with biomethane production growing in line with sector development. In 2016 alone, energy production derived from biomethane increased by 4971 GWh (+40%) within the European countries reviewed [54]. The key to future research is thus the optimization of process param- eters that affect efficiency. Temperature change, for example, is known to affect microbial activity and growth rates. Higher digestion temperatures, for instance in the thermophilic range, have been demonstrated to lead to higher biogas produc- tivities, but thermophilic digestion represents a higher investment due to energy costs. On the other hand, digestion of simple substrates often results in a nutrient imbalance that affects the stability of the process. Thus, C/N ratio optimization by co-digestion has been widely tested with good results taking advantage of the synergies between different substrates. This strategy represents the most economi- cal way to improve process productivity nowadays. The use of multiple steps in AD has also been observed to be an interesting solution for achieving the best use of different substrates [55–58]. The integration of anaerobic digestion with microalgae cultivation presents potential benefits [59, 60]. From an economic point of view, costs can be substan- tially reduced by using the digestate from AD as a source of nutrients for algae growth. However, several barriers will have to be overcome before the scale-up of the process. The main obstacle identified in the reviewed research was the need to find a robust microalga strain capable of binding with organic and inorganic carbon and tolerate extremes of pH. 2.8 Fermentation Fermentation is an anaerobic metabolic process, in which microorganisms (bacteria, yeast) turn carbohydrates into fatty acids, alcohols, and gaseous products such as H2 and CO2 (Figure 5). The most common industrial products resulting from fermenta- tion are ethanol, acetic acid, and citric acid (2-hydroxypropane-1,2,3-tricarboxylic 10 Review of Biofuel Technologies in WtL and WtE DOI: http://dx.doi.org/10.5772/intechopen.84833 acid). The conversion of sugars into ethanol is the most well-known form of fermenta- tion, producing alcoholic beverages such as wine, beer, and cider. Interestingly, the same fermentation occurs in the production of bread, yogurt, and other foods fer- mented by the formation of lactic acid (2-hydroxypropanoic acid). In addition, there have been significant advances in the production of bioethanol, biobutanol (butan- 1-ol), and bio-hydrogen (molecular hydrogen), among other high-valued chemicals. Continuous fermentation of syngas using fixed-bed drip reactors for ethanol pro- duction has been proven as a valid concept with the highest ethanol concentration (13.2 g L−1) obtained during co-current continuous syngas fermentation at a dilution rate of 0.012 h−1 [61]. However, despite being a promising technology, the process has encountered some difficulties in its development on an industrial scale. Besides fixed-bed bioreactors, other efforts related with reactor design have been focused on membranes combined with the formation of biofilms due to enhances in mass trans- fer. Studies on the production of bio-hydrogen have been focused on bio-photolysis of water using algae and cyanobacteria, photodecomposition of organic compounds by photosynthetic bacteria [62], and dark fermentation of organic compounds with anaerobes [63]. For dark fermentation, special attention has to be given to inhibitors such as the excess of substrate, micronutrients, macronutrients and metal ions, high temperatures, acidic pH levels, and competition from other microorganisms [63]. Figure 5. Example scheme of fermentation technology. Review of Biofuel Technologies in WtL and WtE DOI: http://dx.doi.org/10.5772/intechopen.84833 Figure 5. Example scheme of fermentation technology. Figure 5. Example scheme of fermentation technology. g 5 Example scheme of fermentation technology. g 5 Example scheme of fermentation technology. acid). The conversion of sugars into ethanol is the most well-known form of fermenta- tion, producing alcoholic beverages such as wine, beer, and cider. Interestingly, the same fermentation occurs in the production of bread, yogurt, and other foods fer- mented by the formation of lactic acid (2-hydroxypropanoic acid). In addition, there have been significant advances in the production of bioethanol, biobutanol (butan- 1-ol), and bio-hydrogen (molecular hydrogen), among other high-valued chemicals. 2.8 Fermentation ), y g ( y g ), g g Continuous fermentation of syngas using fixed-bed drip reactors for ethanol pro- duction has been proven as a valid concept with the highest ethanol concentration (13.2 g L−1) obtained during co-current continuous syngas fermentation at a dilution rate of 0.012 h−1 [61]. However, despite being a promising technology, the process has encountered some difficulties in its development on an industrial scale. Besides fixed-bed bioreactors, other efforts related with reactor design have been focused on membranes combined with the formation of biofilms due to enhances in mass trans- fer. Studies on the production of bio-hydrogen have been focused on bio-photolysis of water using algae and cyanobacteria, photodecomposition of organic compounds by photosynthetic bacteria [62], and dark fermentation of organic compounds with anaerobes [63]. For dark fermentation, special attention has to be given to inhibitors such as the excess of substrate, micronutrients, macronutrients and metal ions, high temperatures, acidic pH levels, and competition from other microorganisms [63]. 2.9 Enzyme treatment Enzymes are macromolecular biological catalysts which accelerate chemical reactions. In 1897, Eduard Buchner resorted to enzymes extracted from yeasts grown in his lab to ferment ethanol, a seminal work for which he received the Nobel Prize for Chemistry in 1907. Industrially, their application lies either in converting substrates into greater value products or as pretreatment for energy recovery and biofuel production. Nowadays, nearly all types of commercially available enzymes are produced by fermentation, being part in virtually every aspect of our lives, from the pharmaceutical industry to laundry detergents. In 2016, an industrial unit including an enzymatic pretreatment started to operate within a perspective of energy recovery from MSW. Specifically, enzymes degrade a fraction of the organ- ics present in MSW so that they can be easily digested anaerobically. The facility is located in Northwich, England, and produces 5 MWe consuming 15 Mg h−1 of MSW [64]. Another commercial application with good future perspective is enzymatic saccharification which can be used to produce bioethanol at a low cost. Some stud- ies on bioethanol production from bamboo, for example, indicate that increasing the amount of the enzyme yields little improvement in the process highlighting 11 Elements of Bioeconomy the need for optimization depending on the waste to be transformed [65]. Other experiments have focused on process enhancement via salt pretreatment. Addition of inorganic salts, for instance, has been reported to improve reducing sugar yields of sugarcane leaf wastes and mustard stalk and straw [66, 67]. 2.10 Microbial electrolysis Microbial electrolysis is a bioelectrochemical transformation where hydrogen or methane is produced from various wastes and wastewaters. Microbial electrolysis cells (MEC) use the metabolic activity of exoelectrogenic bacteria to catalyze redox reactions and promote the flow of electrons between the electrodes [68]. Specifically, the bacteria convert biodegradable substrates at the anode, releasing electrons and protons (Figure 6). The electrons are then transferred to the cath- ode (where hydrogen is produced) inducing an electrical current with electrical potential values (0.2–0.8 V) lower than in traditional electrolysis (1.8–3.5 V) [69]. Microbial electrolysis cells (MEC) have the potential to become one of the most important WtE technologies. However, electrode materials are still costly, and fur- ther developments are needed. In this regard, the use of biochar-based electrodes seems to compose an interesting research route [70–72]. Currently, coupling with other technologies for energy generation seems to be its leading application. The use of microbial electrolysis as a pretreatment for AD, for example, has been explored recently with interesting results. In a study focused on the valorization of highly concentrated FW [73], MEC was found to accelerate methane production rate and stabilization. As another example, post-processing of wastewater resulting from hydrothermal liquefaction for recovered hydrogen has also been demonstrated with effective results [74, 75]. As a technology, MEC are still in the early phase of devel- opment, and further progress is expected with the use of novel electrode materials and new reactor configurations. 2.11 Hydrolysis Hydrolysis is probably the most prevalent chemical reaction in multiple WtE and WtL technologies. Hydrolysis is the chemical reaction where the addition of Figure 6. Example scheme of microbial electrolysis technology. Figure 6. Figure 6. Example scheme of microbial electrolysis technology. 12 Review of Biofuel Technologies in WtL and WtE DOI: http://dx.doi.org/10.5772/intechopen.84833 a water molecule breaks the chemical bond of another molecule and the resulting molecules bind to H+ and OH− ions. In 1819, Henri Braconnot discovered that he could produce sugars from cellulose through hydrolysis with sulfuric acid. This hydrolyzed sugar could then be processed and fermented to produce ethanol. The production of ethanol by hydrolysis began extensively at the beginning of the twen- tieth century, with maximum yields of 190 L Mg−1 of biomass. In the former USSR during the 1930s, the industrial growth at the time needed to develop processes of ethanol production that did not use food sources. In 1934, six pilot reactors were built with the objective of optimizing different hydrolysis technologies, not only to produce ethanol but also other products such as xylitol and furfural. After the First World War, this process was no longer economically viable against more conven- tional methods. With the advances of the last two decades, enzymatic hydrolysis seems to be the most promising application regarding hydrolysis techniques. 2.13 Transesterification Transesterification is the main process used in the production of biodiesel in which vegetable oils are broken into methyl or ethyl esters by reacting with an alcohol and catalysts (acids, alkalis, and enzymes) with glycerol as the only by- product. Biodiesel production has increasingly been seen as a carbon mitigation tool, assuming increasing importance in promoting sustainability in European countries. Since January 1, 2010, for example, all commercial diesel fuel sold in Portugal has a 7% incorporation of biodiesel. Biodiesel production is a controversial issue due to the use, availability, and cost of raw materials, as well as greenhouse gases emission and food competition. In this context, the use of waste oils and nonfood crops seems to compose the best option for the widespread production of biodiesel in the future [80]. In Europe, it is estimated that about 4 million Mg of waste cooking oil are to be collected annually, seven times more than the current collected amount [81]. This underdeveloped collection chain led to record level imports in the first 8 months of 2018 with more than 235,000 Mg of waste cooking oil entering the EU from China. Biodiesel market thus does not show signs of slowing down [82]. Although already mature and well established commercially, biodiesel still needs a lot of research and development to achieve significant improvements in its production [83, 84]. In this regard, continued interest in the use of biodiesel as an alternative fuel has led to increased efforts to develop a new generation of biofuels. Heterogeneous catalysts have been increasingly tested since they offer process improvements over homoge- neous catalyzed commercial production employing liquid bases. In more detail, the use of solid catalyst facilitates post-process separation and fuel purification, along with the continuous synthesis of biodiesel. The increasing use of low-grade waste cooking oil remains a challenge for existing heterogeneous catalysts since the high concentration of impurities (acid, moisture, and heavy metals) induces rapid deac- tivation in flow and requires purification. The development of more robust catalyst formulations tolerant to such components is, therefore, a necessity [85]. Cement was recently tested in the transesterification of Pongamia pinnata and sunflower oil with somewhat low conversion rates (76%), but research should continue in upcoming years [86]. In terms of process coupling, the blend of biodiesel with pyrolysis oil derived from lignocellulosic wastes is an attractive route as an alternative to diesel fuel [87]. 2.13 Transesterification Microalgae are also considered an attractive feedstock alternative to reduce costs in the extraction and conversion of this renewable fuel. 2.12 Solvent extraction Solvent extraction is a relatively modern technology used in the extraction of products from its substrates (Figure 7). By choosing a solvent that best dissolves the wanted product, this process usually results in higher yields when compared with other methods. The separation is quick and efficient and most of the solvent can be reused. Extraction of oils via this technology is a common application in the industry, normally used after mechanical extraction. Hexane is the most used solvent, but ethanol and isopropanol have also been proposed as alternative options. The Soxhlet extractor is often the preferred method for lipid extraction due to the simplicity of operation, relative safety, and ease of replicating results on an industrial scale [76]. From research, organic solvents such as chloroform, ethanol, and hexane were found to produce the best results when performing lipid extrac- tion from microalgae. Solvent mixtures were also observed to yield better results Figure 7. Example scheme of solvent extraction technology. Figure 7. Example scheme of solvent extraction technology. Figure 7. Example scheme of solvent extraction technology. 13 Elements of Bioeconomy than mono-solvent extractions, with a 50/50 mixture of chloroform and ethanol leading to 11.76% lipid extracts. As a mono-solvent, chloroform resulted in the highest quantity of lipids extracted at 10.78% with 3 h showing the best extraction efficiency [77]. Solvent extraction also has the potential to be integrated with other processes like supercritical extraction or pyrolysis in order to produce higher value chemicals from bio-oils [78, 79]. Figure 8. Figure 8. Example scheme of supercritical conversion technology. Figure 8. Example scheme of supercritical conversion technology. the reviewed research generally suggests that, for example, supercritical gasifica- tion in both biorefineries and cogeneration has enormous potential. Supercritical water gasification of olive oil mill wastewater, for example, has been investigated recently with different alkali catalyst types [88]. The tests proved that an increment in catalyst concentration would improve hydrogen yield to a maximum of 76.73 mol H2 kg−1 in specific conditions. Extraction with supercritical carbon dioxide for biodiesel production is another process investigated [89]. In the example study reviewed, the best productivity was 0.312 kg of oil per kg of seed−1 at a pressure of 500 bar and 40°C. Fatty acid content was observed to decrease with increasing pressure. In fact, the extraction of fatty acids and transesterification in a single step are considered one of the greatest potentials for this technology [90]. 2.14 Supercritical conversion Supercritical conversion is a new technique that uses high temperature and high- pressure fluids, above their critical point, to achieve the transformation of waste. Compared with conventional WtE and WtL technologies, this method may ignore drying or dehydration pretreatments, reduce reaction temperature, shorten reac- tion times, and increase product yield. In recent decades, supercritical conversion has gained interest not only for chemical extraction, but also in chemical conversion by replicating processes such as transesterification, gasification, hydrolysis, and others (Figure 8). Studies with real biomasses and at larger scales are lacking, but 14 Review of Biofuel Technologies in WtL and WtE DOI: http://dx.doi.org/10.5772/intechopen.84833 Review of Biofuel Technologies in WtL and WtE DOI: http://dx.doi.org/10.5772/intechopen.84833 Figure 8. Example scheme of supercritical conversion technology. 3. Economics of WtE The most apparent barrier for the implementation of WtE technologies is capital cost, specifically the upfront expense of building and installing the energy genera- tion system. While not enabling a detailed view of project economics, an assessment of capital costs offers simple and clear information which can be used to evaluate the status of different commercial technologies. Figure 9 shows estimates of capital cost for a range of WtE power generation technologies. Capital costs are low for mature technologies such as cocombustion and anaerobic digestion integrated with ICE or gas turbine (GT). For early-stage technologies, the capital costs are extremely uncertain, and as such many were not included in the analysis. Pyrolysis, plasma arc gasification, and refused-derived fuel (RDF) direct combustion, for 15 Elements of Bioeconomy Figure 9. Capital cost (in $/kW) for selected WtE power generation technologies [91–95]. Figure 9. Figure 9. Capital cost (in $/kW) for selected WtE power generation technologies [91–95]. example, have higher capital costs due to technical hurdles and novelty status. As for integrated gasification combined cycle (IGCC), the costs vary widely as the process is still not established with significant cost reductions expected. The size of cost decline, however, is likely to be very dependent on geographical location and in line to the support given by global policy-makers and national frameworks regard- ing each technology [91]. example, have higher capital costs due to technical hurdles and novelty status. As for integrated gasification combined cycle (IGCC), the costs vary widely as the process is still not established with significant cost reductions expected. The size of cost decline, however, is likely to be very dependent on geographical location and in line to the support given by global policy-makers and national frameworks regard- ing each technology [91]. 4. Conclusions This chapter explored the possibility of using postprocess residues as abundant biorenewable and low-cost resources in future waste biorefineries. Available waste streams have a complex and varied composition according to its source, requiring new logistic platforms of assortment and valorization. With the exhaustion of the “collection and disposal” linear economy, new waste handling methods are unavoid- able in the long term. As such, waste biorefineries that produce green energy and make virtually zero-waste high-value products in a “closed loop” and “up-cycling” approach are the “landfills” of the future. They are expected to be crucial in taking sustainable waste management into the real world allowing game-changing economic growth under the concept of circular economy. However, from the reviewed tech- nologies, it can be concluded that single WtL and WtE processes are almost always limited in their scope, producing many times unwanted products. In this regard, the technology with more potential and scope in single applications is by far gasification; nonetheless, even this process has drawbacks such as reactor design, feeding system, and tar production that require costly posttreatment and/or further technologi- cal developments. Conversely, combining multiple WtE and WtL processes in an integrated waste biorefinery will allow the mitigation and elimination of each single process drawbacks. In gasification, for example, some of the unwanted substances generated may be utilized and valued by subsequent chemical processing, and even syngas can be upgraded. This novel waste valuation pyramid will create opportunities 16 Review of Biofuel Technologies in WtL and WtE DOI: http://dx.doi.org/10.5772/intechopen.84833 Review of Biofuel Technologies in WtL and WtE DOI: http://dx.doi.org/10.5772/intechopen.84833 Review of Biofuel Technologies in WtL and WtE DOI: http://dx.doi.org/10.5772/intechopen.84833 for niche technologies such as explosive decompression and torrefaction to make it into practical application by enhancing other technologies that are already well established when used in an integrated approach. Future research should primarily focus on the establishment of a hierarchy of processes to produce the highest value products and then progress gradually to low-cost products and energy production. For this vision to be a reality, however, an increased effort on the part of the research- ers will be required with a combined continuous and sustained support from all potential stakeholders. 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Propuesta metodológica para el desarrollo de productos desechables catalogados como sustentables en México

Proceedings INNODOCT/20. International Conference on Innovation, Documentation and Education

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INNODOCT 2020 Valencia, 11th-13th November 2020 DOI: http://dx.doi.org/10.4995/INN2020.2020.11887 Propuesta metodológica para el desarrollo de productos desechables catalogados como sustentables en México Ing. Carlos E Vázquez Peñaa, Dr. David Cortés Sáenzb a Ing. en Aeronáutica por la UACJ, estudiante de la maestría en diseño de producto en la UACJ, [email protected], , b Dr. en proyectos de innovación tecnológica, coordinador de la maestría en diseño de producto en laUACJ, [email protected] Resumen Actualmente los efectos del impacto ambiental son evidentes. Las decisiones de inversión tanto de los productores como de los países, y el comportamiento que se tiene por parte de los consumidores, siguen siendo ampliamente focalizados a resolver problemas locales y a corto plazo. El diseño ha fomentado este tipo de modelo de consumo, dando un énfasis a crear productos para un mercado donde las alternativas sustentables han recibido poca atención. Un ejemplo claro son los desechables, utilizados para el transporte de alimentos. Esta investigación tiene como objetivo identificar aspectos de metodologías existentes, para desarrollar una propuesta aplicable a los productos desechables utilizados en el sector alimenticio en México y con un enfoque al ciclo de vida del producto y la economía circular. Palabras clave: Ecodiseño, ACV, economía circular, diseño de producto Abstract The effect of environmental impact around the glove is now evident. Inversion decisions from countries and companies and the behavior of consumers are still widely focalized and directed to solve problems localized and in the short term. In this context, design and designers had been part of this consumption model, empathizing on the creation of products in a market, where the sustainable alternatives had a low presence. One example is the disposable products in the food industry. This research aims to identify aspects of existing methodologies, to develop a proposal applicable to disposable products used in the food sector in Mexico and with a focus on the product life cycle and the circular economy. Keywords: Ecodesign, Life Cycle Assessment, circular economy, product design Editorial Universitat Politècnica de València 643 Propuesta metodológica para el desarrollo de productos desechables catalogados como sustentables en México 1. Introducción Durante el siglo XX el diseño de productos ha servido para desarrollar el mercado global, buscando dar soluciones a las problemáticas y así mejorar las condiciones de vida de las personas. Para esto, las cadenas de producción global han sido fundamentales, logrando disminuir el costo de los productos y ampliando las redes de distribución. Sin embargo, la búsqueda del crecimiento económico ha restado importancia al impacto de las actividades humanas en el ecosistema (Bastidas Pacheco & Hernández, 2019). A partir de los años 70’s y con un creciente auge en lo que va del siglo XXI, se ha fortalecido la toma de conciencia y la realización de acciones que pretenden disminuir el impacto al ecosistema (García & Cárcova, 2019). La Organización de las Naciones Unidas (ONU) a través del Programa de las Naciones Unidas para el Desarrollo (PNUD) ha establecido 17 objetivos para el desarrollo sostenible, los cuales se muestran en la Fig. 1 (United Nations Development Programme, 2015). Estos objetivos son la base del “plan para una ciudad sostenible Juárez 2030” (Gobierno Municipal de Ciudad Juárez et al., 2018). En donde se denotan acciones específicas a nivel local. Fig. 1 Objetivos para el desarrollo sostenible (United Nations Development Programme, 2015) Esta investigación parte del objetivo número 12, en el cual se propone buscar medios productivos y de consumo responsables con el medio ambiente, aportando a las siguientes metas:   Para el 2030, reducir la generación de los desechos a través de actividades de prevención, reducción, reciclado y reutilización Ayudar a los países a fortalecer su capacidad científica y tecnológica para crear modalidades de consumo y producción que sean más sostenibles. Dentro del “plan para una ciudad sostenible Juárez 2030”, se crearon indicadores que buscan cumplir las metas descritas por la ONU a través de actividades de prevención, Editorial Universitat Politècnica de València 644 Ing. Carlos E Vázquez Peña, Dr. David Cortés Sáenz reducción, reciclado y reutilización. De estos indicadores son relevantes para esta investigación los siguientes (Gobierno Municipal de Ciudad Juárez et al., 2018):    12.1.1 Reducir a 1 kg o menos la cantidad de basura recolectada per cápita. 12.1.2 Incrementar al 100% el porcentaje de basura del relleno sanitario que se recicla y que se reutiliza. 12.1.3 Incrementar al 80% el porcentaje de viviendas particulares habitadas que separa sus residuos en orgánicos e inorgánicos para su entrega al servicio público de recolección o en un contenedor. El objetivo del presente artículo, es identificar aspectos claves en distintas metodologías existentes, los cuales puedan ser utilizados en una propuesta que este dirigida a los productos desechables. Contaminación A través de las actividades que realiza el hombre se pueden encontrar distintos tipos de desechos, dentro de los cuales se encuentran los residuos sólidos urbanos. En 2015 se generaron aproximadamente 31.5 millones de toneladas en México, mostrando un incremento del 25% desde el 2003. De estas, solo el 5% fue reciclado (INECC, 2018; SEMARNAT, 2016), y el resto termina en los rellenos sanitarios municipales. La secretaria de desarrollo urbano del estado de chihuahua realizó un diagnóstico para conocer la situación del manejo de residuos sólidos urbanos en los distintos municipios que conforman esta entidad. En dicho estudio, se encontró que en Ciudad Juárez se generaron 1,200 toneladas diarias y solo 199 toneladas de basura pasaban a un centro de reciclado (INECC, 2018). Dentro de estos desperdicios se pueden encontrar una cantidad significativa de artículos desechables, los cuales después de un corto tiempo de uso (al finalizar la comida o terminar una bebida) son desechados en la basura. Economía circular y análisis de ciclo de vida. Hablar de economía circular refiere al uso de sistemas donde se busca que la materia prima, productos, procesos y desechos, puedan ser Repensados, Rediseñados, Refabricados, Reparados, Redistribuidos, Reducidos, Reutilizados, Reciclados y Recuperados (conocidas como las “9 R’s”). Algunos beneficios que se obtienen al utilizar la economía circular son: la reducción del uso de materiales y energía nuevos, la reducción de desperdicios y emisiones, nuevas oportunidades de empleo, la reutilización de los recursos, entre otros. (Korhonen, Honkasalo, & Seppälä, 2018). El análisis de ciclo de vida (ACV) es un método que sirve para cuantificar la huella medioambiental de un producto, en el cual se toma en consideración el material, flujo de energía durante cada paso de su ciclo de vida. Este tipo de análisis produce un estimado del Editorial Universitat Politècnica de València 645 Propuesta metodológica para el desarrollo de productos desechables catalogados como sustentables en México consumo de energía, emisiones de gases de efecto invernadero (GEI), y métricos de otros impactos medioambientales (Snyder, 2016). Algunas ventajas de la aplicación de este método, según lo expuesto por María Bernatene (2019), son:      La identificación de procesos donde ocurren el mayor uso de materiales, energía y emisiones, buscando implementar mejoras. El análisis del proceso del producto desde “la cuna” hasta su desecho. El Lograr diferenciar entre una diseño sustentable y estrategias superficiales. Permitir ahorros, tanto de material, recursos económicos y abaratamiento de los costos. Es necesario si se desea implementar eco etiquetado y declaraciones ambientales. 2. metodologías existentes Para formular una metodología, es necesario partir de propuestas ya existentes, como la de Ulrich y Eppinger (2003) mostrada en la Fig. 2, en donde se describe un proceso lineal y se presentan las etapas clásicas del desarrollo de un producto. Esta es un punto de referencia, siendo crucial incluir aspectos ambientales donde se considere el ciclo de vida en su totalidad. Fig. 2 Proceso genérico de desarrollo de product Fuente: imagen obtenida del libro “Diseño y Desarrollo de Productos” (Ulrich & Eppinger, 2003) Un ejemplo de metodología desarrollada con la finalidad de facilitar herramientas para la mejora ambiental, es la propuesta por el IHOBE (2000), la cual consta de 7 etapas centradas en el ecodiseño. Esta ha sido aplicada principalmente en proyectos de mejora, donde se busca detectar los impactos ambientales y posteriormente desarrollar propuestas que busquen minimizarlos. Un ejemplo donde se han obtenido resultados exitosos es en la mesa de oficina GENIUS, desarrollada por Ofita, S.A.M.M., donde se logró una reducción del 52.32% del volumen de transporte, 4.5 kg por mesa en el consumo de acero y 5.6 kg por mesa en el uso de madera (IHOBE, 2000). Editorial Universitat Politècnica de València 646 Ing. Carlos E Vázquez Peña, Dr. David Cortés Sáenz Es importante resaltar que esta metodología no está centrada en la reutilización de los materiales y energías, siendo estos factores significativos para la propuesta que se busca realizar. Sin embargo, tiene relevancia ya que plantea un precedente en el uso del análisis del ciclo de vida del producto para la búsqueda de la reducción del impacto ambiental a lo largo de las distintas etapas del desarrollo de un producto. Otro ejemplo de metodología es la propuesta en el artículo “Criterios de sostenibilidad en metodologías de diseño” (Alvarado Nieto, Roa López, & Zuleta Ortiz, 2015) donde se expone la metodología ARZ, en la cual se detectaron 45 criterios enfocados a facilitar el diseño de productos sostentables. En la Fig. 3 se muestra los pasos correspondientes a la metodología ARZ. Fig. 3 Método de diseño ARZ para productos sostenibles Fuente: tomado de (Alvarado Nieto et al., 2015) Esta metodología se comparó con otras 7 por medio de una prueba piloto. Se les pidió a estudiantes de diseño industrial que crearan un objeto, donde cada uno de los grupos utilizó una metodología distinta. Las propuestas resultantes se mostraron a un grupo de posibles usuarios y clientes, siendo la desarrollada a base de la metodología ARZ la segunda favorita (Alvarado Nieto et al., 2015). Se resaltan factores relevantes de esta metodología, como la validación en el reúso y post consumo, siendo fundamentales dentro de la economía circular. El diseño para la sustentabilidad (D4S), incluye factores ambientales, sociales y económicos (Crul & Diehl, 2009). Esta metodología sostiene que para considerar un producto como sustentable, debe lograr innovar en las expectativas sociales, lograr una Editorial Universitat Politècnica de València 647 Propuesta metodológica para el desarrollo de productos desechables catalogados como sustentables en México distribución equitativa de las ganancias a lo largo de toda la cadena de valor y disminuir el impacto a los ecosistemas que se encuentran relacionados a lo largo de todo el ciclo de vida del producto. Algunos de los retos que se busca resolver a través del D4S, son: reducir la desigualdad en las ganancias, crear nuevas fuentes de empleo, uso de energías renovables, reducción y tratamiento de las emisiones industriales, valor agregado para compañías, proveedores y clientes (Stivale, 2020). El diseño para la economía circular tiene como objetivo principal la reutilización de los materiales y energía para la generación de productos, buscando mantener un ciclo cerrado (Medkova & Fifield, 2016). Para que esta metodología pueda ser implementada es necesario que, con cada producto, se desarrolle un nuevo modelo de negocio que asegure la circularidad de los materiales, convirtiéndose en una estrategia fundamental tanto para usuarios como para las compañías. 3. Conclusiones Partiendo de estas experiencias previas, la problemática actual en materia medioambiental y manejo de residuos a nivel nacional y local, la necesidad de pasar a un modelo circular de producción y consumo y tomando como punto de partida las herramientas en las distintas metodologías; se plantea en la Fig. 4 la propuesta de metodología enfocada al desarrollo de alternativas de productos desechables sustentables. Fig. 4 Metodología para el análisis de ciclo de vida de materiales desechables Fuente: imagen de elaboración propia Editorial Universitat Politècnica de València 648 Ing. Carlos E Vázquez Peña, Dr. David Cortés Sáenz En este artículo se expone la necesidad de contar con una metodología que permita analizar las emisiones y residuos actuales de los productos desechables utilizados en el sector alimenticio, con la finalidad de poder desarrollar alternativas a los ya existentes. La metodología planteada toma en cuenta las problemáticas generadas por las actuales formas de producción y consumo, la contaminación generada por las mismas y los objetivos planteados por la ONU, los cuales se convirtieron en referente para estrategias a nivel internacional, nacional y local. Se analizaron diversas metodologías centradas en el medio ambiente, que nos permiten detectar aspectos clave en el proceso de diseño como lo son el ecodiseño, la economía circular y el análisis de ciclo de vida. Esta propuesta metodológica forma parte de un proyecto en desarrollo, donde el ACV es fundamental para conocer los aspectos ambientales más relevantes, siendo estos los datos de entrada para la identificación de las necesidades y el desarrollo de las propuestas. Referencias Alvarado Nieto, G. A., Roa López, P. A., & Zuleta Ortiz, D. L. (2015). Criterios de sostenibilidad en metodologías de diseño. Iconofacto, 11(17), 112–132. https://doi.org/10.18566/iconofac.v11n17.a07 Bastidas Pacheco, G. A., & Hernández, R. (2019). Cambio climático algunos aspectos a considerar para la supervivencia del ser vivo: revisión sistemática de la literatura. Revista Cuidarte, 10(3), 2135–2144. https://doi.org/10.15649/cuidarte.v10i3.664 Bernatene, M. del R., & Canale, G. J. (2019). Innovación sustentable en Diseño a partir de la integración del análisis de Ciclo de Vida (ACV) con Cadenas Globales de Valor (CGV). Cuadernos Del Centro de Estudios de Diseño y Comunicación, 22(69), 151–174. https://doi.org/10.18682/cdc.vi69.1106 Crul, M., & Diehl, J. C. (2009). Design for Sustainability A Step-by-step Approach. In United Nations Environment Programme. Retrieved from http://wedocs.unep.org/bitstream/handle/20.500.11822/8742/DesignforSustainability.pdf?sequenc e=3&isAllowed=y García, A., & Cárcova, D. (2019). Del diseño industrial al design thinking . Perspectiva histórica de una disciplina en construcción. Centro de Estudios En Diseño y Comunicación, 94, 89–98. Gobierno Municipal de Ciudad Juárez, Gobierno del Estado de Chihuahua, El Colegio de la Frontera Norte, Universidad Autónoma de Ciudad Juárez, Plan Estrategíco de Juárez, & United Nations Development Programme. (2018). Juárez 2030 Plan para una Ciudad Sostenible. Retrieved from https://juarez2030.mx/wp-content/uploads/2018/11/juarez2030_291118.pdf IHOBE. (2000). Manual práctico de ecodiseño. 182. Retrieved Editorial Universitat Politècnica de València 649 from Propuesta metodológica para el desarrollo de productos desechables catalogados como sustentables en México http://www.valledelcauca.gov.co/agricultura/descargar.php?id=1756 INECC. (2018). Panorama de los Residuos en Mexico. In Instituto Nacional de Ecologia y Cambio Climatico. Ciudad de México. Korhonen, J., Honkasalo, A., & Seppälä, J. (2018). Circular Economy: The Concept and its Limitations. Ecological Economics, 143, 37–46. https://doi.org/10.1016/J.ECOLECON.2017.06.041 Medkova, K., & Fifield, B. (2016). Circular Design - Design for Circular Economy. Lahti Cleantech Annual Review 2016, (February), 32–47. SEMARNAT. (2016). Informe de la Situación del Medio Ambiente en Mexico. Compendio de Estadísticas Ambientales. Indicadores Clave y de Desempeño Ambiental. In Secretaría de Medio Ambiente y Recursos Naturales (2015th ed.). Retrieved from http://www.semarnat.gob.mx Snyder, S. W. (2016). An introduction to commercializing biobased products: Opportunities, challenges, benefits, and risks. In RSC Green Chemistry (Vol. 2016-Janua). https://doi.org/10.1039/9781782622444-00001 Stivale, S. (2020). Los caminos del Diseño Sustentable y sus vinculaciones con la investigación en diseño. Cuadernos Del Centro de Estudios de Diseño y Comunicación, 21(80), 77–90. Retrieved from http://search.ebscohost.com/login.aspx?direct=true&db=asu&AN=129882213&lang=es Ulrich, K. T., & Eppinger, S. D. (2003). Diseño y desarrollo de productos. In Diseño y desarrollo de productos. https://doi.org/10.1017/CBO9781107415324.004 United Nations Development Programme. (2015). Objetivos de Desarrollo Sostenible. Retrieved June 12, 2020, from https://www.undp.org/content/undp/es/home/sustainable-development-goals.html Editorial Universitat Politècnica de València 650 

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https://www.researchsquare.com/article/rs-56069/v1.pdf

English

miR-548j-5p regulates angiogenesis in peripheral artery disease

Scientific reports

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Original investigation Keywords: microRNAs, angiogenesis, peripheral artery disease Posted Date: September 9th, 2020 DOI: https://doi.org/10.21203/rs.3.rs-56069/v1 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 Scientific Reports on January 17th, 2022. See the published version at https://doi.org/10.1038/s41598-022-04770-6. Page 1/18 Page 1/18 Abstract Background: Peripheral artery disease (PAD) is a vascular disease involving diffuse atherosclerosis, which is associated with increased cardiovascular mortality and morbidity. Critical limb ischemia is the most severe complication of PAD. In addition to medical and interventional treatment, therapeutic angiogenesis is a novel therapy for PAD. Circulating microRNAs (miRNAs) are considered to be key regulators of gene expression, but their role in ischemic-induced angiogenesis is poorly characterized. There is little knowledge of specific miRNAs associated with PAD. Methods: To determine the regulation of miRNAs, we obtained miRNA profiles using RNA isolated from patients with PAD and a control group. The effect of the specific miRNA in angiogenesis were evaluated by in vitro angiogenic function of endothelial progenitor cells (EPCs), in vivo angiogenesis assay and a hind-limb ischemic model. Results: Circulating miR-548j-5p was significantly reduced in patients with PAD compared with the controls. miR-548j-5p promoted EPC angiogenesis by enhancing migration and tube formation. The endothelial nitric oxide synthase and stromal cell-derived factor (SDF)-1 signaling pathways appeared to be potential targets for miR-548j-5p. Furthermore, a directed in vivo angiogenesis assay of EPCs and a hind-limb ischemia mouse model demonstrated that miR-548j-5p enhanced capillary density and blood flow recovery in hind-limb ischemia. Conclusion: Taken together, our data indicates that up-regulation of miR-548j-5p promotes angiogenesis in ischemic tissue and might be a novel therapeutic approach for PAD. MiRNA library construction and next-generation sequencing RNA was isolated according to the manufacturer’s protocol. Total RNA from samples was further constructed into libraries using an Illumina TruSeq Small RNA Library Prep kit (Cat. RS-200-0012; Illumina, San Diego, CA, USA). In brief, 1 μg of high-quality total RNA from each sample was ligated with adapters using T4 RNA ligase. The adapter-ligated RNA samples were reverse-transcribed to cDNA, amplified by primers containing a specific sequence index, and then size-validated using an Agilent 2100 bioanalyzer (Cat. G2943, Agilent, Santa Clara, CA, USA) loaded with a DNA 1000 kit (Agilent, cat. 5067- 1504). The size-checked libraries were loaded onto Novex TBE gels (Cat. EC6265BOX, Thermo Fisher Scientific, Inc., Waltham, MA, USA), and then size-selected and gel-eluted to obtain proper fragments. The eluted libraries were qualified using an Agilent 2100 bioanalyzer loaded with a DNA 1000 kit, and quantified using Qubit (Thermo Fisher Scientific, Inc., Cat. Q33216) and real-time PCR (Q-PCR). Each library was diluted in equal concentrations and the same volumes were taken for pooling. Pooled libraries were sequenced for 10 M reads/samples with a high-throughput, 50-bp single-end sequencing reagent on an Illumina MiSeq sequencing system. Background Peripheral artery disease (PAD) is an atherosclerotic disease, which increases the risk of cardiovascular mortality and morbidity [1]. The prevalence of PAD is greater than 20% in individuals over 60 years of age and incidence rates have been predicted to increase with aging populations [2]. Critical limb ischemia (CLI) is the most severe clinical presentation of PAD, and causes intermittent claudication, ulceration and gangrene of the foot; it is associated with amputation and cardiovascular death [3]. The pathophysiology of PAD is a complex process involving endothelial dysfunction, oxidative stress, platelet activation and inflammation [4]. Despite pharmacological control of risk factors, the clinical outcomes for patients with PAD are poor. Thus, the development of new therapeutic targets for patients with PAD is an important issue. Recently, microRNAs (miRNAs) have emerged as novel regulators of vascular biology. miRNAs are small, non-coding RNAs that regulate gene expression at the post-transcriptional level to repress expression [5]. miRNAs play key roles in inflammation, angiogenesis, endothelial function and restenosis associate with PAD [6]. Previous studies have shown that specific miRNA expression profiles in patients with PAD may serve as prognostic predictors [7]. In addition, some miRNAs, such as miR-93 [8] and miR let-7g [9], have Page 2/18 Page 2/18 been shown to mediate angiogenesis through various molecular pathways. However, the association between miRNAs and PAD has not yet been fully characterized. been shown to mediate angiogenesis through various molecular pathways. However, the association between miRNAs and PAD has not yet been fully characterized. The present study aimed to explore specific miRNAs relevant to PAD and investigate their angiogenic effects and mechanisms on endothelial progenitor cell (EPC) migration, tube formation and angiogenesis in PAD, in the hope of providing a novel therapeutic strategy for patients with PAD. Study population A total of 40 subjects were included in the current study, including 25 patients with PAD who were diagnosed via the ankle-brachial index or angiography. Patients with acute coronary syndrome, acute ischemic limb or malignancy were excluded. A total of 15 healthy individuals without any evidence of PAD served as the control group. Blood sampling was performed and samples were subjected to RNA isolation (FocusGenomics Biotech Co., Ltd, Taiwan). Following miRNA array analysis, we selected specific miRNAs for comparison between the patients with PAD and the control group. This study was approved by the Taipei Veterans General Hospital’s Institutional Review Board for Research (approval no. 2013-08-020B#3), and written informed consent was provided by all the participants prior to their inclusion within the study. Directed in vivo angiogenesis assay (DIVAA) Angiogenesis in vivo was evaluated using a DIVAA kit (Trevigen, Gaithersburg, MD, USA). Briefly, angioreactors were filled with EPCs transfected with 50,000 miR-548j-5p mimic, negative control, miR- 548j-5p antagomir or scramble control and VEGF/FGF1 embedded in 20 μl basement membrane extract [11]. Angioreactors were incubated at 37°C for 1 h. For positive controls, angioreactors were filled with BME supplemented with VEGF (12.5 ng/ml) plus FGF1 (37.5 ng/ml). Two angioreactors were implanted in each immunocompromised nude mouse (eight- week-old male nude mice) subcutaneously in the dorsal region. The angioreactors were removed 14 days after implantation and photographed. The presence of blood vessels was assessed using FITC-Lectin detection, and fluorescence was determined using a plate reader in mean relative fluorescence units. Western blot analysis EPCs were lysed in a lysis buffer (62.5 mM Tris-HCl, 2% sodium dodecyl sulfate, 10% glycerol, 0.5 mM phenylmethanesulfonyl fluoride, 2 μg/mL aprotinin, pepstatin, and leupeptin). Proteins in the cell lysates were separated using sodium dodecyl sulfate-polyacrylamide (10%) gel electrophoresis, followed by transfer onto poly (vinylidene fluoride) membranes. The membranes were then probed with monoclonal antibodies against phosphorylated eNOS (Upstate Biotechnology, Lake Placid, NY, USA), SDF-1 and actin. Protein band densitometry was performed using ImageQuant software (Promega, Madison, WI, USA). Scratch injury model and EPCs Human EPC isolation, cultivation and characterization were performed as previously described [10]. EPC migration was evaluated using a scratch injury model. EPCs were transfected with miR-548j-5p mimic or Page 3/18 antagomir, or a control. After serum-starvation of EPCs overnight, a scratch injury was applied with a scalpel, and EPC sprouting was examined before and 12/24 h after scratching. Tube formation assay An EPC tube formation assay was performed using an in vitro angiogenesis assay kit (Chemicon, Temecula, CA, USA). EPCs transfected with miR-548j-5p mimic or antagomir were placed onto a matrix with medium for 16 h. Tubule formation was inspected by inverted light microscopy. Six random fields were used to calculate the average number of complete tubes formed by cells using Image-Pro Plus software (Media Cybernetics, Rockville, MD, USA). Ischemic hind-limb model and EPC transplantation Eight-week-old male nude mice were purchased from the BioLASCO Taiwan Co., Ltd. After 2 weeks observation, unilateral hind-limb ischemia was induced by left femoral artery ligation. EPCs transfected with miR-548j-5p mimic (n=6) or antagomir (n=6) or a control (n=6) were injected intramuscularly at six different sites on the ischemic limb distal to the arterial occlusion site. The blood flow of the hind limb was measured using a Laser Doppler perfusion imaging system (Moor Instruments Limited, Devon, UK) before and after surgery, and then weekly. The results were expressed as the ratio of perfusion in the ischemic vs. the non-ischemic limb. Page 4/18 Page 4/18 The mice were sacrificed 3 weeks after surgery and the limbs were fixed overnight in methanol. The ischemic muscles were embedded in paraffin, and then deparaffinized to incubate them with rat monoclonal antibodies against murine CD31 (BD PharMingen, San Diego, CA, USA). New capillaries were identified based on morphology and positive staining for CD31 using the avidin-biotin-complex technique and Vector Red Chromogenic substrate (Vector Laboratories, Burlingame, CA, USA) after counterstaining with hematoxylin. The visible capillaries were counted under 10 random fields, and the capillary density was expressed as the number of capillaries/mm. All animals were housed and handled in accordance with the criteria outlined in the National Institutes of Health ‘‘Guide for Care and Use of Laboratory Animals”. The study protocol was approved by the Institutional Animal Care and Use Committee of Taipei Veterans General Hospital, Taipei, Taiwan (approval number: IACUC_2013–076). Statistical analysis Data are expressed as the mean ± standard error of the mean (SEM). Statistical analysis was performed using the unpaired Student’s t-test or one-way analysis of variance, followed by Scheffe’s multiple comparison post hoc test using Statistical Package of the Social Sciences software (version 14; SPSS, Inc., Chicago, IL, USA). P<0.05 was considered to indicate a statistically significant difference. Detection of circulating miRNAs The baseline clinical characteristics of the PAD group and control group are shown in Table 1. In total, 40 subjects were studied; 25 patients with documented PAD and 15 individuals without evidence of PAD (control group) from Taipei Veterans General Hospital, Taipei, Taiwan, R.O.C. The mean age of participants was 74 years. A total of 17 patients were male, and the median body mass index was 23.19±2.82. There were 19 patients with hypertension and 15 with diabetes mellitus. Coronary artery disease and chronic kidney disease were present in 5 and 10 patients, respectively. Table 1. Baseline characteristics of the study population. Table 1. Baseline characteristics of the study population. Page 5/18 Page 5/18 PAD group (n = 25) Control group (n = 15) Demographics     Age (years) 74 44 Male sex 17 (8) 13 (2) BMI 23.19 ± 2.82 25.5 ± 0.64 Risk factors     Hypercholesterolemia 3 (22) 0 Hypertension 19 (6) 1 (14) Diabetic mellitus 15 (10) 1 (14) Coronary artery disease 5 (20) 0 Chronic kidney disease 10 (15) 0 PAD group (n = 25) Control group (n = 15) PAD group (n = 25) Control group (n = 15) The concentration of several circulating miRNAs was measured. The PAD group had a significantly decreased expression of miR-548j-5p (AAAAGUAAUUGCGGUCUUUGGU) compared with the control group (Fig. 1). The concentration of several circulating miRNAs was measured. The PAD group had a significantly decreased expression of miR-548j-5p (AAAAGUAAUUGCGGUCUUUGGU) compared with the control group (Fig. 1). miR-548j-5p promotes the angiogenic potential of EPCs according to DIVAA The angiogenic effects of miR-548j-5p were further analyzed by DIVVA, as shown in Fig. 4. Transfection of miR-548j-5p mimics into EPCs significantly increased the invasion of vessels in the angioreactors compared with the negative control. However, transfection of miR-548j-5p antagomir into EPCs decreased the invasion of vessels in the angioreactors compared with the scramble control. These findings indicate that miR-548j-5p could promote the angiogenic capacity of EPCs. miR-548j-5p enhances migration and tube formation in EPCs Mobilization and tube formation in EPCs in the ischemic area are important for neovascularization. Compared with the control group, transfection of miR-548j-5p mimics into EPCs significantly increased their migration, as shown in Fig. 2; however, transfection of miR-548j-5p antagomir into EPCs decreased their migration. The effect of miR-548j-5p on tube formation in EPCs was also investigated, as shown in Fig. 3. Transfection of miR-548j-5p mimics into EPCs significantly increased their tube formation compare The effect of miR-548j-5p on tube formation in EPCs was also investigated, as shown in Fig. 3. The effect of miR-548j-5p on tube formation in EPCs was also investigated, as shown in Fig. 3. Transfection of miR-548j-5p mimics into EPCs significantly increased their tube formation compared with the control group; thus, miR-548j-5p could modulate the angiogenic activities of EPCs. Transfection of miR-548j-5p mimics into EPCs significantly increased their tube formation compared with the control group; thus, miR-548j-5p could modulate the angiogenic activities of EPCs. miR-548j-5p promotes blood flow recovery in hind-limb ischemia in a mouse model To evaluate the angiogenic effect of miR-548j-5p, a hind-limb ischemia model was developed. Compared with the control group, the mice with miR-548j-5p mimic transfection of EPCs exhibited significantly enhanced flow recovery. However, the mice with miR-548j-5p antagomir transfection showed delayed blood flow recovery after surgery (Fig. 6A and B). Anti-CD31 immunostaining showed a decreased capillary density in mice with miR-548j-5p antagomir transfection of EPCs compared with the control mice, but the miR-548j-5p mimic mice exhibited significantly increased capillary density in muscles (Fig. 6C). Therefore, miR-548j-5p could enhance the angiogenic activities of EPCs. miR-548j-5p regulates EPC function via targeting of eNOS and SDF-1 miR-548j-5p regulates EPC function via targeting of eNOS and SDF-1 Page 6/18 Page 6/18 EPCs with miR-548j-5p mimic transfection exhibited significantly upregulated eNOS expression, as shown in Fig. 5. In addition, miR-548j-5p mimic transfection of EPCs upregulated SDF-1 expression; however, miR-548j-5p antagomir transfection of EPCs downregulated eNOS and SDF-1 expression. These results suggest that miR-548j-5p could regulate EPC function via the eNOS and SDF-1 signaling pathways. EPCs with miR-548j-5p mimic transfection exhibited significantly upregulated eNOS expression, as shown in Fig. 5. In addition, miR-548j-5p mimic transfection of EPCs upregulated SDF-1 expression; however, miR-548j-5p antagomir transfection of EPCs downregulated eNOS and SDF-1 expression. These results suggest that miR-548j-5p could regulate EPC function via the eNOS and SDF-1 signaling pathways. Conclusion The present study demonstrated that miR-548j-5p contributes to angiogenesis by promoting migration and tube formation in EPCs, which are associated with eNOS and SDF-1 expression. Up-regulation of miR-548j-5p improved the neovascularization in hind-limb ischemic mice. Thus, miR-548j-5p may have therapeutic potential for PAD. Further studies are needed to evaluate the therapeutic effects and to examine the clinical implications of these findings. Discussion These results revealed that miR-548j-5p enhanced migration and tube formation in EPCs by targeting the eNOS signaling pathway. There are some limitations in our studies such as in vitro and in vivo experiments not conducted with control miRNAs. We also need more cases to confirm the importance of miR-548j-5p in the future. Discussion To the best of our knowledge, this is the first study to report down-regulation of miR-548j-5p in PAD. Transfection of EPCs with miR-548j-5p could enhance the activity of eNOS and SDF-1 and improve EPC functions, such as migration and tube formation. Whereas, miR-548j-5p antagomir transfection impaired the angiogenic activities of EPCs. Transfection of EPCs with miR-548j-5p also improved blood flow recovery in a hind-limb ischemic mouse model. These results suggest that miR-548j-5p could induce angiogenesis. Recently, circulating miRNAs have served as novel biomarkers for cardiovascular disease [12]. In patients with PAD, individual miRNAs have been found to be differentially-expressed in circulation. Bogucka-Kocka et al. [13] used dysregulation of 26 miRNAs to propose novel biomarkers for lower-extremity arterial disease. In patients with CLI, higher levels of circulating miR15a and miR16 predicted a poorer prognosis compared with diabetic individuals [14]. Hazarika et al. [15] found that miR-93 was differentially- expressed in the ischemic hind-limb muscle of mice. Overexpression of miR-93 enhances cell proliferation and endothelial cell tube formation, which suggests that miR-93 modulated ischemic-induced angiogenesis independent of HIF-1α regulated angiogenic genes [15]. miR-146a was found to be a regulator of vascular remodeling. Heuslein et al. [16] found that suppression of miR-146a enhanced arteriogenesis in muscular collateral circulation via upregulation of pro- inflammatory endothelial activation. miR29a inhibited the expression of endothelial cell metalloproteinase domain-containing protein 12 in ischemia in hyperglycemia. Modulation of miR29a improved impaired post-ischemic angiogenesis in diabetes [17]. In the present study, miR-548j-5p was observed to facilitate migration and tube formation in EPCs, indicating that miR-548j-5p may contribute to angiogenesis in PAD by regulating the angiogenic activities of EPCs. Page 7/18 Nitric oxide (NO) is a key role for angiogenesis in ischemic disease and eNOS synthetization of NO could promote migration of EPCs [18,19]. SDF-1 is a chemokine that has a strong chemotactic effect on EPCs. After vascular injury, SDF-1 induces migration of EPCs to the injured area and promotes endothelialization [20,21]. Under hyperglycemic conditions miR-133a was found to be up-regulated and induced endothelial dysfunction through inhibition of eNOS [22]. Chen et al. [23] reported that the diabetes-induced expression of miR-133a, impaired angiogenesis in PAD via a reduction in NO synthesis. miR-133a antagonism improved post-ischemic angiogenesis. In the current study, we found that up- regulation of miR-548j-5p induced the expression of eNOS and SDF-1 in EPCs. Abbreviations CLI, critical limb ischemia CLI, critical limb ischemia eNOS, endothelial nitric oxide synthase EPC, endothelial progenitor cell FGF1, fibroblast growth factor-1 miRNA, microRNA NO, nitric oxide PAD, peripheral artery disease SDF-1, stromal cell-derived factor-1 SEM, standard error of the mean VEGF, vascular endothelial growth factor Consent for publication Not applicable. Acknowledgements This work was assisted in part by the Division of Experimental Surgery of the Department of Surgery, Taipei Veterans General Hospital. Authors' contributions CYL and TCW contributed to the study conception and design, literature review and preparation of the manuscript. SHL and TCW contributed to the study conception and design, drafted the manuscript, revised it critically for important intellectual content, and gave final approval. All authors have read and agreed to the published version of the manuscript. Declarations Page 8/18 Page 8/18 Availability of data and material All data generated or analyzed during this study are included in this published article [and its supplementary information files]. Conflicts of interest The authors declare no conflicts of interest. Funding This study was partially supported by grants V105C-108 and V107C-172 (to Tao-Cheng Wu) from Taipei Veterans General Hospital, Taipei, Taiwan, R.O.C. Ethical approval This study was approved by the Taipei Veterans General Hospital’s Institutional Review Board for Research (approval number 2013-08-020B#3), and the animal study protocol was approved by the Institutional Animal Care and Use Committee of Taipei Veterans General Hospital, Taipei, Taiwan (approval number: IACUC_2013–076) Consent to participate Written informed consent was provided by all the participants prior to their inclusion within the study. Consent for publication Written informed consent was provided by all the participants prior to their inclusion within the study. References 1. Criqui MH, Aboyans V. Epidemiology of peripheral artery disease. Circ Res. 2015; doi:10.1161/CIRCRESAHA.116.303849. 2. Stock JK. The challenge of peripheral arterial disease: How do we improve outcome? Atherosclerosis. 2018; doi:10.1016/j.atherosclerosis.2017.12.031. 3. Abu Dabrh AM, Steffen MW, Undavalli C, Asi N, Wang Z, Elamin MB, et al. The natural history of untreated severe or critical limb ischemia. J Vasc Surg. 2015; doi:10.1016/j.jvs.2015.07.065. 4. Morisaki K, Yamaoka T, Iwasa K. Risk factors for wound complications and 30-day mortality after major lower limb amputations in patients with peripheral arterial disease. Vascular. 2018; doi:10.1177/1708538117714197. 5. Quiat D, Olson EN. MicroRNAs in cardiovascular disease: from pathogenesis to prevention and treatment. J Clin Invest. 2013; doi:10.1172/JCI62876. 5. Quiat D, Olson EN. 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Mol Med Rep. 2019; doi:10.3892/mmr.2019.10196. 8. Shu X, Mao Y, Li Z, Wang W, Chang Y, Liu S, et al. MicroRNA93 regulates angiogenesis in peripheral arterial disease by targeting CDKN1A. Mol Med Rep. 2019; doi:10.3892/mmr.2019.10196. 9. Hsu PY, Hsi E, Wang TM, Lin RT, Liao YC, Juo SH. MicroRNA let-7g possesses a therapeutic potential for peripheral artery disease. J Cell Mol Med. 2017; doi:10.1111/jcmm.12997. 9. Hsu PY, Hsi E, Wang TM, Lin RT, Liao YC, Juo SH. MicroRNA let-7g possesses a therapeutic potential for peripheral artery disease. J Cell Mol Med. 2017; doi:10.1111/jcmm.12997. 10. Wu TC, Chan JS, Lee CY, Leu HB, Huang PH, Chen JS, et al. Rivaroxaban, a factor Xa inhibitor, improves neovascularization in the ischemic hindlimb of streptozotocin-induced diabetic mice. Cardiovasc Diabetol. 2015; doi:10.1186/s12933-015-0243-y. 10. Wu TC, Chan JS, Lee CY, Leu HB, Huang PH, Chen JS, et al. Page 9/18 Page 9/18 References MicroRNA-381 Favors Repair of Nerve Injury Through Regulation of the SDF-1/CXCR4 Signaling Pathway via LRRC4 in Acute Cerebral Ischemia after Cerebral Lymphatic Blockage. Cell Physiol Biochem. 2018; doi:10.1159/000488821. 20. Piao JM, Wu W, Yang ZX, Li YZ, Luo Q, Yu JL. MicroRNA-381 Favors Repair of Nerve Injury Through Regulation of the SDF-1/CXCR4 Signaling Pathway via LRRC4 in Acute Cerebral Ischemia after Cerebral Lymphatic Blockage. Cell Physiol Biochem. 2018; doi:10.1159/000488821. 21. Yu G, Liu P, Shi Y, Li S, Liu Y, Zhu W. Sitagliptin Stimulates Endothelial Progenitor Cells to Induce Endothelialization in Aneurysm Necks Through the SDF-1/CXCR4/NRF2 Signaling Pathway. Front Endocrinol (Lausanne). 2019; doi:10.3389/fendo.2019.00823. 21. Yu G, Liu P, Shi Y, Li S, Liu Y, Zhu W. Sitagliptin Stimulates Endothelial Progenitor Cells to Induce Endothelialization in Aneurysm Necks Through the SDF-1/CXCR4/NRF2 Signaling Pathway. Front Endocrinol (Lausanne). 2019; doi:10.3389/fendo.2019.00823. 22. Li P, Yin YL, Guo T, Sun XY, Ma H, Zhu ML, et al. Inhibition of Aberrant MicroRNA-133a Expression in Endothelial Cells by Statin Prevents Endothelial Dysfunction by Targeting GTP Cyclohydrolase 1 in Vivo. Circulation. 2016; doi:10.1161/CIRCULATIONAHA.116.017949. 22. Li P, Yin YL, Guo T, Sun XY, Ma H, Zhu ML, et al. Inhibition of Aberrant MicroRNA-133a Expression in Endothelial Cells by Statin Prevents Endothelial Dysfunction by Targeting GTP Cyclohydrolase 1 in Vivo. Circulation. 2016; doi:10.1161/CIRCULATIONAHA.116.017949. 23. Chen L, Liu C, Sun D, Wang T, Zhao L, Chen W, et al. MicroRNA-133a impairs perfusion recovery after hindlimb ischemia in diabetic mice. Biosci Rep. 2018; doi:10.1042/BSR20180346. 23. Chen L, Liu C, Sun D, Wang T, Zhao L, Chen W, et al. MicroRNA-133a impairs perfusion recovery after hindlimb ischemia in diabetic mice. Biosci Rep. 2018; doi:10.1042/BSR20180346. References Rivaroxaban, a factor Xa inhibitor, improves neovascularization in the ischemic hindlimb of streptozotocin-induced diabetic mice. Cardiovasc Diabetol. 2015; doi:10.1186/s12933-015-0243-y. 11. Flores-Perez A, Marchat LA, Rodriguez-Cuevas S, Bautista-Pina V, Hidalgo-Miranda A, Ocampo EA, et al. Dual targeting of ANGPT1 and TGFBR2 genes by miR-204 controls angiogenesis in breast cancer. Sci Rep. 2016; doi:10.1038/srep34504. 11. Flores-Perez A, Marchat LA, Rodriguez-Cuevas S, Bautista-Pina V, Hidalgo-Miranda A, Ocampo EA, et al. Dual targeting of ANGPT1 and TGFBR2 genes by miR-204 controls angiogenesis in breast cancer. Sci Rep. 2016; doi:10.1038/srep34504. 12. Fichtlscherer S, Zeiher AM, Dimmeler S. Circulating microRNAs: biomarkers or mediators of cardiovascular diseases? Arterioscler Thromb Vasc Biol. 2011; doi:10.1161/ATVBAHA.111.226696. 12. Fichtlscherer S, Zeiher AM, Dimmeler S. Circulating microRNAs: biomarkers or mediators of cardiovascular diseases? Arterioscler Thromb Vasc Biol. 2011; doi:10.1161/ATVBAHA.111.226696. 13. Bogucka-Kocka A, Zalewski DP, Ruszel KP, Stepniewski A, Galkowski D, Bogucki J, et al. Dysregulation of MicroRNA Regulatory Network in Lower Extremities Arterial Disease. Front Genet. 2019; doi:10.3389/fgene.2019.01200. 13. Bogucka-Kocka A, Zalewski DP, Ruszel KP, Stepniewski A, Galkowski D, Bogucki J, et al. Dysregulation of MicroRNA Regulatory Network in Lower Extremities Arterial Disease. Front Genet. 2019; doi:10.3389/fgene.2019.01200. 14. Spinetti G, Fortunato O, Caporali A, Shantikumar S, Marchetti M, Meloni M, et al. MicroRNA-15a and microRNA-16 impair human circulating proangiogenic cell functions and are increased in the proangiogenic cells and serum of patients with critical limb ischemia. Circ Res. 2013; doi:10.1161/CIRCRESAHA.111.300418. Page 10/18 15. Hazarika S, Farber CR, Dokun AO, Pitsillides AN, Wang T, Lye RJ, et al. MicroRNA-93 controls perfusion recovery after hindlimb ischemia by modulating expression of multiple genes in the cell cycle pathway. Circulation. 2013; doi:10.1161/CIRCULATIONAHA.112.000860. 16. Heuslein JL, McDonnell SP, Song J, Annex BH, Price RJ. MicroRNA-146a Regulates Perfusion Recovery in Response to Arterial Occlusion via Arteriogenesis. Front Bioeng Biotechnol. 2018; doi:10.3389/fbioe.2018.00001. 17. Chen L, Okeke E, Ayalew D, Wang D, Shahid L, Dokun AO. Modulation of miR29a improves impaired post-ischemic angiogenesis in hyperglycemia. Exp Biol Med (Maywood). 2017; doi:10.1177/1535370217716424. 18. Aicher A, Heeschen C, Mildner-Rihm C, Urbich C, Ihling C, Technau-Ihling K, et al. Essential role of endothelial nitric oxide synthase for mobilization of stem and progenitor cells. Nat Med. 2003; doi:10.1038/nm948. 19. Yang CY, Chen C, Lin CY, Chen YH, Lin CY, Chi CW, et al. Garcimultiflorone K inhibits angiogenesis through Akt/eNOS- and mTOR-dependent pathways in human endothelial progenitor cells. Phytomedicine. 2019; doi:10.1016/j.phymed.2019.152911. 20. Piao JM, Wu W, Yang ZX, Li YZ, Luo Q, Yu JL. Figures Page 11/18 Figure 1 Expression of microRNAs in patients with PAD compared with the control group. Candidate miRNAs in the PAD and the control group were analyzed. miR-548j-5p was significantly decreased in patients with PAD group compared to controls. (*P<0.05 vs. the control group). Figure 1 Expression of microRNAs in patients with PAD compared with the control group. Candidate miRNAs in the PAD and the control group were analyzed. miR-548j-5p was significantly decreased in patients with PAD group compared to controls. (*P<0.05 vs. the control group). Expression of microRNAs in patients with PAD compared with the control group. Candidate miRNAs in the PAD and the control group were analyzed. miR-548j-5p was significantly decreased in patients with PAD group compared to controls. (*P<0.05 vs. the control group). Page 12/18 Figure 2 Effect of miR-548j-5p on human EPC tube formation performed using ECMatrix gel. (A) Human EPCs tran impaired tube formation compared with the negative miR-548j-5p mimic exhibited enhanced tube formatio are presented as the mean ± SEM. (*P<0.05 vs. the c gure 2 Figure 2 Effect of miR-548j-5p on human EPC tube formation. An in vitro angiogenesis assay for EPCs was performed using ECMatrix gel. (A) Human EPCs transfected with miR-548j-5p antagomir exhibited impaired tube formation compared with the negative control in vitro. (B) Human EPCs transfected with miR-548j-5p mimic exhibited enhanced tube formation compared with the negative control in vitro. Data are presented as the mean ± SEM. (*P<0.05 vs. the control, n=6 for each experiment). Page 13/18 Figure 3 Effect of miR-548j-5p on human EPC migration. The migratory function of EPCs was evaluate scratch injury model. (A) Human EPCs transfected with miR-548j-5p antagomir exhibited supp Figure 3 Effect of miR-548j-5p on human EPC migration. The migratory function of EPCs was evaluated using a scratch injury model. (A) Human EPCs transfected with miR-548j-5p antagomir exhibited suppressed EPC mobilization compared with the negative control in vitro. (B) Human EPCs transfected with miR-548j-5p mimic exhibited enhanced EPC mobilization compared with the negative control in vitro. Data are presented as the mean ± SEM. (*P<0.05 vs. the control, n=6 for each experiment). Figure 3 Figure 3 Effect of miR-548j-5p on human EPC migration. The migratory function of EPCs was evaluated using a scratch injury model. (A) Human EPCs transfected with miR-548j-5p antagomir exhibited suppressed EPC mobilization compared with the negative control in vitro. (B) Human EPCs transfected with miR-548j-5p mimic exhibited enhanced EPC mobilization compared with the negative control in vitro. Data are presented as the mean ± SEM. (*P<0.05 vs. the control, n=6 for each experiment). Page 14/18 Page 14/18 Figure 4 Effect of miR-548j-5p on blood vessel formation in vivo, assessed using a directed in vivo angiogenesis assay. Representative images of angioreactors extirpated from nude mice 9 days after implantation. miR- 548j-5p enhanced blood vessel formation. Data are presented as the mean ± SEM. (*P<0.05 vs. the control, n=6 for each experiment). Figure 4 Effect of miR-548j-5p on blood vessel formation in vivo, assessed using a directed in vivo angiogenesis assay. Representative images of angioreactors extirpated from nude mice 9 days after implantation. miR- 548j-5p enhanced blood vessel formation. Data are presented as the mean ± SEM. (*P<0.05 vs. the control, n=6 for each experiment). Page 15/18 Page 15/18 Figure 5 Figure 5 Effect of miR-548j-5p on eNOS and SDF-1 production in EPCs. (A) Impaired expression of eNOS and SDF- 1 in EPCs was observed in EPCs transfected with miR-548j-5p antagomir compared with the scramble control. (B) Enhanced expression of eNOS and SDF-1 was observed in EPCs transfected with miR-548j-5p mimic compared with the negative control. Data are presented as the mean ± SEM. (*P<0.05 vs. the control, n=6 for each experiment). Figure 5 Effect of miR-548j-5p on eNOS and SDF-1 production in EPCs. (A) Impaired expression of eNOS and SDF- 1 in EPCs was observed in EPCs transfected with miR-548j-5p antagomir compared with the scramble control. (B) Enhanced expression of eNOS and SDF-1 was observed in EPCs transfected with miR-548j-5p mimic compared with the negative control. Data are presented as the mean ± SEM. (*P<0.05 vs. the control, n=6 for each experiment). Page 16/18 Page 16/18 Figure 6 Figure 6 Effect of miR-548j-5p on hind-limb perfusion. (A) Serial Doppler analysis of hind-limb perfusion before and 3 weeks after hind-limb ischemia surgery in nude mice that received transplants of EPCs transfected with miR-548j-5p mimic, miR-548j-5p antagomir, negative control or scramble control. The color scale illustrates blood flow variation from minimal (dark blue) to maximal (red) values. Arrows indicate the ischemic limb after hind-limb ischemia surgery. (B) Quantification analysis of perfusion recovery using Page 17/18 Page 17/18 the laser Doppler perfusion imaging ratio (ischemia/normal hind-limb) in the different groups. (C) Mice were sacrificed 3 weeks after surgery and capillaries in the ischemic muscles were visualized using anti- CD31 immunostaining. Data are presented as the mean ± SEM. (*P<0.05 vs. 0 weeks, #P<0.05 vs. miR- 548j-5p antagomir, n=6 for each experiment). the laser Doppler perfusion imaging ratio (ischemia/normal hind-limb) in the different groups. (C) Mice were sacrificed 3 weeks after surgery and capillaries in the ischemic muscles were visualized using anti- CD31 immunostaining. Data are presented as the mean ± SEM. (*P<0.05 vs. 0 weeks, #P<0.05 vs. miR- 548j-5p antagomir, n=6 for each experiment). the laser Doppler perfusion imaging ratio (ischemia/normal hind-limb) in the different groups. (C) Mice were sacrificed 3 weeks after surgery and capillaries in the ischemic muscles were visualized using anti- CD31 immunostaining. Data are presented as the mean ± SEM. (*P<0.05 vs. 0 weeks, #P<0.05 vs. miR- 548j-5p antagomir, n=6 for each experiment). Page 18/18 Page 18/18

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https://www.frontiersin.org/articles/10.3389/fcell.2017.00014/pdf

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Sonic Hedgehog Signaling in Limb Development

Frontiers in cell and developmental biology

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REVIEW published: 28 February 2017 doi: 10.3389/fcell.2017.00014 Sonic Hedgehog Signaling in Limb Development Cheryll Tickle 1* and Matthew Towers 2* 1 Department of Biology and Biochemistry, University of Bath, Bath, UK, 2 Department of Biomedical Science, The Bateson Centre, University of Sheffield, Western Bank, Sheffield, UK Edited by: Andrea Erika Münsterberg, University of East Anglia, UK Reviewed by: Megan Davey, University of Edinburgh, UK Robert Hill, University of Edinburgh, UK Susan Mackem, National Cancer Institute, USA *Correspondence: Cheryll Tickle [email protected] Matthew Towers [email protected] The gene encoding the secreted protein Sonic hedgehog (Shh) is expressed in the polarizing region (or zone of polarizing activity), a small group of mesenchyme cells at the posterior margin of the vertebrate limb bud. Detailed analyses have revealed that Shh has the properties of the long sought after polarizing region morphogen that specifies positional values across the antero-posterior axis (e.g., thumb to little finger axis) of the limb. Shh has also been shown to control the width of the limb bud by stimulating mesenchyme cell proliferation and by regulating the antero-posterior length of the apical ectodermal ridge, the signaling region required for limb bud outgrowth and the laying down of structures along the proximo-distal axis (e.g., shoulder to digits axis) of the limb. It has been shown that Shh signaling can specify antero-posterior positional values in limb buds in both a concentration- (paracrine) and time-dependent (autocrine) fashion. Currently there are several models for how Shh specifies positional values over time in the limb buds of chick and mouse embryos and how this is integrated with growth. Extensive work has elucidated downstream transcriptional targets of Shh signaling. Nevertheless, it remains unclear how antero-posterior positional values are encoded and then interpreted to give the particular structure appropriate to that position, for example, the type of digit. A distant cis-regulatory enhancer controls limb-bud-specific expression of Shh and the discovery of increasing numbers of interacting transcription factors indicate complex spatiotemporal regulation. Altered Shh signaling is implicated in clinical conditions with congenital limb defects and in the evolution of the morphological diversity of vertebrate limbs. Keywords: Sonic hedgehog, limb, digits, mouse, chick, positional information Specialty section: This article was submitted to Signaling, a section of the journal Frontiers in Cell and Developmental Biology Received: 07 December 2016 Accepted: 08 February 2017 Published: 28 February 2017 Citation: Tickle C and Towers M (2017) Sonic Hedgehog Signaling in Limb Development. Front. Cell Dev. Biol. 5:14. doi: 10.3389/fcell.2017.00014 INTRODUCTION Over 20 years ago the first evidence was presented that Sonic hedgehog (Shh), an orthologue of the Drosophila Hedgehog (Hh) gene, encodes the long sought after morphogen that specifies anteroposterior pattern in developing vertebrate limbs (Riddle et al., 1993). Grafting experiments in chick wing buds in the 1960s revealed that a group of morphologically indistinguishable mesenchyme cells at the posterior margin of the wing bud (the margin nearest the tail), later known as the polarizing region (or zone of polarizing activity), is an important cell-cell signaling center that controls development across the antero-posterior axis (Saunders and Gasseling, 1968). Tissue transplanted from the posterior margin of one chick wing bud to the anterior margin of another was shown to have the striking ability to duplicate the pattern of three digits, so that another set develop in mirror-image symmetry to the normal set. Based on these observations it was proposed that the Frontiers in Cell and Developmental Biology | www.frontiersin.org 1 February 2017 | Volume 5 | Article 14 Tickle and Towers Shh in Developing Limbs polarizing region produces a diffusible morphogen that specifies antero-posterior positional values (Wolpert, 1969). These positional values are interpreted so that a structure, such as a digit with an appropriate identity, develops in the correct position. The key pieces of evidence that Shh is the polarizing morphogen are that Shh transcripts were found to be localized to the polarizing region of the chick wing bud (Figures 1a–f) and that Shh-expressing cells grafted to the anterior margin of chick wing buds can produce the same effects as grafts of the polarizing region (Riddle et al., 1993). Earlier experiments revealed that tissue from the posterior margin of mammalian limb buds grafted to the anterior margin of chick wing buds could duplicate the pattern of chick wing digits (Tickle et al., 1976; Fallon and Crosby, 1977). This is explained by the finding that Shh is expressed at the posterior margin of mammalian limb buds (Echelard et al., 1993; Odent et al., 1999). Shh has now been shown to be expressed at the posterior margin of the limb buds of all vertebrates studied to date, including the fin buds of the most primitive chondrichthyan fishes such as the shark (Dahn et al., 2007). Experiments in which the polarizing region was grafted to the anterior margin of another chick wing bud showed that polarizing region signaling also plays a role in controlling the width of the limb bud and that widening of the bud is required to specify a complete set of new antero-posterior positional values (Tickle et al., 1975; Smith and Wolpert, 1981). The earliest detected effect of a polarizing region graft was an increase in cell proliferation in adjacent mesenchyme in the host wing bud (Cooke and Summerbell, 1980). In addition, it was proposed that the polarizing region controls the production of a factor by the mesenchyme that maintains the apical ectodermal ridge over the region of the wing bud that will give rise to distal structures including the digits (Zwilling and Hansborough, 1956). The apical ectodermal ridge is a signaling region that rims the bud and is required for proximal-distal patterning and outgrowth and the laying down of structures along this axis; the extent of the apical ectodermal ridge across the antero-posterior axis controls the width of the wing bud and determines the number of digits that can form. The effects of the polarizing region on the apical ectodermal ridge also link antero-posterior and proximo-distal pattern formation. This explains the observation that polarizing region grafts made at later stages of development affect the antero-posterior pattern of more-distal structures (Summerbell, 1974). Early experiments highlighted the complex relationship between the polarizing region and apical ectodermal ridge. In order for a polarizing region to signal, it has to contact the apical ectodermal ridge (Tickle et al., 1975) and this interaction is required in order for the polarizing region to maintain production of the apical ridge maintenance factor by the mesenchyme that will form distal structures. In addition, in the chick wing bud, the polarizing region itself demarcates the posterior limit of the apical ectodermal ridge and grafts of the polarizing region placed under the apical ectodermal ridge flatten it (Saunders and Gasseling, 1968). Interestingly, it has also been shown that the dorsal ectoderm of the wing bud, which produces a signal controlling the development of the dorsal pattern of structures (e.g., extensor muscles), is also required Frontiers in Cell and Developmental Biology | www.frontiersin.org for the polarizing region to signal (Yang and Niswander, 1995). Thus, signaling along all three axes of the developing limb bud is integrated. It has now been shown that Shh affects cell proliferation in the chick wing bud by controlling expression of genes encoding cell cycle regulators including D cyclins independently of the apical ectodermal ridge (Towers et al., 2008). Work on mouse limb development has shown that Shh controls expression of the Gremlin1 gene, which encodes the BMP antagonist that acts as the apical ridge maintenance factor (Zuniga et al., 1999). In addition, it has also been demonstrated that short-range Shh signaling can flatten the apical ridge above the polarizing region (Bouldin et al., 2010). Experiments on chick wing buds have identified FGFs as the apical ectodermal ridge signals that promote outgrowth and also maintain Shh expression in the polarizing region (Laufer et al., 1994; Niswander et al., 1994). Genetic experiments in mouse have identified Wnt7a as the dorsalizing signal that also contributes to regulating Shh expression (Parr and McMahon, 1995). Loss of Wnt7a function in the mouse limb results in the transformation of dorsal to ventral fates and loss of posterior digits (Parr and McMahon, 1995). This second phenotype is consistent with a function for Wnt7a in controlling Shh expression since no digits form in the fore-limbs of Shh−/− mouse embryos and only a single digit—considered to be an anterior digit 1—is present in hind-limbs (Chiang et al., 1996). In this review, we will emphasize the parallel contributions that experimental chick embryology and mouse genetics have played in providing the current picture of Shh function in the limb. We will provide an in-depth picture of how Shh specifies antero-posterior positional values in the limb buds of these two main vertebrate models and how this is integrated with its role in growth. We will consider how Shh expression in the limb is initiated, maintained and eventually extinguished and how cells respond to the Shh signal. We will finally review clinical conditions affecting the limb and examples of evolutionary diversification of limb morphology that are associated with changes in Shh signaling. SPECIFICATION OF ANTERO-POSTERIOR PATTERN Chick Wing Detailed embryological experiments on the chick wing bud have been crucial in establishing the signaling parameters of the polarizing region morphogen. The polarizing region was first discovered in the chick wing bud, where it overlaps with a region of programmed cell death, known as the posterior necrotic zone (Saunders and Gasseling, 1962). Indeed, the original grafting experiments were designed to investigate how this region of cell death is controlled (Saunders and Gasseling, 1968). Tissue from the posterior margin of a chick wing bud was grafted to the anterior margin of a second wing bud and this resulted in a mirror-image pattern of digits across the antero-posterior axis. The normal chick wing has three digits (designated at this time as 2, 3, and 4) but following a polarizing region graft to the 2 February 2017 | Volume 5 | Article 14 Tickle and Towers Shh in Developing Limbs FIGURE 1 | Shh as a morphogen in the chick wing bud. (a) Sonic hedgehog (Shh) expression in the polarizing region at the posterior margin of the early chick wing bud (Riddle et al., 1993). (b) A gradient of Shh in the chick wing bud (blue shaded numbers) specifies antero-posterior positional values for three digits (1,2, and 3) in cells adjacent to polarizing region over 12 h. (c) Chick wing digit skeleton with polarizing region descendants fate-mapped by GFP-expression (green) (Towers et al., 2011). Digits form in tissue adjacent to descendants of the polarizing region that form narrow strip of cells along posterior wing margin. (d) Chick wing bud with anterior polarizing region graft expresses Shh at both anterior and posterior margins (Towers et al., 2011). (e) Mirror-image symmetrical positional values specified as in (b) as a result of Shh being produced by both graft and host. (f) Chick wing digit skeleton pattern with grafted polarizing region (d) and progeny fate mapped by GFP expression (Towers et al., 2011). Six digits form in an anterior to posterior pattern 3-2-1-1-2-3 and grafted polarizing region descendants form narrow strip of cells along anterior wing margin. In all cases, data shown is representative of data in the original cited papers. threshold concentrations govern digit identity, with the highest threshold concentration in tissue closest to the polarizing region specifying the most-posterior digit, digit 3, and the lowest threshold concentration in tissue further away specifying the most-anterior digit, digit 1. Thus, any candidate molecule for the polarizing region morphogen must act in a concentrationdependent manner (Tickle, 1981) and provide a long-range signal (Honig, 1981). The first defined molecule found to mimic the duplicating activity of polarizing region grafts was the vitamin A derivative, retinoic acid (Tickle et al., 1982, 1985) but it was subsequently shown that retinoic acid acts indirectly (Noji et al., 1991; Wanek et al., 1991) by inducing Shh expression (Riddle et al., 1993). There is now good evidence that Shh acts in a concentrationdependent fashion to induce digit duplications. When Shh– expressing cells, or beads soaked in bacterially produced ShhN protein (the active N-terminal fragment produced by autocatalytic cleavage of the large precursor Shh protein), are placed at the anterior margin of a chick wing bud, the extent of digit duplication depends on the number of Shh– expressing cells grafted or the concentration of ShhN protein in which the beads are soaked (Yang et al., 1997). Fewer Shhexpressing cells or lower concentrations of Shh elicit duplication of only the anterior digit 1 (Yang et al., 1997). Grafts of Shh-expressing cells that induce full digit duplications were also shown to result in two ulnae developing in the forearm anterior margin, six digits can develop in the pattern 4-3-2-23-4. Note that recent evidence supports numbering of the digits as 1, 2, and 3 (Towers et al., 2011), and this numbering system is now generally accepted and will be used in this review. This grafting experiment provided an assay for polarizing activity and antero-posterior pattern that could readily be scored by the distinct skeletal morphology of each of the three digits of the chick wing. It should be noted that grafts of the polarizing region also affect the antero-posterior pattern of the wing forearm skeleton and soft tissues (Shellswell and Wolpert, 1977; Robson et al., 1994). Thus, following a polarizing region graft, two ulnae develop and the pattern of muscles is also duplicated. The myogenic cells of the muscle originate in the somites and migrate into the limb bud but the pattern of the wing muscles is dictated by the connective tissue, which is derived from the lateral plate mesoderm (Chevalier and Mauger, 1977). Therefore, the duplicated pattern of muscles following a polarizing region graft will be based on the response of the cells that give rise to the muscle connective tissue. The experimental parameters determined for polarizing region signaling in the chick wing (reviewed in Towers and Tickle, 2009) are consistent with the suggestion that the polarizing region produces a long-range morphogen that sets up a concentration gradient across the antero-posterior axis of the wing bud and specifies positional values (Wolpert, 1969). According to this model, the positional values at particular Frontiers in Cell and Developmental Biology | www.frontiersin.org 3 February 2017 | Volume 5 | Article 14 Tickle and Towers Shh in Developing Limbs together with a duplicated pattern of muscles (Duprez et al., 1999). The original model for how antero-posterior values are specified in the chick wing bud did not consider the dynamic nature of the process, although experiments showed that the extent of duplication following a polarizing graft depended on the length of time that the graft was left in place (Smith, 1980). A similar time dependency was subsequently seen with Shh–soaked beads (Yang et al., 1997). Furthermore, fate mapping experiments showed that cells near a Shh-soaked-bead give rise to an anterior digit 1 when the bead is removed after a short time, but give rise to a more posterior digit (2) if the bead is left in place for longer (Yang et al., 1997). This process by which positional values of cells change over time in response to an increasing concentration of morphogen is known as promotion (see also (Gurdon et al., 1995). An alternative process in which wing bud cells acquire a stable positional value depending on the duration of Shh signaling and then are displaced by growth can be ruled out because an anterior digit 1 has been shown to arise in tissue which was not originally adjacent to a polarizing region graft (see Tickle, 1995). The parameters of polarizing region discussed above were determined in experiments in which additional digits were induced following polarizing region grafts to the anterior margin. But what is the evidence that Shh acts long range and how does Shh signaling specify antero-posterior positional values during normal development of the chick wing? Measurements of Shh activity in slices taken from different positions across the bud using an in vitro cell-differentiation assay are consistent with there being a concentration gradient of Shh across the bud, with Shh activity of a posterior slice being 5–6 times higher than that of a middle slice (Zeng et al., 2001). Another indication that Shh spreads across the wing bud and provides a long range signal is that high levels of the transcripts of known direct gene targets of Shh signaling, including Ptch1 (encoding the main receptor for Shh), and Gli1 (encoding a transcriptional effector of Shh signaling) encompass the posterior two-thirds of the wing bud, including adjacent tissue in addition to the polarizing region (Marigo et al., 1996). It should also be noted that following a polarizing region graft or implantation of an Shh bead to the anterior margin of the chick wing, there is a burst of high level Ptch1 expression in the anterior part of the wing bud, which then subsides and is later followed by the establishment of a stable domain of high level Ptch1 expression (Drossopoulou et al., 2000). This suggests that cells could respond to and interpret two waves of Shh signaling; the first defining the size of the domain that can give rise to digits, and the second, promoting the growth of this domain and specifying positional values. The temporal specification of positional values specified by Shh in normal wing development has been directly addressed by applying cyclopamine, a small molecule inhibitor of Hh signaling at the level of Smoothened to chick embryos, at a series of short time intervals after the onset of Shh expression in wing buds (Towers et al., 2011). Smoothened, a member of the Gprotein coupled receptor superfamily, is normally activated upon Shh binding to Ptch1, and this triggers of activation of the Gli family of transcription factors (see section on Mechanisms of Shh Frontiers in Cell and Developmental Biology | www.frontiersin.org signaling). Application of cyclopamine about 4 h after the onset of Shh expression results in the development of just the anterior digit 1, the anterior and middle digits (1 and 2) develop when cyclopamine is added at 8 h while a complete set of digits (1, 2, and 3) develop when cyclopamine is added at 12 h (Towers et al., 2011). Furthermore, fate mapping experiments show that promotion is occurring with cells next to the polarizing region first being specified to form the anterior digit 1, then being promoted to form the middle digit 2 and finally the posterior digit 3 (Figure 2A). The effects of Shh signaling on antero-posterior growth must be included in any comprehensive model for specification of antero-posterior pattern in the chick wing. Application of cyclopamine in the experiments described above demonstrated that Shh signaling has effects on both specification of anteroposterior positional values and growth because this treatment not only prevented promotion but also expansion of the region of the wing bud that will give rise to distal structures leading to the development of fewer digits (Towers et al., 2008, 2011). When growth alone is targeted by adding trichostatin A or colchicine, and following over-expression of the cyclin-dependent kinase inhibitor—p21Cip1 —at a similar series of time points, fewer digits also develop, but because specification of positional values and promotion by Shh signaling are unaffected, the digits that develop are posterior digits (Towers et al., 2008). These experiments show that specification of antero-posterior positional values in the early chick wing bud is coupled with growth that determines the width of the wing bud. The cyclopamine experiments also show that antero-posterior values are specified over a relatively short time period during early wing bud development. However, these values will not be interpreted in terms of digit identity until much later in development when the digit condensations develop (Figure 2A). When the Shh-expressing region is completely removed from the early wing bud at the time when the positional values that specify two digits are specified, truncated wings develop with posterior structures being preferentially lost (Pagan et al., 1996), showing the crucial importance of Shh signaling in stimulating anteroposterior expansion and maintaining the apical ectodermal ridge. Resulting skeletons bear resemblance to those of the wings of the chicken mutant Oligozeugodactyly (Ozd) that develop devoid of Shh (Ros et al., 2003). It is unclear why Shh continues to be expressed at the posterior margin of the chick wing bud long after the antero-posterior values have been specified (Figure 2A see section Termination of Shh expression). Chick Leg The chick leg has four morphologically distinct digits (numbered 1, 2, 3, and 4 in antero-posterior sequence). Early grafting experiments demonstrated that chick leg buds also have a polarizing region but it was noted that when the leg polarizing region was grafted to a chick wing bud, a toe frequently developed in the duplicated wings (Summerbell and Tickle, 1977). It has since been demonstrated using grafts from the Green Fluorescent Protein-expressing transgenic chicken to make fate maps of the polarizing region that the chick leg polarizing region gives rise to the most posterior digit 4, whereas in the chick wing all the digits 4 February 2017 | Volume 5 | Article 14 Tickle and Towers Shh in Developing Limbs FIGURE 2 | Comparison of models of Shh function in chick and mouse limbs. (A) Chick wing promotion model. Positional values of digits 1, 2, and 3 specified adjacent to polarizing region (blue shading) and promoted over 12 h through a series of increasingly posterior positional values by a concentration gradient of paracrine Shh signaling (graded blue shading—note coloring of polarizing region also shows strength of Shh expression. Shh terminated at around 60 h as digit condensations form by self-organization (black numbers). Colors of developing digits indicate a different positional value that cells were specified with. (B) Chick leg promotion model. Positional values of digits 1, 2, and 3 specified as (A) but polarizing region cells promoted through progressively anterior positional values over 16 h in response to time of autocrine Shh signaling (red numbers) and form digit 4. Shh terminated at around 60 h. (C) Mouse limb temporal expansion. Positional values of digits 1, 2, and 3 specified adjacent to the polarizing region by a gradient of paracrine Shh signaling over approximately 24 h– it is unclear whether promotion is involved (see A). Positional values of digits 4 and 5 specified in polarizing region sometime before Shh terminates at 60 h according to duration of autocrine Shh signaling. Shh terminates at around 60 h. (D) Mouse limb biphasic model. Positional values of digits 1, 2, 3, 4, and 5 specified by Shh, possibly by a gradient of paracrine signaling from the polarizing region in approximately 6 h. It is unclear whether promotion is involved and is possible in this time (see A), or if Shh levels can reach concentrations predicted required to specify posterior positional values. Shh signaling over the next 16 h required for specified digit progenitor cells to proliferate and form condensations in the order digit 1, 4, 2, 5, and 3 (purple numbers). (E) Mouse limb promotion model. Positional values of digits 1, 2, 3, and 4 specified as (B) and polarizing region enlarges sufficiently to give rise to digits 4 and 5 by self-organization. Note promotion model does not easily explain digit 5 patterning that requires a shorter exposure to form than digit 3 (see D). (F) Mouse limb truncated promotion model. Anterior positional values specified (1 and 2) specified by autocrine and paracrine signaling and then cells become refractory to further posterior promotion. Digits form by self-organization: 1, 2, and 3 from cells adjacent to polarizing region, digits 4 and 5 from the polarizing region. Frontiers in Cell and Developmental Biology | www.frontiersin.org 5 February 2017 | Volume 5 | Article 14 Tickle and Towers Shh in Developing Limbs (Kruger et al., 2001) Experiments in which Smoothened activity is deleted specifically in the prospective myogenic cells show that Shh signaling has direct effects on these cells; timing myogenic differentiation, promoting slow muscle differentiation and controlling their migration into the distal part of the limb (Anderson et al., 2012; Hu et al., 2012). In chick limbs, antero-posterior positional values clearly relate to the identity of a digit that develops in an appropriate position. However, this is not readily observable in the mouse limb due to the difficulties in determining which digits are present in mouse limbs conditionally lacking Shh function. Therefore, there is currently no general consensus about the model which best reflects how positional values are specified in the mouse limb bud. The various models are now discussed below (also see Figures 2C–F). The first formal model to be proposed for the mouse limb was the temporal expansion model (Harfe et al., 2004). In this model, anterior positional values for digit 2 (and in part for digit 3) are specified in a concentration-dependent fashion by paracrine Shh signaling and then, posterior positional values (for digits 4 and 5) by the duration of autocrine Shh signaling, which is governed by the proliferative expansion and then displacement of cells from the polarizing region (Figure 2C; specification of digit 1 is considered to be Shh-independent in the hind-limb). Consistent with the model, the restriction of paracrine signaling in a Dispatched mutant (see later section on Mechanisms of Shh signaling) resulted in loss of one digit, suggested to be digit 2. This model also gained support from the finding that when Shh expression was curtailed in the developing mouse limb, this resulted in only three digits developing. The authors identified these digits as being 1, 2, and 3 consistent with the prediction that digits should be lost in a posterior to anterior sequence (Scherz et al., 2007). A particular feature of this model is that it takes considerable time for all the antero-posterior positional values to be specified (Figure 2C), rather than over a short time in the early limb bud. Moreover, it does not take into account promotion through a transitory series of anterior to posterior positional values, which has been demonstrated to occur in the limb buds of the chick. A later model was proposed by Zhu et al. (2008) based on the results of a more extensive set of experiments, in which Shh function was deleted at a series of different stages in mouse limb development. Again, digits were lost with progressively fewer digits developing when Shh function was deleted at earlier and earlier stages. However in this case, the authors suggested that the sequence of digit loss reflects the order in which digits form, with digits that form last being lost first. Thus, for example, they identified the digits in limbs with three digits as being 1, 2, and 4. If their identification of the digits is correct, a posterior digit has formed adjacent to an anterior digit, an outcome not predicted by any previous model. Based on their findings, they proposed a biphasic model for digit patterning—in which Shh has two functions (Figure 2D). In the first phase, Shh specifies positional values across the antero-posterior axis of the very early limb bud, possibly via a concentration gradient, while in the second phase Shh is required to support proliferation and survival of cells that will form the digits (Zhu et al., 2008). It is not clear whether this come from tissue anterior to the polarizing region (Towers et al., 2011; see Figure 1c). Shh is expressed at the posterior margin of chick leg buds for a similar duration to its expression in chick wing buds. Furthermore, it has been demonstrated by treating leg buds with cyclopamine that the positional values that specify the three anterior digits of the chick leg are promoted in response to paracrine Shh signaling in an identical fashion to those that specify the three digits of the chick wing (Towers et al., 2011). However, the positional value for the most posterior digit 4 is promoted in response to autocrine Shh signaling (Figure 2B). Thus, when Shh signaling was attenuated in the chick leg bud by cyclopamine 4 h after onset of Shh expression, two toes with digit 1 identities arose—one from the polarizing region, the other from adjacent anterior tissue, while when Shh signaling was attenuated after 8 h, three digits develop, toes with digit 2 identities from the polarizing region and adjacent cells and a toe with a digit 1 identity from cells further away, and so on, until by 16 h, all the antero-posterior positional values in the leg bud have been specified (Towers et al., 2011). These observations show that although it takes slightly longer to specify antero-posterior positional values in the leg compared to the wing, this process is nevertheless accomplished in the early leg bud, and, as in the wing bud, some considerable time elapses before these positional values are interpreted (Figure 2B). It should be noted that, in the Ozd chicken mutant, a single digit 1 forms in the leg (Ros et al., 2003). Mouse Limb The mouse limb has five digits (1, 2, 3, 4 and 5 in antero-posterior sequence) and digits 2–5 all have three phalanges making them morphologically very similar. Fate maps of the mouse limb polarizing region made by tracing genetically labeled cells that have expressed Shh show that the two posterior digits of the mouse limb are entirely derived from the polarizing region, and while there is some contribution to digit 3, the two anterior digits come from cells outside of the polarizing region (Harfe et al., 2004). Shh is expressed at the posterior margin of limb buds of mouse embryos between E9.5–E12.0 (60 h; Zhu et al., 2008, Figure 2C, note expression is between E10-E12.5 in hind-limbs). At E10.5, a graded distribution of Shh across the posterior third of the mouse hind limb bud has been detected by immunohistochemical analysis (Gritli-Linde et al., 2001) in keeping with paracrine Shh signaling specifying antero-posterior positional values as in the chick wing. Shh is expressed not only at the posterior margin but also at the anterior of the limbs of several polydactylous mouse mutants (Masuya et al., 1995) consistent with Shh functioning as a polarizing signal in mouse limbs. In contrast, in mouse embryos lacking Shh function, the limbs taper toward the tip, and only one digit-like structure (interpreted as digit 1) develops in the hind-limb, while no digits develop in the fore-limb (Chiang et al., 1996). This indicates that Shh is required for the outgrowth of the limb and for the development of structures distal to the elbow/knee in the mouse limb. It should also be noted that in mouse embryos lacking Shh function the development of muscles in this distal region of the limb is severely compromised Frontiers in Cell and Developmental Biology | www.frontiersin.org 6 February 2017 | Volume 5 | Article 14 Tickle and Towers Shh in Developing Limbs 1989) and for original paper on reaction-diffusion (Turing, 1952). The first indications that such a self-organization mechanism might be involved in limb development came from experiments in which it was shown that recombinant limb buds formed from disaggregated single cells, re-aggregated and placed back in an ectodermal jacket could still form digits (Zwilling, 1964; Pautou, 1973). Indeed, based on this latter study, one of the first computer simulations of limb development was developed (Wilby and Ede, 1975). Further experiments showed that when recombinant limbs were made from chick mesenchyme cells from the anterior halves of early chick leg buds, which would not include a polarizing region, and which would not normally give rise to digits, two or three morphologically similar digit-like structures developed (Hardy et al., 1995; Elisa Piedra et al., 2000). When a polarizing region was grafted into such recombinant limbs, however, the digits that developed had recognizable identities (MacCabe et al., 1973). These experiments elegantly revealed that positional information and self-organization are integrated in limb development. There is evidence that a self-organization mechanism also operates in mouse limb buds, as the limbs of mutant mouse embryos in which the Shh signaling pathway is non-functional have many morphologically similar digits (Litingtung et al., 2002; te Welscher et al., 2002; see Section– Measurement of Shh concentration and duration of signaling). Indeed, recent studies in the mouse limb have suggested that this mechanism is based on WNT signals acting as inhibitors and BMP signals as activators, that together, converge on the transcription factor Sox9 to generate a repeated series of digit condensations (Raspopovic et al., 2014). Since digits 2–5 have similar morphologies in the mouse limb, particularly in regard to phalangeal count, one proposal is that self-organization plays a dominant process (Delgado and Torres, 2016). This scenario could for account for difficulties in applying a positional information model to the five digits of the mouse limb. Moreover, a recent study on developing chick wings has revealed how positional information and self-organization can interact and this could be relevant to understanding how the mouse digit pattern is specified. If chick wing buds are treated with cyclopamine under conditions in which the promotion of antero-posterior values is truncated, a series of morphologically similar digit 2s in a pattern 1-22-2 can develop by self-organization (Pickering and Towers, 2016). It should be noted that the digit 2s were not of identical morphologies and sizes suggesting other factors control these finer aspects of development. In wings with multiple digit 2s, the most-posterior of these digits arises from cells of the polarizing region. An interpretation of these findings is that antero-posterior expansion mediated by a posteriorly extended apical ectodermal ridge has enabled a small pool of cells specified with the same positional value to produce a series of digit 2s by self-organization (Pickering and Towers, 2016). In extrapolating these data to the mouse limb, it has been suggested that a similar mechanism could account for the patterning of digits 1 through to 4 (Pickering and Towers, 2016; Figure 2F). In addition, the apical ectodermal ridge of the mouse limb completely overlies the polarizing region (Pickering and Towers, 2016), and an latter function is a separate direct function of Shh signaling or reflects an essential role of Shh signaling in maintaining sufficient apical ectodermal ridge signaling. According to this model, the resultant digit patterns when Shh function is deleted are due to loss of Shh compromising survival and proliferation of specified digit progenitor cells rather than failure to specify anteroposterior positional values (Zhu et al., 2008). Furthermore, positional values would have to be specified in the early mouse limb bud over a period of approximately 6 h (based on Ptch1 expression), which suggests that this process is not integrated with growth as in the chick wing. The ability to observe promotion in chick limbs gives insights into the time required to specify positional values, but in the mouse limb, in which promotion is not readily observed, it is difficult to distinguish between the effects of Shh signaling on specification of positional values and survival and proliferation of the cells that will form the digit condensations. Indeed the time required for digit specification proposed by Zhu et al. does not appear consistent with a model in which antero-posterior positional values are promoted in response to the concentration and/or duration of Shh signaling. However, if one were to take promotion into account, a unifying model can be proposed (Towers et al., 2011). According to this proposal, positional values would be specified early in the mouse limb as suggested in the biphasic model. However, these would only be anterior positional values, which would then be promoted to posterior values by both paracrine and autocrine Shh signaling operating in parallel. Thus, the pattern of digits specified would depend on how far positional values have been promoted at the time at which Shh function is deleted in keeping with more conventional models for digit patterning. The digits that develop in the threedigit mouse limb when Shh signaling is curtailed would therefore be predicted to be 1, 2, and 2—a pattern that is readily observed in cyclopamine-treated chick legs (Towers et al., 2011), and occasionally in wings (Pickering and Towers, 2016). However, there are difficulties in applying a promotion model to the specification of digit 5 of the mouse limb as this would imply that it is the last digit of the pattern to be specified (Figure 2F), when in fact it forms before more-anterior digits (Figure 2D, see also discussion in Towers et al., 2011). INTERACTION BETWEEN POSITIONAL INFORMATION AND A TURING-TYPE MECHANISM Although, it has been shown that Shh is the critical signal in controlling development across the antero-posterior axis of the limb, there is evidence that the periodic condensation of cells that will form the digits depends on an underlying Turing type selforganization mechanism independent of graded Shh signaling. In the basic Turing model, diffusible signals—one operating as an inhibitor, the other as an activator—interact to produce the pattern of digits and interdigits. Positional information and self-organization have been presented as competing models of digit development, when in fact the power of both processes operating together has been long recognized (see (Wolpert, Frontiers in Cell and Developmental Biology | www.frontiersin.org 7 February 2017 | Volume 5 | Article 14 Tickle and Towers Shh in Developing Limbs autocrine Shh signaling. Therefore, the crucial questions are how a graded distribution of Shh arises, how the range of Shh signaling is controlled and how cells measure the concentration of Shh and the duration of Shh signaling. intriguing suggestion is that this could enable the cells of the polarizing region to expand sufficiently to give rise to two digits (4 and 5) by self-organization (Figure 2F; Pickering and Towers, 2016). The specification of the same positional value during mouse limb development could occur if cells become refractory to the levels/duration of Shh signaling at a certain point (Figure 2F). In support of such a mechanism operating in the mouse, there is not a simple linear relationship between position and level of positive Shh signaling in the limb bud as expected in a classical positional information model (Ahn and Joyner, 2004). However, even though mouse digits 2–5 are morphologically similar, it is clear that they still have different identities, with the cells of digit 4 being characterized by having many more receptors for both testosterone and estrogen than digit 2 thus determining the sexual dimorphism in digit length (Zheng and Cohn, 2011). Indeed, digit 5 in particular, has quite a different morphology to the other digits. Taken together, even if the cells that give rise to mouse digits 2–5 are specified with the same positional value that is interpreted so that they have the same phalanx number, other factors operate to give the digits their individual morphologies and hence identities. Additional support for a model in which loss of Shh signaling can increase digit number and also result in posterior digits developing with anterior traits has been provided by work on the fore-limbs and hind-limbs of the amphibian Xenopus tropicalis. Inhibition of Shh signaling at a series of developmental stages resulted in forelimbs occasionally developing with five digits rather than four (Stopper et al., 2016). In addition, hind-limbs often developed terminal claws on all five digits whereas in normal development claws are only present on digits 1, 2, and 3. Additional work is required to determine if other characteristics of these posterior digits are anteriorised such as phalange number. The work of Pickering and Towers further highlights the complex relationship between the polarizing region and the apical ridge already mentioned (Niswander et al., 1994), and the importance of short-range reciprocal signaling between these structures in the formation of posterior digits in particular as observed in the mouse limb (Zuniga et al., 1999; Bouldin et al., 2010). Thus, in the chick wing, Shh signaling inhibits the overlying apical ridge and the polarizing region fails to produce digits, yet in the mouse limb, the overlying apical ridge is less sensitive to Shh signaling than in the chick wing (see also (Bouldin et al., 2010), and in persisting posteriorly, allows two digits to form—the chick leg appears to have an intermediate relationship allowing one digit to form. Such dynamic interplay between the polarizing region and apical ridge could have contributed to patterns of posterior digit loss during limb evolution (see Section on Evolutionary aspects of Shh signaling in the limb). Long-Range Shh Signaling and Gradient Formation Studies in developing mouse limbs have revealed general mechanisms that modulate the distribution of Shh protein in tissues. One factor is the addition of lipids. Following its autocatalytic conversion, Shh is secreted by cells as a modified form of ShhN with cholesterol added at the C-terminus and a palmitoyl group (as part of a thiol ester) at the N-terminus (known as ShhNp; p indicating that ShhN is processed; reviewed (Lee et al., 2016). In limb buds of mouse embryos in which the C-terminal processing domain of Shh is conditionally deleted so that the polarizing region produces ShhN instead of ShhNp, ShhN spreads further across the limb bud and additional digits develop anteriorly (Li et al., 2006). It should be noted that previous analyses also suggested that cholesterol modification extends the range of paracrine Shh signaling. Thus, mice limbs expressing ShhN that lacks cholesterol failed to form digits 2 and 3 (Lewis et al., 2001) consistent with a role for paracrine Shh signaling in specifying these digits (Harfe et al., 2004). Other data however are consistent with cholesterol modification restricting the spread of Shh. Thus, mice deficient in SREBP-2 that encodes a sterol regulatory element binding protein that regulates cholesterol production failed to up-regulate Ptch1, consistent with impaired Shh transport (Vergnes et al., 2016). Similar studies on mutant mice that are unable to palmitoylate Shh show that this modification is essential for long range signaling (Chen et al., 2004). Intriguingly, cholesterol has also recently been shown to be the endogenous activator of Smoothened (Huang P. X. et al., 2016). Because cholesterol plays such important roles in Shh signaling, changes in the availability of cholesterol can impact on the development of the limb and might explain the subtle alterations in the spacing of the digits that have been observed in the limbs of mice with a mutation in a gene encoding a protein required for cholesterol metabolism (Schmidt et al., 2009) and in the limbs of rat embryos treated with triparanol, an inhibitor of cholesterol biosynthesis (Gofflot et al., 2003). The membrane protein Dispatched1 is required for paracrine signaling by cholesterol–modified Shh (Tian et al., 2005). The restriction of the spread of the ligand in a Dispatched1 mouse mutant resulted in the loss of a digit, which was interpreted as being digit 2, and as already mentioned, provided crucial evidence for the temporal expansion model (Harfe et al., 2004). Another mechanism that influences the range of Shh signaling is the binding of Shh to cell surface and extracellular proteins. A generic response to Shh in all tissues is transcriptional upregulation of genes encoding cell surface proteins such as Ptch1 and Hhip that bind Shh. The resultant increase in their expression in response to Shh creates negative feedback loops, that not only limit the spread of Shh by sequestering it at the cell surface, but also, in the case of Ptch1, because it inhibits Smoothened activity, dampens activation of the Shh pathway. In mice in which Ptch1 MECHANISMS OF Shh SIGNALING As indicated in the models outlined above, positional values in developing limbs are specified by paracrine Shh signaling, in which Shh acts as a long-range graded signal and in a concentration/time dependent fashion, or by the duration of Frontiers in Cell and Developmental Biology | www.frontiersin.org 8 February 2017 | Volume 5 | Article 14 Tickle and Towers Shh in Developing Limbs the required range of Shh signaling. Furthermore, Boc and Cdo have been visualized in discrete microdomains on a subset of filopodia extending from Shh-responding cells. However, it is not clear whether this transport mechanism could produce robust graded signaling and indeed whether filopodia are required. The involvement of filopodia could however explain the apparently anomalous finding that grafts of cells expressing a membranetethered form of Shh (generated by fusing the integral membrane protein CD4 to the C-terminus of ShhN) can duplicate digits in the chick wing (Yang et al., 1997). is conditionally inactivated in the limbs (Butterfield et al., 2009), and therefore the signaling pathway is activated independently of Shh, the hind-limbs have extra digits, but the fore-limbs have fewer digits. This difference between hind-limbs and fore-limbs appears to be due to the timing of activation of the signaling pathway, which is earlier in the mutant fore- limbs (Zhulyn et al., 2014). In contrast to Ptch1 and Hhip1, the genes Cdo (CAMrelated/downregulated by oncogene), Boc (brother of Cdo) and Gas1 (growth arrest specific 1) encoding membrane associated proteins that bind Shh, are expressed in the anterior region of early limb buds and their expression is negatively regulated by Shh. Analysis of limb development in single or double mouse mutants suggest that Gas1 and Boc sustain paracrine Shh signaling at a distance from the polarizing region (Allen et al., 2007). ShhNp can also bind to heparan sulfate proteoglycans and the distribution of these and other extracellular proteins in the developing limb will affect the distribution of Shh. In Drosophila, the hydrolase notum that cleaves glypicans, a subfamily of heparan sulfate proteoglycans, promotes high-level Hh signaling in the wing. Interestingly, in the chick wing bud, Notum was identified in microarray experiments as being downstream of Shh signaling (Bangs et al., 2010), suggesting possible functional conservation. One way in which Shh could spread across the limb bud is by diffusion (see Muller et al., 2013, for discussion on mechanisms of morphogen transport), although it has been questioned whether simple diffusion would be a sufficiently robust mechanism to generate a stable concentration gradient (Kerszberg and Wolpert, 2007). Mathematical modeling however showed that specification of positional values for the three digits of the chick wing can be simulated by simple diffusion of Shh from the polarizing region (Woolley et al., 2014). In the model, based on the results of (Drossopoulou et al., 2000), Shh specifies the initial size of the domain that will give rise to the digits and then provides positional information. The model incorporates promotion of positional values in a dose-dependent fashion over the observed time frame in a growing domain of the correct dimensions as determined experimentally (Towers et al., 2008). The model can be extended successfully to the specification of the positional values in the chick leg, even though digit 4 arises from the polarizing region. However, it is unclear whether Shh levels in the polarizing region could reach the predicted concentration required to specify digit 4 (assumed to be double that required to specify digit 3) and whether indeed there is a simple graded response to Shh signaling in the leg. It is therefore more plausible that digit 4 is specified by length of time that cells express Shh. The model cannot however be extended further to simulate easily specification of the fifth digit of the mouse limb. Live imaging of chick wing buds showed that Shh can be transported along the external surface of specialized filopodia (similar structures in insects are called cytonemes). These filopodia extend up to 150 microns away from the polarizing region and a similar distance away from the receiving cell (Sanders et al., 2013) equating to about 300 microns, the initial size of the chick wing digit-forming field (Vargesson et al., 1997; Towers et al., 2008). Thus, direct cell-cell contacts can span Frontiers in Cell and Developmental Biology | www.frontiersin.org Measurement of Shh Concentration and Duration of Signaling It has been proposed that limb bud cells respond to paracrine Shh signaling in a concentration dependent fashion although length of exposure to the Shh signal also plays a role. So how do cells measure the concentration of Shh? The mechanism depends on the Shh-dependent processing of full-length Gli proteins, which act as transcriptional activators; in the absence of Shh signaling, Gli proteins are processed to short forms, which act as transcriptional repressors (reviewed in Lee et al., 2016). In normal chick and mouse limb buds, anterior cells not exposed to Shh contain high levels of Gli repressor, while in the posterior region of the limb, there is a gradient in the ratio of Gli activator/Gli repressor, higher posteriorly than anteriorly, reflecting the response to the Shh gradient across this part of the limb (Wang et al., 2000). There are three Gli genes, Gli1, Gli2, and Gli3 with the protein encoded by Gli1 acting exclusively as an transcriptional activator as it does not undergo processing into a repressor form. While functional inactivation of Gli1 and Gli2 in mice has little effect on limb development (Mo et al., 1997; Park et al., 2000), when Gli3, the major contributor to transcriptional repression, is functionally inactivated, Shh is expressed anteriorly and several additional morphologically similar digits form anteriorly while posterior digits are less affected (Wang et al., 2000). Unexpectedly, the limbs of Gli3 and Shh double knockout embryos are identical to the Gli3−/− limb buds showing that the function of Shh in the limb is to relieve repression by Gli3 and allow a patterned set of digits to develop from the posterior part of the limb (Litingtung et al., 2002; te Welscher et al., 2002). In the mouse limb, the gradient of Gli3 activity could only specify at most digits 1, 2, and 3 because Gli3 is not expressed in the polarizing region itself (Buscher and Ruther, 1998). Instead the initial response to autocrine Shh signaling would have to be mediated by Gli2, and consistent with this hypothesis, removing the function of Gli2 in a Gli3 mutant background, thus effectively inactivating all Gli function, results in the digits appearing morphologically similar (Bowers et al., 2012). This suggests that Gli3 mediates the response of cells in the limb bud to paracrine Shh signaling and Gli2 to autocrine Shh signaling. It should also be noted that the digits that form in single Gli3−/− mouse limbs (and also in compound Shh−/− /Gli3−/− mouse limbs) are thinner and more closely spaced together than in normal limbs, suggesting that Gli3 plays a role in regulating the digit period (Sheth et al., 2012, see section Interaction Between Positional Information and a Turing-type Mechanism). 5′ Hoxa/d 9 February 2017 | Volume 5 | Article 14 Tickle and Towers Shh in Developing Limbs are not present in the mouse embryos in which the ZRS is deleted. The ZRS is of general interest as an example of a long-range enhancer—a cluster of three similar long-range enhancers also regulates Shh expression in the epithelial linings of the pharynx, the lung and the gut respectively (Sagai et al., 2009). 3D FISH and chromatin configuration assays showed close associations between the ZRS and the Shh locus in mouse limb bud cells compared to cells from other tissues (Amano et al., 2009). Curiously, transcriptional activity was not seen in all polarizing region cells suggesting that the cells may express Shh in pulses. One possibility is that Shh is expressed periodically during the cell cycle. In support of this, Shh expression is lost in chick wing buds treated with aphidicolin—an inhibitor of progression through Sphase (Ohsugi et al., 1997). More recently FISH and chromatin configuration assays together with super-resolution microscopy have revealed that the Shh locus loops out of its chromosome territory to make contacts with the ZRS in polarizing region cells in the mouse limb bud at the time Shh expression is activated (Williamson et al., 2016). The ZRS provides an excellent reference point for deciphering the gene network that controls Shh expression in the limb and contains binding sites for the transcription factors, Hand2 (heart and neural crest derivatives 2; (Galli et al., 2010) and 5′ Hoxd proteins. The genes encoding these transcription factors are expressed in the posterior region of the early limb bud and when they are deleted in the mouse limb, Shh is not expressed. Conversely, when Hoxd13 is expressed throughout the mouse limb bud, there is an ectopic Shh domain and polydactylous limbs result (Zakany et al., 2004). Expression of Hand2 and Hoxd genes is restricted to the posterior part of mouse limb buds by Gli3. In the mouse fore-limb-forming region, Hand2 expression is also repressed anteriorly by the Hox5 paralogous group genes (Xua et al., 2013), while Hand2 expression in the posterior region of the fore-limb-forming region is dependent on the Hox9 paralogous group genes, thus providing antero-posterior polarity prior to the transcriptional activation of the Shh gene (Xu and Wellik, 2011). Recently, it has emerged that GATA family transcription factors also contribute to supressing anterior expression of Shh (Kozhemyakina et al., 2014) as conditional removal of Gata4/6 in limbs of mouse embryos results in pre-axial polydactyly. Two distinct mechanisms have been proposed. One is that GATA transcription factors in complex with FOG co-factors bind directly to the ZRS enhancer while the other is that GATA6 may interact directly with GLI3 to promote repression of the vertebrate Hedgehog pathway and this may explain the formation of an additional anterior digit in the hindlimb (Hayashi et al., 2016). Shh expression in the polarizing region is also controlled by FGF signaling from the apical ridge and FGF signaling has been shown to regulate the expression of the genes encoding the ETS translocation variant transcription factors ETV4 and ETV5. The genes encoding these transcription factors are expressed beneath the entire extent of the apical ectodermal ridge and suppress Shh expression outside of the polarizing region. These ETV transcription factors bind directly to sites in the ZRS. In the function also seems to be involved since the progressive titration of 5′ Hox genes in the Gli3−/− background increases digit number and decreases the digit period still further (Sheth et al., 2012). Surprisingly, chemical mutagenic screens to identify mutations causing polydactyly in mouse identified genes required for formation and functioning of primary cilia (Huangfu et al., 2003; Weatherbee et al., 2009; Ashe et al., 2012). In such mutants, many morphologically similar digits develop and this is because Gli processing takes place on primary cilia in vertebrate cells. Thus, absence of cilia is equivalent to functional inactivation of all three Gli genes. The classical chicken mutant, talpid3, with a range of defects including polydactylous limbs (Ede and Kelly, 1964) was found to have a mutation in a gene encoding a centrosomal protein required for formation of a primary cilium (Davey et al., 2006), and functionally inactivating the talpid3 gene in a mouse limb, leads to the development of many morphologically similar digits (Bangs et al., 2011). Another chicken mutant, talpid2, with the same range of defects including polydactylous limbs, was found to have a mutation in a gene encoding another ciliary protein—C2CD3 (Chang et al., 2014). For autocrine Shh signaling, the duration of signaling is the most important parameter. Timing appears to be a general way of specifying positional values, but how cells in embryos measure time is little understood. Interestingly, a timing mechanism involving a cell cycle clock has been proposed to specify proximodistal positional values in the chick wing bud (Saiz-Lopez et al., 2015), although the most proximal positional values may be specified by retinoic acid signaling (Cooper et al., 2011; RoselloDiez et al., 2011). The molecular nature of intrinsic timers is currently unknown and presents a widespread problem in developmental biology. INITIATION OF Shh EXPRESSION A key discovery in understanding how Shh expression is localized to the posterior margin of the limb bud was identification of a cisregulatory element that controls limb-specific expression (Lettice et al., 2002). Analysis of Sasquatch, an insertional mouse mutant with limb polydactyly, in which Shh was expressed anteriorly as well as posteriorly in the limb, showed that the exogenous DNA construct had serendipitously disrupted an enhancer (Sharpe et al., 1999). This 1.7 Kb enhancer, which has become known as the ZRS (zone of polarizing activity regulatory sequence), is unexpectedly located in intron 5 of the LMBR1 (limb region 1) gene, which is almost 1 MB upstream of the promoter of the Shh gene. It is still not clear why insertion of the transgene into this particular region of the ZRS in Sasquatch leads to anterior Shh expression in the limb bud. In contrast, deletion of the entire ZRS region in mouse embryos results in loss of Shh expression in the limb buds resulting in limb truncations similar to those found in mouse embryos lacking Shh function (Sagai et al., 2005). It should be noted however, that the many other defects seen in mouse embryos lacking Shh function, which reflect the widespread functions of Shh signaling in organogenesis, Frontiers in Cell and Developmental Biology | www.frontiersin.org 10 February 2017 | Volume 5 | Article 14 Tickle and Towers Shh in Developing Limbs wing polarizing region intrinsically times the duration of Shh expression irrespective of the extrinsic signaling environment (Chinnaiya et al., 2014). Indeed, Shh expression has been shown to terminate on time if the separation of Grem1 and Shh expressing cells is prevented (Towers et al., 2008). Furthermore, the inhibition of Shh signaling with cyclopamine in the chick wing leads to the premature loss of Shh expression in the presence of an Fgf4-expressing apical ectodermal ridge and Grem1 expression extending into the posterior part of the wing bud, thus suggesting that Shh autoregulates its own transcription in the polarizing region (Pickering and Towers, 2016). The mechanism by which this is achieved has not yet been elucidated. polarizing region, posteriorly expressed ETS1/GABPα binds to other sites in the ZRS and over-rides this inhibition and allows expression of Shh (Lettice et al., 2012). Wnt7a signaling from the dorsal ectoderm also contributes to controlling Shh expression but the mechanism is not yet known (Yang and Niswander, 1995). The activity of the ZRS not only determines the location of cells expressing Shh in the developing limb bud but also the size of the Shh expression domain. In addition, an autoregulatory mechanism has been discovered in which Shh controls the number of polarizing region cells by regulating the size of the posterior necrotic zone (Sanz-Ezquerro and Tickle, 2000) via BMP2 signaling (Bastida et al., 2009) Taken together these mechanisms have the crucial function of controlling the levels of Shh signaling. Lastly, retinoic acid derived from the flank also appears to be required for initiating Shh expression in limb buds. Shh expression is greatly reduced in the limb buds of vitamin A deficient quails (Stratford et al., 1999) and in chick wing buds following treatment with inhibitors of retinoic acid synthesis (Stratford et al., 1996). Mouse embryos in which a gene encoding an enzyme that generates retinoic acid was functionally inactivated died early and lacked fore-limbs. When these embryos were provided with retinoic acid so that development can proceed further, Shh was not restricted posteriorly in the rescued fore-limb buds suggesting that retinoic acid plays a role in determining antero-posterior polarity prior to activation of Shh expression (Niederreither et al., 2002; Zhao et al., 2009). RESPONSE TO Shh SIGNALING IN THE LIMB Many studies have provided information about the expression of individual genes that are affected by Shh signaling in the limb. For example, changes in gene expression have been observed in chick limb buds treated with Shh or cyclopamine, and in mouse limb buds in which Shh or Gli3 is functionally inactivated, or in which Gli3 processing does not occur, e.g., mutants with defective cilia. Microarray analyses have been carried out in both chick and mouse limbs (Vokes et al., 2008; Bangs et al., 2010). It has been estimated from one microarray study that 10% of the genes expressed in the early limb bud (about 1,000 genes) are downstream of Shh signaling (Bangs et al., 2010). Putative direct targets of Gli3 repression have been identified by ChIP seq analysis of limb bud nuclear extracts using transgenic mice expressing a tagged form of the Gli3 protein (Vokes et al., 2008). Further analysis has involved RNAseq (Lewandowski et al., 2015). Analysis of this information has begun to uncover the gene regulatory network underlying the response to the Shh signaling pathway in the limb in addition to the generic suite of genes that encode proteins that enable or modulate Shh signaling. The genes in the network include those that are expressed posteriorly either due to positive regulation by Shh or because Shh relieves Gli3 repression; also those that are expressed anteriorly either due to negative regulation by Shh or because they are downstream of Gli3 repression (Bangs et al., 2010). A study involving analysis of gene expression patterns in the limb buds of Shh−/− , Gli3−/− double mouse mutants indicated that the expression of nearly all the putative Gli target genes identified by ChIP seq in the posterior mesenchyme of E10.5 mouse limb buds depends on Gli repressor activity rather than Gli activator activity (Lewandowski et al., 2015). One generic class of potential target genes already mentioned comprises genes encoding cell cycle regulators such as N-myc and Cyclin D1 that are predominantly expressed posteriorly and Cyclin D2 that is expressed in the polarizing region, and that are likely mediate the effects of Shh on proliferation (Towers et al., 2008; Welten et al., 2011). Shh has also been shown to promote vascularisation of the chick wing bud via regulating expression genes encoding pro-angiogenic factors such as VEGF (Davey et al., 2007). There is evidence in the mouse limb, that transcription factor genes including 5′ genes in the Hoxa TERMINATION OF Shh EXPRESSION The failure of the positive feedback loop between the polarizing region and the apical ectodermal ridge has been proposed to terminate the duration of Shh expression in the chick wing. In this model, Shh up-regulates Grem1 by paracrine signaling, but cells displaced from the polarizing region by proliferative expansion are then unable to up-regulate Grem1 (the apical ridge maintenance factor; Scherz et al., 2004). This is proposed to create a tissue barrier that results in Shh being no longer able to up-regulate Grem1 at a distance, leading to de-repressed BMP signaling suppressing Fgf4 expression in the apical ectodermal ridge, that in turn, leads to loss of Shh expression in the polarizing region (Scherz et al., 2004). Tbx2 is proposed to be the factor that suppresses the posterior up-regulation of Grem1 in and around the polarizing region (Farin et al., 2013). In the absence of Tbx2, Grem1 expression expands posteriorly resulting in prolonged Shh expression and extra tissue growth indicated by the bifurcation of digit 4. It is unclear why this only occurs in the hind-limbs of these Tbx2 knockout mice. An alternative model for the mouse limb is that increased FGF signaling inhibits Grem1 expression leading to termination of the feedback loop (Verheyden and Sun, 2008). A clock linked with the cell cycle has also been shown to be involved in timing the duration of Shh expression in the polarizing region of the chick wing bud with the clock being set once retinoic acid concentrations fall below a certain level. Thus, tissue transplantation experiments have shown that the chick Frontiers in Cell and Developmental Biology | www.frontiersin.org 11 February 2017 | Volume 5 | Article 14 Tickle and Towers Shh in Developing Limbs the development of other tissues. However, point mutations in the ZRS enhancer that would be predicted to lead to ectopic SHH expression specifically in the limb bud are found in human patients with pre-axial polydactyly type 1 (PPD1—OMIM 174400) and triphalangeal thumb polysyndactyly syndrome (TPTPS OMIM 174500) (see review Hill and Lettice, 2013). In TPTPS, additional digits can arise post-axially as well as pre-axially, suggesting that the normal regulation of SHH expression at the posterior margin of the limb is also perturbed. It remains to be determined how these point mutations affect the regulation of endogenous SHH expression. One possibility is that the levels and/or duration of SHH expression are increased and these lead not only to an additional digit pre-axially but also to overgrowth of the polarizing region and its subsequent development into additional post-axial digits—perhaps by self-organization (see section on Interaction between positional information and a Turing-type mechanism). A point mutation at a particular position in the ZRS is associated with Werner mesomelic syndrome in which there are distal arm and leg bone defects in addition to extra digits (VanderMeer et al., 2014). Unexpectedly, duplications of the ZRS have also been reported in individuals with TPTPS as well as the related condition Haastype polysyndactyly (OMIM 186200). Microduplications of the ZRS have also been detected in patients with Laurin-Sandrow syndrome OMIM 13750); the limb phenotype of these patients overlaps with the Haas-type polysyndactyly phenotype but can be distinguished by mirror-image polysyndactyly of the feet and duplication of the fibula (Lohan et al., 2014). In contrast, patients with a deletion involving exon 4 and portions of introns 3 and 4 of the LMBR1 gene, a region distinct from the ZRS, have a condition known as acheiropodia (OMIM 200500) in which elements distal to the elbow/knee fail to form in all four limbs. This condition not only resembles the phenotype of the limb buds of mouse embryos lacking Shh function but also that of the limbs of Ozd mutant chickens in which it has now been shown that a large part of the ZRS sequence is deleted (Maas and Fallon, 2004). Inborn errors in cholesterol metabolism can lead to limb anomalies, as might be expected given the importance of cholesterol in Shh signaling as already discussed. For example, post-axial polydactyly is found in patients with Smith-Lemli-Opitz syndrome (OMIM 270400) in which a mutation deactivates the function of 7-dehydrocholesterol reductase, which is the final enzyme in the metabolic pathway that generates cholesterol. Post-axial polydactyly is also seen at low frequencies in patients with other syndromes in which cholesterol biosynthesis is altered (Gofflot et al., 2003). Why post-axial polydactlyly occurs however is not clear. Defects in the response to Shh signaling are found in syndromes that include polydactyly. For instance, the Pallister-Hall (OMIM 146510, Hill et al., 2007) and Grieg Cephalopolysyndactyly (OMIM 175700- Kalff-Suske et al., 1999) syndromes present with pre-axial and post-axial polydactyly and are caused by mutations in the GLI3 gene. The effects of these mutations are likely due to the de-repression of the Shh signaling pathway in the anterior part of the limb. Since the processing of full-length Gli3, occurs in primary cilia, syndromes known as ciliopathies, in which cilia function/structure is and Hoxd clusters, Sall1, and Tbx2/Tbx3 are putative direct targets of Shh and would be predicted to encode the positional information conferred by the autocrine/paracrine Shh signaling (Vokes et al., 2008). Experiments with cultured mouse limb buds suggest that Shh signaling is required for robust and continued expression of 5′ members of the Hoxd cluster (Panman et al., 2006; Lewandowski et al., 2015) while mis-expression of Tbx2 and Tbx3 genes in the chick leg bud in the embryo has been reported to change digit identity (Suzuki et al., 2004). Other putative direct Gli3 targets are genes involved in BMP signaling; Gremlin encoding the apical ridge maintenance factor and Bmp 2 expressed together with Bmp7, in the posterior region of the early limb bud (Vokes et al., 2008). There is a close relationship between Shh and Bmp2 expression elsewhere in vertebrate embryos, which is also conserved in Drosophila. For instance in the Drosophila wing imaginal disc, Hh secreted from the posterior compartment induces expression of the Bmp2 orthologue, Dpp, that encodes a long range signaling molecule regulating position-dependent expression of transcription factors such as Spalt and Omb, orthologues of Sall1 and Tbx2/3 respectively. Experiments in chick wing buds show that Bmpsoaked beads placed at the anterior margin of a chick limb do not induce digit duplications (Drossopoulou et al., 2000). However, when a bead soaked in a BMP antagonist was implanted at the anterior margin of the wing bud following implantation of an Shh-soaked bead, a series of morphologically similar digits developed anteriorly suggesting that BMP signaling is involved in digit promotion (Drossopoulou et al., 2000). In chick leg buds, BMP signaling is graded across the tip of the bud at the stage at which the digit condensations form in the so-called phalanxforming region (PFR—Suzuki et al., 2008). Grafting interdigital tissue to different positions between digit condensations and manipulating BMP signaling alters the morphology of the digits in terms of phalange number suggesting that it is BMPs produced by interdigital regions that are directly responsible for realizing digit-specific morphology (Dahn and Fallon, 2000). Recently, evidence has been presented that interdigital signaling may also be involved in regulating the morphogenesis of the digit condensations in mouse limbs (Huang B. L. et al., 2016). CLINICAL ASPECTS OF Shh SIGNALING IN THE LIMB The increasing understanding of the molecular basis of anteroposterior pattern formation has led to insights into congenital malformations that affect the limb. Unsurprisingly, defects in Shh function have been found to underlie several inherited disorders. In particular, these include polydactyly: pre-axial polydactyly in which additional digits arise from the thumb-side of the hand, and post-axial polydactyly in which the additional digits arise from the little finger-side (Biesecker, 2011). Often these conditions are associated with syndactyly (fusion of the soft tissues between the digits). Alterations in the coding sequence of the SHH locus are not known to form the basis of any congenital malformation of the limb—presumably because such lesions are not compatible with Frontiers in Cell and Developmental Biology | www.frontiersin.org 12 February 2017 | Volume 5 | Article 14 Tickle and Towers Shh in Developing Limbs compromised, include polydactyly as part of their spectrum of defects—examples being, Bardet-Biedl syndrome (BBS– OMIM 209900, Forsythe and Beales, 2013) and Meckel-Gruber syndrome (OMIM 249000, Shaheen et al., 2013). Recently mutations in the TALPID3 gene, required for formation of cilia have been discovered in patients with Joubert syndrome (OMIM 21330) although these patients rarely show limb defects (Roosing et al., 2015; Stephen et al., 2015). Homozygous mutations in the TALPID3 gene have however been found in families affected by lethal ciliopathies associated with polydactyly (Alby et al., 2015), phenotypes more akin to those of the homozygous chicken mutants already mentioned in which the talpid3 gene was first identified. Several clinical conditions are associated with mutations in putative gene targets of Shh signaling in the limb (see previous section on Response to Shh signaling, also reviewed Pickering and Towers, 2014). Sall1 encoding a transcription factor is expressed in the posterior region of the early chick and mouse limb buds but more widely at the base of the digital plate at later stages (Buck et al., 2001; Fisher et al., 2011). Mutations in SALL1 that produce a truncated protein with dominant negative activity have been detected in patients with Townes-Brockes syndrome characterized in the limb by pre-axial polydactyly and triphalangeal thumb (Kohlhase et al., 1998). A transgenic mouse model in which a truncated SALL1 protein is produced mimics the human limb phenotype (Kiefer et al., 2003). Inactivating mutations in the gene encoding the transcription factor Tbx3, which is expressed at high levels in stripes at both anterior and posterior margins of early chick and mouse limb buds (Tumpel et al., 2002; Emechebe et al., 2016) are seen in patients with Ulnar-mammary syndrome (OMIM 181450); the defects affect the development of posterior structures in the upper limb and include missing ulna, missing posterior digits and post-axial polydactyly. The same limb phenotype is seen in mouse Tbx3 mutant embryos (Davenport et al., 2003; Emechebe et al., 2016). Finally mutations in HOXD13 are associated with many clinical conditions in which there are digital abnormalities including polydactyly, syndactyly (fused digits) and brachydactly (short digits). Hoxd13 is another putative gene target of Shh signaling identified in the mouse limb and is expressed in the posterior region of early chick and mouse limb buds and then throughout the digital plate at later stages (Nelson et al., 1996). A complex spectrum of mutations in HOXD13-polyanaline tract expansions, truncating mutations and point mutations leading to amino acid substitutions have been identified (reviewed Goodman, 2002). Hoxd13 is likely to have several roles in digit development and the challenge is to understand how a particular genetic change leads to a particular phenotype. relevant in the context of stimulating growth and repair of tissues in damaged limbs. Following amputation of a newt limb, a mound of undifferentiated cells called the blastema forms at the stump surface and proliferation of blastemal cells replenishes the missing limb structures. Shh is expressed in posterior part of the newt limb blastema recapitulating embryonic expression in the limb bud (Imokawa and Yoshizato, 1997), and when regenerating salamander limbs were treated with cyclopamine, only one digit-like structure formed—similar to hind-limbs of Shh mutant mice (Chiang et al., 1996). Recently, it has been demonstrated that Shh, which is expressed in the posterior part of the salamander blastema is part of a reciprocal feedback loop via Grem1 and Fgf8 that are expressed in the anterior part of the blastema (Nacu et al., 2016). This feedback loop is required for outgrowth of the blastema and closely recapitulates the epithelial-mesenchyme signaling network that drives embryonic limb development. The demonstration that two signals, which can act at a distance—Shh and Fgf8—drive limb regeneration is at odds with a long standing model in which direct cell-cell interactions stimulate intercalary growth to even out disparate positional identities between anterior and posterior parts of the blastema (French et al., 1976). The size of the limb blastema is about 10 times that of embryonic limb buds, therefore it is not clear whether these signals could indeed act over the large distances involved. Fate maps of the blastema showing which cells give rise to the digits and experiments addressing timing of specification of antero-posterior positional values could give important insights into whether digit regeneration is comparable to embryonic development. One possibility is that cells within a blastema maintain memory of their position along the antero-posterior axis and restore missing structures by a timing mechanism linked to proliferation. Evidence for such a cellular memory based on epigenetic modifications has been obtained in regenerating limb buds of Xenopus embryos (Hayashi et al., 2015). A timing model would dispense with difficulties in scaling long range gradients over considerable distances to restore missing positional values during regeneration and the role of Shh and Fgfs would be to maintain the outgrowth and the width of the blastema. It would also be useful to know the fate of polarizing region cells from embryonic urodele limb buds in adult limbs and regenerating limbs. Unlike urodeles, anuran amphibians can only regenerate their limbs during embryonic stages. Interestingly, increased methylation of the ZRS enhancer during Xenopus development correlates with reduced capacity to regenerate the limb in the adult suggesting that epigenetic mechanisms limit this process by preventing re-expression of Shh (Yakushiji et al., 2007). Shh SIGNALING AND LIMB REGENERATION EVOLUTIONARY ASPECTS OF Shh SIGNALING IN THE LIMB Adult urodele amphibians (newts and salamanders) can regenerate their limbs after amputation. Shh signaling occurs in adult urodele limbs during regeneration and understanding how Shh expression is activated in these adult tissues may be The ZRS element located in the fifth intron of Lmbr1 gene that drives limb-specific Shh expression is well conserved at the sequence level in many vertebrates. The ZRS is an excellent candidate for evolutionary modifications that have resulted in Frontiers in Cell and Developmental Biology | www.frontiersin.org 13 February 2017 | Volume 5 | Article 14 Tickle and Towers Shh in Developing Limbs in limbs of the theropod ancestors of birds (Vargas and Wagner, 2009). However, the Green Fluorescent Protein fate-mapping experiments in chick wings (see Figure 1c) showed that in fact digits with the identities 1, 2, and 3 arise from embryonic positions 1, 2, and 3 that are found in tissue adjacent to the polarizing region (Towers et al., 2011). Therefore, it is not necessary to invoke a frameshift and suggests that the digits 4 and 5 of the dinosaur hand were simply lost and that bird wing digits should be numbered 1, 2, and 3 in line with the fossil record, as is now generally accepted. As already mentioned, in the chick wing bud, the posterior necrotic zone overlaps with the polarizing region. In the chick wing bud, the posterior necrotic zone is much larger than the corresponding zone in chick leg and mouse limb buds (Fernandez-Teran et al., 2006). Therefore, the loss of the two posterior digits in birds might be based on evolutionary changes in Shh signaling, in particular the autoregulatory mechanisms by which Shh signaling regulates apoptosis in the posterior necrotic zone of the wing bud (Sanz-Ezquerro and Tickle, 2000) and also proliferation (Chinnaiya et al., 2014). Interestingly, a recent study showed that an extension of the posterior part of the apical ectodermal ridge in the absence of Shh signaling was sufficient to enable the polarizing region to give rise to a digit in the chick wing. In such buds, the posterior necrotic zone was lost and this was accompanied by a dramatic increase in proliferation of polarizing region cells (Pickering and Towers, 2016). Shh has also been implicated in digit loss in cow limbs in which only two digits form (3 and 4). It was revealed that Ptch1 is expressed in the very posterior of the bud and at low levels in response to Shh signaling, because of the degeneration of a cisregulatory enhancer. As a consequence, it is suggested that Shh fails to be sequestered and restricted to the posterior part of the cow limb bud resulting in more-or-less uniform Shh signaling which results in symmetrical and distally restricted anteroposterior gene expression patterns (Lopez-Rios et al., 2014). As a result, the two digits of the cow limb are also symmetrical and lateral digits are lost because the apical ectodermal ridge fails to extend sufficiently to support their outgrowth. Similarly, Ptch1 is also restricted to the posterior of the limb buds of pigs that develop four digits, two of which are prominent (digits 3 and 4; Cooper et al., 2014). However, camels do not display a posterior restriction and down-regulation of Ptch1 in their developing limb buds although they also produce two digits (3 and 4), suggesting another mechanism of digit loss (Cooper et al., 2014). An additional case of digit loss involves the limbs of different species of the Australian skink, Hemiergis (Shapiro, 2002). The shortened duration of Shh expression in these lizards correlates well with the extent of digit reduction—species with five digits express higher levels of Shh for a longer time than those with only two digits (Shapiro et al., 2003). Interestingly, digit reduction correlates with a reduction in cell proliferation. One possibility is that factors other than reduced Shh signaling could be involved. As yet no mutations have been reported in ZRS sequences of various Hemiergis clades. However, as further studies are required to understand how positional values are specified by Shh signaling in mammals and lizards, this means that it is difficult to interpret some of the patterns of evolutionary digit loss discussed in this section. changes in limb morphology because the rich diversity of limb morphologies could have evolved without affecting other features of the body plan. In support of this, mutations in the ZRS at a conserved ETS1 binding site in pythons have been described that appear to be responsible for the early loss of Shh expression and subsequent failure of limb bud outgrowth (Kvon, 2016; Leal, 2016). CRISPR/CAS9 gene editing approaches, in which the mouse ZRS was replaced by the python ZRS sequence, resulted in limb truncations similar to those obtained upon the complete removal of Shh function in the mouse limb (Kvon, 2016). As in pythons, Shh fails to be up-regulated in the hind-limbs of the spotted dolphin and is associated with reduced outgrowth, although the molecular basis of this has not been examined (Thewissen et al., 2006). Many described ZRS mutations to date, however, result in ectopic expression of Shh in the anterior part of the limb, and therefore the development of additional digits as in domesticated animals; for instance, Dorking’s (Bouldin and Harfe, 2009) and Silkie chickens (Dunn et al., 2011) have an additional anterior digit in the leg and dogs and cats (notably Hemingway cats) have extra anterior digits in their fore-paws (Lettice et al., 2008). Limbs with more than five digits have not been selected for during evolution suggesting there is little benefit in increasing digit number. Interestingly, the limbs of the earliest Devonian tetrapods such as Acanthostega and Icthyostega had up to eight digits (Clack, 2002). The mechanism by which such digit patterns would have been specified is of considerable interest. In having several digits, the limbs of such tetrapods superficially resemble the limbs of mouse Gli mutants, which have many digits that form by self-organization. However, the digits in these Devonian tetrapods display differences in phalangeal number suggestive of antero-posterior positional values specified by Shh in the early limb bud. Once pentadactyly was established in tetrapods, this has remained the basic plan, although occasionally limbs with so-called “sixth digits” have evolved. These sixth digits are in fact, adaptations of other limb bones, such as the overgrown wrist bone in the case of the mole’s “paddle-like” limb (Mitgutsch et al., 2012). The chick leg has retained the basic pentadactyl phalangeal pattern in digits 1–4 and therefore is of special interest to the evolution of digit patterns. As we discussed earlier, a model in which Shh signaling specifies different positional values is sufficient to explain chick leg patterning. Thus, any deviations away from this model in the mouse limb would therefore suggest a derived mode of patterning digits 1–4 in the mammalian lineage. Digit loss has commonly occurred over the course of evolution and alterations in Shh expression and response to Shh have been implicated. A striking example is seen in the wings of birds and the fore-limbs of their basal theropod dinosaur ancestors in which two digits have been lost during evolution (Sereno, 1999). Understanding this mode of digit loss has puzzled investigators for over 150 years because theropods appeared to have had digit identities 1, 2, and 3, but in the embryo at least, bird digits appear to arise from positions 2, 3, and 4 (Burke and Feduccia, 1997). Therefore, it was suggested in the so-called “frameshift” model that digits with the identities 1, 2, and 3 arise from positions 2, 3, and 4 of the bird wing (Wagner and Gauthier, 1999; Tamura et al., 2011), perhaps due to reduced Shh signaling levels/duration Frontiers in Cell and Developmental Biology | www.frontiersin.org 14 February 2017 | Volume 5 | Article 14 Tickle and Towers Shh in Developing Limbs FUTURE DIRECTIONS important to fill this gap in knowledge in order to apply the principles to developing human limbs and gain deeper insights into the basis of congenital limb defects and to evolutionary alteration in digit pattern. The analysis of the function of Shh in new animal models of limb development could help resolve issues regarding the relationship between positional values and digit identity. Further development of the CRISPR/Cas9 system should facilitate this. An issue of general relevance is the mode of Shh transport in the limb and how a graded distribution of Shh is established. This may require further refinement of in vivo imaging techniques to visualize directly the distribution of Shh in real time. It also seems clear that the timing of Shh expression is another critical parameter that still needs to be addressed. Disentangling the relationship between autoregulatory mechanisms of intrinsic timing of Shh expression and extrinsic mechanisms could shed light on processes that ensure robustness of limb development and pattern scaling between different species. It is now established that Shh has a pivotal function in vertebrate limb development and many details have been uncovered. Surprisingly however, there is still no consensus about how Shh specifies antero-posterior positional values in the limb. It remains possible that different combinations of transcription factors govern antero-posterior positional values, but it has been difficult to identify them because all the digits are made up of the same differentiated cell types. Therefore, a generegulatory network such as one operating downstream of Shh in the neural tube to specify distinct neural fates is unlikely to operate during limb development (Balaskas et al., 2012). It is also likely that the temporal regulation of the same sets of genes could contribute to specifying positional values. For instance, there is a clear relationship between Hoxd expression and thumb (digit 1) development, with cells that give rise to thumb the only cells that express Hoxd13 and not Hoxd12 (Vargas and Fallon, 2005). Therefore, since the cells that give rise to all the other digits express Hoxd12 and Hoxd13, a simple Hox code is unlikely to specify the digits, and perhaps timing of expression is the important determinant. 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Biol. 25, 1–47. doi: 10.1016/S0022-5193(69)80016-0 Wolpert, L. (1989). Positional information revisited. Development 107, 3–12. Frontiers in Cell and Developmental Biology | www.frontiersin.org Conflict of Interest Statement: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Copyright © 2017 Tickle and Towers. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. 19 February 2017 | Volume 5 | Article 14 

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The intrinsically disordered SARS-CoV-2 nucleoprotein in dynamic complex with its viral partner nsp3a

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To cite this version: Luiza Mamigonian Bessa, Serafima Guseva, Aldo Camacho-Zarco, Nicola Salvi, Damien Maurin, et al.. The intrinsically disordered SARS-CoV-2 nucleoprotein in dynamic complex with its viral partner nsp3a. Science Advances , 2022, 8 (3), pp.eabm4034-1-eabm4034-12. ￿10.1126/sciadv.abm4034￿. ￿hal- 03621049￿ The intrinsically disordered SARS-CoV-2 nucleoprotein in dynamic complex with its viral partner nsp3a Luiza Mamigonian Bessa, Serafima Guseva, Aldo Camacho-Zarco, Nicola Salvi, Damien Maurin, Laura Mariño Perez, Maiia Botova, Anas Malki, Max Nanao, Malene Ringkjøbing Jensen, et al. y p p p Luiza Mamigonian Bessa, Serafima Guseva, Aldo Camacho-Zarco, Nicola Salvi, Damien Maurin, Laura Mariño Perez, Maiia Botova, Anas Malki, Max Nanao, Malene Ringkjøbing Jensen, et al. INTRODUCTION replication and transcription remains poorly understood. It has been shown in mouse hepatitis virus (MHV) that this colocalization de- pends on an essential interaction with the N-terminal component of nonstructural protein 3 (nsp3) (15, 16). Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the origin of the respiratory coronavirus disease 2019 (COVID-19) pandemic, is a member of the betacoronavirus genus, also including Middle East respiratory syndrome (MERS)–CoV and SARS-CoV. In response to the rapid spread of this virus, it has become urgent to develop viral inhibitors that can reduce or eradicate symptoms. The processes of replication and transcription of viral RNA represent important targets for viral inhibition, and the development of ratio- nal strategies to achieve this end requires a molecular understand- ing of the viral replication cycle. nloaded from https://www.science.org at JOINT ILL-ESRF on April 08, 2022 p p Coronaviral N is highly dynamic, and three of the five domains (numbered N1 to N5 here; see Fig. 1A) are intrinsically disordered regions (IDRs), intercalated with two folded domains [N2 and N4, also known as N- and C-terminal domains (NTD and CTD)] (17). The structural features of betacoronaviral N are strongly conserved, with 90% sequence identity between SARS-CoV and SARS-CoV-2. N is dimeric and highly basic, and RNA binding is thought to be cooperative and to involve all five domains of N from SARS-CoV, where it plays a role in both chaperoning and RNA packaging (8, 18). Electron tomography identified numerous N-RNA complexes within SARS-CoV-2 virions, apparently associated with different segments of the 35-kb genomes (19, 20). The N3 IDR (175 to 263) comprises a serine-arginine (SR)–rich domain that is phosphorylated in in- fected cells, a modification that plays a role in both function and localization of SARS-CoV N (21). Coronaviridae are enveloped positive-sense single-strand RNA viruses that express their own replication machinery. Replication of the SARS-CoV-2 genome is carried out by the RNA-dependent RNA polymerase complex, whose functional modes have been investigated by cryo–electron microscopy (1–3). Betacoronaviral replication-­ transcription complexes (RTCs) are associated with membrane networks in the form of viral replication organelles called double-­ membrane vesicles (DMVs) constituted from membranes partici- pating in the host secretory pathway (4). DMVs have been shown to constitute active sites of viral RNA synthesis (5, 6). JOINT ILL-ESRF on April 08, 2022 The folded domains N2 and N4 are not thought to interact with each other in SARS-CoV (17). HAL Id: hal-03621049 https://hal.univ-grenoble-alpes.fr/hal-03621049v1 Submitted on 13 Jul 2022 L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. SCIE N C E A D V A NCES | RESEA R CH A RT ICL E The intrinsically disordered SARS-CoV-2 nucleoprotein in dynamic complex with its viral partner nsp3a Luiza Mamigonian Bessa1†, Serafima Guseva1†, Aldo R. Camacho-Zarco1†, Nicola Salvi1†, Damien Maurin1, Laura Mariño Perez1, Maiia Botova1, Anas Malki1, Max Nanao2, Malene Ringkjøbing Jensen1, Rob W. H. Ruigrok1, Martin Blackledge1* The processes of genome replication and transcription of SARS-CoV-2 represent important targets for viral inhibition. Betacoronaviral nucleoprotein (N) is a highly dynamic cofactor of the replication-transcription complex (RTC), whose function depends on an essential interaction with the amino-terminal ubiquitin-like domain of nsp3 (Ubl1). Here, we describe this complex (dissociation constant - 30 to 200 nM) at atomic resolution. The interaction implicates two linear motifs in the intrinsically disordered linker domain (N3), a hydrophobic helix (219LALLLLDRLNQL230) and a disordered polar strand (243GQTVTKKSAAEAS255), that mutually engage to form a bipartite interaction, folding N3 around Ubl1. This results in substantial collapse in the dimensions of dimeric N, forming a highly compact molecular chaperone, that regulates binding to RNA, suggesting a key role of nsp3 in the association of N to the RTC. The identification of distinct linear motifs that mediate an important interaction between essential viral factors provides future targets for development of innovative strategies against COVID-19. C O R ONAVIRUS Copyright © 2022 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution License 4.0 (CC BY). 1Université Grenoble Alpes, CNRS, CEA, IBS, F-38000 Grenoble, France. 2Structural Biology Group, European Synchrotron Radiation Facility, F-38000 Grenoble, France. *Corresponding author. Email: [email protected] †These authors contributed equally to this work. Bessa et al., Sci. Adv. 8, eabm4034 (2022) 19 January 2022 1Université Grenoble Alpes, CNRS, CEA, IBS, F-38000 Grenoble, France. 2Structural Biology Group, European Synchrotron Radiation Facility, F-38000 Grenoble, France. *Corresponding author. Email: [email protected] †These authors contributed equally to this work. SCIE N C E A D V A NCES | RESEA R CH A RT ICL E Fig. 1. Figurative representation of N and nsp3a SARS-CoV-2 proteins. (A) Domain representation of N. Domain N1 is intrinsically disordered. Domain N2 [otherwise known as N-terminal domain (NTD) or RNA binding domain], whose recently determined x-ray crystallographic structure is shown here [PDB code 6m3m (23)]. Domain N3 is also intrinsically disordered, comprising a serine-arginine (SR)–rich region, followed by a short helix. Domain N4 [also known as the C-terminal domain (CTD] is re- sponsible for dimerization of N [structure shown is taken from the crystal structure (28), PDB code 3wzo]. The basic domain N5 is also intrinsically disordered. (B) Nsp3a. The N-terminal domain of nsp3a has a ubiquitin-like fold [the NMR structure of nsp3a of SARS-CoV (PDB code 2idy) is shown here (49)]. The C-terminal domain of nsp3a is intrinsically disordered and strongly acidic. (C) ITC of sUbl1 (16 to 111) in complex with sN3 (191 to 263) (235F or alpha variant). These domains represent the necessary domains for interaction between N and nsp3a (fig. S1 and table S1). Fig. 1. Figurative representation of N and nsp3a SARS-CoV-2 proteins. (A) Domain representation of N. Domain N1 is intrinsically disordered. Domain N2 [otherwise known as N-terminal domain (NTD) or RNA binding domain], whose recently determined x-ray crystallographic structure is shown here [PDB code 6m3m (23)]. Domain N3 is also intrinsically disordered, comprising a serine-arginine (SR)–rich region, followed by a short helix. Domain N4 [also known as the C-terminal domain (CTD] is re- sponsible for dimerization of N [structure shown is taken from the crystal structure (28), PDB code 3wzo]. The basic domain N5 is also intrinsically disordered. (B) Nsp3a. The N-terminal domain of nsp3a has a ubiquitin-like fold [the NMR structure of nsp3a of SARS-CoV (PDB code 2idy) is shown here (49)]. The C-terminal domain of nsp3a is intrinsically disordered and strongly acidic. (C) ITC of sUbl1 (16 to 111) in complex with sN3 (191 to 263) (235F or alpha variant). These domains represent the necessary domains for interaction between N and nsp3a (fig. S1 and table S1). ownloaded from https: https://www.science.org at JOINT ILL-ESRF on April 08, 2022 which are present in N3 (30). N2 and full-length N have been shown to bind to different RNAs (25, 35, 36). different domains of nsp3 as interaction partners (51). SCIE N C E A D V A NCES | RESEA R CH A RT ICL E Notably, de- letion of Ubl1 results in abrogation of viral replication (15) in MHV, while the conserved highly acidic IDR was dispensable for replica- tion. The interaction between nsp3a and N from MHV was investi- gated using NMR (50), revealing apparent cooperative binding of N2 and N3, and a possible inhibitory role of N1. Nsp3a was also shown to bind single-stranded RNA in SARS-CoV (49), and inter- action between the transcription regulatory sequence RNA (52) and N was suggested to inhibit the N:nsp3a interaction (50). Ubl1 from SARS-CoV-2 was recently found to partition into liquid droplets comprising N and RNA (39). X-ray crystallography revealed that the 3D structure of Ubl1 in SARS-CoV-2 [Protein Data Bank (PDB) code 7kag] is very similar to that found in SARS-CoV and MHV, while NMR shows that the structure is retained in solution and that the acidic domain is disordered (53). N from SARS-CoV-2 has been shown to undergo liquid-liquid phase separation (LLPS), forming biomolecular condensates upon mixing with RNA (31, 34, 35, 37–41). LLPS, mediated by dynamic interactions involving IDRs or RNA, offers an efficient mechanism to spatially and temporally control biochemical processes in the cell (42). In negative-sense single-stranded RNA viruses, such as rabies (43) and measles (44), components involved in viral replication have been shown to form functional membraneless condensates. In positive-sense RNA coronaviruses, there is currently no direct evi- dence that LLPS is related to either replication or the formation of DMVs, although it has been shown that SARS-CoV-2 RNA poly- merase can localize to N:RNA droplets (34). Phosphorylation of N3 in the SR region was also shown to modulate the biophysical properties of these droplets (35, 39–41). Nsp3 is one of the nonstructural viral proteins that are essential for formation of DMVs in MERS-CoV (54), and is thought to play a role in trafficking N to the RTC, colocalizing with N in the vicinity of DMVs (12). Recent investigation of DMVs using cellular cryo– electron tomography (4) identified molecular pores that allow the import and export of the replication/transcription substrate and product, further highlighting the importance of this interaction. The pores are shown to be constituted of nsp3 oligomers, with nsp3a protruding into the cytosol in the vicinity of putative N oligomers, suggesting that interaction with N may regulate RNA exit and en- capsidation (4). INTRODUCTION N4 is responsible for homodimeriza- tion (17) and, together with N5, is thought to contribute to the for- mation of higher-order assemblies that constitute the nucleocapsid (22). N2 has been investigated in isolation by x-ray crystallography (23, 24) and nuclear magnetic resonance (NMR) spectroscopy (25), and its interaction with RNA has been investigated by NMR (25–27). The structure of N4 has been determined by x-ray crystallography (24, 28), and N was shown to form dimers or higher-order oligomers under different experimental conditions (28–30). Molecular modeling and single-molecule fluorescence resonance energy transfer (31) were used to probe the dynamic nature of N. The disordered character of domains N1 and N3 was verified using NMR spectroscopy (32, 33), revealing the presence of a central helical element in N3 and the flexible nature of both domains in the presence of N2. A recent study also characterized the isolated SR region of N3 in its phos- phorylated and nonphosphorylated forms (34). Mass spectrometry was used to identify a number of autocatalytic sites in N, two of The nucleoprotein of SARS-CoV-2 (N) is an essential cofactor of the replication machinery (7, 8), encapsidating the viral genome, providing protection from the host cell environment, and playing an essential role in regulating gene transcription (9). N is produced at high levels in infected cells, making it an important marker for infection, and has also been linked to the perturbation of numerous host processes (8). Understanding the conformational behavior and function of N is therefore important for both potential therapeutic and diagnostic treatment of COVID-19 (10) as well as vaccine development (11). N colocalizes to the RTC in betacoronaviruses (12–14), although the molecular basis of its role in regulation of 1 of 12 1 of 12 Bessa et al., Sci. Adv. 8, eabm4034 (2022) 19 January 2022 SCIE N C E A D V A NCES | RESEA R CH A RT ICL E Bessa et al., Sci. Adv. 8, eabm4034 (2022) 19 January 2022 Intrinsically disordered N3 folds around Ubl1 via two separate linear motifs 15 13 A series of 15N- and 13C-edited 3D NOESY (nuclear Overhauser effect spectroscopy) experiments were performed to identify inter- and intramolecular contacts (Fig. 2G). Although exchange occurring on different time scales affected spectral quality, it was possible to ex- tract both intra- and intermolecular distance restraints that allowed us to determine the structure of the complex. Approximately 50% of side chains were assigned, and 54 intermolecular distance restraints and 543 intramolecular restraints were extracted. The alpha variant of SARS-CoV-2 comprises a mutation at position S235F in N. CSPs between sN3 and sUbl1 are not affected by this mutation (fig. S4), although NOESYs were slightly higher quality. Data were therefore combined with those of the ancestral form to determine the struc- tures of the complex. Additional distance information was derived from site-specific 13C labeling of A220I (Fig. 2G). The Ubl1 domain is slightly better defined [root mean square deviation (rmsd), 1.41 ± 0.26 Å] compared to the two binding sites (1.80 ± 0.42 Å) (Fig. 3, A and B). The structured part of the complex, defined as Ubl1(17 to 106), N(219 to 232), and N(245 to 254), has a backbone rmsd of 0.70 Å with respect to the medoid structure. oaded from https://www.science.org at JOINT ILL-ESRF on April 08, 2022 SCIE N C E A D V A NCES | RESEA R CH A RT ICL E The N cofactor nsp3 is the largest protein encoded in the SARS- CoV-2 genome, comprising 1945 amino acids, constituting up to 16 distinct domains (45), and playing an essential role in replication and transcription (46). The amino-terminal domains of nsp3, comprising a ubiquitin-like domain (Ubl1) and a highly acidic IDR, together con- stitute nsp3a (Fig. 1B) (47). Ubl1 was implicated in binding to the SR region of N in MHV (48), while a yeast two-hybrid approach implicated a hydrophobic linear motif slightly upstream of the SR region (16). The three-dimensional (3D) structures of Ubl1 from SARS-CoV (49) and MHV (50) have been determined by NMR. Yeast two-­ hybrid and glutathione S-transferase (GST) pull-down experiments identified viral proteins nsp8 and nsp9—components of the RTC—and The N cofactor nsp3 is the largest protein encoded in the SARS- CoV-2 genome, comprising 1945 amino acids, constituting up to 16 distinct domains (45), and playing an essential role in replication and transcription (46). The amino-terminal domains of nsp3, comprising a ubiquitin-like domain (Ubl1) and a highly acidic IDR, together con- stitute nsp3a (Fig. 1B) (47). Ubl1 was implicated in binding to the SR region of N in MHV (48), while a yeast two-hybrid approach implicated a hydrophobic linear motif slightly upstream of the SR region (16). The three-dimensional (3D) structures of Ubl1 from SARS-CoV (49) and MHV (50) have been determined by NMR. Yeast two-­ hybrid and glutathione S-transferase (GST) pull-down experiments identified viral proteins nsp8 and nsp9—components of the RTC—and Here, we use solution-state NMR, combined with isothermal titration calorimetry (ITC) and small-angle x-ray scattering (SAXS), 2 of 12 2 of 12 Bessa et al., Sci. Adv. 8, eabm4034 (2022) 19 January 2022 SCIE N C E A D V A NCES | RESEA R CH A RT ICL E all binding sites in both proteins (vide infra). Unless stated, these components were therefore used in subsequent assignment, relaxation, and structure determination experiments. to describe the interaction of N with nsp3a from SARS-CoV-2. We show that the interaction is mediated uniquely by the disordered N3 domain, which folds on the surface of Ubl1 via a bipartite interac- tion involving two distinct linear motifs. The two proteins form a high-affinity complex implicating a hydrophobic helix, which docks into a hydrophobic groove on Ubl1, and a second interaction site in the proximity of N4. RESULTS ITC id i ITC identifies the thermodynamic nature of the interaction Interaction between N and nsp3a was initially investigated using ITC. Different constructs of N were tested, comprising domains N123, N3, N234, N45, and short N3 (sN3, 191 to 263), in interaction with full- length nsp3a (1 to 206), Ubl1 (1 to 111), and short Ubl1 (sUbl1, 16 to 111) (Fig. 1, A and B). In all cases where interaction is observed, the thermodynamic nature of complex formation is conserved (fig. S1). The interaction exhibits dissociation constants in the range of 30 to 200 nM, depending on the exact constructs used (table S1). The enthalpic component dominates (20 to 30 kcal mol−1), with a much smaller and always negative entropic contribution (Fig. 1C and fig. S1). Very similar entropic and enthalpic contributions are mea- sured for the complexes comprising constructs containing sN3 and Ubl1, suggesting that the interaction site present in sN3 dominates the thermodynamics of complex formation. NMR identifies the interaction interfaces of the dynamic complex The backbone resonances of free N1 and N3 have been assigned (32), confirming the disordered nature of the two domains. NMR re- vealed the existence of a helical region in N3 (1), spanning residues 219 to 230 with local propensities varying between 35 and 70% [1 to 2 ppm (parts per million) secondary C shift] and no secondary structure elsewhere (six residues in 1 could not be assigned in the free form of N3). With the exception of the first 15 residues, including the SR region, resonances from N3 superpose with those from N123, suggesting that N3 is equally flexible in the isolated form and in the presence of N1 and N2. p The complex is mediated by two linear motifs of N3, the first comprises the central helix 1, that binds in the hydrophobic groove between C (65 to 70) and A (33 to 39) of Ubl1 (Fig. 3, C and D). The second motif, 13 amino acids downstream of 1, follows the polyQ strand and comprises a QTVT (glutamine-threonine-valine-­ threonine) motif followed by six residues (249 to 255) that fold into an -helical conformation (2) upon binding, located in the vicinity of the loop between the  sheet and the N terminus of Ubl1 helix B. These same residues are seen to form an  helix in the N-terminal region of the crystal structure of N4 (24, 28) and were shown to adopt an -helical conformation in solution studies of N4 (55). The structure of sUbl1 compares closely with the crystal structure (PDB 7kag) of Ubl1 (1.7 ± 0.2 Å; fig. S5), with the main differences located in the C and N termini of helices A and C, respectively. The backbone resonance assignment of nsp3a was also recently published (53), confirming the presence of a folded domain exhib- iting the same secondary structure as the crystal structure of Ubl1 and a C-terminal domain that is essentially devoid of secondary structure. 15N relaxation confirms the disordered nature of this do- main (fig. S2). The 15–amino acid N-terminal region of Ubl1 is also highly flexible in solution. All NMR experiments measured on N:nsp3a complexes were hindered by substantial exchange broadening. Highest-quality spec- tra were recorded on complexes comprising an N-terminal truncated Ubl1 (sUbl1: 16 to 111) and sN3, molecular constructs that comprise The 1 N3 interaction site is stabilized by extensive hydrophobic contacts with Ubl1. SCIE N C E A D V A NCES | RESEA R CH A RT ICL E The strand between the two binding sites and the remainder of N3 remain flexible in the complex, and domains N1, N2, N4, and N5, as well as the SR region, are not directly impli- cated in the interaction. Nevertheless, the wrapping of N3 around Ubl1 leads to the massive compaction of the otherwise highly flexi- ble N protein, suggesting that nsp3a is involved in trafficking and chaperoning N before encapsidation. Binding of short RNAs is also found to be abrogated in the compact Ubl1-bound form of N. The identification of linear motifs and binding sites that are important for viral function will provide active targets for the development of peptide-based viral inhibitors. NMR chemical shift perturbations (CSPs) were measured to map the interaction sites between the two proteins (Fig. 2). The inter- action is in the slow exchange regime on the NMR chemical shift time scale, compatible with the relatively tight affinity observed by ITC. CSPs in N3 are localized to two continuous regions of the IDR, helix 1 and the region following the poly-glutamine (polyQ) strand, spanning residues 243 to 255 (2) (Fig. 2C). Assignment of the backbone resonances of sN3 in complex with sUbl1 reveals that 1 binds in an -helical conformation, while 2 adopts a less well-defined conformation followed by a binding-induced  helix (Fig. 2D). 15N relaxation measured on sN3 in the presence of sUbl1 (Fig. 2, E and F) supports the existence of bipartite binding motifs, with elevated R2 and reduced R1 uniquely in 1 and 2. The disordered regions, in- cluding the SR-rich and polyQ regions, therefore remain flexible in the complex and do not appear to be directly involved in binding. 1 Addition of sN3 to 15N-labeled sUbl1 reveals extensive CSPs that implicate helices A (33 to 39) and C (69 to 75), as well as the sol- vent-exposed strand of the  sheet and helix B (51 to 66) (fig. S3). Bessa et al., Sci. Adv. 8, eabm4034 (2022) 19 January 2022 NMR identifies the interaction interfaces of the dynamic complex Methyl groups in the N-terminal poly-leucine 3 of 12 3 of 12 Bessa et al., Sci. Adv. 8, eabm4034 (2022) 19 January 2022 SCIE N C E A D V A NCES | RESEA R CH A RT ICL E NMR characterization of the sN3:sUbl1 complex. (A) 15N-1H correlation spectra showing chemical shift perturbations (CSPs) of the sUbl1 spectrum upon binding Exchange between free (blue) and bound (red) sUbl1 resonances is in the slow exchange regime. The spectrum shows a 1:1 complex at a concentration of 500 M K and was measured at 850 MHz. (B) 15N-1H correlation spectra showing CSP of the sN3 spectrum upon binding of sUbl1. Exchange between free (blue) and bound N3 resonances is again in the slow exchange regime. The spectrum shows a 1:1 complex at a concentration of 500 M at 298 K and was measured at 850 MHz. 1H CSP plotted as a function of sequence of sN3. Experimental conditions as in (B). Orange shading denotes the region of sN3 that could not be assigned in the m of the protein. (D) Secondary structure propensity of sN3 in 1:1 complex with sUbl1. 13C chemical shifts of approximately +3.0 ppm correspond to a fully formed E and F) 15N spin relaxation of sN3 in 1:1 complex with sUbl1 (500 M), measured at 950 MHz. Both transverse (R2, xy) and longitudinal (R1) relaxation identifies two nding sites separated by a highly flexible linker. (G) Sample strips from 15N- and 13C-edited NOESY-HSQC experiments, showing both inter- and intramolecular eaks reporting on the structure of the sUbl1-sN3 complex All mixing times shown were between 100 and 120 ms Downloaded from https://www.science.org at JOINT ILL-ESRF on April 08, 2022 Downloaded from https://www.science.org at JOINT ILL-ESRF on April 08, 2022 OINT ILL-ESRF on April 08, 2022 Fig. 2. NMR characterization of the sN3:sUbl1 complex. (A) 15N-1H correlation spectra showing chemical shift perturbations (CSPs) of the sUbl1 spectrum upon binding of sN3. Exchange between free (blue) and bound (red) sUbl1 resonances is in the slow exchange regime. The spectrum shows a 1:1 complex at a concentration of 500 M at 298 K and was measured at 850 MHz. (B) 15N-1H correlation spectra showing CSP of the sN3 spectrum upon binding of sUbl1. Exchange between free (blue) and bound (red) sN3 resonances is again in the slow exchange regime. NMR identifies the interaction interfaces of the dynamic complex The spectrum shows a 1:1 complex at a concentration of 500 M at 298 K and was measured at 850 MHz. (C) 15N-1H CSP plotted as a function of sequence of sN3. Experimental conditions as in (B). Orange shading denotes the region of sN3 that could not be assigned in the free form of the protein. (D) Secondary structure propensity of sN3 in 1:1 complex with sUbl1. 13C chemical shifts of approximately +3.0 ppm correspond to a fully formed helix. (E and F) 15N spin relaxation of sN3 in 1:1 complex with sUbl1 (500 M), measured at 950 MHz. Both transverse (R2, xy) and longitudinal (R1) relaxation identifies two rigid binding sites separated by a highly flexible linker. (G) Sample strips from 15N- and 13C-edited NOESY-HSQC experiments, showing both inter- and intramolecular cross peaks, reporting on the structure of the sUbl1-sN3 complex. All mixing times shown were between 100 and 120 ms. motif (219LALLLL224) of 1 interact with hydrophobic residues at the base of the groove, partially burying the helix (Fig. 3E) such that mainly polar and charged residues are exposed to the solvent. R225 and D226 of N3, situated on opposing sides of helix 1, point in the direction of Ubl1 E70 and K38, respectively, on either side of the groove, while the terminal residue E231 is adjacent to K63, suggesting that electrostatic interactions also play an important role in stabilizing binding (Fig. 3F). The second binding site involves the polar strand 244QTVT247 of N3, which binds in a weakly defined extended conformation, adjacent to the external  strand in Ubl1 (41 to 44), similar to mechanisms adopted by small ubiquitin-like modifier (SUMO)–interacting motifs (56). Interaction with Ubl1 results in large-scale compaction of N Interaction with Ubl1 results in large-scale compaction of N We investigated the structural nature of longer constructs of N and the impact of the interaction with Ubl1 on intrinsic conformational sampling. NMR spectroscopy of dimeric N234, comprising both folded domains (636 amino acids in total), exhibits a well-resolved 15N-1H correlation spectrum (Fig. 4A), allowing measurement of spin relaxation that identifies distinct dynamic properties of N2, N3, and N4 in the context of the longer construct (Fig. 4B). Not surprisingly, the RNA binding domain, N2, exhibits increased dynamics (lower transverse relaxation rates) compared to the di- merization domain N4, which shows the highest relaxation rates, while the disordered linker N3 exhibits the highest level of dynamics (considerably lower relaxation rates). The chemical shifts of N2 and N4 are indistinguishable from the chemical shifts of the isolated domains (fig. S11) (25, 55). These observations are consistent with the perception of N234 as folded domains tethered by a highly flexible SAXS was also measured on the N3:Ubl1 complex. An ensemble of conformations was generated, on the basis of the NMR bundle with the remaining region of N3 (175 to 215) sampling coil confor- mations in agreement with NMR chemical shifts and relaxation. This pool of conformers represents a more extended complex than mea- sured experimentally (fig. S8). Selection of a sub-ensemble of con- formers in agreement with the experimental SAXS data demonstrates that the complex is more compact, apparently involving additional transient contacts between the region 175 to 215 and the surface of Ubl1 (Fig. 3G and fig. S8). Cooperative binding of the two linear motifs forming a highly dynamic complex The terminal residue E231 is adjacent to K63. (G) Representation of the N3:Ubl1 complex that is in agreement with experimental SAXS data. The unfolded domain of N3 appears to transiently interact with the surface of Ubl1 to make a compact complex. (H) Repre- sentation of regions showing broadening on sUbl1 in the presence of spin-labeled C235. Orange surface represents region with PREs lower than 0.5. Position of C235 in the flexible loop is shown in red. aded from https://www.science.org at JOINT ILL-ESRF on April 08, 2022 .science.org at JOINT ILL-ESRF on April 08, 2022 N3 linker (234 to 243). Exchange spectroscopy (EXSY) experiments also reveal the presence of slow exchange of the second binding mo- tif for nine consecutive amino acids (243 to 251) between free and bound states, on a time scale of 40 to 100 s−1 (figs. S6 and S7 and Materials and Methods). Slow exchange is not seen for the first he- lix, indicating that the second binding site exchanges between free and bound states, while 1 is bound, consistent with the 1 site having a higher affinity, such that binding of the second site (2) is coop- erative and dependent on binding of 1. broadening observed on Ubl1 in the concave face formed between helices A (33 to 39) and C (65 to 70) and the  sheet (Fig. 3H and fig. S9). In addition, a truncated construct of sN3 (215 to 263) showed indistinguishable affinity for sUbl1 but induced slight CSPs relative to the longer construct, located uniquely on the opposing face of the protein (fig. S10), again supporting the determined orientation of the helix. N3 linker (234 to 243). Exchange spectroscopy (EXSY) experiments also reveal the presence of slow exchange of the second binding mo- tif for nine consecutive amino acids (243 to 251) between free and bound states, on a time scale of 40 to 100 s−1 (figs. S6 and S7 and Materials and Methods). Slow exchange is not seen for the first he- lix, indicating that the second binding site exchanges between free and bound states, while 1 is bound, consistent with the 1 site having a higher affinity, such that binding of the second site (2) is coop- erative and dependent on binding of 1. OINT ILL-ESRF on April 08, 2022 Cooperative binding of the two linear motifs forming a highly dynamic complex In total, the two interaction sites implicate a stretch of around 40 N3 amino acids when binding to Ubl1, although NMR spin relaxation shows that the two binding motifs are decoupled by a highly dynamic Bessa et al., Sci. Adv. 8, eabm4034 (2022) 19 January 2022 SCIE N C E A D V A NCES | RESEA R CH A RT ICL E Fig. 3. sN3 folds around sUbl1 upon binding via a bipartite binding motif. (A and B) Representation of the bundle of 10 structures determined from 594 experimen- tal nOes measured on 1:1 complexes of sN3:sUbl1. Two binding sN3 sites are identified and shown in blue (sUbl1 in green). The first site (1) binds in a hydrophobic groove. The second (2) binds to the solvent-accessible strand of the Ubl1  sheet (41AYTVE45) and then folds into a helix located in the vicinity of the N terminus of Ubl1 helix B. The disordered strand between the two binding sites on sN3 is shown in yellow. (C and D) Definition of the position of 1 (blue) in the hydrophobic groove situ- ated between helices A, the C terminus of B and C on sUbl1 (green). (E) Stabilization of the leucine-rich 1 helix of sN3 on sUbl1 via hydrophobic interactions with hy- drophobic side chains on the surface of sUbl1 (orange shading). (F) Electrostatic stabilization of the interaction site. R225 and D226 on opposing sides of sN3 helix 1 point in the direction of E70 and K38, respectively, on either side of the groove. The terminal residue E231 is adjacent to K63. (G) Representation of the N3:Ubl1 complex that is in agreement with experimental SAXS data. The unfolded domain of N3 appears to transiently interact with the surface of Ubl1 to make a compact complex. (H) Repre- sentation of regions showing broadening on sUbl1 in the presence of spin-labeled C235. Orange surface represents region with PREs lower than 0.5. Position of C235 in the flexible loop is shown in red. Fig. 3. sN3 folds around sUbl1 upon binding via a bipartite binding motif. (A and B) Representation of the bundle of 10 structures determined from 594 experimen- tal nOes measured on 1:1 complexes of sN3:sUbl1. Two binding sN3 sites are identified and shown in blue (sUbl1 in green). The first site (1) binds in a hydrophobic groove. Cooperative binding of the two linear motifs forming a highly dynamic complex The second (2) binds to the solvent-accessible strand of the Ubl1  sheet (41AYTVE45) and then folds into a helix located in the vicinity of the N terminus of Ubl1 helix B. The disordered strand between the two binding sites on sN3 is shown in yellow. (C and D) Definition of the position of 1 (blue) in the hydrophobic groove situ- ated between helices A, the C terminus of B and C on sUbl1 (green). (E) Stabilization of the leucine-rich 1 helix of sN3 on sUbl1 via hydrophobic interactions with hy- drophobic side chains on the surface of sUbl1 (orange shading). (F) Electrostatic stabilization of the interaction site. R225 and D226 on opposing sides of sN3 helix 1 point in the direction of E70 and K38, respectively, on either side of the groove. The terminal residue E231 is adjacent to K63. (G) Representation of the N3:Ubl1 complex that is in agreement with experimental SAXS data. The unfolded domain of N3 appears to transiently interact with the surface of Ubl1 to make a compact complex. (H) Repre- sentation of regions showing broadening on sUbl1 in the presence of spin-labeled C235. Orange surface represents region with PREs lower than 0.5. Position of C235 in the flexible loop is shown in red. Fig. 3. sN3 folds around sUbl1 upon binding via a bipartite binding motif. (A and B) Representation of the bundle of 10 structures determined from 594 experimen- tal nOes measured on 1:1 complexes of sN3:sUbl1. Two binding sN3 sites are identified and shown in blue (sUbl1 in green). The first site (1) binds in a hydrophobic groove. The second (2) binds to the solvent-accessible strand of the Ubl1  sheet (41AYTVE45) and then folds into a helix located in the vicinity of the N terminus of Ubl1 helix B. The disordered strand between the two binding sites on sN3 is shown in yellow. (C and D) Definition of the position of 1 (blue) in the hydrophobic groove situ- ated between helices A, the C terminus of B and C on sUbl1 (green). (E) Stabilization of the leucine-rich 1 helix of sN3 on sUbl1 via hydrophobic interactions with hy- drophobic side chains on the surface of sUbl1 (orange shading). (F) Electrostatic stabilization of the interaction site. R225 and D226 on opposing sides of sN3 helix 1 point in the direction of E70 and K38, respectively, on either side of the groove. Validation of the sN3:sUbl1 structure The theoretically accessible conformational sampling of Ubl1-bound N234 was modeled by superimposing the common 2 helix from our NMR ensemble and the crystal structure of N4 (Fig. 5C), the crystal structure of N2, and building the flexible, NMR-visible domains (174 to 217 and 231 to 243) (Fig. 5, D and E). Ten thousand such conformers were initially compared individually to the experimental SAXS data. SAXS from the bound state can only be reproduced by highly compact individual models of the complex, exhibiting interdomain contacts between N2 and either Ubl1 or N4 (Fig. 5F). SAXS is not capable of distin- guishing between such models, and the level of domain flexibility within this framework is still substantial in particular, N(174 to 217) and (231 to 243) remain highly dynamic. Notably, however, the available degrees of conformational freedom of the flexible region of N3 in the N234:sUbl1 complex would in theory allow a broad dis- tribution of radii of gyration (Fig. 5B), which is evidently not populated in solution. On the basis of the calculated distribution, the probability of such compact conformations being sampled by unbiased statistical coil sampling of the flexible region of N3 is estimated to be less than 1 in 103. While further detail of the physical basis of this compaction will We investigated the interaction of Ubl1 with dimeric N234, forming a complex of 860 amino acids in total, by observing 15N-labeled N234 in complex with unlabeled Ubl1. Although ITC measurements show a slightly reduced affinity (Fig. 1), possibly due to reduced accessibility of N3 to the binding site in the presence of N2 and N4, the interaction is maintained in the context of this lon- ger construct, with only amino acids preceding the 1 binding site and between the 1 and 2 binding sites remaining visible by NMR (Fig. 4, A and C). The remainder of the complex is undetected under our conditions, consistent with binding to a much larger ob- ject, causing extreme line broadening of amide resonances from N2, N4, and the two binding sites. The dynamic model of the unbound N234 dimer is supported by SAXS (Fig. 5A). The conformational behavior of free N234 was modeled using crystal structures of N4 (57) and N2 (24) and sam- pling of N3 in agreement with NMR chemical shifts (Fig. 5D). SAXS data were predicted from the ensemble of conformers rep- resenting this state. Bessa et al., Sci. Adv. 8, eabm4034 (2022) 19 January 2022 Validation of the sN3:sUbl1 structure of the linker domain with negligible contacts between N2 and N4 (29). The distribution of simulated radii of gyration varies in a range from 40 to 95 Å with a maximum population around 52 Å (Fig. 5B). On the basis of SAXS, binding of dimeric N234 to Ubl1 pro- vokes a major reduction of radius of gyration (Fig. 5A) (from 50 to 39 Å according to Guinier analysis). The theoretically accessible conformational sampling of Ubl1-bound N234 was modeled by superimposing the common 2 helix from our NMR ensemble and the crystal structure of N4 (Fig. 5C), the crystal structure of N2, and building the flexible, NMR-visible domains (174 to 217 and 231 to 243) (Fig. 5, D and E). Ten thousand such conformers were initially compared individually to the experimental SAXS data. SAXS from the bound state can only be reproduced by highly compact individual models of the complex, exhibiting interdomain contacts between N2 and either Ubl1 or N4 (Fig. 5F). SAXS is not capable of distin- guishing between such models, and the level of domain flexibility within this framework is still substantial in particular, N(174 to 217) and (231 to 243) remain highly dynamic. Notably, however, the available degrees of conformational freedom of the flexible region of N3 in the N234:sUbl1 complex would in theory allow a broad dis- tribution of radii of gyration (Fig. 5B), which is evidently not populated in solution. On the basis of the calculated distribution, the probability of such compact conformations being sampled by unbiased statistical coil sampling of the flexible region of N3 is estimated to be less than 1 in 103. While further detail of the physical basis of this compaction will of the linker domain with negligible contacts between N2 and N4 (29). The distribution of simulated radii of gyration varies in a range from 40 to 95 Å with a maximum population around 52 Å (Fig. 5B). (29). The distribution of simulated radii of gyration varies in a range from 40 to 95 Å with a maximum population around 52 Å (Fig. 5B). On the basis of SAXS, binding of dimeric N234 to Ubl1 pro- vokes a major reduction of radius of gyration (Fig. 5A) (from 50 to 39 Å according to Guinier analysis). Validation of the sN3:sUbl1 structure The orientation of helix 1 in the hydrophobic groove was inde- pendently verified using paramagnetic tags attached to cysteine labels in the flexible 232 to 243 linker (M234C and S235C), resulting in 5 of 12 Bessa et al., Sci. Adv. 8, eabm4034 (2022) 19 January 2022 SCIE N C E A D V A NCES | RESEA R CH A RT ICL E Fig. 4. Dynamics of N234 in free and Ubl1-bound forms. (A) 15N-1H correlation spectrum showing free (light blue) and Ubl1-bound (red) dimeric N234. All signals from N2, N4, and the two binding sites (1 and 2) are broadened beyond detection when bound to Ubl1. Spectra recorded at 850 MHz and at 298 K. (B) Transverse 15N spin relaxation measured on free N234 (850 MHz, 380 M), showing characteristic dynamic properties of the three domains. N4 exhibits the slowest rotational correlation time, while N2 retains substantial degrees of conformational flexibility relative to this, via the dynamic behavior of N3, which behaves like an intrinsically disordered domain. (C) Comparison of the sN3-sUbl1 and N234-sUbl1 1:1 complexes. The remaining observable resonances can be identified to correspond to the flexible regions of N3 (from 175 to 215 and 232 to 242). p 4 Dynamics of N234 in free and Ubl1-bound forms (A) 15N-1H correlation spectrum showing free (light blue) and Ubl1-bound (red) dimeric N234 All signals from Downloaded fro aded from https://www.science.org at JOINT ILL-ESRF on April 08, 2022 Fig. 4. Dynamics of N234 in free and Ubl1-bound forms. (A) 15N-1H correlation spectrum showing free (light blue) and Ubl1-bound (red) dimeric N234. All signals from N2, N4, and the two binding sites (1 and 2) are broadened beyond detection when bound to Ubl1. Spectra recorded at 850 MHz and at 298 K. (B) Transverse 15N spin relaxation measured on free N234 (850 MHz, 380 M), showing characteristic dynamic properties of the three domains. N4 exhibits the slowest rotational correlation time, while N2 retains substantial degrees of conformational flexibility relative to this, via the dynamic behavior of N3, which behaves like an intrinsically disordered domain. (C) Comparison of the sN3-sUbl1 and N234-sUbl1 1:1 complexes. The remaining observable resonances can be identified to correspond to the flexible regions of N3 (from 175 to 215 and 232 to 242). linker (sampling statistical coil conformations except for the 1 he- lical element), with no significantly populated interaction between these domains in solution. DISCUSSION require additional interdomain information, these data unambigu- ously reveal that Ubl1 binding results in a massive collapse of the conformational space available to N234. The molecular mechanisms underlying the function, intracellular trans- port, and molecular colocalization of the different components of the replication machinery of SARS-CoV-2 remain poorly described but represent major targets for inhibitory strategies to treat COVID-19. Using NMR, SAXS, and ITC, we describe the dynamic interaction between nsp3a and N from SARS-CoV-2 that plays a vital role in this process. Nsp3 is one the viral proteins that are critical for formation of DMVs (54), while N is essential for genome encap- sidation, protection of the viral genome from host immunity, as well as regulation of replication (8) and transcription (9). Nsp3 is thought to play a role in trafficking N to the RTC and colocalizes with N in the vicinity of DMVs (12). In MHV, N and nsp3a were shown to interact (15, 16, 50), an interaction mediated by the Ubl1 domain (15), which was also shown to be essential for viral replication. We also observe CSPs and local NMR relaxation induced in the disordered domain of nsp3a in the presence of N234 (fig. S12). This indicates that the charged regions in the acidic domain of nsp3a interact transiently with N234, possibly implicating the SR region of N3 or RNA binding sites on N2 and N4 (57). Validation of the sN3:sUbl1 structure This direct comparison (nonfitted) broadly reproduces experimental data (Fig. 5A) (the data suggest that the ensemble may even be slightly more extended than this), again demonstrating that all experimental data measured on the unbound N234 dimer are consistent with extensive conformational sampling 6 of 12 SCIE N C E A D V A NCES | RESEA R CH A RT ICL E Fig. 5. Binding of Ubl1 causes massive compaction of N234. (A) SAXS of N234, free (green) and Ubl1-bound (orange). Experimental data are broadly reproduced by a model with N3 sampling statistical coil conformations [see (E)] [blue curve (D)]. Binding of Ubl1 provokes a collapse in conformational sampling. These data can only be reproduced by compact conformations of N234:Ubl1 [examples in (F)] shown by the red curve. (B) Distribution of radii of gyration (Rg) of 10,000 models of N234 (red), randomly sampling conformational space defined by statistical coil sampling for N3 (except helix 1) (D). Blue: Distribution Rg of 10,000 models of N234 bound to Ubl1, randomly sampling conformational space defined by statistical coil sampling for the dynamic region of N3 [175 to 215 and 232 to 242 (E)]. Green dashed line shows the Rg measured from experimental SAXS, in agreement with highly compact models of N234:Ubl1 complex (F). (C) Representation of the core scaffold, comprising the sN3:sUbl1 complex and N4 dimer [PDB code 6wzo (28)]. Relative positions of N4 and sN3:sUbl1 were assembled by superposition of helix 2. (D) Statistically available conformational sampling of free N234 described in (B). Colors as in Fig. 1: magenta, N2 domains; pink, N4; orange, N4; red, N3. (E) Statistically available sampling of Ubl1-bound N234 described in (B). Colors as in (D): green, Ubl1. (F) Models that reproduce experimental SAXS data of Ubl1-bound N234. On the basis of our current experimental data, we cannot distinguish between different compact conformations. Color legend as in (E). Downloaded from https://www.science.org at JOINT ILL-ES Downloaded from https://www.science.org at JOINT ILL-ESRF on April 08, 2022 s://www.science.org at JOINT ILL-ESRF on April 08, 2022 Fig. 5. Binding of Ubl1 causes massive compaction of N234. (A) SAXS of N234, free (green) and Ubl1-bound (orange). Experimental data are broadly reproduced by a model with N3 sampling statistical coil conformations [see (E)] [blue curve (D)]. Binding of Ubl1 provokes a collapse in conformational sampling. Validation of the sN3:sUbl1 structure These data can only be reproduced by compact conformations of N234:Ubl1 [examples in (F)] shown by the red curve. (B) Distribution of radii of gyration (Rg) of 10,000 models of N234 (red), randomly sampling conformational space defined by statistical coil sampling for N3 (except helix 1) (D). Blue: Distribution Rg of 10,000 models of N234 bound to Ubl1, randomly sampling conformational space defined by statistical coil sampling for the dynamic region of N3 [175 to 215 and 232 to 242 (E)]. Green dashed line shows the Rg measured from experimental SAXS, in agreement with highly compact models of N234:Ubl1 complex (F). (C) Representation of the core scaffold, comprising the sN3:sUbl1 complex and N4 dimer [PDB code 6wzo (28)]. Relative positions of N4 and sN3:sUbl1 were assembled by superposition of helix 2. (D) Statistically available conformational sampling of free N234 described in (B). Colors as in Fig. 1: magenta, N2 domains; pink, N4; orange, N4; red, N3. (E) Statistically available sampling of Ubl1-bound N234 described in (B). Colors as in (D): green, Ubl1. (F) Models that reproduce experimental SAXS data of Ubl1-bound N234. On the basis of our current experimental data, we cannot distinguish between different compact conformations. Color legend as in (E). OINT ILL-ESRF on April 08, 2022 Formation of the Ubl1:N complex abrogates binding to short RNA strands (A) 1D NMR spectra of 100 M polyA hexameric RNA free (blue) and upon addition of 50 M N234 (red). Clear chemical shifts report on binding of RNA to N234. (B) One-­ dimensional NMR spectra of 100 M polyA hexameric RNA free (blue) and upon addition of 50 M N234-Ubl1 complex (orange). Formation of the complex abro- gates binding. It is interesting to speculate on the role of this interaction in traf- ficking N to the RTC. Two colocalization processes are thought to accompany replication of the viral genome and transcription of sub­ genomic regions. First, replication of betacoronaviral RNA has been shown to occur principally in DMVs (5, 6), formed from remodeling of host membranes upon interaction with viral proteins, including nsp3. A recent study using electron tomography revealed the presence of molecular pores in DMVs and identified nsp3 as a constituent of the architecture of the pore (4). Nsp3a was located at the terminus of prongs extending into the cytosolic side of the pore, with six copies of the protein protruding away from the pore at symmetry-related positions on the rim of the channel. Increased density in electron tomograms suggested accumulation of N localized at the exterior of the DMV, possibly to allow rapid encapsidation and/or participa- tion in the RTC. In view of the narrow dimensions of the exit chan- nel, it seems plausible that naked genomic RNA may exit the DMVs following synthesis, with encapsidation by N occurring in the im- mediate vicinity of the exit channel. An interaction between N and nsp3a may facilitate this process. between N and nsp3a, identifying N3 and Ubl1 as the essential com- ponents. The interaction has a relatively strong affinity, exhibiting dissociation constants between 30 and 200 nM depending on the exact constructs, and is dominated by enthalpic contributions. The essential N3 domain comprises a long intrinsically disordered SR-rich N terminus and a polar C terminus, flanking a central hydrophobic strand that exhibits strongly helical propensity (1), which appears to be conserved over betacoronaviridae (58). loaded from https://www.science.org at JOINT ILL-ESRF on April 08, 2022 ( ) NMR CSP and spin relaxation map the interaction site of N for Ubl1 to two distinct linear motifs in the central and C-terminal re- gions of N3. Formation of the Ubl1:N complex abrogates binding to short RNA strands The conformation of Ubl1 resembles that of the recently determined crystal structure (PDB code 7kag) and the NMR struc- tures of SARS-CoV (49) and MHV (50), with local differences in the N-binding sites. The structure of the complex reveals a bipartite interaction, which folds N3 around Ubl1, with the 1 helix binding in the hydrophobic groove formed between helices C (65 to 70) and A (33 to 39). The 1 interaction is further stabilized by electro- static contacts with surface residues of Ubl1. The extent of inter- molecular interaction corroborates the strongly enthalpic nature of complex formation. The 1 binding site is linked to the second (2) binding site via a flexible linker. 2 forms an extended con- formation that binds to the external strand of the  sheet of Ubl1 (41 to 44) and comprises a final helix whose orientation is less well defined. 2 coincides with the short helix found in the N terminus of the crystal structure of N4, suggesting that Ubl1 and N4 would lie in close proximity in the complex of dimeric N234 with Ubl1. In summary, 1 and 2 mutually engage to form a bipartite inter- action, folding N3 around Ubl1. The expected sampling radius of the remaining N-terminal SR-rich domain is also reduced, apparently due to transient interactions with Ubl1. OINT ILL-ESRF on April 08, 2022 y Second, N has been shown to colocalize in the vicinity of the RTC, forming membraneless organelles, either in the presence of viral RNA or under certain conditions in its isolated form. This process of LLPS has been associated with colocalization of other components of the replication machinery, for example, the RNA polymerase was recently found to partition into membraneless organelles comprising N and RNA (34). Although it is not yet known how droplet forma- tion may be related to the formation of DMVs and SARS-CoV-2 replication, the concentration of components of the RTC may present advantages in terms of RNA synthesis and protection from host de- fense strategies, as well as efficient encapsidation. In this respect, it is interesting to note that Ubl1 can be recruited into droplets formed by N in vitro (39), suggesting a mechanism by which a high- ly concentrated reservoir of N could be maintained in the vicinity of the DMV pore. Formation of the Ubl1:N complex abrogates binding to short RNA strands One of the key roles of N in viral function is to bind the viral RNA genome, forming nucleocapsid-like structures. We have investigated the impact of complex formation with Ubl1 on RNA binding to the N234 construct by adding short, polymeric RNA (polyadenosine, polyA). Interaction of RNA with N234 is observed by 1D NMR (Fig. 6A), an interaction that is abolished in the presence of Ubl1 (Fig. 6B). ITC demonstrates that N1, N2, N4, and N5 do not contribute significantly to the binding enthalpy or entropy of interaction Bessa et al., Sci. Adv. 8, eabm4034 (2022) 19 January 2022 7 of 12 7 of 12 SCIE N C E A D V A NCES | RESEA R CH A RT ICL E Fig. 6. Formation of the N234-Ubl1 complex abrogates binding of short RNAs. (A) 1D NMR spectra of 100 M polyA hexameric RNA free (blue) and upon addition of 50 M N234 (red). Clear chemical shifts report on binding of RNA to N234. (B) One-­ dimensional NMR spectra of 100 M polyA hexameric RNA free (blue) and upon addition of 50 M N234-Ubl1 complex (orange). Formation of the complex abro- gates binding. NMR measurements of domains N234, which is dimeric in solu- tion, reveal a flexible multidomain molecule, with the RNA binding (N2) and dimerization (N4) domains connected by highly dynamic N3 linker domains. SAXS of the N234:Ubl1 complex reveals that Ubl1 binding causes a massive contraction of the conformational space sampled by dimeric N234 in solution (Fig. 7). Modeling of the possible conformational sampling of N234 in the N234:Ubl1 com- plex demonstrates that the N2 domains must lie predominantly in close proximity to the folded domains of N4 or Ubl1. Such confor- mations are statistically highly unlikely when the remainder of N3 is flexible in the complex and therefore must be stabilized by as-yet unidentified interactions between N234 and Ubl1 domains. NMR data demonstrate that the complex, while compact, still exhibits considerable flexibility, with the linker regions, including the SR re- gion and the polyQ strand, remaining highly dynamic. It is not clear whether the exchange of the 2 binding site is maintained in the large complex, although this would be compatible with the existence of visible signals in the loop between 1 and 2. Fig. 6. Formation of the N234-Ubl1 complex abrogates binding of short RNAs. Bessa et al., Sci. Adv. 8, eabm4034 (2022) 19 January 2022 Formation of the Ubl1:N complex abrogates binding to short RNA strands It has recently been suggested that the region com- prising residues 210 to 246 is essential for phase separation (40) and that N4 and residues 210 to 246 provide the multivalency associated with LLPS. This is intriguing because this latter region coincides partially with the two linear motifs forming the interaction surface identified here (218 to 255), and tight interaction with nsp3a might be expected to interfere with the network of weak interactions sta- bilizing the droplet. The 2 site appears to bind weaker than 1, as revealed by the presence of exchange between bound and free states, while 1 remains bound, suggesting cooperative binding driven by the 1 binding site. We note that slow conformational exchange was also recently observed in helix 2 in the context of N4 in solution (55). It is not clear what role the two distinct binding sites play, although freeing the 2 site of nsp3a temporarily from the surface of N4 would allow extensive remodeling of the complex, for example, upon RNA binding. 8 of 12 Bessa et al., Sci. Adv. 8, eabm4034 (2022) 19 January 2022 SCIE N C E A D V A NCES | RESEA R CH A RT ICL E Fig. 7. Impact of binding of nsp3a on the conformational sampling of N234. Cartoon representation of the collapse in conformational sampling that occurs upon folding and binding of N3 on the surface of Ubl1, accompanied by further collapse of the position of N2 in the vicinity of the core scaffold formed by N3, N4, and Ubl1. Color legend as in Fig. 1: magenta, N2 domains; pink, N4; orange, N4; red, N3; green, nsp3a. Fig. 7. Impact of binding of nsp3a on the conformational sampling of N234. Cartoon representation of the collapse in conformational sampling that occurs upon folding and binding of N3 on the surface of Ubl1, accompanied by further collapse of the position of N2 in the vicinity of the core scaffold formed by N3, N4, and Ubl1. Color legend as in Fig. 1: magenta, N2 domains; pink, N4; orange, N4; red, N3; green, nsp3a. The remarkable transformation of the highly flexible N, into a much more compact conformation upon binding Ubl1, may suggest a role for nsp3 as a chaperone of N before encapsidation of the viral genome at the replication site, similarly to the role of phosphopro- teins in the negative-sense RNA paramyxoviridae (59). Formation of the Ubl1:N complex abrogates binding to short RNA strands This chap- eroning role could be related to the observed abrogation of RNA binding upon formation of the complex. Our experimental data demonstrate that the behavior of the N2 domains is strongly affected by formation of the compact complex with nsp3a, placing them in the vicinity of the N34:Ubl1 core of the complex and possibly pro- tecting available RNA binding sites. It is also possible that nsp3a bind- ing inhibits assembly of N into higher-order structures that form the nucleocapsids. In this dynamic complex, the long disordered acidic tail of nsp3a also interacts transiently with either N4 or N2, a mechanism that may provide a protection against nonspecific, or untimely, binding to RNA. Netherlands). Single-point mutations (sN3 A220I, M234C, S235C, and S235F) were made in-house by site-directed mutagenesis. Trans- formation was performed by heat shock, and proteins were expressed in E. coli AI cells (Thermo Fisher Scientific) for 5 hours at 37°C after induction at an optical density of 0.6 with 1 mM isopropyl--d-­ thiogalactopyranoside (IPTG). Cells were harvested by centrifuging at 5000 rpm and resuspended in 20 mM tris (pH 8.0) and 500 mM NaCl buffer; 1 M NaCl was used for N1–419, lysed by sonication, and centrifuged again at 18,000 rpm at 4°C. The supernatant was sub- jected to standard Ni purification. Proteins were eluted with 20 mM tris (pH 8), 500 mM NaCl, and 500 mM imidazole. Samples were then dialyzed against 20 mM tris (pH 8), 500 mM NaCl, and 5 mM 2-mercaptoethanol at room temperature overnight. Following TEV cleavage and removal of the excess N-terminal tag and TEV by Ni affinity, samples were concentrated and subjected to size exclusion chromatography (SEC; Superdex 75/200) in 50 mM Na-phosphate (pH 6.5), 250 mM NaCl, and 2 mM dithiothreitol (DTT) buffer (NMR and ITC buffer). ded from https://www.science.org at JOINT ILL-ESRF on April 08, 2022 In conclusion, we have determined the structure of the molecular complex between SARS-CoV-2 N and nsp3 proteins, which are both associated with viral replication and transcription. We identify two dis- tinct linear motifs that combine to wrap N3 around Ubl1, resulting in substantial collapse in the dimensions of N when bound to nsp3a. N3 remains locally dynamic, with the 12 site exchanging between free and bound conformations while 1 is bound. Formation of the Ubl1:N complex abrogates binding to short RNA strands Combination with the recently published structure of N4 dimer allows us to propose a model for the compaction of N resulting from interaction with Ubl1, providing a mechanism of chaperoning or colocalization of N be- fore encapsidation. Formation of this complex regulates binding to RNA, which may again play a chaperoning role in trafficking of N to the RTC. These observations, allied with the ability of nsp3a to localize in condensates formed by N, allow us to speculate on the role of the interaction in the replication cycle. The identification of two linear motifs and two interaction surfaces that are important for viral function provides previously unidentified active targets for the development of peptide-based strategies to combat COVID-19. The primary sequence of nsp3a from SARS-CoV-2 was extracted from National Center for Biotechnology Information (NCBI) genome entry NC_045512.2 [GenBank entry MN908947.3], and a gene codon-optimized for expression in E. coli was commercially synthesized (GenScript) and subcloned in a pET21b(+) vector. Two constructs corresponding to residues 1 to 111 (Ubl1) or 1 to 206 (nsp3a) were synthesized. Hexahistidine, GST, and TEV cleavage tags were introduced at the N terminus for the purposes of protein purification. sUbl1 (16 to 111) was cloned into a pESPRIT plasmid as described for the N constructs. The plasmids were trans- formed into BL21 (DE3) E. coli cells. Cells were grown at 37°C until OD600 (optical density at 600 nm) of 0.6 to 0.8, induced with IPTG, and incubated for 12 hours at 22°C. Bacteria were harvested by cen- trifugation, and the cell pellet was resuspended in buffer A [50 mM tris-HCl (pH 8.0) and 150 mM NaCl] with protease inhibitors (cOmplete, Roche). Cell lysis was performed by sonication, followed by centrifugation (45  min, 18,000  rpm at 5°C). Supernatant was subjected to a standard Ni purification. The protein was purified by affinity chromatography on Ni-NTA agarose (Thermo Fisher Scientific), washed with buffer A supplemented with 20 mM imidazole, and eluted with buffer A supplemented with 500 mM imidazole. Following TEV cleavage, samples were concentrated and subjected to SEC with a Hiload 16/600 Superdex 75 column (GE Healthcare) in NMR buffer [50 mM Na-phosphate (pH 6.5), 250 mM NaCl, 2 mM DTT]. For 15N and 13C isotope labeling, cells were grown in M9 minimal medium supplemented with 15N-NH4-Cl and MATERIALS AND METHODS Protein expression and purification Structure determination NMR structure calculations were performed using CNS software, using previously described simulated annealing and restrainted molecular dynamics protocols (71). Restraints were identified from 15N and 13C NOESY HSQC spectra in the presence of unlabeled partner proteins. Backbone dihedral angles in secondary structural elements were identified from 13C secondary chemical shift analysis, and hydrogen bonding in  sheet of sUbl1 was identified from interstrand nOes. Statistical analyses of the NMR ensemble were carried out by wwPDB dedicated software upon structure deposition in the PDB and BMRB. SAXS data measured on N3:Ubl1 were simulated by construct- ing disordered regions (residues 1 to 15 for Ubl1 and 175 to 217 and 258 to 263 for N3) onto the 10 members of the NMR ensemble (from residues 218 to 257 for N3), using the statistical coil algo- rithm flexible-meccano (72). SAXS curves were predicted using the program Crysol (see table S3) (73). Ensembles of conformations were selected from a pool of 10,000 such structures of the Ubl1:N3 complex using the algorithm ASTEROIDS (74). Data were also calculated from the entire ensemble for comparison. SAXS data measured on N234:Ubl1 were simulated by first constructing the core scaffold of the complex, comprising the sN3:sUbl1 complex and N4 dimer (PDB code 6wzo) (28). The relative positions of N4 and sN3:sUbl1 were assembled by superposition of helix 2. The proce- dure was repeated for each of the 10 NMR models that have slightly different helical orientations. Disordered regions of N3 (175 to 217) NMR structure calculations were performed using CNS software, using previously described simulated annealing and restrainted molecular dynamics protocols (71). Restraints were identified from 15N and 13C NOESY HSQC spectra in the presence of unlabeled partner proteins. Backbone dihedral angles in secondary structural elements were identified from 13C secondary chemical shift analysis, and hydrogen bonding in  sheet of sUbl1 was identified from interstrand nOes. Statistical analyses of the NMR ensemble were carried out by wwPDB dedicated software upon structure deposition in the PDB and BMRB. rg at JOINT ILL ESRF on Apri 13C- and 15N-edited 3D NOESY-HSQC experiments (64) were run with 15N and 15N, 13C-labeled sN3 in 1:1 complex with un- labeled sUbl, with 15N and 15N, 13C-labeled sUbl1 in 1:1 complex with unlabeled sN3 and 15N, 13C, 2D-labeled sN3 in 1:1 complex with unlabeled sUbl, and 15N and 15N, 13C, 2D-labeled Ubl1 in 1:1 com- plex with unlabeled N3. Small-angle x-ray scattering Before measurement, concentrated samples were dialyzed into fresh NMR buffer overnight. Samples of 1:1 complexes of N3(175 to 263):Ubl1(1 to 111) and N234(47 to 364):Ubl1(1 to 111) and N234(47 to 364) were diluted to 0.5, 1.0, and 2.0 mg/ml. SAXS mea- surements were acquired at the European Synchrotron Research Facility in Grenoble, France, on the beamline BM29 (see table S3) (70). Paramagnetic relaxation enhancement (PRE) effects were measured from the peak intensity ratios by comparing 15N-heteronuclear single-­ quantum coherence (HSQC) 2D spectra recorded on samples labeled with 2,2,6,6-tetramethylpiperidine 1-oxyl (TEMPO) and samples reduced by the addition of 2 mM ascorbic acid. Cysteine mutants of sN3 at positions 234 and 235 were reduced with 10 mM DTT at 4°C for 12 hours and dialyzed into 50 mM phosphate buffer (pH 7.0) containing 250 mM NaCl without DTT. Tenfold molar excess of 4-maleimido-TEMPO dissolved in dimethyl sulfoxide was added to the reduced cysteine mutants. The reaction was incubated for 2 hours at room temperature and then dialyzed into NMR buffer at 4°C for 12 hours to eliminate the excess of TEMPO. NMR EXSY An NOE buildup curve was measured by repeating a 2D NOESY experiment with the mixing time varying from 10 to 700 ms. The NOE buildup was followed by measuring the intensity of resolved exchange cross peaks and fitted using standard expressions describing longitudinal exchange processes (69). Buildups of nine exchange cross peaks associated with residues in the 2 binding site were fitted simultaneously, extracting common apparent exchange rate of 42 ± 13 s−1 and 1H longitudinal relaxation rate (R1) of 18 ± 3 s−1. Using expressions in (69) to account for the effect on the low (~1%) population of unbound 2 on the apparent exchange rate, we esti- mate that on-off dynamics of the 2 binding site is characterized by a rate constant kex ~ 100 s−1. NMR experiments All NMR experiments were done in 50 mM Na-phosphate (pH 6.5), 250 mM NaCl, and 2 mM DTT or 50 mM Na-phosphate (pH 6.5), 250 mM NaCl, and 1 mM tris(2-carboxyethyl)phosphine (TCEP) at 25°C unless stated otherwise. Experiments were acquired on Bruker spectrometers with 1H frequencies of 600, 700, 850, 950, and 1000 MHz. Spectra were processed with NMRPipe (61) and visual- ized using NMRFAM-Sparky (62). 1 1 g p y ( ) 15N R1 relaxation was measured at 298 K and a 1H frequency of 950 MHz using a spin lock of 1.5 kHz (63). Typical relaxation delays of 1, 20, 50, 70, 70, 90, 120, 160, and 200 ms and 0, 0.08, 0.2, 0.4, 0.6, 0.6, 0.8, 1.04, 1.4, and 1.8 s were used for 15N R1 and 15N R1 and included repetition of one delay. Relaxation rates were fitted using in-house soft- ware, and errors were estimated with noise-based Monte Carlo simulation. Paramagnetic relaxation enhancement (PRE) effects were measured from the peak intensity ratios by comparing 15N-heteronuclear single-­ quantum coherence (HSQC) 2D spectra recorded on samples labeled with 2,2,6,6-tetramethylpiperidine 1-oxyl (TEMPO) and samples reduced by the addition of 2 mM ascorbic acid. Cysteine mutants of sN3 at positions 234 and 235 were reduced with 10 mM DTT at 4°C for 12 hours and dialyzed into 50 mM phosphate buffer (pH 7.0) containing 250 mM NaCl without DTT. Tenfold molar excess of 4-maleimido-TEMPO dissolved in dimethyl sulfoxide was added to the reduced cysteine mutants. The reaction was incubated for 2 hours at room temperature and then dialyzed into NMR buffer at 4°C for 12 hours to eliminate the excess of TEMPO. 15N R1 relaxation was measured at 298 K and a 1H frequency of 950 MHz using a spin lock of 1.5 kHz (63). Typical relaxation delays of 1, 20, 50, 70, 70, 90, 120, 160, and 200 ms and 0, 0.08, 0.2, 0.4, 0.6, 0.6, 0.8, 1.04, 1.4, and 1.8 s were used for 15N R1 and 15N R1 and included repetition of one delay. Relaxation rates were fitted using in-house soft- ware, and errors were estimated with noise-based Monte Carlo simulation. SCIE N C E A D V A NCES | RESEA R CH A RT ICL E 13C6-d-glucose. For selective labeling of the isoleucine C1 methyl group in the sN3 A220I mutant, 2-ketobutyrate-4-13C (50 mg/liter; Sigma-­Aldrich) was added to unlabeled M9 medium 1 hour before induction (60). the shorter sN3 and sUbl1 constructs. To obtain a complete assign- ment of the 1 helix of sN3 in the complex, the S235F mutant of sN3 was used, for which 3D correlation spectra were of highest quality. 13C chemical shifts were compared to random coil values using the program SSP (68). Isothermal titration calorimetry Statistical analyses of the NMR ensemble were carried out by wwPDB dedicated software upon structure deposition in the PDB and Biological Magnetic Resonance Data Bank (BMRB). NMR struc- tures have been deposited in the Brookhaven PDB under the accession code 7pku. Assignment of NMR resonances has been deposited in the BMRB under the accession codes 51052 and 34661. ITC was measured using MicroCal iTC200 (GE healthcare) at 25°C. The titration experiments were performed by adding 2.0 l of aliquots of 200 M of the different N constructs (sN3, N3, N45, N234, and N123) into the microcalorimeter cell filled with 20 M of sUbl1 (16 to 111), Ubl1 (1 to 111), or Nsp3a (1 to 206). N constructs were titrated through 20 injections at 180-s intervals; the reaction mixture was continuously stirred at 750 rpm. The titration curve was fitted to the experimental data using Origin version 7.0 with the ITC plugin from MicroCal. All experiments were performed with both components in NMR and ITC buffer (see above). Protein expression and purification ll d h h All proteins were expressed in Escherichia coli BL21 (DE3) Novagen) or AI (Invitrogen). All N constructs, N3 (175 to 263), sN3 (191 to 263), xsN3 (215 to 419), N45 (255 to 419), N (1 to 419), and N234 (47 to 364), were cloned into a pESPRIT vector between the AatII and Nott1 cleavage sites with His6-tag and TEV (tobacco etch virus) protease cleavage sites at the N terminus (GenScript Biotech, The Bessa et al., Sci. Adv. 8, eabm4034 (2022) 19 January 2022 9 of 12 SCIE N C E A D V A NCES | RESEA R CH A RT ICL E REFERENCES AND NOTES Bárcena, A molecular pore spans the double membrane of the coronavirus replication organelle. Science 369, 1395–1398 (2020). 4. G. Wolff, R. W. A. L. Limpens, J. C. Zevenhoven-Dobbe, U. Laugks, S. Zheng, A. W. M. de Jong, R. I. Koning, D. A. Agard, K. Grünewald, A. J. Koster, E. J. Snijder, M. Bárcena, A molecular pore spans the double membrane of the coronavirus replication organelle. Science 369, 1395–1398 (2020). 28. Q. Ye, A. M. V. West, S. Silletti, K. D. Corbett, Architecture and self-assembly of the SARS- CoV-2 nucleocapsid protein. 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SCIE N C E A D V A NCES | RESEA R CH A RT ICL E SCIE N C E A D V A NCES | RESEA R CH A RT ICL E were built using the statistical coil algorithm flexible-meccano (72) and terminated with the known structure of N2. Twenty thousand conformations were individually compared to experimental SAXS data using Crysol (table S3) (73). and replication characterized by in situ cryo-electron tomography. Nat. Commun. 11, 5885 (2020). 21. T.-Y. Peng, K.-R. Lee, W.-Y. Tarn, Phosphorylation of the arginine/serine dipeptide-rich motif of the severe acute respiratory syndrome coronavirus nucleocapsid protein modulates its multimerization, translation inhibitory activity and cellular localization. FEBS J. 275, 4152–4163 (2008). Structure determination 13C-edited NOESY-HSQC was performed on selectively 13C isoleucine 1 methyl-labeled A220I mutant of sN3, and 2D NOESY experiments were performed on sUbl1 in 1:1 com- plex with sN3. NOESY mixing times of 75, 100, 120, and 150 ms were used. Side-chain assignment was achieved using HCC(CO)NNH (65) and HCCH-TOCSY (66) experiments. p p SAXS data measured on N3:Ubl1 were simulated by construct- ing disordered regions (residues 1 to 15 for Ubl1 and 175 to 217 and 258 to 263 for N3) onto the 10 members of the NMR ensemble (from residues 218 to 257 for N3), using the statistical coil algo- rithm flexible-meccano (72). SAXS curves were predicted using the program Crysol (see table S3) (73). Ensembles of conformations were selected from a pool of 10,000 such structures of the Ubl1:N3 complex using the algorithm ASTEROIDS (74). Data were also calculated from the entire ensemble for comparison. SAXS data measured on N234:Ubl1 were simulated by first constructing the core scaffold of the complex, comprising the sN3:sUbl1 complex and N4 dimer (PDB code 6wzo) (28). The relative positions of N4 and sN3:sUbl1 were assembled by superposition of helix 2. The proce- dure was repeated for each of the 10 NMR models that have slightly different helical orientations. Disordered regions of N3 (175 to 217) Backbone resonance assignment of resonances of the complex was initially obtained on 15N, 13C, 2H-labeled samples of N3 and Ubl1 in 1:1 complex with their respective unlabeled partners, using band-­ selective excitation short-transient (BEST)–type triple resonance experiments (67). These assignments were directly transferrable to 10 of 12 Bessa et al., Sci. Adv. 8, eabm4034 (2022) 19 January 2022 REFERENCES AND NOTES 1. Y. Gao, L. Yan, Y. Huang, F. Liu, Y. Zhao, L. Cao, T. Wang, Q. Sun, Z. Ming, L. Zhang, J. Ge, L. Zheng, Y. Zhang, H. Wang, Y. Zhu, C. Zhu, T. Hu, T. Hua, B. Zhang, X. Yang, J. Li, H. Yang, Z. Liu, W. Xu, L. W. Guddat, Q. Wang, Z. Lou, Z. Rao, Structure of the RNA- dependent RNA polymerase from COVID-19 virus. Science 368, 779–782 (2020). 24. Y. Peng, N. Du, Y. Lei, S. Dorje, J. Qi, T. Luo, G. F. Gao, H. 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Acknowledgments Funding: This work was supported by the European Research Council Advanced Grant DynamicAssemblies under the European Union’s Horizon 2020 research and innovation program (grant agreement number 835161) to M.B., GRAL (ANR-10-LABX-49-01) to M.B, and Agence Nationale de Recherche SARS2NUCLEOPROTEIN to M.B. The work used the platforms of the Grenoble Instruct-ERIC centre (ISBG; UMS 3518 CNRS-CEA-UJA-EMBL) with support from FRISBI (ANR-10-INSB-05-02) and GRAL (ANR-10-LABX-49-01) within the Grenoble Partnership for Structural Biology (PSB). A.R.C.-Z. received funding from the European Union’s Horizon 2020 research and innovation programme under Marie Skłodowska-Curie grant agreement no. 796490 and HFSP postdoctoral HFSP fellowship LT001544/2017. This work is carried out in the context of the COVID-19-NMR consortium. L.M.P. was funded by the Fondation pour la Recherche Medicale via a postdoctoral fellowship (contract SPF201909009258). Financial support from the IR-RMN-THC Fr3050 CNRS for conducting the research is gratefully acknowledged. The Institut de Biologie Structurale (IBS) acknowledges integration into the Interdisciplinary Research Institute of Grenoble (IRIG, CEA). This work used the BM29 Bio-SAXS beamline at the ESRF. Author contributions: S.G., L.M.B., A.R.C.-Z., N.S., and M.Bl. conceived the project and planned its execution. S.G., L.M.B., A.R.C.-Z., M.Bo., and N.S. ran NMR experiments. D.M., M.Bo., A.R.C.-Z., S.G., and L.M.B. prepared protein samples. M.Bl. and L.M.B. analyzed NOESY and determined NMR structure. A.R.C.-Z. ran ITC experiments. L.M.B., N.S., S.G., L.M.P., and M.Bl. assigned spectra. A.M. measured SAXS data. M.Bl. modeled SAXS data in terms of molecular ensembles. R.W.H.R., M.R.J., and M.N. provided support and advice on strategy. M.Bl. supervised the study and wrote the article. All authors contributed to the final version. 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Use of this article is subject to the Terms of service The intrinsically disordered SARS-CoV-2 nucleoprotein in dynamic complex with its viral partner nsp3a The intrinsically disordered SARS-CoV-2 nucleoprotein in dynamic complex with its viral partner nsp3a The intrinsically disordered SARS-CoV-2 nucleoprotein in dynamic complex with its viral partner nsp3a Luiza Mamigonian BessaSerafima GusevaAldo R. Camacho-ZarcoNicola SalviDamien MaurinLaura Mariño PerezMaiia BotovaAnas MalkiMax NanaoMalene Ringkjøbing JensenRob W. H. RuigrokMartin Blackledge Luiza Mamigonian BessaSerafima GusevaAldo R. Camacho-ZarcoNicola SalviDamien MaurinLaura Mariñ BotovaAnas MalkiMax NanaoMalene Ringkjøbing JensenRob W. H. RuigrokMartin Blackledge Sci. Adv., 8 (3), eabm4034. • DOI: 10.1126/sciadv.abm4034 Acknowledgments Baumeister, High-resolution structure and biophysical characterization of the nucleocapsid phosphoprotein dimerization domain from the Covid-19 severe acute respiratory syndrome coronavirus 2. 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Effect of lithium on ventricular remodelling in infarcted rats via the Akt/mTOR signalling pathways

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c⃝2017 The Author(s). This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons Attribution Licence 4 0 (CC BY) Tsung-Ming Lee1,2,3,4, Shinn-Zong Lin5 and Nen-Chung Chang4,6 1Department of Medicine, Cardiology Section, An Nan Hospital, China Medical University, Tainan, Taiwan; 2Department of Medicine, China Medical University, Taichung, Taiwan ; 3Cardiovascular Research Laboratory, China Medical University Hospital, Taichung, Taiwan.; 4Division of Cardiology, Department of Internal Medicine, School of Medicine, College of Medicine, Taipei Medical University, Taipei, Taiwan; 5Bioinnovation Center, Tzu Chi foundation; Department of Neurosurgery, Buddhist Tzu Chi General hospital, Tzu Chi University, Hualien, Taiwan; 6Division of Cardiology, Department of Internal Medicine, Taipei Medical University Hospital, Taipei, Taiwan Correspondence: Nen-Chung Chang ([email protected]) Activation of phosphoinositide 3-kinase (PI3K)/Akt signalling is the molecular pathway driv- ing physiological hypertrophy. As lithium, a PI3K agonist, is highly toxic at regular doses, we assessed the effect of lithium at a lower dose on ventricular hypertrophy after my- ocardial infarction (MI). Male Wistar rats after induction of MI were randomized to either vehicle or lithium (1 mmol/kg per day) for 4 weeks. The dose of lithium led to a mean serum level of 0.39 mM, substantially lower than the therapeutic concentrations (0.8–1.2 mM). Infarction in the vehicle was characterized by pathological hypertrophy in the remote zone; histologically, by increased cardiomyocyte sizes, interstitial fibrosis and left ventricu- lar dilatation; functionally, by impaired cardiac contractility; and molecularly, by an increase of p-extracellular-signal-regulated kinase (ERK) levels, nuclear factor of activated T cells (NFAT) activity, GATA4 expression and foetal gene expressions. Lithium administration mit- igated pathological remodelling. Furthermore, lithium caused increased phosphorylation of eukaryotic initiation factor 4E binding protein 1 (p-4E-BP1), the downstream target of mam- malian target of rapamycin (mTOR). Blockade of the Akt and mTOR signalling pathway with deguelin and rapamycin resulted in markedly diminished levels of p-4E-BP1, but not ERK. The present study demonstrated that chronic lithium treatment at low doses mitigates pathological hypertrophy through an Akt/mTOR dependent pathway. Bioscience Reports (2017) 37 BSR20160257 DOI: 10.1042/BSR20160257 Received: 16 July 2016 Revised: 23 December 2016 Accepted: 23 January 2017 c⃝2017 The Author(s). This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons Attribution Licence 4.0 (CC BY). access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons Attribution Introduction Pathological hypertrophy is associated with cardiomyocyte hypertrophy, interstitial fibrosis, cardiac dysfunction, left ventricular dilatation and increased expression of foetal genes such as atrial natriuretic peptide (ANP), β-myosin heavy chain (β-MHC) and skeletal α-actin [8,9]. Lithium has been the mainstay of treatment for bipolar disorder for more than 60 years. Lithium has been rec- ognized for its neuroprotective effects against diverse insults, such as ischaemia, both in vitro and in vivo [10,11]. Recently, lithium has been shown to activate insulin-like growth factor-1 [5], which in turn triggered PI3K/Akt sig- nalling pathways [12]. However, the mechanism whereby PI3K activation by lithium mediates ventricular remod- elling after MI is unknown. In contrast, previous studies have shown that lithium has an additive effect on cardiac hypertrophy in a model of abdominal aortic banding, a pathological hypertrophy [13]. The effect of lithium after MI on physiological compared with pathological hypertrophy is unknown. Lithium is highly toxic at regular doses and whether the subtherapeutic concentration is enough for optimal efficacy and acceptable toxicity remains controver- sial. Thus, the purpose of the present study was: (i) to investigate how lithium chloride (LiCl) at a low dose affects physiological or pathological hypertrophy during ventricular remodelling and (ii) to assess the axis of Akt/mTOR systems in a rat MI model. Animals Part 1 Male Wistar rats (250–300 g) were intubated and the anterior descending artery was ligated using a 6-0 silk, resulting in infarction at the left ventricle (LV) as previously described [4]. For surgery, haemodynamics measurements, elec- trophysiological studies and sacrifice, rats were intraperitoneally anaesthetized with ketamine (90 mg/kg body weight (BW)) and xylazine (9 mg/kg). Anaesthesia monitoring was by rear-foot reflexes before and during procedures, ob- servation of respiratory pattern and responsiveness to manipulations throughout the procedures. Twenty-four hours after ligation, rats were randomly assigned into either saline group (NaCl) or LiCl (1 mmol/kg per day). The drug was given orally by gastric gavage once a day. The drug was started 24 h after MI; during this window, the drug can max- imize benefits while minimizing the possibility of a direct effect on infarct size [14]. For chronic lithium treatment, rats were given water and saline ad libitum to prevent hyponatraemia caused by lithium-induced increased excretion of sodium. To evaluate general toxicity of lithium, BW was monitored weekly. Mortality rate and general conditions of the animals were also observed daily throughout the whole experiment. The study duration was designed to be 4 weeks because the majority of the myocardial remodelling process in the rat (70–80%) is complete within 3 weeks [14]. Sham rats underwent the same procedure except the suture was passed under the coronary artery and then removed. Sham operation served as controls. Introduction Ventricular remodelling is associated with cardiac physiological or pathological hypertrophy after myocardial infarction (MI), depending on interventional drugs [1]. Distinct signalling pathways are responsible for the development of cardiac pathological and physiological hypertrophy. Phys- iological hypertrophy is mediated primarily by the insulin-like growth factor-1/phosphoinositide 3-kinase (PI3K (p110α)) pathway [2]. Akt, a downstream target of PI3K, phosphorylates and ac- tivates the mammalian target of rapamycin (mTOR), which is central to cardiac physiological hy- pertrophy. Transgenics with a dominant-negative mutant of the PI3K subunit p110α or a disrup- tion of the Akt1 gene have virtually no signs of hypertrophy in response to exercise training [3], a kind of cardiac physiological hypertrophy. In contrast, pathological hypertrophy is mediated by G-protein-coupled receptors (GPCRs) following stimulation by hormones such as angiotensin II and endothelin-1, both of which are increased after MI [4]. Activation of GPCRs results in a number of downstream signalling events, such as activation of mitogen-activated protein kinases (MAPKs) (e.g. extracellular-signal-regulated kinase (ERK) 1/2 (ERK1/2)) and dephosphorylation of nuclear fac- tor of activated T cells (NFAT) transcription factors by calcineurin [5]. NFAT is not activated by Received: 16 July 2016 Revised: 23 December 2016 Accepted: 23 January 2017 Accepted Manuscript Online: 23 January 2017 Version of Record published: 27 March 2017 1 Bioscience Reports (2017) 37 BSR20160257 DOI: 10.1042/BSR20160257 physiologic stimuli, suggesting that activation of NFAT may specifically regulate pathological remodelling of the my- ocardium [6]. Thus, the PI3K/Akt axis seems more linked to physiological hypertrophy, whereas MAPK signalling and NFAT pathways participate in the development of the pathological hypertrophy. Physiological hypertrophy shows a normal cardiac structure with a relatively normal pattern of cardiac gene expression and improved cardiac function [7]. Pathological hypertrophy is associated with cardiomyocyte hypertrophy, interstitial fibrosis, cardiac dysfunction, left ventricular dilatation and increased expression of foetal genes such as atrial natriuretic peptide (ANP), β-myosin heavy chain (β-MHC) and skeletal α-actin [8,9]. physiologic stimuli, suggesting that activation of NFAT may specifically regulate pathological remodelling of the my- ocardium [6]. Thus, the PI3K/Akt axis seems more linked to physiological hypertrophy, whereas MAPK signalling and NFAT pathways participate in the development of the pathological hypertrophy. Physiological hypertrophy shows a normal cardiac structure with a relatively normal pattern of cardiac gene expression and improved cardiac function [7]. Materials and methods All rats received humane care and the experiment was approved and conducted in accordance with local institutional guidelines of the China Medical University for the care and use of laboratory animals and conformed with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. c⃝2017 The Author(s). This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons Attribution Licence 4.0 (CC BY). Morphometric determination of myocyte size and interstitial fibrosis Because ventricular remodelling after infarction is a combination of reactive fibrosis and myocyte hypertrophy, we measured cardiomyocyte sizes in addition to myocardial weight to avoid the confounding influence of non-myocytes on cardiac hypertrophy. For a detailed method, please refer to the Supplementary Material online. Laboratory measurement y Blood samples were collected from rats at the end of the study from the ascending aorta and serum was separated by centrifugation for the estimation of lithium levels using an EEL-flame photometer. The NFAT activity was analysed by ELISA according to the manufacturer’s instructions (TransAM NFAT Family Transcription Factor Assay Kit; Active Motif). Briefly, nuclear extracts were added to the wells of a 96-well plate that contained the immobilized oligonucleotide carrying an NFAT consensus site, 5′-AGGAAA-3′. Proteins bound to this immobilized oligonucleotide were detected by incubating with a primary antibody that recognizes active NFAT, followed by horseradish peroxidase–conjugated secondary antibody and were quantified by spectrophotometry at 450 nm with a reference wavelength of 650 nm. g Histological collagen results were confirmed by hydroxyproline assay adapted from Stegemann and Stalder [18]. The samples from remote areas were immediately placed in liquid nitrogen and stored at −80◦C until measurement of the hydroxyproline content. The results were calculated as hydroxyproline content per weight of tissue. Echocardiogram At 28 days after operation, rats were lightly anaesthetized with intraperitoneal injection of ketamine (45 mg/kg) and xylazine (5 mg/kg). Echocardiographic measurements were done using the GE Healthcare Vivid 7 Ultra-sound System (Milwaukee, WI) equipped with a 14-MHz probe. M-mode tracing of the LV was obtained from the parasternal long-axis view to measure LV end-diastolic diameter dimension (LVEDD), LV end-systolic diameter dimension (LVESD) and fractional shortening (FS, %). The wall tension index (WTI) was defined as the ratio (LVEDD/2*posterior wall thickness) as described previously [17]. WTI was measured in order to indirectly assess myocardial wall stress. After this, the rats quickly underwent haemodynamic measurement after systemic hepariniza- tion. Real-time RT-PCR of GATA4, ANP, β-MHC and skeletal α-actin mRNAs were quantified by real-time RT-PCR with cyclophilin as a loading control. The cardiac-specific transcription factor, GATA4, plays important roles in cardiac hypertrophy. For a detailed method, please refer to the Supplementary Material online. Western blot analysis of Ser473-p-Akt1, Akt1, Thr37/46-p-4E-BP1, 4E-BP1, Thr202/Tyr204-p-ERK1/2 and ERK1/2 Western blot analysis of Ser473-p-Akt1, Akt1, Thr37/46-p-4E-BP1, 4E-BP1, Thr202/Tyr204-p-ERK1/2 and ERK1/2 Western blot analysis of Ser473-p-Akt1, Akt1, Thr37/46-p-4E-BP1, 4E-BP1, Thr202/Tyr20 and ERK1/2 Samples were obtained from the remote zone at week 4 after infarction. Experiments were replicated three times and results were expressed as the mean value as described in detail in the Supplementary Material online. p pp y The primary antibodies used were as follows: p-Akt1 (Ser473), p-4E-BP1 (Thr37/46), total-4E-BP1, p-ERK1/2 (Thr202/Tyr204) and total ERK1/2 antibody (Cell Signaling Technology, Beverly, MA) and Akt1 and β-actin (Santa Cruz Biotechnology, Santa Cruz, CA). Real-time RT-PCR of GATA4, ANP, β-MHC and skeletal α-actin mRNAs were quantified by real-time RT-PCR with cyclophilin as a loading control. The cardiac-specific transcription factor, GATA4, plays important roles in cardiac hypertrophy. For a detailed method, please refer to the Supplementary Material online. Haemodynamics and infarct size measurements Haemodynamic parameters and infarct size were measured in anaesthetized rats at the end of the study as described in detail in the Supplementary Material online. Statistical analysis Results were presented as mean +−S.D. Comparisons among groups were assessed for significance by one-way ANOVA. When significant differences were detected, individual mean values were compared by Bonferroni’s post hoc test (SPSS, version 18.0, Chicago, IL). Probability values were two-tailed and P<0.05 was considered to be statis- tically significant. Part 2 Although results of the above study showed that LiCl significantly increased ventricular hypertrophy after infarction (see ‘Results’), the involved mechanism remained unclear. To rule out non-specific effect of lithium and confirm the importance of Akt and mTOR signalling in LiCl-induced hypertrophy, we employed deguelin (a specific Akt inhibitor) and rapamycin (an mTORC1 inhibitor) in an ex vivo experiment. Four weeks after induction of MI by coronary ligation, infarcted rat hearts were isolated and subjected to saline (NaCl), LiCl (0.4 mM) or a combination of LiCl and deguelin (10 μM, Sigma, St. Louis, MO) or LiCl and rapamycin (0.4 μM, Sigma, St. Louis, MO). Each heart was perfused with a non-circulating modified Tyrode’s solution as previously described [15]. Drugs were infused for 1 h. The doses of LiCl, deguelin and rapamycin used were as previously described [2,5,16]. At the end of the study, all hearts (n=5 per group) were used for Western blot of eukaryotic initiation factor 4E binding protein 1 (4E-BP1) and ERK in the remote zone (>2 mm outside the infarct). 2 c⃝2017 The Author(s). This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons Attribution Licence 4 0 (CC BY) Bioscience Reports (2017) 37 BSR20160257 DOI: 10.1042/BSR20160257 Bioscience Reports (2017) 37 BSR20160257 DOI: 10.1042/BSR20160257 Bioscience Reports (2017) 37 BSR20160257 DOI: 10.1042/BSR20160257 Lithium affects ventricular remodelling Lithium affects ventricular remodelling Differences in mortality rates between saline- and lithium-treated infarcted groups were not found throughout the study. Most of the mortalities occurred within the first hours after ligation, with deaths due to excessive infarction 3 c⃝2017 The Author(s). This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons Attribution Licence 4 0 (CC BY) c⃝2017 The Author(s). This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons Attribution Licence 4.0 (CC BY). Bioscience Reports (2017) 37 BSR20160257 DOI: 10.1042/BSR20160257 Table 1 Cardiac morphology, haemodynamics and serum lithium concentrations at the end of the study Sham Infarction treated with Parameters Saline LiCl Saline LiCl Number of rats 10 10 11 12 BW, g 418 +−11 355 +−17* 410 +−13 328 +−18† Heart rate, bpm 393 +−15 407 +−14 410 +−20 404 +−16 LVESP, mm Hg 104 +−5 105 +−5 97 +−8 96 +−7 LVEDP, mm Hg 5 +−2 5 +−2 20 +−6* 18 +−6* +dp/dt, mm Hg/s 7831 +−285 7918 +−312 2382 +−265* 3182 +−259*† –dp/dt, mm Hg/s 6723 +−302 6982 +−267 2281 +−287* 2871 +−254*† Infarct size, % . . . . . . 40 +−3 41 +−3 LVW/tibia, mg/cm 242 +−20 245 +−19 297 +−21* 286 +−22* RVW/tibia, mg/cm 67 +−12 62 +−14 92 +−14* 89 +−15* LungW/tibia, mg/cm 392 +−31 381 +−35 498 +−32* 450 +−29*† Li, mM . . . 0.35 +−0.05 . . . 0.39 +−0.06 Values are mean +−S.D. LVW, left ventricular weight; RVW, right ventricular weight. *P<0.05 compared with respective sham; †P<0.05 compared with saline-treated infarcted group. Table 2 Echocardiographic findings at the end of the study Sham Infarction treated with Parameters Saline LiCl Saline LiCl LVEDD, mm 5.6 +−0.2 5.5 +−0.2 8.9 +−0.3* 8.5 +−0.3*† LVESD, mm 3.4 +−0.1 3.5 +−0.1 7.1 +−0.2* 6.5 +−0.2*† LVPW, mm 1.4 +−0.1 1.5 +−0.2 1.9 +−0.2* 2.1 +−0.2* FS, % 39 +−2 36 +−2 19 +−3* 24 +−3*† WTI 2.01 +−0.08 1.83 +−0.07‡ 2.34 +−0.10* 2.02 +−0.09*† Values are mean +−S.D. Abbreviations are as in Table 1. LVPW, left ventricular posterior wall. *P<0.05 compared with respective sham; †P<0.05 compared with saline-treated infarcted group; ‡P<0.05 compared with saline-treated sham. Lithium affects ventricular remodelling Consistent with a previous study [19], the gain in BW in lithium-treated rats was less than that in the saline-treated rats despite there being no difference in weight at the start of the study. Four weeks after infarction, the infarcted area of the LV was very thin and was totally replaced by fully differentiated scar tissue. The weight of the LV inclusive of the septum remained essentially constant for 4 weeks between the two infarcted groups. The lung weight (LungW)/tibia ratio, an index of lung oedema, was significantly lower in the LiCl-treated infarcted group compared with that in the saline-treated infarcted group. The values of +dp/dt and –dp/dt were significantly higher in the LiCl-treated infarcted group compared with those in the saline-treated infarcted group. LV end-systolic pressure (LVESP), LV end-diastolic pressure (LVEDP) and infarct size did not differ between the two infarcted groups. and arrhythmia. No animals died due to lithium treatment. Relative heart weights corrected for tibia length at the end of the experimental period (12 weeks of age) are presented in Table 1. Consistent with a previous study [19], the gain in BW in lithium-treated rats was less than that in the saline-treated rats despite there being no difference in weight at the start of the study. Four weeks after infarction, the infarcted area of the LV was very thin and was totally replaced by fully differentiated scar tissue. The weight of the LV inclusive of the septum remained essentially constant for 4 weeks between the two infarcted groups. The lung weight (LungW)/tibia ratio, an index of lung oedema, was significantly lower in the LiCl-treated infarcted group compared with that in the saline-treated infarcted group. The values of +dp/dt and –dp/dt were significantly higher in the LiCl-treated infarcted group compared with those in the saline-treated infarcted group. LV end-systolic pressure (LVESP), LV end-diastolic pressure (LVEDP) and infarct size did not differ between the two infarcted groups. To characterize the cardiac hypertrophy on a cellular level, morphometric analyses of LV sections were performed (Figure 1a). Compared with saline-treated sham, saline-treated infarcted rats showed structural changes such as in- creased cardiomyocyte sizes (Figure 1b-b’), consistent with LV remodelling. LiCl-treated infarcted rats had a further increase in cardiomyocyte size compared with saline-treated infarcted rats. Fibrosis of the LV from the remote area was examined in tissue sections after Sirius red staining, as shown in Figure 1c-c’. c⃝2017 The Author(s). This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons Attribution Licence 4.0 (CC BY). Lithium affects ventricular remodelling Table 1 Cardiac morphology, haemodynamics and serum lithium concentrations at the end of the study Sham Infarction treated with Parameters Saline LiCl Saline LiCl Number of rats 10 10 11 12 BW, g 418 +−11 355 +−17* 410 +−13 328 +−18† Heart rate, bpm 393 +−15 407 +−14 410 +−20 404 +−16 LVESP, mm Hg 104 +−5 105 +−5 97 +−8 96 +−7 LVEDP, mm Hg 5 +−2 5 +−2 20 +−6* 18 +−6* +dp/dt, mm Hg/s 7831 +−285 7918 +−312 2382 +−265* 3182 +−259*† –dp/dt, mm Hg/s 6723 +−302 6982 +−267 2281 +−287* 2871 +−254*† Infarct size, % . . . . . . 40 +−3 41 +−3 LVW/tibia, mg/cm 242 +−20 245 +−19 297 +−21* 286 +−22* RVW/tibia, mg/cm 67 +−12 62 +−14 92 +−14* 89 +−15* LungW/tibia, mg/cm 392 +−31 381 +−35 498 +−32* 450 +−29*† Li, mM . . . 0.35 +−0.05 . . . 0.39 +−0.06 Values are mean +−S.D. LVW, left ventricular weight; RVW, right ventricular weight. *P<0.05 compared with respective sham; †P<0.05 compared with saline-treated infarcted group. y, haemodynamics and serum lithium concentrations at the end of the study Values are mean +−S.D. LVW, left ventricular weight; RVW, right ventricular weight. *P<0.05 compared with respective sham; †P<0.05 compared with saline-treated infarcted group. Table 2 Echocardiographic findings at the end of the study Sham Infarction treated with Parameters Saline LiCl Saline LiCl LVEDD, mm 5.6 +−0.2 5.5 +−0.2 8.9 +−0.3* 8.5 +−0.3*† LVESD, mm 3.4 +−0.1 3.5 +−0.1 7.1 +−0.2* 6.5 +−0.2*† LVPW, mm 1.4 +−0.1 1.5 +−0.2 1.9 +−0.2* 2.1 +−0.2* FS, % 39 +−2 36 +−2 19 +−3* 24 +−3*† WTI 2.01 +−0.08 1.83 +−0.07‡ 2.34 +−0.10* 2.02 +−0.09*† Values are mean +−S.D. Abbreviations are as in Table 1. LVPW, left ventricular posterior wall. *P<0.05 compared with respective sham; †P<0.05 compared with saline-treated infarcted group; ‡P<0.05 compared with saline-treated sham. Table 2 Echocardiographic findings at the end of the study Values are mean +−S.D. Abbreviations are as in Table 1. LVPW, left ventricular posterior wall. *P<0.05 compared with respective sham; †P<0.05 compared with saline-treated infarcted group; ‡P<0.05 compared with saline-treated sham. and arrhythmia. No animals died due to lithium treatment. Relative heart weights corrected for tibia length at the end of the experimental period (12 weeks of age) are presented in Table 1. Lithium affects ventricular remodelling Compared with sham, infarcted rats treated with saline had significant increased fibrosis, as evi- denced by increased collagen staining. The lithium-treated infarcted rats showed attenuated cardiac fibrosis com- pared with saline-treated infarcted rats. Measurement of hydroxyproline content mirrored the histological observa- tion (3.26 +−0.96% dry weight tissue in saline-treated infarcted rats compared with 2.48 +−0.67% dry weight tissue in lithium-treated infarcted rats, P<0.05). LV functional parameters were studied by echocardiography 28 days after surgical procedure (Table 2, Figure 2). Compared with sham-operated hearts, MI hearts showed structural changes such as increased LV diastolic and sys- tolic diameters, consistent with LV remodelling. Both LVEDD and LVESD in rats with MI were significantly reduced 4 ce Reports (2017) 37 BSR20160257 1042/BSR20160257 1. Western analysis of RhoA membrane fraction and cytosolic fraction from the border zone at day 3 after MI. resentative Masson trichrome-stained section of a vehicle-treated heart at 4 weeks after infarction (blue colour, from 12 to 4 o’c mm. (b) Representative cardiomyocytes (magnification 400×) and quantitative analysis of the cardiomyocyte sizes in the r taining with FITC-labelled wheat germ haemagglutinin of cross-sectional sections of myocardium; Bar =50 μm. (c) Repre Bioscience Reports (2017) 37 BSR20160257 DOI: 10.1042/BSR20160257 Bioscience Reports (2017) 37 BSR20160257 DOI: 10.1042/BSR20160257 Figure 1. Western analysis of RhoA membrane fraction and cytosolic fraction from the border zone at day 3 after MI. a) Representative Masson trichrome-stained section of a vehicle-treated heart at 4 weeks after infarction (blue colour, from 12 to 4 o’clock); Bar =2 mm. (b) Representative cardiomyocytes (magnification 400×) and quantitative analysis of the cardiomyocyte sizes in the remote one. Staining with FITC-labelled wheat germ haemagglutinin of cross-sectional sections of myocardium; Bar =50 μm. (c) Representa- ve sections from the remote area with Sirius Red staining (red, magnification 400×) at 4 weeks after infarction; Bar =50 μm. (S-Sa), aline-treated sham; (S-LiCl), LiCl-treated sham; (MI-Sa), saline-treated infarcted rat; (MI-LiCl), LiCl-treated infarcted rat. Each column and ar represents mean +−S.D. (n=5–6 per group). *P<0.05 compared with saline-treated sham; †P<0.05 compared with LiCl-treated sham; P<0.05 compared with saline-treated infarcted group analysis of RhoA membrane fraction and cytosolic fraction from the border zone at day 3 after MI. Figure 1. Western analysis of RhoA membrane fraction and cytosolic fraction from the border zone at da (a) Representative Masson trichrome-stained section of a vehicle-treated heart at 4 weeks after infarction (blue colour, from 12 to 4 o’clock); Bar =2 mm. Lithium affects ventricular remodelling (b) Representative cardiomyocytes (magnification 400×) and quantitative analysis of the cardiomyocyte sizes in the remote zone. Staining with FITC-labelled wheat germ haemagglutinin of cross-sectional sections of myocardium; Bar =50 μm. (c) Representa- tive sections from the remote area with Sirius Red staining (red, magnification 400×) at 4 weeks after infarction; Bar =50 μm. (S-Sa), saline-treated sham; (S-LiCl), LiCl-treated sham; (MI-Sa), saline-treated infarcted rat; (MI-LiCl), LiCl-treated infarcted rat. Each column and bar represents mean +−S.D. (n=5–6 per group). *P<0.05 compared with saline-treated sham; †P<0.05 compared with LiCl-treated sham; ‡P<0.05 compared with saline-treated infarcted group 5 c⃝2017 The Author(s). This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons Attribution Licence 4.0 (CC BY). c⃝2017 The Author(s). This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons Attribution Licence 4.0 (CC BY). Bioscience Reports (2017) 37 BSR20160257 DOI: 10.1042/BSR20160257 Figure 2. Representative M-mode image Representative M-mode image reveals a hypokinetic-to-akinetic anterior wall and LV dilation in the infarcted hearts treated with either saline (c) or LiCl (d). There are markedly dilated LVEDD and LVESD in the saline-treated infarcted group compared with those in the LiCl-treated infarcted group. Figure 2. Representative M-mode image g p g Representative M-mode image reveals a hypokinetic-to-akinetic anterior wall and LV dilation in the infarcted hearts treated with either saline (c) or LiCl (d). There are markedly dilated LVEDD and LVESD in the saline-treated infarcted group compared with those in the LiCl-treated infarcted group. Representative M-mode image reveals a hypokinetic-to-akinetic anterior wall and LV dilation in the infarcted hearts treated with either saline (c) or LiCl (d). There are markedly dilated LVEDD and LVESD in the saline-treated infarcted group compared with those in the LiCl-treated infarcted group. by LiCl compared with saline (P<0.05). LV FS was significantly higher in the LiCl-treated infarcted group compared with saline. A significant decrease in WTI was observed in the LiCl-treated infarcted group compared with saline (P<0.05). These data were corroborated by the results that +dp/dt and –dp/dt were significantly improved in the LiCl-treated infarcted group compared with saline. by LiCl compared with saline (P<0.05). LV FS was significantly higher in the LiCl-treated infarcted group compared with saline. A significant decrease in WTI was observed in the LiCl-treated infarcted group compared with saline (P<0.05). Lithium affects ventricular remodelling These data were corroborated by the results that +dp/dt and –dp/dt were significantly improved in the LiCl-treated infarcted group compared with saline. c⃝2017 The Author(s). This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons Attribution Licence 4.0 (CC BY). Lithium increases phosphorylation of Akt and 4E-BP1 Western blot showed that lithium treatment resulted in a significant increase (P<0.05) in relative p-Akt level of 32 +− 8% compared with 25 +−4% for relative level of p-Akt in saline-treated infarcted rats (Figure 3). Treatment with LiCl enhanced the 4E-BP1 phosphorylation by 138% (P<0.01) in the infarcted rats compared with the saline-treated rats. This effect of lithium treatment on the levels of 4E-BP1 phosphorylation was completely blocked in the presence of deguelin or rapamycin (Figure 4), implying the axis of Akt/mTOR in regulating 4E-BP1 activity. Lithium inhibits NFAT and ERK activities As expected, MI significantly increased NFAT-dependent transcription compared with sham (Figure 5a). Lithium administration significantly reduced the NFAT activity by 21% (P<0.05) in the infarcted rats compared with the saline-treated rats. These data indicate that a low concentration of lithium is efficient at selectively inhibiting impor- tant regulators involved in pathological hypertrophy (such as NFAT). In addition, the MI-induced up-regulation of the p-ERK levels was attenuated in the presence of lithium (Figure 3). Finally, to further assess the role of Akt/mTOR pathway in lithium-attenuated p-ERK levels, Western blot was performed on infarcted hearts treated with deguelin or rapamycin in an ex vivo model. As shown in Figure 4, neither deguelin nor rapamycin affected the ERK phosphorylation compared with lithium alone, implying the attenuated ERK levels after adding lithium is not related to Akt/mTOR pathway. 6 6 c⃝2017 The Author(s). This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons Attribution Licence 4.0 (CC BY). Bioscience Reports (2017) 37 BSR20160257 DOI: 10.1042/BSR20160257 Figure 3. Western blot in an in vivo study (Part 1 experiment) Western blots show the expression of p-Akt, p-4E-BP1 and p-ERK. There was a significant increase in the levels of p-Akt and p-4E-BP1 in the LiCl-treated infarcted rats compared with the saline-treated infarcted rats. Bar graphs represent the quantitative analysis and difference in the expression of p-Akt and p-4E-BP1, after they were normalized with corresponding total proteins respectively, in arbitrary units. The values are mean +−S.D. (n=5–6 per group). Experiments were replicated three times and results expressed as the mean value. S, sham; Sa, saline. *P<0.05 compared with saline-treated sham; †P<0.05 compared with LiCl-treated sham; ‡P<0.05 compared with saline-treated infarcted group. Figure 3. Western blot in an in vivo study (Part 1 experiment) Western blots show the expression of p-Akt, p-4E-BP1 and p-ERK. There was a significant increase in the levels of p-Akt and p-4E-BP1 in the LiCl-treated infarcted rats compared with the saline-treated infarcted rats. Bar graphs represent the quantitative analysis and difference in the expression of p-Akt and p-4E-BP1, after they were normalized with corresponding total proteins respectively, in arbitrary units. The values are mean +−S.D. (n=5–6 per group). Experiments were replicated three times and results expressed as the mean value. S, sham; Sa, saline. *P<0.05 compared with saline-treated sham; †P<0.05 compared with LiCl-treated sham; ‡P<0.05 compared with saline-treated infarcted group. Figure 4. Lithium inhibits GATA4 expression and foetal gene expressions of ANP, β-MHC and skeletal α-actin MI increased gene expressions of GATA4, ANP, β-MHC and skeletal α-actin (Figure 5b–e), as expected. Saline-treated infarcted hearts were found to significantly increase GATA4 expression as compared with saline-treated sham (1.28 +−0.11 compared with 0.76 +−0.15 in sham, P<0.05, Figure 3). LiCl treatment in post-infarcted hearts resulted in a decrease in GATA4 compared with saline treatment. The foetal gene expressions of ANP, β-MHC and skeletal α-actin showed similar changes to GATA4. Discussions Our data indicate for the first time that lithium at a low dose could be utilized to alleviate the pathological develop- ment of hypertrophy and improve adaptive physiological cardiac growth. These results were concordant for beneficial effects of lithium, as documented structurally by increase in myocyte sizes, molecularly by myocardial Akt/4E-BP1 levels and functionally by improvement of cardiac contractility. Our results were consistent with previous observation that enhanced PI3K activity by pharmacological intervention that had a beneficial impact against subsequent pressure overload by inhibiting pathological processes [20]. Thus, lithium acts as an activator of physiological hypertrophy and inhibited pathological hypertrophy. The present study provides several novel findings that increase our understanding of the signal transduction mech- anism of the cardioprotection afforded by ventricular remodelling. A low dose of lithium is efficient at selectively in- hibiting regulators involved in pathological hypertrophy (such as NFAT, ERK and GATA4), while enhancing pathways involved in physiological hypertrophy (Akt and 4E-BP1) as evidenced by three observations (Figure 6). Lithium inhibits NFAT and ERK activities NFAT activity and RT-PCR of GATA4, ANP, β-MHC and skeletal α-actin Each mRNA was corrected for an mRNA level of cyclophilin. Each column and bar represents mean +−S.D. *P<0.05 compared with saline-treated sham; †P<0.05 compared with LiCl-treated sham; ‡P<0.05 compared with saline-treated infarcted group. gure 5. NFAT activity and RT-PCR of GATA4, ANP, β-MHC and skeletal α-actin Figure 5. NFAT activity and RT-PCR of GATA4, ANP, β-MHC and skeletal α-actin Figure 5. NFAT activity and RT-PCR of GATA4, ANP, β-MHC and skeletal α-actin Each mRNA was corrected for an mRNA level of cyclophilin. Each column and bar represents mean +−S.D. *P<0.05 compared with saline-treated sham; †P<0.05 compared with LiCl-treated sham; ‡P<0.05 compared with saline-treated infarcted group. Each mRNA was corrected for an mRNA level of cyclophilin. Each column and bar represents mean +−S.D. *P<0.05 compared with saline-treated sham; †P<0.05 compared with LiCl-treated sham; ‡P<0.05 compared with saline-treated infarcted group. orrected for an mRNA level of cyclophilin. Each column and bar represents mean +−S.D. *P<0.05 compared with †P<0.05 compared with LiCl-treated sham; ‡P<0.05 compared with saline-treated infarcted group. Each mRNA was corrected for an mRNA level of cyclophilin. Each column and bar represents mean +−S.D. *P<0. saline-treated sham; †P<0.05 compared with LiCl-treated sham; ‡P<0.05 compared with saline-treated infarcted group Lithium inhibits NFAT and ERK activities Western blot in an ex vivo study (Part 2 experiment) Western blot analysis of 4E-BP1 and ERK to furthermore confirm the Akt and mTOR on kinase activity in homogenates of the LV from the remote zone in a rat-isolated infarcted heart model. A significantly increased p-4E-BP1 level is noted in the LiCl-treated group compared with that seen in the saline-treated group, which was attenuated after administering deguelin (a specific Akt inhibitor) and rapamycin (an mTORC1 inhibitor). The values are mean +−S.D. (n=5 per group). Experiments were replicated three times and results expressed as the mean value. *P<0.05 compared with saline-, LiCl-deguelin-, and LiCl-rapamycin-treated infarcted rats; †P<0.05 compared with saline-treated infarcted rats. c⃝2017 The Author(s) This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons Attribut Figure 4 Western blot in an ex vivo study (Part 2 experiment) Figure 4. Western blot in an ex vivo study (Part 2 experiment) Western blot analysis of 4E-BP1 and ERK to furthermore confirm the Akt and mTOR on kinase activity in homogenates of the LV from the remote zone in a rat-isolated infarcted heart model. A significantly increased p-4E-BP1 level is noted in the LiCl-treated group compared with that seen in the saline-treated group, which was attenuated after administering deguelin (a specific Akt inhibitor) and rapamycin (an mTORC1 inhibitor). The values are mean +−S.D. (n=5 per group). Experiments were replicated three times and results expressed as the mean value. *P<0.05 compared with saline-, LiCl-deguelin-, and LiCl-rapamycin-treated infarcted rats; †P<0.05 compared with saline-treated infarcted r t 7 7 c⃝2017 The Author(s). This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons Attribution Licence 4 0 (CC BY) c⃝2017 The Author(s). This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons Attribution Licence 4.0 (CC BY). Bioscience Reports (2017) 37 BSR20160257 DOI: 10.1042/BSR20160257 Figure 5. NFAT activity and RT-PCR of GATA4, ANP, β-MHC and skeletal α-actin Each mRNA was corrected for an mRNA level of cyclophilin. Each column and bar represents mean +−S.D. *P<0.05 compared with saline-treated sham; †P<0.05 compared with LiCl-treated sham; ‡P<0.05 compared with saline-treated infarcted group. Figure 5. c⃝2017 The Author(s). This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons Attribution Licence 4.0 (CC BY). Lithium shows beneficial effects at a low dose Recommendations for target serum lithium concentrations (0.8–1.2 mM) appear to have been originally derived from studies of the effect of lithium on various indexes such as mania recurrence [21]. Despite the obvious advan- tages of chronic lithium therapy, its clinical use is often curtailed by its narrow therapeutic index and its devastating 8 Bioscience Reports (2017) 37 BSR20160257 DOI: 10.1042/BSR20160257 Figure 6. Signalling cascades leading to physiological and pathological hypertrophy after infarction The diagram summarizes the histological, molecular and pharmacological evidence. Inhibition of these signalling pathways by their respec- tive inhibitors is indicated by the vertical lines. Our data suggest that lithium is efficient at selectively inhibiting important regulators involved in pathological hypertrophy (such as NFAT and ERK), while augmenting pathways involved in physiological hypertrophy (such as 4E-BP1). Figure 6. Signalling cascades leading to physiological and pathological hypertrophy after infarction The diagram summarizes the histological, molecular and pharmacological evidence. Inhibition of these signalling pathways by their respec- tive inhibitors is indicated by the vertical lines. Our data suggest that lithium is efficient at selectively inhibiting important regulators involved in pathological hypertrophy (such as NFAT and ERK), while augmenting pathways involved in physiological hypertrophy (such as 4E-BP1). overdose-induced toxicity. Furthermore, it should be noted that in spite of therapeutic lithium serum levels, wide vari- ations between serum lithium levels and intracellular concentrations of lithium have been reported [22,23]. However, low dose levels have scantly been assessed in animal and clinical studies. The present study showed lithium at a low dose can provide beneficial biological effects after MI. c⃝2017 The Author(s). This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons Attribution Licence 4.0 (CC BY). c⃝2017 The Author(s). This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons Attribution Licence 4 0 (CC BY) Lithium inhibits pathological remodelling p g g PI3K pathway can inhibit pathological growth in addition to promoting physiological growth. Akt is activated by PI3K (p110α) to induce physiological hypertrophy but is also activated in response to GPCR agonists, e.g. endothelin-1 via another PI3K isoform (p110γ) that induces pathological hypertrophy [2]. That is why the p-Akt levels were significantly higher after inducing MI (Figure 3). Furthermore, PI3K (p110α) signalling negatively reg- ulated GPCR-stimulated extracellular responsive kinase and Akt (via PI3K, p110) activation [16]. Thus, although there was similar activation of Akt between the two groups of lithium-treated sham rats and vehicle-treated infarcted rats, we assessed ERK1/2 activation. p-ERK was significantly increased in infarcted hearts but not changed in the lithium-treated sham, implying different downstream signalling pathways. Finally, in the infarcted rats, lithium ad- ministration reduced the p-ERK levels and NFAT activity compared with the saline group, implying the inhibitory effect of lithium on pathological hypertrophy. Our results were consistent with the notion that the PI3K/Akt axis is more linked to physiological hypertrophy, whereas MAPK signalling, in collaboration with the NFAT pathway, participates in the development of the pathological hypertrophy [30]. To more directly address this interpretation, molecular markers of pathological cardiac hypertrophy were analysed by mRNA. The data showed that ventricular remodelling was associated with the expression of ANP, β-MHC and skeletal α-actin in the heart. Dephosphorylated NFAT (increased activity) enters the nucleus where it interacts with GATA4 and causes transcriptional activation of hypertrophic foetal genes leading to cardiomyocyte hypertrophy [31]. These foetal gene expressions are inhibited after lithium administration, consistent with the results that lithium inhibited pathological remodelling. Other mechanisms Although the present study suggests that the mechanisms of lithium-induced physiological ventricular remodelling may be related to an Akt/mTOR axis, other pathways may take part in the effect of lithium. It may be supposed that lithium elicits cardioprotection, in part, through its ability to inhibit GSK-3β by increasing GSK-3β phosphorylation. Previous studies have shown that the lack of GSK-3β phosphorylation in response to pressure overload is associated with reduced hypertrophy and development of dilated cardiomyopathy, highlighting the important role of GSK-3β phosphorylation in the development of compensatory hypertrophy [32]. Thus, lithium may increase physiological cardiac hypertrophy by inhibiting GSK-3β activity. Lithium enhances physiological remodelling mTORC1 has been shown to play a crucial role in the regulation of 9 Bioscience Reports (2017) 37 BSR20160257 DOI: 10.1042/BSR20160257 Bioscience Reports (2017) 37 BSR20160257 DOI: 10.1042/BSR20160257 Bioscience Reports (2017) 37 BSR20160257 DOI: 10.1042/BSR20160257 cellular homoeostasis, growth and response to stress. However, its functional role is still under debate because differ- ent roles of mTORC1 have been suggested under various experimental conditions. The data from the pharmacolog- ical modulators of mTOR and the animal models with genetic modifications of the components of mTOR signalling pathway will be expected to be different because the degree of mTORC1 activation among models is different. The degree of mTORC1 activation and the mTORC1 physiological functions to be preserved to convert mTORC1 activa- tion from detrimental into beneficial during cardiac stress remains unclear. Indeed, our results were consistent with the previous findings, showing that the activation of mTORC1/4E-BPs axis plays a role in physiological hypertrophy [29]. c⃝2017 The Author(s). This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons Attribution Licence 4.0 (CC BY). c⃝2017 The Author(s). This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons Attribution Licence 4.0 (CC BY). Lithium enhances physiological remodelling Both lithium- and infarction-induced hypertrophy are associated with an increase in myocyte size but with distinct molecular and histological phenotypes. During ventricular remodelling after MI, pathological cardiac hypertrophy is characterized molecularly, by the induction of foetal genes, such as ANP, β-MHC and skeletal α-actin; histologically, by increased interstitial fibrosis and left ventricular dilatation; and functionally, by impaired cardiac contractility. Given the reduction in LV dilatation, better-preserved systolic function, reduction in WTI and decreased expression of foetal genes in the lithium treatment, this hypertrophy may be viewed as more ‘physiological’ than ‘pathological’. Given p-4E-BP1 is activated in the development of physiological hypertrophy, lithium-increased p-4E-BP1 levels are blunted to the level similar to the vehicle group when Akt1 is inhibited by deguelin, implying that Akt is essential for the development of physiological hypertrophy. Previously, Chun et al. [24] reported that deguelin treatment had only mimimal effects on the MAPK pathway. In our study, we also found no significant differences in phosphorylation levels of ERK, suggesting that deguelin application does not have a sufficient effect on MAPK signalling after MI. Whether Akt-induced cardiac hypertrophy is physiological or pathological is complex [17]. Activation of PI3K/Akt1 signalling is required for exercise-induced hypertrophy [16]. Others also point out that Akt1 is a critical mediator of pathological cardiac hypertrophy [25,26]. These latter conclusions, however, are derived from transgenic mouse models overexpressing constitutively active Akt1 at 15-fold higher than the physiological levels. Overexpres- sion of Akt1 to this extent can overtake the function of other Akt isoforms and also can lead to off-target effects because of non-physiological protein–protein interactions and aberrant intracellular localization. Indeed, Akt1 has a dichotomous role in cardiac remodelling by mediating physiological compared with pathological signalling based upon the duration, the intensity and the type of stress. Our study answered the question of the effect of chronic pharmacological activation of Akt with lithium on cardiac hypertrophy after MI. Our results do not seem consistent with previous studies, showing that chronic Akt1 activation, which activates mTORC1, has been shown to worsen aging-induced cardiac hypertrophy and impair myocardial contractile [27]. mTOR exerts its main cellular functions by interacting with specific adaptor proteins to form two distinct multipro- tein complexes, mTORC1 and mTORC2 [28]. an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons Attribution Study limitations There are some limitations in the present study that have to be acknowledged. First, our studies on PI3K/Akt signalling were obtained by pharmacological inhibition. Thus, we can not exclude the non-specific actions of drugs. Second, lithium may affect the serum sodium levels [33] and subsequently modulate the hypertrophy. We did not measure the serum sodium levels. The LiCl administration regulates serum sodium levels in a dose-dependent manner [33]. Previous studies have shown in rats that chronic administration of LiCl at the dose of 50 mg/kg per day, higher than that used in the present study (1 mmol/kg per day of LiCl, equivalent to 42 mg/kg per day), did not significantly change the serum sodium levels [33]. Thus, although we did not measure the sodium levels, it is logical to speculate that the serum sodium levels would not significantly change at the subtherapeutic dose. In conclusion, our findings suggest that in the setting of MI, a dysbalance towards pathological pathways occurs and leads to a deterioration of cardiac remodelling and function, which can be corrected by lithium administration through an Akt/mTOR pathway. Lithium may constitute a new therapeutic option for mending the infarcted my- ocardium and its clinical efficacy needs to be tested in clinical trials. Funding This work was supported by the An-Nan Hospital [grant numbers ANHRF 104-02, ANHRF 105-01]; and the Ministry of Science and Technology, Taiwan [grant numbers MOST 104-2314-B-039-021, MOST 105-2314-B-039-042]. Clinical implications p The present study was undertaken to explore the possibility that lithium might have clinical efficacy for the treatment after MI. Traditional therapeutics to prevent post-MI remodelling (e.g. angiotensin-converting enzyme inhibitors and angiotensin-receptor antagonists) are effective to some degree but progression to congestive heart failure or death, despite standard approaches, is common. Novel signalling pathways involved in the cardiac remodelling after MI, like Akt/mTOR signalling, need to be explored. Induction of physiological cardiac hypertrophy may be a poten- tial therapeutic strategy for the treatment of heart failure. Lithium’s ability to induce hypertrophy would reduce LV WTI according to Laplace’s law, and its effects on contractility would enhance the function of the non-infarcted my- ocardium, both of which would be of particular benefit if applied, while the process of remodelling was beginning. Our findings may have potential implications as a therapeutic agent for treatment of patients post-MI. Activation of PI3K (p110α), via exercise training or pharmacological approaches, offers a novel therapeutic strategy for preventing LV remodelling in patients at the risk of developing heart failure. While angiotensin converting enzyme inhibitors and angiotensin receptor blockers slow LV remodelling by targeting pathological hypertrophy signalling pathways, activation of PI3K (p110α) attenuates LV remodelling by activating physiological hypertrophy signalling pathways as well as inhibiting pathological signalling pathways. 10 Bioscience Reports (2017) 37 BSR20160257 DOI: 10.1042/BSR20160257 Abbreviations ANP, atrial natriuretic peptide; BW, body weight; ERK, extracellular-signal-regulated kinase; ERK1/2, ANP, atrial natriuretic peptide; BW, body weight; ERK, extracellular-signal-regulated kinase; ERK1/2, extracellular-signal-regulated kinase 1/2; FS, fractional shortening; GPCR, G-protein-coupled receptor; LiCl, lithium chlo- ride; LungW, lung weight; LV, left ventricle; LVEDD, LV end-diastolic diameter dimension; LVEDP, LV end-diastolic pressure; LVESD, LV end-systolic diameter dimension; LVESP, LV end-systolic pressure; MAPK, mitogen-activated protein kinase; MI, myocardial infarction; mTOR, mammalian target of rapamycin; NFAT, nuclear factor of activated T-cells; PI3K, phosphoinositide 3-kinase; WTI, wall tension index; 4E-BP1, eukaryotic initiation factor 4E binding protein 1; β-MHC, β-myosin heavy chain. Author contribution T.-M.L. and S.-Z.L. operated the experiment, collected the data and performed the analysis. N.-C.C. edited and reviewed the manuscript. All authors read and approved the final manuscript. Competing interests p g The authors declare that there are no competing interests associated with the manuscript. References 1 Pantos, C., Mourouzis, I. and Cokkinos, D.V. (2010) Rebuilding the post-infarcted myocardium by activating ‘physiologic’ hypertrophic signaling pathways: the thyroid hormone paradigm. Heart Fail. Rev. 15, 143–154 1 Pantos, C., Mourouzis, I. and Cokkinos, D.V. (2010) Rebuilding the post-infarcted myocardium by activating ‘physiologic’ hypertrophic signaling pathways: the thyroid hormone paradigm. Heart Fail. Rev. 15, 143–154 1 Pantos, C., Mourouzis, I. and Cokkinos, D.V. (2010) Rebuilding the post-infarcted myocardium by activating ‘physiologic’ hypertrophic signaling pathways: the thyroid hormone paradigm. Heart Fail. 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Global expression analysis of the brown alga Ectocarpus siliculosus (Phaeophyceae) reveals large-scale reprogramming of the transcriptome in response to abiotic stress Simon M Dittami, Delphine Scornet, Jean-Louis Petit, Béatrice Segurens, Corinne da Silva, Erwan Corre, Michael Dondrup, Karl-Heinz Glatting, Rainer König, Sterck Lieven, et al. To cite this version: Simon M Dittami, Delphine Scornet, Jean-Louis Petit, Béatrice Segurens, Corinne da Silva, et al.. Global expression analysis of the brown alga Ectocarpus siliculosus (Phaeophyceae) reveals large-scale reprogramming of the transcriptome in response to abiotic stress. Genome Biology, 2009, 10, pp.R66. ￿10.1186/gb-2009-10-6-r66￿. ￿hal-01806423￿ Open Access 2009 Dittami et al. Volume 10, Issue 6, Article R66 Research Global expression analysis of the brown alga Ectocarpus siliculosus (Phaeophyceae) reveals large-scale reprogramming of the transcriptome in response to abiotic stress Simon M Dittami*†, Delphine Scornet*†, Jean-Louis Petit‡§¶, Béatrice Ségurens‡§¶, Corinne Da Silva‡§¶, Erwan Corre¥, Michael Dondrup#, Karl-Heinz Glatting**, Rainer König**, Lieven Sterck††, Pierre Rouz醆, Yves Van de Peer††, J Mark Cock*†, Catherine Boyen*† and Thierry Tonon*† Open Access Addresses: *UPMC Univ Paris 6, UMR 7139 Végétaux marins et Biomolécules, Station Biologique, 29680 Roscoff, France. †CNRS, UMR 7139 Végétaux marins et Biomolécules, Station Biologique, 29680 Roscoff, France. ‡CEA, DSV, Institut de Génomique, Génoscope, rue Gaston Crémieux, CP5706, 91057 Evry, France. §CNRS, UMR 8030 Génomique métabolique des genomes, rue Gaston Crémieux, CP5706, 91057 Evry, France. ¶Université d'Evry, UMR 8030 Génomique métabolique des genomes, 91057 Evry, France. ¥SIG-FR 2424 CNRS UPMC, Station Biologique, 29680 Roscoff, France. #Center for Biotechnology (CeBiTec), University of Bielefeld, 33594 Bielefeld, Germany. **German Cancer Research Center (DKFZ), Im Neuenheimer Feld 580, 69120 Heidelberg, Germany. ††VIB Department of Plant Systems Biology, Ghent University, 9052 Ghent, Belgium. Correspondence: Simon M Dittami. Email: [email protected]. Thierry Tonon. Email: [email protected] Received: 19 November 2008 Revised: 4 February 2009 Accepted: 16 June 2009 Published: 16 June 2009 The electronic version of this article is the complete one and can be found online at http://genomebiology.com/2009/10/6/R66 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. B l t i t i Th b l it E t ili l /it lik t t i l l t d t i i f it t i t d i th li ti t ild bi ti t / HAL Id: hal-01806423 L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. Background For this, we have developed an EST-based microarray along with several tools and annotations (availa- ble on our Ectocarpus transcriptomics homepage [22]), and used this array to study the transcriptomic response of E. siliculosus to three forms of abiotic stress: hyposaline, hyper- saline, and oxidative stress. Hypersaline stress is a stress experienced by intertidal seaweeds - for example, in rock- pools at low tide (due to evaporation) or due to anthropogenic influences - and is comparable to desiccation stress. Hyposa- line stress is also common in the intertidal zone, and can arise, for example, due to rain. Furthermore, organisms with a high tolerance to saline stress can inhabit a wide range of habitats. E. siliculosus strains have been isolated from loca- tions covering a wide range of salinity. A specimen was found in a highly salt-polluted area of the Werra river in Germany, where chloride concentrations at times reached 52.5 grams per liter [23]. At the same time, E. siliculosus can be found in estuaries, in the Baltic sea, and one strain of E. siliculosus was isolated from freshwater [24]. Oxidative stress is commonly experienced by living organisms. Reactive oxygen species (ROSs) are produced intracellularly in response to various stressors due to malfunctioning of cellular components, and have been implicated in many different signaling cascades in plants [25]. In algae, several studies have demonstrated the production of ROSs in response to biotic stress (reviewed in [26]). Therefore, protection against these molecules is at the basis of every stress response and has been well studied in many organisms. We simulated this stress by the addition of hydrogen peroxide to the culture medium. In order to fill this knowledge gap, Ectocarpus siliculosus, a small, cosmopolitan, filamentous brown alga (see [11] for a recent review) has been chosen as a model [12], mainly because it can complete its life cycle rapidly under laboratory conditions, is sexual and highly fertile, and possesses a rela- tively small genome (200 Mbp). Several genomic resources have been developed for this organism, such as the complete sequence of its genome and a large collection of expressed sequence tags (ESTs). Although Ectocarpus is used as a model for developmental studies [13,14], no molecular stud- ies have been undertaken so far to study how this alga deals with the high levels of abiotic stress that are a part of its nat- ural environment. Background ulation of stress-response genes in the red alga Chondrus crispus after treatment with methyl jasmonate [19] and sug- gesting that hypersaline and hyposaline stress are similar to important stressors in natural environments [20]. Further- more, in the brown alga Laminaria digitata, Roeder et al. [21] performed a comparison of two EST libraries (sporo- phyte and protoplasts) and identified several genes that are potentially involved in the stress response, including the brown alga-specific vanadium-dependent bromoperoxidases and mannuronan-C5-epimerases, which are thought to play a role in cell wall modification and assembly. These studies have provided valuable information about the mechanisms and pathways involved in algal stress responses, but they were nevertheless limited by the availability of sequence information for the studied organisms at the time. g The brown algae (Phaeophyceae) are photosynthetic organ- isms, derived from a secondary endosymbiosis [1], that have evolved complex multicellularity independently of other major groups such as animals, green plants, fungi, and red algae. They belong to the heterokont lineage, together with diatoms and oomycetes, and are hence very distant phyloge- netically, not only from land plants, animals, and fungi, but also from red and green algae [2]. Many brown algae inhabit the intertidal zone, an environment of rapidly changing phys- ical conditions due to the turning tides. Others form kelp for- ests in cold and temperate waters as well as in deep-waters of tropical regions [3,4]. Brown algae, in terms of biomass, are the primary organisms in such ecosystems and, as such, rep- resent important habitats for a wide variety of other organ- isms. As sessile organisms, brown algae require high levels of tolerance to various abiotic stressors such as osmotic pres- sure, temperature, and light. They differ from most terrestrial plants in many aspects of their biology, such as their ability to accumulate iodine [5], the fact that they are capable of syn- thesizing both C18 and C20 oxylipins [6], their use of lami- narin as a storage polysaccharide [7], the original composition of their cell walls, and the associated cell wall synthesis pathways [8-10]. Many aspects of brown algal biol- ogy, however, remain poorly explored, presenting a high potential for new discoveries. With the tools and sequences available for the emerging brown algal model E. siliculosus, we are now in a position to study the stress response of this alga on the level of the whole transcriptome. Abstract Background: Brown algae (Phaeophyceae) are phylogenetically distant from red and green algae and an important component of the coastal ecosystem. They have developed unique mechanisms that allow them to inhabit the intertidal zone, an environment with high levels of abiotic stress. Ectocarpus siliculosus is being established as a genetic and genomic model for the brown algal lineage, but little is known about its response to abiotic stress. Results: Here we examine the transcriptomic changes that occur during the short-term acclimation of E. siliculosus to three different abiotic stress conditions (hyposaline, hypersaline and oxidative stress). Our results show that almost 70% of the expressed genes are regulated in response to at least one of these stressors. Although there are several common elements with terrestrial plants, such as repression of growth-related genes, switching from primary production to protein and nutrient recycling processes, and induction of genes involved in vesicular trafficking, many of the stress-regulated genes are either not known to respond to stress in other organisms or are have been found exclusively in E. siliculosus. Conclusions: This first large-scale transcriptomic study of a brown alga demonstrates that, unlike terrestrial plants, E. siliculosus undergoes extensive reprogramming of its transcriptome during the acclimation to mild abiotic stress. We identify several new genes and pathways with a putative function in the stress response and thus pave the way for more detailed investigations of the mechanisms underlying the stress tolerance ofbrown algae. Genome Biology 2009, 10:R66 Genome Biology 2009, Volume 10, Issue 6, Article R66 Dittami et al. Dittami et al. R66.2 http://genomebiology.com/2009/10/6/R66 Background This is also true for intertidal seaweeds in general, where very few studies have addressed this question. In the 1960s and 1970s several studies (reviewed in [15]) examined the effects of abiotic stressors such as light, temper- ature, pH, osmolarity and mechanical stress on algal growth and photosynthesis. However, only a few of the mechanisms underlying the response to these stressors - for example, the role of mannitol as an osmolyte in brown algae [16,17] - have been investigated so far. Developing and applying molecular and biochemical tools will help us to further our knowledge about these mechanisms - an approach that was suggested 12 years ago by Davison and Pearson [18]. Nevertheless, it was only recently that the first transcriptomic approaches were undertaken to investigate stress tolerance in intertidal sea- weeds. Using a cDNA microarray representing 1,295 genes, Collén et al. [19,20] obtained data demonstrating the up-reg- Intracellular osmolarity and Na+ concentration Intracellular osmolarity and Na+ concentration Apart from the photosynthetic activity, we also measured intracellular osmolarity and Na+ concentrations (Figure 2). After 6 hours of exposure to different salinities, the intracel- lular osmolarity was always about 500 mOsm higher than that of the extracellular medium. The intracellular Na+ con- centration was about 500 mM lower than in the extracellular medium under hypersaline stress, 60 mM lower under con- trol conditions, and the same under hyposaline stress. Oxida- tive stress had no detectable effect on the intracellular ion composition or osmolarity (data not shown). 0 0.1 0.2 0.3 0.4 0.5 0.6 0 5 10 15 20 25 Time [h] Quantum yield 100 % salinity 50 % salinity 25 % salinity 12.5 % salinity 0 % salinity 0 0.1 0.2 0.3 0.4 0.5 0.6 0 5 10 15 20 25 100% salinity 50% salinity 25% salinity 12.5% salinity 0% salinity Oxidative stress 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0 10 20 30 40 50 Time [h] Quantum yield Control H2O2 0.1 mM H2O2 0.5 mM H2O2 1 mM H2O2 10 mM Oxidative stress 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0 10 20 30 40 50 Control H2O2 0.1 mM H2O2 0.5 mM H2O2 1 mM H2O2 10 mM Determination of sub-lethal stress conditions Hypersaline stress 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0 5 10 15 20 25 Time [h] Quantum yield 450 mM NaCl 900 mM NaCl 1,470 mM NaCl 1,900 mM NaCl Hypersaline stress 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0 5 10 15 20 25 450 mM NaCl 900 mM NaCl 1,470 mM NaCl 1,900 mM NaCl Hyposaline stress 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0 5 10 15 20 25 Time [h] Quantum yield 100 % salinity 50 % salinity 25 % salinity 12.5 % salinity 0 % salinity Hyposaline stress 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0 5 10 15 20 25 100% salinity 50% salinity 25% salinity 12.5% salinity 0% salinity Effects of saline and oxidative stress of different intensities on the photosynthetic efficiency (quantum yield) of E. siliculosus Figure 1 Effects of saline and oxidative stress of different intensities on the photosynthetic efficiency (quantum yield) of E. siliculosus. The conditions in red (1,470 mM NaCl, 12.5% seawater, and 1 mM H2O2) were the conditions chosen for the microarray analysis. Determination of sub-lethal stress conditions Determination of sub-lethal stress conditions The aim of this study was to determine the mechanisms that allow short-term acclimation to abiotic stress. To be sure to monitor the short-term response to stress rather than just cell death, the intensity of the different stresses needed to be cho- Genome Biology 2009, 10:R66 Genome Biology 2009, Volume 10, Issue 6, Article R66 Dittami et al. R66.3 http://genomebiology.com/2009/10/6/R66 Dittami et al. R66.3 sen carefully. Using a pulse amplitude modulation fluorome- ter (see Materials and methods), we measured the effects of different stress intensities on photosynthesis. Figure 1 shows the change in quantum yield of photosynthesis in response to different intensities of the different stresses, where values of over 0.5 indicate low stress. The quantum yield can vary dur- ing the course of the day even under controlled conditions, as changes in light have a strong impact on this parameter. Stress conditions were chosen to have a clear effect on the photosynthesis rate, but to be sub-lethal, allowing the alga to acclimate and recover. The conditions that corresponded best to these criteria were 1.47 M NaCl (hypersaline condition, approximately three times the salinity of normal seawater), 12.5% seawater, and 1 mM H2O2 (oxidative stress condition), although, for this last stressor, we can assume that the H2O2 concentration in the medium decreases over the course of the experiment. Each stress was applied for 6 hours because this corresponds to the time span between high and low tide. In addition, experiments carried out on land plants [27] and red algae [19] have indicated that the application of stress for 6 hours induces the most marked changes in transcription. Initially, we had considered a fourth stress condition, 2 M sorbitol in artificial sea water (ASW), to imitate the osmotic pressure of the hypersaline treatment without the possible effects of the salts. However, this treatment was not included in the final experiment because cultures did not survive this treatment for 6 hours. For the other stresses, we observed 100% recovery of photosynthesis after about 6 days, even after 24 hours of stress (Additional data file 1). Determination of sub-lethal stress conditions Hypersaline stress 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0 5 10 15 20 25 Time [h] Quantum yield 450 mM NaCl 900 mM NaCl 1,470 mM NaCl 1,900 mM NaCl Hypersaline stress 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0 5 10 15 20 25 450 mM NaCl 900 mM NaCl 1,470 mM NaCl 1,900 mM NaCl Hyposaline stress 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0 5 10 15 20 25 Time [h] Quantum yield 100 % salinity 50 % salinity 25 % salinity 12.5 % salinity 0 % salinity Hyposaline stress 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0 5 10 15 20 25 100% salinity 50% salinity 25% salinity 12.5% salinity 0% salinity Oxidative stress 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0 10 20 30 40 50 Time [h] Quantum yield Control H2O2 0.1 mM H2O2 0.5 mM H2O2 1 mM H2O2 10 mM Oxidative stress 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0 10 20 30 40 50 Control H2O2 0.1 mM H2O2 0.5 mM H2O2 1 mM H2O2 10 mM Hypersaline stress 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0 5 10 15 20 25 Time [h] Quantum yield 450 mM NaCl 900 mM NaCl 1,470 mM NaCl 1,900 mM NaCl Hypersaline stress 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0 5 10 15 20 25 450 mM NaCl 900 mM NaCl 1,470 mM NaCl 1,900 mM NaCl The E. siliculosus microarray represents 17,119 sequences We designed a microarray based on 90,637 ESTs obtained by sequencing clones from 6 different cDNA libraries: immature sporophyte (normalized and non-normalized), mature sporo- phyte, immature gametophyte, mature gametophyte, and stress (sporophyte). Cleaning and assembly resulted in the generation of 8,165 contigs and 8,874 singletons. In addition, 21 genomic sequences and 231 E. siliculosus Virus 1 (EsV-1) genes were included. The array design file has been deposited under the accession number [ArrayExpress:A-MEXP-1445] and is also available on our Ectocarpus transcriptomics homepage [22]. Oxidative stress 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0 10 20 30 40 50 Time [h] Quantum yield Control H2O2 0.1 mM H2O2 0.5 mM H2O2 1 mM H2O2 10 mM Oxidative stress 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0 10 20 30 40 50 Control H2O2 0.1 mM H2O2 0.5 mM H2O2 1 mM H2O2 10 mM Oxidative stress Oxidative stress Of the 17,119 genes represented on the array, 12,250 gave a significant signal over background in our experiments and were considered to be expressed under the conditions tested. The analysis focused on these 12,250 genes (see Materials and methods). A first comparison with the data obtained from a tiling experiment with E. siliculosus (MP Samanta and JM Cock, personal communication), where 12,600 genes were considered strongly expressed, demonstrates that our array offers a rather complete coverage of at least the highly tran- scribed parts of the E. siliculosus genome, suggesting that we are working at the whole genome scale. Effects of photosynt Figure 1 Effects of saline and oxidative stress of different intensities on the photosynthetic efficiency (quantum yield) of E. siliculosus Figure 1 Effects of saline and oxidative stress of different intensities on the photosynthetic efficiency (quantum yield) of E. siliculosus. The conditions in red (1,470 mM NaCl, 12.5% seawater, and 1 mM H2O2) were the conditions chosen for the microarray analysis. p y y (q y ) g Effects of saline and oxidative stress of different intensities on the photosynthetic efficiency (quantum yield) of E. siliculosus. The conditions in red (1,470 mM NaCl, 12.5% seawater, and 1 mM H2O2) were the conditions chosen for the microarray analysis. Genome Biology 2009, 10:R66 Genome Biology 2009, Volume 10, Issue 6, Article R66 Dittami et al. R66.4 Genome Biology 2009, Volume 10, Issue 6, Article R66 http://genomebiology.com/2009/10/6/R66 Dittami et al. Ribosomal protein genes are among those whose transcript abundances are least affected by stress The 100 most stably expressed genes in these microarray experiments included 51 genes with unknown functions. Nineteen genes code for ribosomal proteins, and 21 genes are known housekeeping genes with functions related to protein turnover (transcription, 4 genes; translation, 3 genes; degra- dation, 3 genes), energy production (6 genes), and the cytoskeleton (5 genes). For a detailed list of these most stably expressed genes, please see Additional data file 3. Classification of stress response genes using automatic annotations Overall, 8,474 genes were identified as being differentially expressed in at least one of the conditions compared to the control, allowing a FDR of 10% (5,812 were labeled significant at an FDR of 5%). As can be seen in Figure 3, the relative change for these genes ranged from 1.2-fold (log2-ratio ≈0.3) to more than 32-fold (log2-ratio >5). Of these 8,474 genes, 2,569 (30%) could be automatically annotated with GO terms using the GO-term Prediction and Evaluation Tool (GOPET) [36] and 1,602 (19%) with KEGG orthology annotations using the KOBAS software [37]. These automatic annotations were analyzed for each stress condition individually, to identify GO categories and KEGG pathways that were significantly over- represented. cDNA synthesis and amplification provided consistent results with both mRNA and total RNA samples For reasons as yet unknown, cDNA synthesis reactions with E. siliculosus are inhibited at high concentrations of RNA. Therefore, we decided to synthesize cDNAs from a small quantity of total RNA or mRNA, and to include a PCR ampli- fication step in the protocol to obtain sufficient double- stranded cDNA (4 μg) for each hybridization. A comparison of the four four-fold replicates synthesized from 30 ng of mRNA and the single four-fold replicate synthesized from 100 ng total RNA showed that these two protocols yielded similar results. All total RNA replicates clustered with the mRNA replicates of the same stress (data not shown). Never- theless, at a false discovery rate (FDR) of 5%, 163 transcripts gave significantly different results with the two types of sam- ple. These transcripts represented mainly constituents of the ribosome, as revealed by a Kyoto Encyclopedia of Genes and Genomes (KEGG) Orthology Based Annotation System (KOBAS) analysis and by an analysis of overrepresented GO terms (Additional data file 2). The E. siliculosus microarray represents 17,119 sequences R66.4 Intracellular versus extracellular osmolarity and Na+ concentration under saline stress Figure 2 Intracellular versus extracellular osmolarity and Na+ concentration under saline stress. Oxidative stress samples are not shown as they did not differ significantly from the control sample. Every point represents the mean of five biological replicates ± standard deviation. 0 500 1000 1500 2000 2500 3000 3500 4000 4500 0 1000 2000 3000 4000 Extracellular osmolarity [mOsm] Intracellular osmolarity [mOsm] 0 300 600 900 1200 1500 1800 0 500 1000 1500 2000 Extracellular Na+ [mM] Intracellular Na+ [mM] isoosmotic line isoosmotic line 1.47 M NaCl 1.47 M NaCl 12.5 % SW 12.5 % SW control control Intracellular versus extracellular osmolarity and Na concentration under saline stress Figure 2 Intracellular versus extracellular osmolarity and Na+ concentration under saline stress. Oxidative stress samples are not shown as they did not differ significantly from the control sample. Every point represents the mean of five biological replicates ± standard deviation. Table 1 Table 1 Comparison of microarray and RT-qPCR results for genes changing expression ID Genome ID Name r Function CL4038Contig1 [Esi0355_0025] HSP70 0.87 HSP70 LQ0AAB7YD09FM1.SCF [Esi0155_0065] NADH 0.94 NADH dehydrogenase CL7513Contig1 [Esi0269_0011] ProDH 0.79 Proline dehydrogenase CL3741Contig1 [Esi0024_0066] TF 0.90 Putative transcription factor LQ0AAB12YN05FM1.SCF [Esi0399_0008] WD_rep 0.66 WD repeat gene CL1Contig3 [Esi0085_0055] CLB1 0.95 Chlorophyll binding protein CL43Contig1 [Esi0199_0054] CLB2 0.98 Fucoxanthin binding protein CL7742Contig1 [Esi0026_0055] TagS 0.69 TAG synthase CL2765Contig1 [Esi0526_0006] NH4-Tr 0.96 Ammonium transporter CL3832Contig1 [Esi0437_0012] FOR 0.67 Phycoerythrobilin:ferredoxin oxidoreductase LQ0AAA16YN10FM1.SCF [Esi0153_0004] Arg-MetTr 0.71 Arginine N-methyltransferase CL7099Contig1 [Esi0018_0111] HSD 0.83 Homoserine dehydrogenase CL6576Contig1 [Esi0107_0059] IGPS 0.97 Indole-3-glycerol-phosphate synthase CL7231Contig1 [Esi0686_0001] CDPK 0.85 cAMP-dependent protein kinase CL4027Contig1 [Esi0122_0054] mGST -0.48 Microsomal glutathione S-transferase CL4274Contig1 [Esi0023_0183] SNR 0.57 SNR (vesicular transport) CL5850Contig1 [Esi0109_0088] mG 0.99 Glycin-rich protein CL455Contig1 [Esi0159_0021] G6PD 0.91 Glucose-6-phosphate 1-dehydrogenase CL6746Contig1 [Esi0116_0065] IF4E 0.91 Eukaryotic initiation factor 4E R is the Pearson correlation coefficient between the microarray and the RT-qPCR expression profile. ID corresponds to the name of the sequence on the array. Comparison of microarray and RT-qPCR results for genes changing expression Genome Biology 2009, 10:R66 Genome Biology 2009, Volume 10, Issue 6, Article R66 Dittami et al. R66.5 Genome Biology 2009, Volume 10, Issue 6, Article R66 Dittami et al. R66.5 http://genomebiology.com/2009/10/6/R66 knowledge, the use of this technique has not been reported with commercial photolithographically synthesized arrays. cDNA synthesis and amplification provided consistent results with both mRNA and total RNA samples Validation of microarray results using quantitative PCR Nineteen genes that exhibited significant changes in their expression patterns in the microarray analysis were analyzed by real time quantitative PCR (RT-qPCR). Eighteen of these had similar expression profiles in both the microarray exper- iment and the RT-qPCR experiment (correlation coefficient r of between 0.57 and 0.99; Table 1). Only one gene, which codes for a microsomal glutathione S-transferase, displayed a different pattern in the two experiments (r = -0.48). Further- more, the seven most stable 'housekeeping genes' as identi- fied by qPCR in a previous report [28] showed only statistically non-significant relative changes of <1.5-fold (log2-ratio <0.58) in the microarray experiment (Table 2). This demonstrated that the protocol for cDNA amplification provided reliable measures of the relative transcript abun- dances. Although this method has been successfully applied in several small-scale expression studies [29-35], to our The KOBAS results (Figure 4; Additional data file 4) indicated that under hyposaline and hypersaline stresses most of the changes involved down-regulation of the synthesis and metabolism of amino acids. More precisely, genes involved in the synthesis of valine, leucine, and isoleucine, as well as that of the aromatic amino acids (phenylalanine, tyrosine, tryp- tophan), and arginine and proline metabolism were affected. This effect on amino acid synthesis was less marked for oxi- dative stress, where glutamate metabolism was the only The table displays the maximum log2-ratio between any stress and the control condition for both the microarray and the RT-qPCR analysis. No RT- qPCR value is available for R26S, as this gene was used for normalization of the RT-qPCR samples. ID corresponds to the name of the sequence on the array Distributio Figure 3 Distribution of observed fold changes (log2 ratios of stress and control samples) Figure 3 Distribution of observed fold-changes (log2-ratios of stress and control samples). All three comparisons between stress and control treatments were considered and the observed frequencies averaged. The color coding shows how many transcripts were labeled as differentially expressed at different FDRs. Not sig., not significant. over-represented among the down-regulated genes. In agree- ment with the down-regulation of amino acid metabolism identified by the KOBAS analysis, we observed a decrease in the abundance of transcripts encoding aminoacyl-tRNA ligases in hypersaline and hyposaline conditions using the GOPET annotations. Also, under hypersaline stress, we observed down-regulation of genes associated with the GO terms RNA binding and translation factor activity, which cor- responds to the KEGG category translation factors, and down-regulation of transcripts coding for proteins with a CTP synthase activity, which are involved in purine and pyrimi- dine metabolism. Under hyposaline stress, we observed that NAD(P)+ transhydrogenases, a number of transferases and oxidoreductases involved in amino acid metabolism, as well as genes with functions in nucleic acid and chlorophyll bind- ing, were most affected, the latter matching well with the pathways 'photosynthesis-antenna proteins' identified by KOBAS. Under oxidative stress, using the GOPET annota- tions, we detected down-regulation of several different cate- gories of transferases, nitrate transporters, oxidoreductases involved in steroid metabolism, and 3-isopropylmalate dehy- dratase-like enzymes that are involved in amino acid metab- amino acid metabolism affected. Under hypersaline condi- tions, there was also an increase in transcripts coding for enzymes that metabolize valine, leucine, and isoleucine. In addition, photosynthesis and vesicular transport seemed to be altered by both hyposaline and oxidative stress. Pathways that appeared to be specifically affected by one stress included the up-regulation of fatty acid metabolism and down-regulation of translation factors under hypersaline stress, the up-regulation of the proteasome and down-regula- tion of nitrogen metabolism under hyposaline stress, and an increase in glycerophospholipid metabolism under oxidative stress (Figure 4). A complete list of the pathways identified is available in Additional data file 4, with possible artifacts aris- ing from the automatic annotation marked in grey. The GOPET analysis (Table 3; Additional data file 5) was focused on the molecular function of the individual genes rather than their role in a specific pathway. Table 2 Comparison of microarray and RT-qPCR results for housekeeping or stable genes ID Genome ID Name Maximum change ARRAY Maximum change QPCR Function LQ0AAB30YA12FM1.SCF [Esi0298_0008] Dyn 0.23 0.77 Dynein CL1914Contig1 [Esi0021_0024] ARP2.1 0.22 0.44 Actin related protein CL3Contig2 [Esi0387_0021] EF1A 0.08 0.46 Elongation factor 1 alpha CL8Contig12 [Esi0053_0059] TUA 0.57 0.91 Alpha tubulin CL1073Contig1 [Esi0054_0059] UBCE 0.22 0.38 Ubiquitin-conjugating enzyme CL29Contig4 [Esi0302_0019] UBQ 0.18 0.82 Ubiquitin CL461Contig1 [Esi0072_0068] R26S 0.22 n/a Ribosomal protein S26 The table displays the maximum log2-ratio between any stress and the control condition for both the microarray and the RT-qPCR analysis. No RT- qPCR value is available for R26S, as this gene was used for normalization of the RT-qPCR samples. ID corresponds to the name of the sequence on the array. Comparison of microarray and RT-qPCR results for housekeeping or stable genes The table displays the maximum log2-ratio between any stress and the control condition for both the microarray and the RT-qPCR analysis. No RT- qPCR value is available for R26S, as this gene was used for normalization of the RT-qPCR samples. ID corresponds to the name of the sequence on the array. Genome Biology 2009, 10:R66 http://genomebiology.com/2009/10/6/R66 Genome Biology 2009, Volume 10, Issu Genome Biology 2009, Volume 10, Issue 6, Article R66 Dittami et al. R66.6 Genome Biology 2009, Volume 10, Issue 6, Article R66 http://genomebiology.com/2009/10/6/R66 Dittami et al. R66.6 Distribution of observed fold-changes (log2-ratios of stress and control samples) Figure 3 Distribution of observed fold-changes (log2-ratios of stress and control samples). All three comparisons between stress and control treatments were considered and the observed frequencies averaged. The color coding shows how many transcripts were labeled as differentially expressed at different FDRs. Not sig., not significant. 0 100 200 300 400 500 600 700 800 900 1000 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2 2.2 2.4 2.6 2.8 3 3.2 3.4 3.6 3.8 4 4.2 4.4 4.6 4.8 >5 log2(fold-change) Number of genes not sig. FDR<0.1 FDR<0.05 FDR<0.01 log2(fold-change) Distribution of observed fold-changes (log2-ratios of stress and control samples) Figure 3 Distribution of observed fold-changes (log2-ratios of stress and control samples). All three comparisons between stress and control treatments were considered and the observed frequencies averaged. The color coding shows how many transcripts were labeled as differentially expressed at different FDRs. Not sig., not significant. Genome Biology 2009, Volume 10, Issue 6, Article R66 Venn diagr stress cond Figure 4 g p y p g p g y p g ( ) g Venn diagram of KEGG pathways identified as over-represented among the transcripts significantly up- or down-regulated (FDR <0.1) in the different stress conditions. Only KEGG pathways with q-values < 0.1 in at least two conditions or for both datasets (FDR of 0.05 and FDR of 0.1) were considered. The general category 'other enzymes' was not included. Further 'SNARE interactions in vesicular transport' includes the category 'SNARE', and 'photosynthesis' includes 'photosynthesis proteins' and 'porphyrin and chlorophyll metabolism'. No pathways were found to be common only to hyposaline and hypersaline stress. SNARE, soluble N-ethylmaleimide-sensitive factor attachment receptor. sponding genome sequence to public protein databases, can be found in Additional data file 3. olism. Here, the KOBAS analysis did not identify any significantly up- or down-regulated pathways. Also in con- trast to the KOBAS results, no GO terms were significantly over-represented among the genes identified as being up- or down-regulated in both oxidative and hypersaline stresses, or in all three stresses at the same time. We identified 519 genes (53.7%) with no homologues in either the National Center for Biotechnology Information (NCBI) databases or other heterokont genomes (e-value > 1e-10). An additional 122 genes (12.6%) code for conserved genes with unknown function. Of these 122 conserved genes, 23 (18.9%) are conserved only within the heterokont lineage. The remaining 325 genes (33.6%) were divided into 12 groups according to their putative functions in amino acid metabo- lism, DNA replication and protein synthesis, protein turno- ver, carbohydrate metabolism, photosynthesis-related processes, fatty acid metabolism, transporters, vesicular traf- ficking and cytoskeleton, classical stress response pathways, autophagy, signaling, and other processes. The following sec- Distributio Figure 3 Only three GO terms were identified as being over-represented among the up-regulated genes: arginase and agmatinase activity under hypersaline conditions, and microtubule motor activity under oxidative stress. Most GO terms were found to be significantly Genome Biology 2009, 10:R66 Genome Biology 2009, Volume 10, Issue 6, Article R66 Genome Biology 2009, Volume 10, Issue 6, Article R66 Dittami et al. R66.7 Dittami et al. R66.7 http://genomebiology.com/2009/10/6/R66 Venn diagram of KEGG pathways identified as over-represented among the transcripts significantly up- or down-regulated (FDR <0.1) in the different stress conditions Figure 4 Venn diagram of KEGG pathways identified as over-represented among the transcripts significantly up- or down-regulated (FDR <0.1) in the different stress conditions. Only KEGG pathways with q-values < 0.1 in at least two conditions or for both datasets (FDR of 0.05 and FDR of 0.1) were considered. The general category 'other enzymes' was not included. Further 'SNARE interactions in vesicular transport' includes the category 'SNARE', and 'photosynthesis' includes 'photosynthesis proteins' and 'porphyrin and chlorophyll metabolism'. No pathways were found to be common only to hyposaline and hypersaline stress. SNARE, soluble N-ethylmaleimide-sensitive factor attachment receptor. Manual classification of stress response genes with the most significant changes in expression To identify the most important mechanisms involved in the stress response, we manually classified and examined in detail 966 genes that exhibited the most significant changes in one of the stress conditions compared to the control (that is, genes that meet both criteria: significance at an FDR <1% and a relative change in expression of more than two-fold). A complete list of these genes, including their putative function, assigned manually based on sequence homology of the corre- Genome Biology 2009, 10:R66 Genome Biology 2009, Volume 10, Issue 6, Article R66 Dittami et al. R66.8 http://genomebiology.com/2009/10/6/R66 Table 3 GO terms identified to be over-represented among the transcripts of significantly up- or down-regulated in the different stress condi- tions Condition Change in expression Category Function GO ID Hyper Down Nucleic acid binding RNA binding (mRNA, rRNA, snoRNA) [GO:0003723]; [GO:0003729]; [GO:0019843]; [GO:0030515] Nucleic acid binding Translation factor activity (elongation and initiation) [GO:0008135]; [GO:0003746]; [GO:0003743] Lyase UDP-glucuronate decarboxylase activity [GO:0048040] Ligase CTP synthase activity [GO:0003883] Isomerase activity Intramolecular oxidoreductase activity [GO:0016860] Up Hydrolase Agmatinase activity [GO:0008783] Hydrolase Arginase activity [GO:0004053] Hyper Hypo Down Ligase Aminoacyl-tRNA ligase activity (inlcuding Pro, Ser, Ile, Glu) [GO:0004812]; [GO:0016876]; [GO:0004828]; [GO:0004829]; [GO:0004822] Hypo Down Oxidoreductase (S, peroxide) Antioxidant activity (glutathione- disulfide reductase and catalase, cytochrome-c peroxidase) [GO:0016209]; [GO:0004362]; [GO:0004096]; [GO:0004130] Nucleic acid binding Structure-specific DNA binding [GO:0000404]; [GO:0032134]; [GO:0000403]; [GO:0032137]; [GO:0032138]; [GO:0032139] Tetrapyrrole binding Chlorophyll binding [GO:0016168] Lyase Carbon-oxygen lyase activity [GO:0016835] Transferase (N) Transaminase activity (including TYR, ASP, histidinol-P, aromatic amino acids) [GO:0008483]; [GO:0004838]; [GO:0004400]; [GO:0008793]; [GO:0004069] Transferase (C1) Aspartate carbamoyltransferase activity [GO:0004070] Transferase (glycosyl) Transferase activity, transferring pentosyl groups [GO:0016763] Oxidoreductase CH-CH Biliverdin reductase activity [GO:0004074] Oxidoreductase (CH-NH2) Glutamate synthase activity [GO:0015930] Isomerase Isomerase activity [GO:0016853] Transporter NAD(P)+ transhydrogenase (B- specific) activity [GO:0003957] Hypo Down Oxidoreductase Oxidoreductase activity [GO:0016491] Oxi Oxidoreductase activity, acting on NADH or NADPH [GO:0016651]; [GO:0016652] Oxidoreductase activity, acting on the CH-OH group of donors, NAD or NADP as acceptor (including L-iditol 2-dehydrogenase activity) [GO:0016616]; [GO:0016614]; [GO:0003939] Oxi Down Lyase 3-Isopropylmalate dehydratase activity [GO:0003861] r-represented among the transcripts of significantly up- or down-regulated in the different stress condi- entified to be over-represented among the transcripts of significantly up- or down-regulated in the differen Hypo Oxi Oxi Genome Biology 2009, 10:R66 Genome Biology 2009, Volume 10, Issue 6, Article R66 Dittami et al. Manual classification of stress response genes with the most significant changes in expression Genes with roles in fatty acid metabolism altered their expression patterns in a similar way under all stress conditions. We were able to distinguish between two groups: three genes involved in the synthesis of fatty acids, which were down-regulated; and genes function- ing in the degradation of fatty acids, among which five of six genes were up-regulated. We further observed that three genes involved in lipid synthesis were up-regulated, and genes involved in inositol metabolism were also affected. tion gives a brief overview of the different groups of genes identified among the most significantly regulated genes. Among genes involved in amino acid metabolism, we found a total of 32 down-regulated genes related to the metabolism of all 20 standard amino acids except aspartic acid. In contrast, nine genes were induced in at least one abiotic stress condi- tion. These were involved in the metabolism of proline, arginine, cysteine, alanine, phenylalanine, tyrosine, tryp- tophan, leucine, isoleucine, and valine. Highly regulated genes involved in the different steps of DNA replication and protein synthesis coded for proteins, including helicases, DNA polymerases and related enzymes, proteins involved in purine and pyrimidine synthesis, DNA repair proteins, tran- scription factors, RNA processing enzymes, proteins involved in translation, ribosomal proteins, and proteins for tRNA syn- thesis and ligation. Most of these genes were down-regulated in all stress conditions, but some genes were up-regulated in response to abiotic stress. These genes include some heli- cases, transcription factors, and DNA repair proteins. We also found seven genes related to protein turnover to be down-reg- ulated and six to be up-regulated in one or more of the stress conditions. Among the up-regulated genes, there were two ubiquitin conjugating enzymes, which play a potential role in targeting damaged proteins to the proteasome, or control the stability, function, or subcellular localization of proteins. With respect to transporters, we identified five genes encod- ing nitrogen transporters (all down-regulated) as well as three genes encoding sugar transporters (all up-regulated). Genes coding for ion transporters were also mainly down-reg- ulated under hypersaline and hyposaline conditions, although two potassium and magnesium transporter genes were up-regulated under hypersaline stress. Among genes responsible for the transport of solutes and proteins to the mitochondrion, we observed an up-regulation mainly in the hyposaline stress condition. Manual classification of stress response genes with the most significant changes in expression R66.9 http://genomebiology.com/2009/10/6/R66 http://genomebiology.com/2009/10/6/R66 Transferase (P) Amino acid kinase activity [GO:0019202] Transporter Nitrate transmembrane transporter activity [GO:0015112] Transferase (C1) S-adenosylmethionine-dependent methyltransferase activity (including nicotinate phosphoribosyltransferase) [GO:0008757] Oxidoreductase (steroids) Steroid dehydrogenase activity, acting on the CH-OH group of donors, NAD or NADP as acceptor [GO:0033764] Transferase (glycosyl) Transferase activity, transferring pentosyl groups [GO:0016763]; [GO:0004853] Transferase (glycosyl) Uracil phosphoribosyltransferase activity [GO:0004845] Transferase (P) Phosphoribulokinase activity [GO:0008974] Up Motor activity Microtubule motor activity [GO:0003777] The table shows only pathways that were labeled significant at an FDR <10% in both sets of significant genes (5% FDR and 10% FDR). GO terms identified to be over-represented among the transcripts of significantly up- or down-regulated in the different stress condi- tions r-represented among the transcripts of significantly up- or down-regulated in the different stress condi- ys that were labeled significant at an FDR <10% in both sets of significant genes (5% FDR and 10% FDR). The table shows only pathways that were labeled significant at an FDR <10% in both sets of significant genes (5% FDR and 10% FDR). regulated in response to abiotic stress included eight chloro- phyll a/c binding proteins as well as genes responsible for the assembly of photosystem 2, electron transport, light sensing, and carotenoid synthesis. Many of these genes were strongly affected in the hypersaline condition, with the majority being down-regulated (17 versus 11 that were up-regulated). There was at least one gene that was up-regulated under one or more stress condition in every group. Genes with roles in fatty acid metabolism altered their expression patterns in a similar way under all stress conditions. We were able to distinguish between two groups: three genes involved in the synthesis of fatty acids, which were down-regulated; and genes function- ing in the degradation of fatty acids, among which five of six genes were up-regulated. We further observed that three genes involved in lipid synthesis were up-regulated, and genes involved in inositol metabolism were also affected. regulated in response to abiotic stress included eight chloro- phyll a/c binding proteins as well as genes responsible for the assembly of photosystem 2, electron transport, light sensing, and carotenoid synthesis. Many of these genes were strongly affected in the hypersaline condition, with the majority being down-regulated (17 versus 11 that were up-regulated). There was at least one gene that was up-regulated under one or more stress condition in every group. Manual classification of stress response genes with the most significant changes in expression Regarding genes related to vesic- ular trafficking and the cytoskeleton, we identified 13 up- and 6 down-regulated genes, many of these genes containing an ankyrin repeat domain and showing strongest changes in transcription under hyposaline and oxidative stress condi- tions. The situation was similar for genes involved in carbohydrate metabolism, where we found both glycolysis- and citric acid cycle-related genes to be strongly down-regulated under all the stresses tested (six and seven genes down-regulated, respectively). However, four genes, encoding a gluconolacto- nase, a xylulokinase, a phosphoglycerate kinase, and an isoc- itrate lyase, were up-regulated. In particular, an isocitrate lyase gene was 19- to 212-fold up-regulated under the differ- ent stress conditions. Photosynthesis-related genes that were We further found several classical stress response genes to be up-regulated. Four genes coding for heat shock proteins (HSPs) were up-regulated mainly under hyposaline and oxi- Genome Biology 2009, 10:R66 Genome Biology 2009, Volume 10, Issue 6, Article R66 Dittam http://genomebiology.com/2009/10/6/R66 Dittami et al. R66.10 dative stress, but there were also two genes coding for a chap- eronin cpn60 and a prefoldin, each of which was down- regulated. In addition, we found genes involved in protection against oxidative stress to be induced. These include a glutar- edoxin (oxidative stress), a methionine sulfoxide reductase (hyposaline stress), and three glutathione peroxidases (mainly hypersaline stress). At the same time, however, a cat- alase-coding gene was down-regulated in all stress condi- tions, most strongly under hyposaline stress. thesis of this polyol could be clearly identified based on sequence homology: mannitol 1-phosphate dehydrogenase (see [38] for a description of the mannitol synthesis pathway in brown algae). Our array contains probes for two genes identified as potential mannitol 1-phosphate dehydroge- nases: one (CL200Contig2 corresponding to Esi0017_0062 in the Ectocarpus genome), which was among the most sig- nificantly regulated genes and six-fold down-regulated in hyposaline condition, and one (CL2843Contig corresponding to Esi0020_0181), which was generally expressed at a very low level but was up-regulated approximately five-fold under hypersaline stress (P = 0.066). Two genes involved in autophagy, one of which is represented by two sequences on the microarray, were up-regulated in all stress conditions and several genes with putative signaling functions were affected. Six protein kinases were among the most significantly up-regulated genes: three equally under all stress conditions, and one each specifically under hyposaline, hypersaline and oxidative stress. Discussion hi d This study presents the first global gene expression analysis of a brown alga. Our goal was to determine the transcriptomic changes in response to short-term hypersaline, hyposaline and oxidative stress - three stresses that play an important role in the natural habitat of many brown algae, the intertidal zone [20,26]. Our results show that almost 70% of the expressed genes had a modified expression pattern in at least one of the examined stress conditions. This is in contrast to what has been observed in flowering plants, where the pro- portion of significantly regulated genes generally ranges from 1% to 30%, depending on types of abiotic stress examined, their number, and the statistical treatment applied (see [27,39,40] for some examples). Our findings demonstrate that, rather than relying on a few specific stress response pro- teins, E. siliculosus responds to abiotic stress by extensive reprogramming of its transcriptome. Stress response genes with unknown function p g All unknown and conserved unknown genes present among the most significantly regulated genes were sorted into groups according to their sequence similarity (Additional data file 6). Among the groups with three or more members, there were three (I to III) that had no known homologs in spe- cies other than E. siliculosus, and three (IV to VI) for which we were able to find homologs in other lineages for most of the sequences. A more detailed description of all of the unknown and unknown conserved stress response genes, including an analysis of conserved protein and transmem- brane domains, is available in Additional data file 6. Manual classification of stress response genes with the most significant changes in expression Furthermore, one protein kinase and one WD-40 domain containing gene were down- regulated under hyper- and hyposaline stress, respectively. Clusters of genes with similar expression patterns Clusters of genes with similar expression patterns Based on a figure of merit (FOM) graph, we decided to divide the set of expressed genes into seven different clusters (A to G). These clusters, along with the GO terms and KEGG path- ways that are over-represented among each of them, are shown in Figure 5. We identified one cluster (A) representing the stably expressed genes, three clusters included mainly up- regulated genes (B-D), and the remaining three clusters included mainly down-regulated genes (E-G). Among both the up- and down-regulated clusters, we found one cluster each that was equally affected by all stress conditions (B and E), one each where gene expression was affected only by hyposaline and oxidative stress conditions (C and G), and one cluster each where gene expression was affected mainly by hypersaline stress (D and F). Most of the principal functions identified for each cluster by GOPET and KOBAS fit well with the results from our earlier analysis of the up- and down-reg- ulated genes. Several other genes are not mentioned here, either because only a very vague prediction of their function was possible, or because they are difficult to put into categories with other genes. More detailed information about these genes can be found in Additional data file 3. Known brown algal stress genes glutathione - disulfide reductase activiy) GO: Oxidoreductase activity (aldehyde or oxo group of donors) GO: Ribosomal- protein-alanine N-acetyltransferase activity GO: Uracil phosphoribosyltransferase activity GO: Phosphoribulokinase activity GO: Transaminase activity (ASP) GO: Intramolecular oxidoreductase activity GO: Inorganic anion transmembrane transporter activity Cluster F (1,565 genes) GO: RNA binding (mRNA) GO: 3'-5'-Exoribonuclease activity GO: TATA-binding protein binding KO: Cell cycle Cluster D (1,311 genes) - Cluster E (881 genes) GO: Amino acid kinase activity GO: Aminoacyl-tRNA ligase activity Hypo Hyper Oxi Hypo Hyper Oxi -7 0 7 -7 0 7 -7 0 7 -7 0 7 Cluster B (574 genes) KO: Benzoate degradation via CoA ligation KO: Photosynthesis - antenna proteins KO: Fatty acid metabolism KO: Riboflavin metabolism KO: 2,4-Dichlorobenzoate degradation Cluster C (1,975 genes) KO: SNAREs + vesicular transport KO: MA PK signaling pathw ay GO: Protein binding Cluster A (4,052 genes) KO: Ribosome GO: Structural constituent of ribosome GO: RNA binding (rRNA) GO: RNA splicing factor activity KO: Proteasome GO: Threonine endopeptidase activity Cluster D (1,311 genes) - Hypo Hyper Oxi Cluster E (881 genes) GO: Amino acid kinase activity GO: Aminoacyl-tRNA ligase activity Cluster C (1,975 genes) KO: SNAREs + vesicular transport KO: MA PK signaling pathw ay GO: Protein binding Cluster D (1,311 genes) Expression Figure 5 e t e by t e ea s a go t dentified by the k-means algorithm. The graphs display the log2-ratio of all stress conditions (hypo = green, hyper = red, ox tion. GO terms and KEGG pathways (KO) identified as over-represented in these clusters (FDR = 10%) are shown next to p g p y g g Expression graphs of clusters identified by the k-means algorithm. The graphs display the log2-ratio of all stress conditions (hypo = green, hyper = red, oxi = blue) with the control condition. GO terms and KEGG pathways (KO) identified as over-represented in these clusters (FDR = 10%) are shown next to the graph. stresses on the cluster analysis and the results from the man- ually analyzed 966 most significantly changing genes. metabolism and growth processes; and activation of energy stores and of genes and pathways involved in 'stress manage- ment'. These findings are summarized in Figure 6. In the fol- lowing sections we will first discuss the differences observed between the different stress conditions that were tested, and then highlight some of the general trends that emerged from our data. Known brown algal stress genes Many of the brown alga-specific stress response genes identi- fied in L. digitata by Roeder et al. [21] were not among the most regulated genes identified in this study. Nevertheless, we decided to examine their expression patterns in more detail. The array used in this study contained probes for one vanadium-dependent bromoperoxidase (CL83Contig2), but this gene was not strongly regulated under the different stress conditions (1.06-fold to 1.4-fold induced, P = 0.75). Twenty- four C5-epimerases were represented, but none of these genes were among the most significantly regulated loci, although several of them were either induced or repressed under the different stress conditions. A detailed list of these genes, including their expression profiles, can be found in Additional data file 7. Finally, we decided to consider genes involved in the synthesis of mannitol, a well-known osmolyte in brown algae [16,17]. Only one enzyme specific to the syn- A more detailed analysis of the manual annotation of the 966 most significantly regulated genes and the results for the GOPET and KOBAS analysis for all three stress conditions, reveals two major themes concerning the short-term stress response of E. siliculosus: down-regulation of primary Genome Biology 2009, 10:R66 Genome Biology 2009, Volume 10, Issue 6, Article R66 Dittami et al. R66.11 http://genomebiology.com/2009/10/6/R66 Expression graphs of clusters identified by the k-means algorithm Figure 5 Expression graphs of clusters identified by the k-means algorithm. The graphs display the log2-ratio of all stress conditions (hypo = green, hyper = red, oxi = blue) with the control condition. GO terms and KEGG pathways (KO) identified as over-represented in these clusters (FDR = 10%) are shown next to the graph. -7 0 7 -7 0 7 -7 0 7 -7 0 7 -7 0 7 -7 0 7 -7 0 7 Cluster B (574 genes) KO: Benzoate degradation via CoA ligation KO: Photosynthesis - antenna proteins KO: Fatty acid metabolism KO: Riboflavin metabolism KO: 2,4-Dichlorobenzoate degradation Cluster C (1,975 genes) KO: SNAREs + vesicular transport KO: MA PK signaling pathw ay GO: Protein binding Cluster A (4,052 genes) KO: Ribosome GO: Structural constituent of ribosome GO: RNA binding (rRNA) GO: RNA splicing factor activity KO: Proteasome GO: Threonine endopeptidase activity Cluster G (1,892) KO: Photosynthesis - antenna proteins GO: Chlorophyll binding KO: Methionine metabolism KO: Carbon fixation KO: One carbon pool by folate GO: Zeaxanthin epoxidase activity GO: Transferase activity (alkyl or aryl groups) GO: Antioxidant activity (incl. Known brown algal stress genes The first and most apparent observation from the clustering was that the changes in gene expression induced by hyposa- line and oxidative stress were more similar to each other than to those observed under hypersaline stress. One explanation for this might be that hypersaline stress, although it is a com- mon stressor in the natural habitat of brown algae, is not likely to occur at the same intensity in the field as that used for our laboratory experiments (about three times the concentra- tion of normal seawater). Even though we did not observe a strong difference in the efficiency of photosynthesis under the different stress conditions, it is possible that hypersaline stress, at the intensities applied in our experiments, repre- sents a condition the alga is less able to adjust to. This hypoth- esis is supported by the fact that in cluster F (down-regulated in hypersaline conditions) cell cycle-related genes were over- represented, indicating that growth was most strongly Major tran Figure 6 ajo t a sc pto c c a ges . s cu osus u e s o t te o at ve a sa e st ess gu e 6 Major transcriptomic changes in E. siliculosus under short-term oxidative and saline stress. This schema summarizes the most important transcriptomic changes discussed in the text. Processes on the left (blue) were repressed, while processes on the right (yellow) were activated. Please note that this graph displays only the general trends; some of the pathways are not regulated in all stress conditions and not all genes of one pathway are always regulated in the same way (see text for details) j p g g Major transcriptomic changes in E. siliculosus under short-term oxidative and saline stress. This schema summarizes the most important transcriptomic changes discussed in the text. Processes on the left (blue) were repressed, while processes on the right (yellow) were activated. Please note that this graph displays only the general trends; some of the pathways are not regulated in all stress conditions and not all genes of one pathway are always regulated in the same way (see text for details). affected under hypersaline stress conditions. Furthermore, other classical stress responses, such as the up-regulation of SNARE (soluble N-ethylmaleimide-sensitive factor attach- ment receptor)-related genes, which are important for cellu- lar transport of vesicles and their fusion with membranes and play a role in plant development and abiotic stress response [41], were observed mainly under oxidative and hyposaline stress. Down-regulation of growth and primary metabolism Many genes involved in several pathways related to growth and primary metabolism were identified to be down-regu- lated by more than one of our analyses (GOPET, KOBAS, k- means clustering, and manual analysis). We observed a decrease in the abundance of transcripts of genes that are important in the synthesis of purine and pyrimidine nucle- otides and, correspondingly, of several genes responsible for the replication of DNA. This function is essential for cell divi- sion, a process affected in all of the stress conditions exam- ined. There are also several smaller differences between oxidative and hyposaline stress, such as the induction of a glutaredoxin gene (Additional data file 2) under oxidative stress condi- tions. Glutaredoxins are known to play a central role in the protection against oxidative damage [42], as they can be oxi- dized by diverse substrates, including ROSs, and are reduced by glutathione. Comparison of stress conditions We have compared each stress condition to the control condi- tion, and analyzed these results using KOBAS and GOPET. Due to the necessity to control the FDR with multiple testing, the chance of beta-errors (that is, the chance of falsely labe- ling a gene or a pathway as not significantly regulated) greatly increased, making a direct comparison of the genes and groups of genes identified as being up- or down-regulated under the different stress conditions prone to false conclu- sions. Therefore, we based our comparison of the different Genome Biology 2009, 10:R66 Genome Biology 2009, Volume 10, Issue 6, Article R66 Dittami et al. R66.12 http://genomebiology.com/2009/10/6/R66 Major tran Figure 6 Other genes did not change expression under oxidative stress, but were specifically regulated under saline stress. Such examples are given in the following paragraphs. A reduction in growth implies a reduction in the require- ments for primary metabolites necessary to fuel this growth. Within our dataset, we found widespread evidence for down- regulation of processes involved in primary metabolism. This was most pronounced in the case of protein synthesis. Several genes encoding enzymes involved in this and related proc- Genome Biology 2009, 10:R66 Genome Biology 2009, Volume 10, Issue 6, Article R66 Dittami et al. R66 http://genomebiology.com/2009/10/6/R66 Dittami et al. R66.13 esses (the synthesis of amino acids, their ligation with the appropriate tRNAs, the production of mRNA (that is, tran- scription), and the actual assembly of polypeptide chains (that is, translation)) were down-regulated under stress con- ditions. Furthermore, genes responsible for the uptake of nitrogen, which is used mainly for the synthesis of amino acids, were also down-regulated. Together, this provides a strong indication that the overall rate of protein synthesis was reduced under the stress conditions examined. bohydrates when non-lipid-derived storage reserves are depleted. Corresponding to this and the up-regulation of autophagy-related genes, we observed an up-regulation of three genes coding for sugar transporters in all stress condi- tions and of genes coding for mitochondrial exchange pro- teins under the hyposaline and oxidative stress conditions. These transporters may direct recycled sugars and nutrients to the mitochondrion, where they can be used for energy pro- duction. However, there were additional primary metabolic processes that were at least partially affected by the stress treatments. These included the synthesis of fatty acids, photosynthesis and pigment synthesis, and carbohydrate metabolism. Again, these changes probably reflected a decreased need for metab- olites for growth. Recently, additional roles of autophagy have been discovered in higher plants, including the degradation and removal of oxidized or damaged proteins during stress [47]. To a certain degree, these roles overlap with the role of the proteasome. Although we found some genes involved in protein turnover to be down-regulated, the KEGG pathway 'proteasome' was identified as up-regulated in the hyposaline stress condition and we identified two genes involved in ubiquitination among the most significantly up-regulated genes under all stress conditions. Ubiquitination is a process in which proteins are labeled with a small polypeptide (ubiquitin) [48], thereby modifying their stability, function, or subcellular localization. Activation of signaling pathways Large-scale transcriptomic reprogramming as observed in our dataset most certainly requires a large number of signals for coordination. We have already discussed a possible regu- latory role of ubiquitination in the previous paragraph. This, however, is not the only regulatory mechanism highlighted by the transcriptomic changes in our dataset. We found several other genes that might play roles in orchestrating the abiotic stress response of E. siliculosus. For example, three protein kinases with a potential role in cell signaling were strongly up-regulated under all stress conditions, while three other members of this large family appeared each to be specific to one particular stress. Furthermore, several potential tran- scription factors were strongly regulated under different stress conditions. Since there is still very little known about the molecules and proteins involved in the stress sensing sig- naling cascades of brown algae, these genes provide particu- larly interesting candidates for more targeted experiments such as targeted mutagenesis and chromatin immunoprecip- itation in the case of the putative transcription factors. Major tran Figure 6 This could serve regulatory purposes (for example, by target- ing transcription factors or other regulatory proteins for deg- radation by the proteasome), accelerate translation of the large-scale transcriptomic changes into changes in protein abundance, and/or, just like autophagy, facilitate nitrogen recycling [49]. One possible explanation for the observed down-regulation of genes involved in growth and primary metabolism can be found in the results of our pulse amplitude modulation fluor- ometer experiment: we observed that the efficiency of photo- synthesis decreased almost immediately after the application of the stress treatments. Photosynthesis is strongly affected by environmental stress [43]. A decrease in photosynthetic efficiency is synonymous with a decrease in energy produc- tion, and we can assume that reducing all of these aspects of primary metabolism might represent a means of conserving energy. This phenomenon is known in higher plants, where Kovtun et al. [44] have reported cross-talk between oxidative stress and auxin signaling, auxin being a major growth hor- mone in higher plants. Most likely, this cross-talk allows plants to shift their energy from growth-related processes to stress protection and survival. This might also be true for E. siliculosus, where the observed down-regulation of growth- and primary metabolism-related genes might represent a way of compensating for reduced energy production under stress conditions and redirecting energy to specific stress response processes. Synthesis of 'classical' stress response proteins The only medium throughput transcriptomic analysis of the abiotic stress response in brown algae so far [21], conducted with protoplasts of L. digitata, reported the transcriptional activation of vanadium-dependent bromoperoxidases and C5-epimerases. Neither of these genes was regulated in our study. While in L. digitata vanadium-dependent bromoper- oxidases (enzymes implicated in the synthesis of halogenated organic compounds associated with defense of seaweeds against biotic stressors [52]) comprise a multigenic family [21], the E. siliculosus genome contains only a single copy of a vanadium-dependent bromoperoxidase, indicating a possi- bly different or subordinate role in this organism. Regarding C5-epimerases, which are enzymes responsible for the modi- fication of brown algal cell walls [10] highly represented in the Ectocarpus genome, we observed differences in regulation between the study of Roeder et al. [21] and our study. This can be explained by the nature of the stress (generation of proto- plasts (that is, removal of the cell wall) in [21] versus milder saline or oxidative stress in our study). An alternative or additional explanation to that of non-tran- scriptional mechanisms regulating the activity of ROS scav- enging enzymes could be that other, yet unknown proteins and mechanisms play more important roles in the defense against ROS in brown algae. One candidate for this kind of protein could be the chlorophyll a/c binding proteins. Thirty chlorophyll a/c binding proteins were represented on our microarray, most of them being down-regulated mainly under hyposaline and oxidative stress conditions. As chloro- phyll a/c binding proteins serve as light-harvesting antennae, this down-regulation is likely to represent a response to the reduced photosynthesis efficiency (quantum yield) under stress conditions. Reducing the amount of energy that reaches the photosynthetic reaction center would also reduce the need for non-photochemical quenching and decrease the risk of the formation of ROS. However, there were also three genes coding for chlorophyll a/c binding proteins among the most significantly up-regulated genes under hyposaline and hypersaline stresses. A similar observation was made by Hwang et al. [55] in the Antarctic diatom Chaetoceros neogracile, where heat stress induced the up-regulation of five and the down-regulation of ten genes coding for chloro- phyll a/c binding proteins. It is possible that these up-regu- lated chlorophyll a/c binding proteins, in spite of their high sequence similarity with the other transitionally down-regu- lated ones, have evolved or are evolving to serve different functions within the heterokont lineage. Activation of protein degradation, energy stores, and nutrient recycling g We observed an up-regulation of two genes involved in autophagy in all three of the stresses examined. Under nutri- ent-limited and under stress conditions, this process has been shown to play a role in the re-allocation of sugar and nutrients to essential biological processes in several organisms [45]. The activation of autophagy-related genes might, therefore, just like the down-regulation of growth-related processes, represent a mechanism of compensating for reduced energy production under stress conditions and provide sugars and nutrients for both core biological processes and synthesis of stress proteins. This coincides with the strong up-regulation of an isocitrate lyase gene under all stress conditions. Isoci- trate lyases are enzymes located in the glyoxysome and cata- lyze a rate-controlling step in the glyoxylate cycle (reviewed in [46]), one function of which is the conversion of lipids to car- In addition to transcription factors and protein kinases, we detected an up-regulation of genes involved in fatty acid metabolism, and more specifically fatty acid catabolism. Fatty acid derivates such as oxylipins have been shown to function in signaling in both terrestrial plants [50] and marine algae [51]. Thus, we can conclude that our data show an up-regula- Genome Biology 2009, 10:R66 Genome Biology 2009, Volume 10, Issue 6, Article R66 Genome Biology 2009, Volume 10, Issue 6, Article R66 Dittami et al. R66.14 Dittami et al. R66.14 http://genomebiology.com/2009/10/6/R66 tion of genes putatively involved in several different signaling pathways. dant activity was slightly repressed (1.17-fold) under hyposa- line stress conditions and slightly induced under hypersaline stress conditions (1.15-fold). One possible explanation for this might lie in the fact that transcriptional regulation is not the most important mechanism regulating the activity of these enzymes. This hypothesis would be compatible with an earlier study by Collén and Davison [54], who found that the cellular activity of ROS scavenging enzymes correlated with vertical zonation of different species of the brown algal order Fucales in the intertidal zone. To our knowledge, it is currently not known whether the activity of the ROS scavengers that were examined also changes upon exposure to abiotic stress. Such studies could greatly aid our understanding of the role of these enzymes in the brown algal stress response. Synthesis of 'classical' stress response proteins Generally, only a few 'classical' stress response genes changed expression in our experiments. In most organisms, up-regu- lation of genes coding for HSPs and other chaperones has been observed under abiotic stress conditions. These mole- cules stabilize proteins and membranes, and have been shown to play a vital role in protecting against stress by re- establishing normal protein conformation and, thus, cellular homeostasis [53]. In E. siliculosus, we observed four HSPs or chaperones to be among the most significantly up-regulated genes in hyposaline and oxidative stress conditions. How- ever, this was not the case under hypersaline stress condi- tions. Moreover, two chaperone-like proteins were down- regulated under all stress conditions. The situation was simi- lar for genes coding for proteins with antioxidant activity. Three genes coding for glutathione peroxidases were up-reg- ulated under hypersaline and oxidative stress, and one glutar- edoxin under hyposaline stress. At the same time, two genes encoding a glutathione S-transferase and a catalase were among the most significantly down-regulated in all stress conditions. Consequently, the GO term 'antioxidant activity' was significantly over-represented among the most down- regulated genes in the hyposaline condition. In flowering plants all of these proteins are known to carry out important functions in the protection against reactive oxygen species [25]. Our finding that these genes were not induced was, at first, surprising, but is in accordance with a transcriptomic analysis of the abiotic stress response of the intertidal red alga C. crispus. Collén et al. [20] have reported that the average expression of HSP-coding genes was only moderately ele- vated (approximately 1.3-fold) under hypersaline and hyposaline conditions. Furthermore, in E. siliculosus, the average expression of genes coding for proteins with antioxi- Conclusions hi d In this study, which presents the first large-scale transcrip- tomic study within the brown algal lineage, we have devel- oped and compiled the tools and protocols necessary to perform microarray experiments in the emerging model brown alga E. siliculosus, and used these tools to study the transcriptional response to three different stresses. Our results show that E. siliculosus undergoes large-scale tran- scriptomic reprogramming during the short-term acclima- tion to abiotic stress. The observed changes include several modifications to transcription, translation, amino acid metabolism, protein turnover, and photosynthesis, and indi- cate a shift from primary production to protein and nutrient recycling. While there is no evidence of the synthesis of additional com- patible osmolytes in the Burma mangrove, we observed an increasing difference between intracellular osmolarity and intracellular Na+ concentration with rising salinity in E. silic- ulosus, demonstrating the accumulation of other osmotically active substances in the cell. Mannitol has frequently been suggested to be a compatible osmolyte in brown alga [16,17], and indeed, one of the two mannitol 1-phosphate-dehydog- enases in E. siliculosus was down-regulated in hyposaline stress, and the other up-regulated in hypersaline stress (though with a weak P-value of 0.066). In addition, under hyposaline conditions, we observed a strong up-regulation of a proline dehydrogenase gene (Additional data file 3), an enzyme responsible for the degradation of proline, which is known as a compatible osmolyte in higher plants [59] and diatoms [60]. Degrading proline under conditions of low salinity might help E. siliculosus to reduce the osmotic pres- sure between the intracellular and extracellular medium. Finally, a possible role of urea as a compatible osmolyte was suggested in diatoms [61]. E. siliculosus possesses the com- plete urea cycle and genes encoding arginases were up-regu- lated in hypersaline conditions. As arginases catalyze the last reaction of this cycle - that is, the degradation of arginine to ornithine and urea - their up-regulation supports the hypoth- esis of urea as a compatible osmolyte in heterokonts. There are, however, other or additional possible roles of arginases: in higher plants, for example, arginases have been shown to play a regulatory role in nitric oxide metabolism, increasing both the synthesis of proline and polyamines [62], both of which, in turn, are part of their osmotic stress response [59]. Ions and potential osmolytes In parallel to the induction of 'classical' stress response genes, we observed the transcriptional regulation of several genes involved in the synthesis or degradation of potential organic osmolytes and the transport of ions under saline stress. Under hypersaline stress, changes in the extracellular salt concentration as well as cell volume are likely to cause imbal- ances in ion concentrations, explaining the need for trans- porters to maintain homeostasis. Two genes coding for a magnesium and a potassium transporter were up-regulated specifically under hypersaline stress. Interestingly, we did not observe transcriptional activation of sodium transporter genes under salt stress, although many glycophytes (non- or Genome Biology 2009, 10:R66 Genome Biology 2009, Volume 10, Issue 6, Article R66 Genome Biology 2009, Volume 10, Issue 6, Article R66 Dittami et al. R66.15 http://genomebiology.com/2009/10/6/R66 Dittami et al. R66.15 moderately salt tolerant terrestrial plants) use these trans- porters to exclude NaCl from their cytosol, allowing a certain degree of salt tolerance [56]. The latter observation suggests that the large quantities of NaCl accumulated upon exposure to saline stress (Figure 2) are stored within the cytoplasm rather than in the vacuole. A similar observation was made by Miyama and Tada [57] in the Burma mangrove. In this tree, exposure to sub-lethal NaCl stress did not cause an activation of Na transporters but led to a slow increase of the NaCl con- centration in the leaves. One possible explanation for this, as proposed by Miyama and Tada [57], is that NaCl itself could serve as an osmolyte within the cells of the Burma mangrove. This may also be true for brown algae - a hypothesis that is strengthened by the observation that, in Ectocarpus as well as in Lamninaria [58] and the Burma mangrove [57], sorbitol, added at the same osmolarity as NaCl, had irreversible toxic effects. not forget that this analysis was based on only a subset of the genes that actually changed expression. Our manual analysis of the 966 of the most significantly regulated genes has shown that 53.7% of these genes, to date, have no known homologs in current databases, including diatoms, and for another 12.6% there is no indication of their function, even though homologs exist in other organisms. This demonstrates both the discovery potential working with E. siliculosus and the amount of work that lies ahead for the phycological commu- nity. Conclusions hi d Additional experiments addressing the question of the possi- ble compatible osmolytes in brown algae - for example, metabolite profiling - are now required to further test these hypotheses. Although E. siliculosus shares many stress responses with flowering plants, for example, the induction of genes involved in vesicular trafficking, some classical stress responses, such as the induction of several ROS scavengers, could not be observed. On the other hand, our data highlighted many novel reactions such as the up-regulation of several genes coding for chlorophyll a/c binding proteins or the regulation of a large percentage of unknown genes, many of which are unique to E. siliculosus. In particular, the latter result, that is, that the functions of two-thirds of the regulated genes are unknown, underlines the fact that many of the molecular mechanisms underlying the acclimation to environmental stresses in brown algae are still entirely unknown. Under- standing these mechanisms is a challenge that will still require much research, and our study provides a valuable starting point to approach this task. Materials and methods Growth conditions, stress treatments, and measurements of osmolarity and Na+ concentration E. siliculosus (Dillwyn) Lyngbye (Ectocarpales, Phaeophyc- eae) unialgal strain 32 (accession CCAP 1310/4, origin San Juan de Marcona, Peru) was cultivated in 10-liter plastic flasks in a culture room at 14°C using filtered and autoclaved natural seawater enriched in Provasoli nutrients [63]. Light was provided by Philips daylight fluorescence tubes at a pho- ton flux density of 40 μmol m-2 s-1 for 14 h per day. Cultures were bubbled with filtered (0.22 μm) compressed air to avoid Automatic annotation and correspondence table Both the superna- tant and a sample of the culture medium were then used to measure the concentration of osmolytes employing a freezing point depression osmometer (Osmometer Type 15, Löser Messtechnik, Berlin, Germany), and to determine the intrac- ellular concentration of Na+ with a FLM3 flame photometer (Radiometer, Copenhagen, Denmark). Immediately after harvesting, about 300 mg (wet weight) of sample were thoroughly ground at room temperature (RT) and centrifuged for 1 minute at 16,000 g. Both the superna- tant and a sample of the culture medium were then used to measure the concentration of osmolytes employing a freezing point depression osmometer (Osmometer Type 15, Löser Messtechnik, Berlin, Germany), and to determine the intrac- ellular concentration of Na+ with a FLM3 flame photometer (Radiometer, Copenhagen, Denmark). Sample preparation, hybridization and verification RNA was extracted from approximately 100 mg (wet weight) of tissue following Apt et al. [68] with modifications as described by Le Bail et al. [28], using a cetyltrimethylammo- nium bromide (CTAB)-based buffer and subsequent phenol- chloroform purification, LiCl-precipitation, and DNAse (Turbo DNAse, Ambion, Austin, TX, USA) steps. RNA quality and quantity was then verified on 1.5% agarose gel stained with ethidium bromide and a NanoDrop ND-1000 spectro- photometer (NanoDrop products, Wilmington, DE, USA). For four of the five flasks, mRNA was isolated from the total RNA using the PolyATtract® mRNA Isolation System III (Promega, Madison, WI, USA). These samples were concen- trated in a SpeedVac concentrator (Savant, Ramsy, MN, USA) and again quantified using the NanoDrop. One replicate was used to verify if our procedure would directly work from total RNA. Automatic annotation and correspondence table Automatic annotation and correspondence table All sequences were automatically annotated with KEGG orthology (KO) numbers using KOBAS [37] and with GO terms [66] using GOPET [36]. Protein sequences correspond- ing to the assembled EST sequences were then predicted using ORF predictor [67]. The automatic annotation of these sequences yielded 2,383 and 3,148 annotated sequences, respectively. As 37.5% of the cDNA sequences that were rep- resented on the array contained mainly, or exclusively, 3' untranslated region sequence, their function could not be assessed directly. In these cases the corresponding genome sequence was annotated, yielding an additional 1,047 KO and 2,743 GO annotations. The correspondence table used to relate the assembled ESTs to supercontigs was generated by blasting all of the assembled EST sequences against the full E. siliculosus genome (coding and non-coding sequences) and selecting the best hit (best identity, longest alignments). Wherever this best hit was part of a predicted coding sequence (CDS), the corresponding CDS was chosen. In cases where the hit region was upstream of only one CDS, this CDS was chosen. In some cases the best hit was located upstream of two CDSs on opposite strands. Here the closest CDS was selected, if the distance to the closer CDS was half as long as or shorter than that to the other CDS. Otherwise no corre- sponding genome sequence was selected. In total, 3,430 (20%) of all represented genes were annotated with KEGG and 5,891 (34%) were annotated with GO terms. In order to monitor the intensity of a stress, we measured the quantum yield, a fluorometric marker for the photosynthetic efficiency, using a Walz Phyto-pulse amplitude modulation fluorometer (Waltz, Effeltrich, Germany) and default param- eters (actinic light intensity 3, approximately 90 μE m-2 s-1; saturation pulse intensity 10, approximately 2,000 μE m-2 s-1, 200 ms) before harvesting the cultures. The stress experiment was started by filtering 20 liters of ASW-acclimated E. siliculosus and transferring approxi- mately 4 g of tissue each to 20 flasks (5 replicates per condi- tion) containing 1 liter of one of the three stress media or the control medium (ASW with Provasoli nutrients). After 6 h the content of each flask was harvested by filtration, dried with a paper towel, and immediately frozen in liquid nitrogen. Immediately after harvesting, about 300 mg (wet weight) of sample were thoroughly ground at room temperature (RT) and centrifuged for 1 minute at 16,000 g. ress response genes with unknown functions Although our study has revealed several major themes under- lying the abiotic stress response of E. siliculosus, we should Genome Biology 2009, 10:R66 Genome Biology 2009, Volume 10, Issue 6, Article R66 Genome Biology 2009, Volume 10, Issue 6, Article R66 Dittami et al. R66. Dittami et al. R66.16 http://genomebiology.com/2009/10/6/R66 CO2 depletion. Ten days before the stress experiments, tissues were transferred to ASW with the following ion composition: 450 mM Na+, 532 mM Cl-, 10 mM K+, 6 mM Ca2+, 46 mM Mg2+, 16 mM SO42-. CO2 depletion. Ten days before the stress experiments, tissues were transferred to ASW with the following ion composition: 450 mM Na+, 532 mM Cl-, 10 mM K+, 6 mM Ca2+, 46 mM Mg2+, 16 mM SO42-. Four 60-mere probes were designed for 17,119 of the 17,332 sequences (132 genes are not represented on the array) by Roche NimbleGen (Madison, WI, USA) and synthesized on 4- plex arrays with 72,000 features per hybridization zone. Roche NimbleGen also carried out cDNA labeling and hybrid- ization as part of their gene expression array service. Three different stress media were prepared based on ASW. For hyposaline stress, ASW was diluted to 12.5% of its original concentration with distilled water, resulting in a final NaCl concentration of 56 mM. For hypersaline stress, ASW with a final concentration of 1.47 M NaCl was used. For oxidative stress, H2O2 (30% w/w; Sigma-Aldrich, St. Louis, MO, USA) was added to the ASW immediately before beginning the stress experiment at a final concentration of 1 mM. Identical quantities of Provasoli nutrients were added in each of these media. Stress response genes with unknown functions Stress response genes with unknown functions Genes were considered unknown stress response genes if they significantly changed expression in at least one stress treat- ment compared to the control (FDR <1%, more than twofold change compared to the control) and when, for both the assembled EST sequence itself and for the corresponding genome sequence, no BLAST hits were found in either the NCBI databases or among the known heterokont genomes (Phytophthora sojae, P. ramosum, Thalassiosira pseudo- nana, and Phaeodactylum tricornutum). Where homologs (e-value < 1e-10) were found, but these homologs had no functional annotations, genes were considered conserved unknown stress response genes. To group these genes, all unknown stress response genes and conserved unknown stress response genes were blasted against themselves using the NCBI BLAST program (version 2.2.18) [78] and a cut-off e-value of 1e-10. If there were several alignments between two genes, only the alignment with the lowest e-value was consid- ered. Self-hits were removed. All groups of genes with homologs among the (conserved) unknown stress response genes in E. siliculosus were then visualized using Cytoscape 2.6.0 [79]. Their subcellular localizations were identified using HECTAR [80] and transmembrane domains were searched for using TMAP [81]. RT-qPCR validation of the microarray was performed as described by Le Bail et al. [28]. Sequence preparation and array design Sequence preparation and array design The 90,637 EST sequences used for the microarray design were cleaned using Phred [64] (trim-cutoff 0.05) and Seq- Clean and assembled using TGICL [65] and default parame- ters. Forty-one sequences that had been removed by Phred were re-included in the dataset, because they had significant BLAST hits with known eukaryotic proteins. In addition, 231 E. siliculosus virus 1 (Es-V1) sequences and 21 genomic intron sequences were included in the design. All assembled sequences are available directly from our homepage [22] and the ESTs have been deposited in public databases [EMBL: FP245546-FP312611]. Relevant accession numbers are listed in Additional data file 8. Double-stranded cDNA was synthesized and amplified with the SMART cDNA synthesis kit (Clontech, Mountain View, CA, USA) and the M-MuLV reverse transcriptase (Finnzymes, Genome Biology 2009, 10:R66 Genome Biology 2009, Volume 10, Issue 6, Article R66 Ditta Dittami et al. R66.17 http://genomebiology.com/2009/10/6/R66 control, considering all mRNA replicates using TigrMEV 4.1. A k-means algorithm [74], 'Euclidian distance', and 'average linkage' were selected. The ideal number of clusters for our dataset (k = 7) was determined using a figure of merit graph [75]. After clustering, the different replicates were averaged to generate the expression graphs. Both the clusters and the over-expressed and repressed genes identified by the t-test (FDR 10%) were analyzed to identify over-represented groups of genes. Over-expressed KEGG categories were identified using the KOBAS web-site [37] and a binomial test. Over-rep- resented GO terms were identified using the GO Local Explo- ration Map (GOLEM) software (version 2.1) [76] and the Benjamini and Yekutieli algorithm to determine the FDR [77]. Additionally, the 966 genes that showed the most signif- icant changes in expression (that is, genes that meet both cri- teria: significance at an FDR <1% and a relative change in expression compared to the control of more than twofold) were annotated and grouped manually. Espoo, Finland) starting from 30 ng of mRNA or 100 ng of total RNA. In this kit, the first strand of cDNA is synthesized using an oligo(dT) primer with an attached SMART priming site. The terminal C-transferase activity of the reverse tran- scriptase will create an oligo(dC) tail at the 5' end of each mRNA, which is used to add a second SMART priming site. Statistical analysis Expression values were generated by Roche NimbleGen using quantile normalization [69], and the Robust Multichip Aver- age algorithm [70,71]. To increase the power of subsequent statistical tests, non- expressed genes were removed from the dataset. This was done by comparing the raw expression values of each gene with those of the random probes included in the array design by Roche NimbleGen. Most of the random probes (>99.66%) had raw expression values inferior to 450, so all genes that had raw expression values over 450 in one of the replicates of the different experimental conditions were considered expressed. The probability p' of labeling a non-expressed gene as being expressed can be calculated according to the laws of a binomial distribution. Since 16 hybridizations were considered, for our dataset p' equals 5.3%, meaning that we have theoretically removed 94.7% of all genes without detect- able expression. Data deposition Microarray data have been deposited in a public database [ArrayExpress:E-TABM-578]. Sequence preparation and array design The two priming sites are then used to produce the second strand of the cDNA and to amplify it by PCR according to the Clontech protocol, using the Advantage2 polymerase (Clon- tech). The optimal number of amplification cycles was deter- mined by semi-quantitative PCR, and ranged between 20 and 25 for our samples. Espoo, Finland) starting from 30 ng of mRNA or 100 ng of total RNA. In this kit, the first strand of cDNA is synthesized using an oligo(dT) primer with an attached SMART priming site. The terminal C-transferase activity of the reverse tran- scriptase will create an oligo(dC) tail at the 5' end of each mRNA, which is used to add a second SMART priming site. The two priming sites are then used to produce the second strand of the cDNA and to amplify it by PCR according to the Clontech protocol, using the Advantage2 polymerase (Clon- tech). The optimal number of amplification cycles was deter- mined by semi-quantitative PCR, and ranged between 20 and 25 for our samples. The PCR reactions were purified by first vortexing with one volume of phenol:chloroform:isoamyl alcohol (25:24:1), then by precipitating the aqueous phase with 0.5 volume of 7.5 M NH4OH, 6 μg of nuclease-free Glycogen (Ambion), and 2.4 volumes of ethanol. After centrifugation at RT (20 minutes at 14,000 g), the pellet was washed with 70% EtOH, centrifuged (10 minutes at 14,000 g and RT) and resuspended in 14 μl of H2O. To finish, cDNAs were once more quantified and checked on an agarose gel to ensure that they met the require- ments of Roche NimbleGen for hybridization (concentration >250 μg l-1, A260/280 ≥ 1.7, A260/230 ≥ 1.5, median size ≥ 400 bp). Additional data files 9. Tonon T, Rousvoal S, Roeder V, Boyen C: Expression profiling of the mannuronan C5-epimerase multigenic family in the brown alga Laminaria digitata (Phaeophyceae) under biotic stress conditions. J Phycol 2008, 44:1250-1256. The following additional data are available with the online version of this paper: a figure showing the recovery of the photosynthetic efficiency (quantum yield) in E. siliculosus cultures after 24 hours of stress treatment (Additional data file 1); a table listing the KEGG pathways and GO terms iden- tified to be over-represented among the transcripts signifi- cantly different in the mRNA and total-RNA-derived samples (Additional data file 2); a table listing the 966 most regulated (FDR <0.01) and the 100 most stable genes on the microarray in all stress conditions (Additional data file 3); a table listing the KEGG pathways identified to be over-represented among the transcripts significantly up- or down-regulated in the dif- ferent stress conditions (Additional data file 4); a table listing the GO categories identified to be over-represented in the dif- ferent conditions at a FDR of 10% (Additional data file 5); description of the unknown stress response genes identified in this study (Additional data file 6); a table listing the C5-epi- merase-genes represented on the microarray and their expression profiles (Additional data file 7); accession num- bers of all sequences used in the design of the E. siliculosus microarray (Additional data file 8). Additional data files Addi i l d fil R f h h h i ffi i ( i ld) i E ili l l f h f Th d di i d h di i d f h i i ( M N Cl % d M H O ) b h d i f h h h h 6 h Cli k h f fil Addi i l d fil KEGG h d GO id ifi d b d h i i ifi l diff i h RNA d l RNA d i d l P i di h b bili f hi h b i ll d i h d ( % FDR d % FDR) Th l FDR f h FDR h li d h id if i h i if i l l d i d f hi l i Cli k h f fil Addi i l d fil C l li f h 66 l d (FDR ) d f h bl h i i ll di i All d di h i i f i Cli k h f fil Addi i l d fil C l li f KEGG h id ifi d b d h i i ifi l d l d i h diff di i Q h l h i h d i f f l i i i d h h h i id d i ifi I i i f h d i i id d b i if i l l d FDR f % d % P h i ifi l hi hli h d i b ldf h k b i E ili l d h i ibl if f h i i h i Cli k h f fil Addi i l d fil C l li f GO i id ifi d b d i h diff di i FDR f % Th l FDR f h FDR h li d h id if i h i ifi l l d i d f hi l i Cli k h f fil Addi i l d fil 6 U k id ifi d i hi d Th fi b i hi l i f id ifi d f k Th l h l f (I VI) f h k h h i i h h i f k I h k h b d l f d i E ili l hil d f d i h i l Ed h i il i b ( l f h BLAST ) Th l h i d b h k l i (Fi ) i i i b ldf F h h i fil f h i h h di i (h li h li d id i ) i i h i h Y ll i di h d i i l di i hil bl k i di h d bl i di h i l d Th h b i ddi i l i f i h di i l i id b ll l l li i f h i h l f h b d i h d h i fil f b h h h i h fi b d h h h ld b i d h diff Cli k h f fil Addi i l d fil C i d h i d h i i fil C i d h i d h i i fil Cli k h f fil Addi i l d fil 8 A i b f ll d i h d i f h E ili l i A i b f ll d i h d i f h E ili l i Cli k h f fil 10. Additional data files Nyvall P, Corre E, Boisset C, Barbeyron T, Rousvoal S, Scornet D, Kloareg B, Boyen C: Characterization of mannuronan C-5-epi- merase genes from the brown alga Laminaria digitata. Plant Physiol 2003, 133:726-735. y 11. Charrier B, Coelho SM, Le Bail A, Tonon T, Michel G, Potin P, Kloareg B, Boyen C, Peters AF, Cock JM: Development and phys- iology of the brown alga Ectocarpus siliculosus: two centuries of research. New Phytol 2008, 177:319-332. 12. Peters AF, Marie D, Scornet D, Kloareg B, Cock JM: Proposal of Ectocarpus siliculosus (Ectocarpales, Phaeophyceae) as a model organism for brown algal genetics and genomics. J Phycol 2004, 40:1079-1088. y 13. Coelho SM, Peters AF, Charrier B, Roze D, Destombe C, Valero M, Cock JM: Complex life cycles of multicellular eukaryotes: new approaches based on the use of model organisms. Gene 2007, 406:152-170. 14. Peters AF, Scornet D, Ratin M, Charrier B, Monnier A, Merrien Y, Corre E, Coelho SM, Cock JM: Life-cycle-generation-specific developmental processes are modified in the immediate upright mutant of the brown alga Ectocarpus siliculosus. Devel- opment 2008, 135:1503-1512. p 15. Soeder C, Stengel E: Physico-chemical factors affecting metab- olism and growth rate. In Algal Physiology and Biochemistry Volume 10. Edited by: Stewart WDP. Oxford, London, Edinburgh, Melbourne: Blackwell Scientific Publications; 1974. [Brunett JH, Baker HG, Beevers H, Whatley FR (Series Editors): Botanical Monographs] y ( ) g p 16. Davison IR, Reed RH: The physiological significance of mannitol accumulation in brown algae: the role of mannitol as a com- patible solute. Phycologia 1985, 24:449-457. p y g 17. Reed RH, Davison IR, Chudek JA, Foster R: The osmotic role of mannitol in the phaeophyta - an appraisal. Phycologia 1985, 24:35-47. Abbreviations Geissler U: Die salzbelastete Flusstrecke der Werra - ein Bin- l d d fü E f id (R h) Kj ll Information; ROS: reactive oxygen species; RT: room tem- perature; RT-qPCR: real time quantitative PCR; SNARE: sol- uble N-ethylmaleimide-sensitive factor attachment receptor. 2. Baldauf SL: The deep roots of eukaryotes. Science 2003, 300:1703-1706. 2. Baldauf SL: The deep roots of eukaryotes. Science 2003, 300:1703-1706. 3. Graham MH, Kinlan BP, Druehl LD, Garske LE, Banks S: Deep-water kelp refugia as potential hotspots of tropical marine diversity and productivity. Proc Natl Acad Sci USA 2007, 104:16576-16580. 4. Santelices B: The discovery of kelp forests in deep-water habi- tats of tropical regions. Proc Natl Acad Sci USA 2007, 104:19163-19164. 3. Graham MH, Kinlan BP, Druehl LD, Garske LE, Banks S: Deep-water kelp refugia as potential hotspots of tropical marine diversity and productivity. Proc Natl Acad Sci USA 2007, 104:16576-16580. 4 Santelices B: The discovery of kelp forests in deep water habi kelp refugia as potential hotspots of tropical marine diversity and productivity. Proc Natl Acad Sci USA 2007, 104:16576-16580. 4. Santelices B: The discovery of kelp forests in deep-water habi- tats of tropical regions. Proc Natl Acad Sci USA 2007, 104:19163-19164. p y 4. Santelices B: The discovery of kelp forests in deep-water habi- tats of tropical regions. Proc Natl Acad Sci USA 2007, 104:19163-19164. Authors' contributions 5. Küpper FC, Carpenter LJ, McFiggans GB, Palmer CJ, Waite TJ, Boneberg E-M, Woitsch S, Weiller M, Abela R, Grolimund D, Potin P, Butler A, Luther GW, Kroneck PMH, Meyer-Klaucke W, Feiters MC: Iodide accumulation provides kelp with an inorganic antioxi- dant impacting atmospheric chemistry. Proc Natl Acad Sci USA 2008, 105:6954-6958. SMD, together with and under supervision of TT, performed the experiments, analyzed the data and wrote the manuscript, with help from CB and JMC; DS, BS and JLP have generated cDNA libraries (normalized and non-normalized); CDS was involved in EST data treatment and data formatting for sequence submissions; EC helped assemble the ESTs and set up the internet site; RK and KHG provided the GOPET anno- tations; MD modified ArrayLIMS and EMMA to work with the Roche NimbleGen arrays; JMC, PR, YVDP, and LS were involved in genome-wide gene annotation; all authors read and approved the manuscript. 6. Ritter A, Goulitquer S, Salaün JP, Tonon T, Correa JA, Potin P: Cop- per stress induces biosynthesis of octadecanoid and eicosa- noid oxygenated derivatives in the brown algal kelp Laminaria digitata. New Phytol 2008, 180:809-821. 7. Bartsch I, Wiencke C, Bischof K, Buchholz CM, Buck BH, Eggert A, Feuerpfeil P, Hanelt D, Jacobsen S, Karez R, Karsten U, Molis M, Roleda MY, Schubert H, Schumann R, Valentin K, Weinberger F, Wiese J: The genus Laminaria sensu lato: recent insights and developments. Eur J Phycol 2008, 43:1-86. p J y 8. Kloareg B, Quatrano RS: Structure of the cell-walls of marine- algae and ecophysiological functions of the matrix polysac- charides. Oceanogr Mar Biol 1988, 26:259-315. Acknowledgements We would like to thank Declan Schroeder for providing the E. siliculosus virus 1 sequences, Maela Kloareg for her advice on the analysis of transcrip- tomic data, Gurvan Michael for his help identifying the E. siliculosus C5-epi- merases, Gildas Le Corguillé for his help installing KOBAS and setting up the website, and Jonas Collén, as well as the anonymous reviewers, for crit- ical reading of the manuscript. Part of this work was performed within the framework of the Network of Excellence 'Marine Genomics Europe' (Euro- pean Commission contract no. GOCE-CT-2004-505403). SD has received funding from the European community's Sixth Framework Programme (ESTeam contract no. MESTCT 2005-020737). 18. Davison IR, Pearson GA: Stress tolerance in intertidal sea- weeds. J Phycol 1996, 32:197-211. J y 19. Collén J, Hervé C, Guisle-Marsollier I, Léger JJ, Boyen C: Expression profiling of Chondrus crispus (Rhodophyta) after exposure to methyl jasmonate. J Exp Bot 2006, 57:3869-3881. y j J p 20. Collén J, Guisle-Marsollier I, Léger JJ, Boyen C: Response of the transcriptome of the intertidal red seaweed Chondrus crispus to controlled and natural stresses. New Phytol 2007, 176:45-55. 21. Roeder V, Collén J, Rousvoal S, Corre E, Leblanc C, Boyen C: Iden- tification of stress gene transcripts in Laminaria digitata (Phaeophyceae) protoplast cultures by expressed sequence tag analysis. J Phycol 2005, 41:1227-1235. 1. Boyen C, Oudot MP, Loiseaux-De Goër S: Origin and evolution of plastids and mitochondria: the phylogenetic diversity of algae. Cah Biol Mar 2001, 42:11-24. Abbreviations The most stable genes were defined by ranking the genes according to the sum of squares of the log2-ratios of all stress conditions with the control. Differentially expressed genes were identified in the reduced dataset by t-test using TigrMEV 4.1 [72] with subsequent calculation of the FDR according to Benjamini and Hochberg [73]. Clustering was performed on the log2-ratios of all expressed genes with the ASW: artificial sea water; CDS: coding sequence; EST: expressed sequence tag; FDR: false discovery rate; GO: Gene Ontology; GOPET: GO-term Prediction and Evaluation Tool; HSP: heat shock protein; KEGG: Kyoto Encyclopedia of Genes and Genomes; KOBAS: KEGG Orthology-Based Anno- tation System; NCBI: National Center for Biotechnology Genome Biology 2009, 10:R66 Genome Biology 2009, Volume 10, Issue 6, Article R66 Genome Biology 2009, Volume 10, Issue 6, Article R66 Dittami et al. R66.18 http://genomebiology.com/2009/10/6/R66 Dittami et al. R66.18 2. Baldauf SL: The deep roots of eukaryotes. Science 2003 300:1703-1706. 3. Graham MH, Kinlan BP, Druehl LD, Garske LE, Banks S: Deep-water kelp refugia as potential hotspots of tropical marine diversity and productivity. Proc Natl Acad Sci USA 2007, 104:16576-16580. 4. Santelices B: The discovery of kelp forests in deep-water habi- tats of tropical regions. Proc Natl Acad Sci USA 2007 104:19163-19164. 5. Küpper FC, Carpenter LJ, McFiggans GB, Palmer CJ, Waite TJ Boneberg E-M, Woitsch S, Weiller M, Abela R, Grolimund D, Potin P Butler A, Luther GW, Kroneck PMH, Meyer-Klaucke W, Feiters MC Iodide accumulation provides kelp with an inorganic antioxi- dant impacting atmospheric chemistry. Proc Natl Acad Sci USA 2008, 105:6954-6958. 6. Ritter A, Goulitquer S, Salaün JP, Tonon T, Correa JA, Potin P: Cop- per stress induces biosynthesis of octadecanoid and eicosa- noid oxygenated derivatives in the brown algal kelp Laminaria digitata. New Phytol 2008, 180:809-821. 7. Bartsch I, Wiencke C, Bischof K, Buchholz CM, Buck BH, Eggert A Feuerpfeil P, Hanelt D, Jacobsen S, Karez R, Karsten U, Molis M Roleda MY, Schubert H, Schumann R, Valentin K, Weinberger F Wiese J: The genus Laminaria sensu lato: recent insights and developments. Eur J Phycol 2008, 43:1-86. 8. Kloareg B, Quatrano RS: Structure of the cell-walls of marine- algae and ecophysiological functions of the matrix polysac- charides. Oceanogr Mar Biol 1988, 26:259-315. 9. Tonon T, Rousvoal S, Roeder V, Boyen C: Expression profiling of the mannuronan C5-epimerase multigenic family in the brown alga Laminaria digitata (Phaeophyceae) under biotic stress conditions. Abbreviations J Phycol 2008, 44:1250-1256. 10. Nyvall P, Corre E, Boisset C, Barbeyron T, Rousvoal S, Scornet D Kloareg B, Boyen C: Characterization of mannuronan C-5-epi- merase genes from the brown alga Laminaria digitata. Plant Physiol 2003, 133:726-735. 11. Charrier B, Coelho SM, Le Bail A, Tonon T, Michel G, Potin P Kloareg B, Boyen C, Peters AF, Cock JM: Development and phys- iology of the brown alga Ectocarpus siliculosus: two centuries of research. New Phytol 2008, 177:319-332. 12. Peters AF, Marie D, Scornet D, Kloareg B, Cock JM: Proposal of Ectocarpus siliculosus (Ectocarpales, Phaeophyceae) as a model organism for brown algal genetics and genomics. Phycol 2004, 40:1079-1088. 13. Coelho SM, Peters AF, Charrier B, Roze D, Destombe C, Valero M Cock JM: Complex life cycles of multicellular eukaryotes: new approaches based on the use of model organisms. Gene 2007 406:152-170. 14. Peters AF, Scornet D, Ratin M, Charrier B, Monnier A, Merrien Y Corre E, Coelho SM, Cock JM: Life-cycle-generation-specific developmental processes are modified in the immediate upright mutant of the brown alga Ectocarpus siliculosus. Devel- opment 2008, 135:1503-1512. 15. Soeder C, Stengel E: Physico-chemical factors affecting metab- olism and growth rate. In Algal Physiology and Biochemistry Volume 10. Edited by: Stewart WDP. Oxford, London, Edinburgh, Melbourne Blackwell Scientific Publications; 1974. 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Climate Change and Inequality: The Effectiveness and Potential Improvements of the Existing Approaches

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Climate Change and Inequality: The Effectiveness and Potential Improvements of the Existing Approaches Zhancheng Liu1,*, Xinzi Lu2a, Ziyi Guo3b, Haonan Ye4c 1Beijing Haidian Foreign Language Shiyan School, Haidian Foreign Language Academy, Beijing, 100195, China 2School of Law, Zhongnan University of Economics and Law, Wuhan, 430073, China 3Colorado College, Colorado Springs, 80903, US 4International Department, Affiliated High School of South China Normal University, Guangzhou, 510630, China Zhancheng Liu1,*, Xinzi Lu2a, Ziyi Guo3b, Haonan Ye4c 1Beijing Haidian Foreign Language Shiyan School, Haidian Foreign Language Academy, Beijing, 100195, China 2School of Law, Zhongnan University of Economics and Law, Wuhan, 430073, China 3Colorado College, Colorado Springs, 80903, US 1Beijing Haidian Foreign Language Shiyan School, Haidian Foreign Language Academy, Beijing, 100195, China 2School of Law, Zhongnan University of Economics and Law, Wuhan, 430073, China 3Colorado College, Colorado Springs, 80903, US 4International Department, Affiliated High School of South China Normal University, Guangzhou, 510630, China p g , , rtment, Affiliated High School of South China Normal University, Guangzhou, 510630, China Abstract: This paper examines the relationship between climate change and inequality, evaluates three existing approaches from both macro principles and micro practices, and proposes the potential improvements for those approaches. Available evidence indicates that climate change exacerbates inequality globally and the existing approaches are insufficient and still need to be more aggressive. More specifically, the principle of Common but Differentiated Responsibilities and Respective Capabilities (CBDR-RC) in the United Nations Framework Convention on Climate Change (UNFCCC) is blunt to effectively address climate change and respond to inequality even by distributing the common responsibilities differently to the individual countries. Developed countries should take the responsibility to finance climate change due to the principle “the polluter pays” and the obligation to protect human rights; however, developed countries have not yet met their climate finance obligations. Similarly, the international carbon market has been viewed as a feasible measure, while additional actions are still needed to respond to the inequalities exacerbated by climate change. Figure 1: GDP Per Capita and Baseline Temperature of Latin America Countries [2] Source: IDB staff calculations based on Burke et al. (2015). SHS Web of Conferences 148, 01026 (2022) ICPRSS 2022 SHS Web of Conferences 148, 01026 (2022) ICPRSS 2022 SHS Web of Conferences 148, 01026 (2022) https://doi.org/10.1051/shsconf/202214801026 © 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/). 1.1. The Inequality Exacerbated by Climate Change Climate change has negatively affected human beings all over the world, but it more negatively impacts developing countries than developed countries. According to the International Monetary Fund (IMF), unmanaged climate change hurts global economic growth by “damaging poverty eradication efforts and disproportionately affecting the poorest regions and people.”[1] As pointed out by Bridget Hoffman, there are two main reasons why climate change brings inequality and poverty[2]: First, climate change and natural disasters affect the economy of poorer countries, regions, and people more than rich countries. Less developed countries and people in poverty tend to lose more money when the natural disasters caused by climate change hit them since many people in poor regions heavily depend on activities that may be vulnerable to climate change including agricultural practices, fisheries, and forestry. Second, poorer countries and people with fewer resources are more vulnerable and less able to respond to the negative consequences of climate shocks than those rich countries, which have more developed technologies and corresponding laws and policies. Figure 1: GDP Per Capita and Baseline Temperature of Latin America Countries [2] Source: IDB staff calculations based on Burke et al. (2015). Not only does climate change bring poverty and inequality to poor countries, but it also exacerbates the existing inequality and poverty. According to Hoffman’s research, GDP per capita is negatively correlated with the baseline temperature of Latin American countries, implying that poorer countries are more likely to be exposed to high temperatures (Figure 1). As temperatures continue to rise due to global warming, those high-poverty regions will be further impacted by high temperatures and other climate-related impacts such as natural disasters, including heavy rainfalls and flooding. In this case, with *Correspondence author email: [email protected] [email protected] [email protected] [email protected] *Correspondence author email: [email protected] [email protected] [email protected] [email protected] SHS Web of Conferences 148, 01026 (2022) ICPRSS 2022 SHS Web of Conferences 148, 01026 (2022) https://doi.org/10.1051/shsconf/202214801026 less developed countries, depending more on agriculture, are more affected than the developed countries are. Exposed to natural disasters, these developing countries are more vulnerable to dealing with economic and ecological loss. Climate change, therefore, does not equally affect everyone, but it makes more negative impacts directly on the people in poor regions. the continuously increasing temperature, these poor regions suffer more from climate change and lose greater wealth. 1.1. The Inequality Exacerbated by Climate Change Brazilian states have the same pattern in which the higher the temperature, the lower the GDP per capita, thus emphasizing that poor states are more likely to be exposed to the consequences of rising temperature (Figure 2). When climate change disasters hit, the economies of those Figure 2 : GDP Per Capita and Baseline Temperature of Brazilian States [2] Source: IDB staff calculations based on the University of Delaware (reconstruction data assembled by Willmott and Matsuura (2018)) and the Brazilian Institute of Geography and Statistics (IBGE) (2010) Figure 2 : GDP Per Capita and Baseline Temperature of Brazilian States [2] Source: IDB staff calculations based on the University of Delaware (reconstruction data assembled by Willmott and Matsuura (2018)) and the Brazilian Institute of Geography and Statistics (IBGE) (2010) 0 0) author has ever contributed to IPCC processes, according to the finding of Corbera et al. (2016).[3] The overrepresentation of the Global North implies the dominance of developed countries in climate science. Figure 3. provides the data about the geographical origin of organizations in the events of climate engineering. Most organizations are based in more developed countries such as the United States, the United Kingdom, and Germany.[3] China, India, and Brazil have 3 to 4 active organizations in the database of the BRICS countries. “With only 12 organizations distributed across Fiji, Indonesia, Jamaica, Kenya, Singapore, and Thailand, other developing countries show much less participation.” In this case, it is clear that the organizations from developed countries dominate in the science of climate change, yet those from developing countries are underrepresented. Since developing countries’ experts do not sufficiently have a voice in the science of climate change, developing countries’ interests, special circumstances, and vulnerabilities will be hardly considered. Thus, to enable developing countries to directly participate in improving their global environmental law and policy, the global community should reinforce equity, justice, and fairness by guaranteeing the access to international negotiations for the representatives from the least developed countries. 1.3. The Importance of Appropriate International Responses to Developing Countries on Climate Change Based on the principle of sovereign equality that grants each country the same vote in international law, the least developed countries have considerable influence in intergovernmental negotiations the same as developed countries do. However, “existing frameworks that aim to promote mitigation and adaptation are inadequate”, argued by vulnerable nations.[4] Therefore, “loss and damage”, an international mechanism dealing with residual climate change impacts, is necessary.[5] “Loss and damage” refers to “the actual and/or potential manifestation of impacts associated with climate change in developing countries that negatively affect human and natural systems, including impacts from extreme events such as heatwaves and slow-onset events such as sea-level rise and glacial retreat.”[4] With fewer resources, the developing countries are less able to adapt to natural disasters caused by climate change and they suffer more than the developed countries do. For instance, building infrastructure such as flooding defenses is essential when flooding hits, but less developed countries are not sufficiently empowered to provide enough time and money for the people to transfer, therefore minimizing the loss and damage. Adaptation may not be sufficient enough to prevent the negative consequences of current and future climate change, as argued by some developing countries.[4] 1.2. Developing Countries’ Underrepresentation of Climate Change Science The issue will be resolved effectively only by explicitly considering inequality and involving the less developed countries in decision-making. Since transnational challenges like climate change cannot be solved by either one developing country or developed country alone, they require collective action coordination and commitment by all states together. 1.2. Developing Countries’ Underrepresentation of Climate Change Science APPROACH ONE: THE PRINCIPLE OF COMMON BUT DIFFERENTIATED RESPONSIBILITIES AND RESPECTIVE CAPABILITIES (CBDR RC) Figure 3: Distribution of countries in the climate engineering discursive process, based on institutions represented at more than one climate engineering event (in the capacity of speakers). Countries are coded based on the International Naming Convention [3] 1.3. The Importance of Appropriate International Responses to Developing Countries on Climate consequences of climate shocks is significant. Developed countries need to assist the most vulnerable when natural disasters hit. The issue will be resolved effectively only by li itl id i i lit d i l i th l Figure 3: Distribution of countries in the climate engineering discursive process, based on institutions represented at more than one climate engineering event (in the capacity of speakers). Countries are coded based on the International Naming Convention [3] 1.3. The Importance of Appropriate International Responses to Developing Countries on Climate Change Based on the principle of sovereign equality that grants each country the same vote in international law the least consequences of climate shocks is significant. Developed countries need to assist the most vulnerable when natural disasters hit. The issue will be resolved effectively only by explicitly considering inequality and involving the less developed countries in decision-making. Since transnational challenges like climate change cannot be solved by either one developing country or developed Figure 3: Distribution of countries in the climate engineering discursive process, based on institutions represented at more than one climate engineering event (in the capacity of speakers). Countries are coded based on the International Naming Convention [3] 1.3. The Importance of Appropriate International Responses to Developing Countries on Climate Change Based on the principle of sovereign equality that grants each country the same vote in international law, the least developed countries have considerable influence in intergovernmental negotiations the same as developed consequences of climate shocks is significant. Developed countries need to assist the most vulnerable when natural disasters hit. The issue will be resolved effectively only by explicitly considering inequality and involving the less developed countries in decision-making. Since transnational challenges like climate change cannot be solved by either one developing country or developed country alone, they require collective action coordination and commitment by all states together. s are coded based on the International Naming Convention [3] consequences of climate shocks is significant. Developed countries need to assist the most vulnerable when natural disasters hit. 1.2. Developing Countries’ Underrepresentation of Climate Change Science Not only is inequality manifested in the economic vulnerability of poor countries, but it is also represented in their limited scientific participation. In addition to the people directly suffering from poverty and inequality, the experts of developing countries have lower involvement in climate mitigation science, which is essential for global environmental law and policy making. Developing countries should play a major role in agenda-setting since they are most vulnerable to climate change. However, their concerns are not sufficiently conveyed in the scientific assessment reports that are politically significant. For instance, Frank Biermann and Ina Möller point out that the experts that write the IPCC reports are mainly based in North America and Europe, according to recent IPCC analyses[3]. Based on the assessment reports published between 1990 and 2007, Ho-Lem et al. (2011) found that “only 3.1% of IPCC authors are from Africa or South America, compared to 35.6% and 37.2% from Europe and North America.”[3] Moreover, in the developing world, the other 45% of all countries, no 2 2 SHS Web of Conferences 148, 01026 (2022) ICPRSS 2022 SHS Web of Conferences 148, 01026 (2022) https://doi.org/10.1051/shsconf/202214801026 Figure 3: Distribution of countries in the climate engineering discursive process, based on institutions represented at more than one climate engineering event (in the capacity of speakers). Countries are coded based on the International Naming Convention [3] 1.3. The Importance of Appropriate International Responses to Developing Countries on Climate Change Based on the principle of sovereign equality that grants each country the same vote in international law, the least developed countries have considerable influence in intergovernmental negotiations the same as developed countries do. However, “existing frameworks that aim to promote mitigation and adaptation are inadequate”, argued by vulnerable nations.[4] Therefore, “loss and damage”, an international mechanism dealing with residual climate change impacts, is necessary.[5] “Loss d d f h l d/ i l consequences of climate shocks is significant. Developed countries need to assist the most vulnerable when natural disasters hit. The issue will be resolved effectively only by explicitly considering inequality and involving the less developed countries in decision-making. Since transnational challenges like climate change cannot be solved by either one developing country or developed country alone, they require collective action coordination and commitment by all states together. 2. 2.3. What Does CBDR Still Lack and How to Improve? The principle of CBDR-RC has been then implemented into multiple future climate change protocols and agreements. In the Kyoto Protocol, the developed countries were asked to reduce their emission to a certain extent while the developing countries only needed to report their emission inventory. The principle of CBDR-RC is blunt and insufficient to effectively address climate change and respond to the concurrent issue of inequality even by distributing the common responsibilities differently to the individual countries based on their social and economic capacities. Table 1: The Categorization and Description of the Parties[7] Category Description Annex I Parties Including the industrialized and relatively more economically developed countries such as the members of the OECD (Organization for Economic Co-operation and Development) in 1992, the countries with economies in transition (the EIT Parties), including the Russian Federation, the Baltic States, and several Central and Eastern European States. They are legally binding to reduce greenhouse gas emissions and are required to report the annual greenhouse gas inventory by the April of every year. Annex II Parties Consisting of some of the Annex I members. Beyond the requirements of Annex I, they are also required to provide financial resources to enable and assist developing countries to undertake mitigation and adaptation under the Convention. With higher overall capacity, they are also responsible to provide funding and promote technological development and transfer to other developing Parties in the Convention. Non-annex I Parties Mostly developing countries, they are either recognized by the Convention as being especially vulnerable to the negative impacts of climate change or vulnerable to the potential negative economic impacts of climate change response measures. Their activities will be supported by the Annex I and Annex II Parties when necessary. Least Developed Countries The 49 Parties classified as least developed countries (LDCs) by the United Nations are given special consideration under the Convention on account of their limited economic and social capacity to enact mitigation and adaptation to climate change. Other parties should support the LDCs through funding and technology transfer. Table 1: The Categorization and Description of the Parties[7] 2.3.1. The Issue of Domestic Social Inequality The main cause of climate change is greenhouse gas emissions, and every individual on earth will contribute to the greenhouse gas emission to some extent. The principle of CBDR-RC does make all parties recognize and alleviate the problem of inequality between countries while responding to climate change by differentiating the responsibilities between them; however, although the inter-countries inequality has been put on the agenda of international discussion, the principle of CBDR-RC is still not sufficient enough to address the climate change and inequality fully and effectively because the domestic “social inequality”[8] has not received as much attention as it should. People within countries have different levels of greenhouse gas emissions due to various factors such as income level. It is important to identify the greenhouse gas emission more specifically and differ the mitigation and adaptation burden to the “individual level” (see, for example, Chakravarty et al. 2007)[9] to improve the effectiveness, which will require much more domestic and international efforts. 2.3.2. The Principle of CBDR-RC, Moral Hazard, and Free Rider 2.1. Introduction Common but Differentiated Responsibilities and Respective Capabilities (CBDR-RC) is a fundamental principle within the UNFCCC that acknowledges the disparity of socioeconomic capabilities and differentiated responsibilities of individual countries in addressing the common existential challenge of humanity—climate change and the concurrent issue—inequality. The principle of CBDR-RC was formalized and enshrined in the UNFCCC of Earth Summit in Rio de Janeiro, 1992, which has called for the widest possible cooperation between the countries to deal with the rising threat of climate change based on the principle of CBDR-RC. The UNFCCC Article 3.1 has stated that: “The Parties should protect the climate system for the benefit of present and future generations of humankind, based on equity and in accordance with their common but differentiated responsibilities and respective capabilities.” Therefore, it has been further added that the developed countries should take the leading responsibilities. “The Paris Agreement reinforced the Warsaw International Mechanism for Loss and Damage as the main vehicle under the UNFCCC process to avert, minimize, and address loss and damage associated with climate change impacts.” (Article 8)[6] Improving less developed countries’ ability to deal with the negative 3 3 SHS Web of Conferences 148, 01026 (2022) https://doi.org/10.1051/shsconf/202214801026 ICPRSS 2022 2.2. Categories Paris Agreement in 2015 required all parties to take certain actions and reduce their greenhouse gas emission, which is still based on the principle of CBDR-RC but amended the responsibilities of developing countries this time since they have gradually become more capable of addressing climate change. (UNFCCC) Paris Agreement in 2015 required all parties to take certain actions and reduce their greenhouse gas emission, which is still based on the principle of CBDR-RC but amended the responsibilities of developing countries this time since they have gradually become more capable of addressing climate change. (UNFCCC) In specific, parties have been categorized into Annex I Parties, Annex II Parties, Non-annex I Parties, and Least Developed Countries based on their different levels of capacities in addressing climate change as mentioned in the UNFCCC Article 4.3, 4.4, and 4.5. Table 1 shows the categorization of the parties with description of their duties. 3. APPROACH TWO: CLIMATE FINANCE The analysis shows that the developed countries should finance climate change due to the “the polluter pays” principle and the commitment to protect human rights; however, developed countries have not yet met their climate finance obligations. Two suggestions are made to promote climate finance. 3.2. Past contributions and necessary resources In 1992, 154 nations signed the UNFCCC, of which Article 4.7 states that developing country Parties rely on the financial resources and technology of developed country Parties to fulfil their pledge. The first implementation of the UNFCCC was the Kyoto Protocol signed in 1997, under which the idea that developed countries should offer financial resources to developing countries. In 2009, developed nations promised to achieve yearly funding of $100 billion by 2020 at the 15th Conference of Parties (COP15). Yet, so far, most developed countries have not achieved this goal [15]. Until 2019, the total amount has just been close to $80 billion [16]. In 2015, the Paris Agreement replaced the Kyoto Protocol. The Paris Agreement requires each country to report contributions, yet it has not required the amount that countries should pay. Countries are asked to submit NDCs to keep improving their contributions[17]. 2.3.2. The Principle of CBDR-RC, Moral Hazard, and Free Rider It should be admitted that the more developed parties are generally the main contributors to recent years’ greenhouse gas emissions with the gain of economic growth, which as the principle of CBDR-RC stated, it is reasonable that as the more capable parties, they should take the lead on addressing the climate change with the legally binding greenhouse gas emission reduction. However, the principle of CBDR-RC may arise the problem of moral hazard in the developing parties because of the lack of incentive to act since they are not legally binding to the greenhouse gas emission reduction and developed parties are mainly responsible to address the issue. In this case, the developing parties will have an “unfair economic advantage.”[10] The development status and the overall capacities of the Parties have been changing over time. Their responsibilities have also been amended. The Convention has been amended, adding Kazakhstan into the Annex I Parties as it proposed for inclusion in 1999. Moreover, the Under the commitments of reduction promised by the developed parties, the developing parties will emit to meet their maximum yield, which will undermine the 4 4 SHS Web of Conferences 148, 01026 (2022) ICPRSS 2022 SHS Web of Conferences 148, 01026 (2022) https://doi.org/10.1051/shsconf/202214801026 3.1.1. “The polluter pays” principle The principle “the polluter pays” relates historical and current greenhouse gas emissions to the money each country should pay to address climate change[19]. The developed countries started the industrial revolution early and therefore have historically emitted climate change pollution for a longer time. Their pollution starting from industrialization to nowadays has resulted in a large amount of climate pollution to the environment, shown by the fact that the developed countries caused 79% of historical carbon emissions[12]. The US and the European States collectively have contributed over half of the total pollution. These historical carbon emissions with the “the polluter pays” principle constitutes the first reason for paying. Additionally, the developing countries are now unequally suffering from climate change. From a human rights perspective, the UN High Commissioner for Human Rights has warned that climate change can prevent people from enjoying their human rights[11]. Health rights, for example, are included in international human rights law as an inclusive right [13]. When facing health-threatening situations like diseases, the developing countries, however, lack the technical expertise and resources, and public health systems to deal with the problems[14]. Since human rights as a universal right that should be protected regardless of nationality, developed countries should promote efforts to protect human rights in developing countries. According to the game theory of prisoner's dilemma, rational parties might not cooperate even though it would yield the best overall result. Parties will prioritize considering their own interest, and every party has an incentive to free ride on greenhouse gas emission reduction in other countries without contributing on its own. In this case, the principle of CBDR-RC is useless since no matter what the responsibilities of the parties are, they might free ride, resulting in the ineffectiveness of international cooperation and the tragedy of commons. In 2010, the Russian Federation indicated that it would not intend to assume a quantitative emission limitation or reduction commitment for the second commitment period of the Kyoto Protocol. 3.3. Mechanism for finance climate change solutions The following analysis shows that the developed countries should take the burden to finance climate change because of the “polluter pays” principle and the obligation to protect human rights. Under the current financial mechanism, according to the UNFCCC, the operation can be delegated to several present international entities, including the Green Climate Fund (GCF) and the Global Environmental Facility (GEF). The current mechanism is accountable to the Conference of the Parties (COP) [18]. 3.1.2. Obligation to protect human rights effectiveness of international cooperation in addressing climate change. It is also important to note that developing countries are at a defining stage of climate change. Their mindsets of energy consumption will determine their future energy transformation from conventional energy use to renewable energy use. Setting a baseline and enhancing the responsibilities are essential to stimulate the developing parties to act on climate change and increase the overall effectiveness of international cooperation. The Paris Agreement in 2015 has shown a good start in improving the principle of CBDR-RC by requiring all parties to take action. effectiveness of international cooperation in addressing climate change. It is also important to note that developing countries are at a defining stage of climate change. Their mindsets of energy consumption will determine their future energy transformation from conventional energy use to renewable energy use. Setting a baseline and enhancing the responsibilities are essential to stimulate the developing parties to act on climate change and increase the overall effectiveness of international cooperation. The Paris Agreement in 2015 has shown a good start in improving the principle of CBDR-RC by requiring all parties to take action. Additionally, the developing countries are now unequally suffering from climate change. 3.4. Problems of Existing Climate Finance The primary problems of existing climate finance are the insufficiency of funds and the ineffectiveness of the climate finance mechanism. Firstly, the finance gap between existing funds and the funds is needed to prevent the most significant impacts from happening and to constrain global warming to 1.5°C. More specifically, a 590% increase in annual climate finance is required to avoid the most dangerous impacts of climate change. Moreover, the adaptation fund, until 2021, is still far from enough [19]. Most of the adaptation fund is from the public sector yet it was only 14% of public finance. The adaptation fund of USD 46 billion in 2019 at 5 SHS Web of Conferences 148, 01026 (2022) ICPRSS 2022 SHS Web of Conferences 148, 01026 (2022) https://doi.org/10.1051/shsconf/202214801026 3.5. Potential Improvements for Climate Finance Referring to some insights from the existing papers, this part gives three suggestions for improving the current situation of climate finance, both in terms of the finance amount and the effectiveness of finance. Countries are still hammering out the rules six years later and are far from reaching an agreement. Fortunately, the United Nations Climate Change Conference in Glasgow in 2021(COP26) ultimately handed us a strong Paris Agreement framework for international collaboration through carbon markets. They urged countries to adopt concrete and immediate steps to combat hazardous climate change. It is an answer to climate change, but this additional ambition will still not be enough to meet the Paris Agreement’s goals. Additional actions should be taken to address the challenges caused by pre-existing inequalities. Firstly, the climate funds should appeal to more donors to join climate finance. The impact of climate finance will depend on the donors’ efforts. It is suggested that if next- to-traditional donors (emerging developing countries) like China also guarantee notable amounts of funding to the GCF, they can pressure the traditional countries to increase their contributions [21]. Secondly, the climate finance mechanisms can give some control over the use of funds for the donors after careful consideration. For now, one of the two climate finance mechanisms GCF requires the fund to be equally distributed for adaptation and mitigation. Moreover, it is country-driven, meaning that the receiving countries will decide the use of funds for programs. It is argued that the lack of control for donors makes them less incentivized to donate [21]. However, it is worth noting that the control given to donors needs to be carefully designed since there should still be a fair balance between adaptation and mitigation efforts. 4.2. The operation of carbon markets under Art.6 Parties to the UNFCCC have fundamentally altered the modalities of international collaboration (via market and non-market measures) with Article 6 of the Paris Agreement. Parties can pursue both cooperative measures involving the use of internationally transferred mitigation outcomes (ITMO) between Parties (Article 6.2) and mitigation and sustainable development mechanism involving public and private sector actors (Article 6.4). Also, the non-market approach is stipulated in Article 6.8 to “boost countries’ mitigation, adaptation, finance, technology transfer, and capacity-building resisting climate change in no trade involved situations”[22]. Before utilizing this approach, countries must prepare a work program on such approaches. Therefore, this paper focuses on those contemporary opportunities in Articles 6.2 and 6.4. Thirdly, it can be considered to reduce the interval between submitting a new NDCs plan. It is very urgent to achieve the 2030 goal of climate change to constrain global warming under 1.5 °C, which means that there will only be two more proposals before 2030. A 5-year gap between two NDCs plans might hinder the promotion of funds needed for developing countries to adapt to climate change or mitigate its impact. On the other hand, if the interval between two NDCs plans reduces, countries can share more information and thus might promote the funds needed to achieve the 2030 goal. Fourthly, developed countries can shift a bigger portion of their budget to make a bigger pledge to provide climate finance. The obligation to protect human rights from being hurt by climate change has not spread vastly, yet it is indeed a huge concern. This obligation can be used together with the “the polluter pays” principle together to call for huger funding in countries. One potential way of providing climate finance is to impose a carbon tax on domestic high-polluting firms and to use that revenue for climate finance, which has the positive externality of disincentivizing high-polluting industries, promoting domestic firms to transform into low-polluting ones. 4.1. Introduction 4.1. Introduction To reduce global greenhouse gas emissions, economists devised carbon markets by creating a financial incentive. The global scheme has evolved from “cap and trade” under the Kyoto Protocol 1997 to the Clean Development Mechanism (CDM). Then in 2015, 190 countries signed up for the Paris Agreement and set emissions reduction targets. They agreed to set up a voluntary global carbon market under Article 6 of the climate treaty, hoping to avoid the flaws that led to the CDM's demise. The notion is that one country might cut its emissions by funding the construction of carbon-reducing initiatives in a second country and counting these reductions toward its goals. 4. APPROACH THREE: GLOBAL CARBON MARKET least needs to triple to reach the minimum estimated cost [19]. Secondly, the existing climate finance mechanism contains internal ineffectiveness. For now, climate finance was transported to developing countries based on individual mitigation or adaptation projects. It is pointed out that this approach is not effective enough when climate finance increases by a significant amount and a sectoral or national plan will be needed [20]. Moreover, since the use of funds is now determined by receiving countries, it disincentivizes the donors as they do not have control or visibility of their funds. 4.3.2. The deficiency: Implementing challenges caused by pre-existing inequalities 4.3.2. The deficiency: Implementing challenges caused by pre-existing inequalities In the context of global cooperation on combating climate change and the establishment of carbon markets, the unequal administrative capacities call upon challenges. Take the literature on the EU’s policy implementation as an example, policy implementation can be separated into three major stages. The first is policy transposition, which requires national governments to incorporate international accords into their domestic legislation. The efforts to operationalize and practice the abstract international agreement and national public policies are the second step. The third stage is defined by the implementation of policies. Depending on the policy in question, this may entail a variety of operations, for instance, on-the-spot inspections, permission issuance, or the coordination of various private and governmental players. Accordingly, well-established administrations are essential to the effective functioning of public policies. In other words, administrative capacities are indispensable for the smooth operation and delivery of policies. The extent to which implementing authorities can carry out the respective activities depends on their human capacity and the financial, technical, and organizational resources available[24]. 4.2.2. New mitigation and sustainable development mechanism Using the recently developed mechanism contributing to the mitigation of greenhouse gas and sustainable development is another option for collaboration according to Article 6.4. Then, the emission reductions can be transferred from the country in which they were achieved to another country and counted towards its NDC, much as the bilateral cooperation options outlined in Article 6.2. Unlike Article 6.2, which enables direct bilateral collaboration, this mechanism will be overseen by a body established by the Conference of the Parties. Furthermore, the Conference of the Parties approved norms, modalities, and processes that must be followed while carrying out Article 6.4 activities. As a result, standardized procedures are followed in the planning, implementation, and verification of emission reduction operations. For example, before activities may be registered with the Supervisory Body after successful validation by an independent verification agency, they must first be permitted by the host country. The mechanism's purpose of mobilizing the private sector to participate in climate change mitigation by offering adequate incentives is another distinctive feature. As a result of the Paris Agreement, the subnational level actors can directly use the system established under Article 6.4. At this stage, low-capacity countries have to implement a completely new policy on the national bureaucracy, together with new administrative structures and procedures. Compared with high-capacity countries, it is unequal to those low-capable countries, which may make it hard to build up the required institutional structures quickly and precisely. For instance, based on their weaker administrative foundation, lack of sufficient degree of expertise, supporting staff, and resources, it is tough to create an eligible policy to participate in carbon markets successfully. What is worse, the unequal basis will, in turn, lead to unequal consequences as well. 4.3. Evaluation of the international carbon market Climate change exacerbates inequality globally, and international cooperation and the existing approaches have to be improved to effectively address the issues. Developing countries overall are more vulnerable and less prepared to address climate change and inequality. The mechanism of the principle of CBDR-RC is a decent start in addressing climate change and the concurrent issue of inequality. However, the differentiation of responsibilities is not sufficient enough and still needs to be refined to avoid the moral hazard and free rider, and effectively address these two tough common issues, which in this case, more international efforts are needed. The analysis shows that climate finance is an essential tool to address climate change since the developing countries do not have enough capability to solve the problems solely on their own. However, the developed countries are not fulfilling their pledge. It is urgent for them to act so that climate change Climate change exacerbates inequality globally, and international cooperation and the existing approaches have to be improved to effectively address the issues. 4.2.1. Cooperative approach Under Article 6.2, parties can directly collaborate with each other. This first enables emission reduction measures to be performed in one nation, and the resulting emission reductions are transferred to another country and credited against the latter’s NDC. This demands a transparent technique as well as a precise emission reduction accounting. The new regulations make it illegal to count emission reductions twice, such as in the nation's 6 SHS Web of Conferences 148, 01026 (2022) ICPRSS 2022 SHS Web of Conferences 148, 01026 (2022) https://doi.org/10.1051/shsconf/202214801026 diffusion, enhanced energy security, and increased competitiveness. diffusion, enhanced energy security, and increased competitiveness. emissions inventory where the reductions are carried out and the country's emissions inventory where the emission reductions are transferred. It also enables national and regional instruments to be linked to similar programs to create a single, cross-border carbon market. While international oversight of these cooperative efforts is not planned, stringent reporting and accounting procedures have been implemented with the goal of ensuring long- term development gains while avoiding undesirable consequences. States engaging in mitigation activities, for example, must demonstrate that the activity is compatible with the host country's sustainable development goals, that negative impacts are minimized, and that human rights and other rights are maintained. 4.3.1. The effectiveness: meet the goals of PA Two explicit goals of the Paris Agreement are supported and contributed by the carbon market under Article 6: (1) delivering emission reductions and (2) mobilizing investments. If countries set up the rules correctly, countries using these relevant measures can meet and beat their carbon emission reduction targets while channeling increased investment in sustainable development, accelerated by Article 6[23]. Such market approaches will aid in decreasing the costs of achieving specific low- carbon goals, mobilizing more local and international resources to sustain low-carbon benefits, and delivering extra benefits such as accelerated technology transfer and 7 SHS Web of Conferences 148, 01026 (2022) SHS Web of Conferences 148, 01026 (2022) ICPRSS 2022 https://doi.org/10.1051/shsconf/202214801026 9. Chakravarty, Shoibal, Ananth Chikkatur, Helen de Coninck, Stephen Pacala, Robert Socolow, and Massimo Tavoni (2007). Climate Policy Based on Individual Emissions, Princeton, NJ, Princeton University Center, Princeton University. will not worsen in the future. The carbon market is a decent answer to the emergent climate change, but this additional ambition is still not sufficient enough to keep the world under a 1.5-2°C temperature rise. Further actions are still essential in propelling the global ambition to meet the Paris Agreement’s goals. For the inequalities of the administration, all climate agreements involving both developed and developing countries and promoting carbon markets should ensure that all parties are able to develop the essential bureaucratic infrastructure to successfully participate in emission trading. Other parties and international organizations should intervene and assist the countries when missing those capacities. Overall, more aggressive and comprehensive international cooperation is needed in order to solve the two defining challenges of humanity. 10. Rajamani, Lavanya. "The Principle of Common but Differentiated Responsibility and the Balance of Commitments under the Climate Regime". Review of European Community & International Environmental Law. 9, 2: 128. 11. Schalatek, L., Neil B. (2016) The Principles and Criteria of Public Climate Finance - A Normative Framework. us.boell.org/sites/default/files/uploads/2016/11/cff1_ 2016_normativeframework_english.pdf. 12. Center for Global Development. Developed Countries Are Responsible for 79 Percent of Historical Carbon Emissions. www.cgdev.org/media/who-caused-climate-change- historically. Acknowledgement All the authors contributed equally to this work and should be considered co-first authors. 13. World Health Organization. Human Rights. www.who.int/health-topics/human-rights#tab=tab_1. References 14. Kasotia, P. The Health Effects of Global Warming: Developing Countries Are the Most Vulnerable. www.un.org/en/chronicle/article/health-effects- global-warming-developing-countries-are-most- vulnerable. 1. Guivarch, Céline., et al. Linking Climate Change and Inequality, IMF, 2021. https://www.imf.org/Publications/fandd/issues/2021/ 09/climate-change-and-inequality-guivarch-mejean- taconet 15. Bos, J, et al, (2021). Are Countries Providing Enough to the $100 Billion Climate Finance Goal?. www.wri.org/insights/developed-countries- contributions-climate-finance-goal. 2. Hoffman, Bridget. How Climate Change Worsens Poverty and Inequality, https://blogs.iadb.org/ideas- matter/en/how-climate-change-worsens-poverty- and-inequality/, 2021. 16. Timperley, J. (2021) The Broken $100-Billion Promise of Climate Finance - and How to Fix It. www.nature.com/articles/d41586-021-02846-3. 3. Biermann, Frank and Möller, Ina, Rich man’s solution? Climate engineering discourses and the marginalization of the Global South, 2019, (153). 3. Biermann, Frank and Möller, Ina, Rich man’s solution? Climate engineering discourses and the marginalization of the Global South, 2019, (153). 17. United Nations. (2015) The Paris Agreement. https://unfccc.int/sites/default/files/resource/parisagr eement_publication.pdf. 4. James, R., Otto, F., Parker, H. et al. Characterizing loss and damage from climate change. Nature Clim Change 4, 938–939 (2014). https://doi.org/10.1038/nclimate2411. 18. United Nations. Introduction to Climate Finance. unfccc.int/topics/climate-finance/the-big- picture/introduction-to-climate-finance. 5. Introduction to Loss and Damage, https://unfccc.int/topics/adaptation-and- resilience/the-big-picture/introduction-to-loss-and- damage. 19. Buckner, B. (2019) Global landscape of Climate Finance. Climate Policy Initiative, London. 20. Haites, E. (2015) Climate change finance." Routledge, Milton Park. 6. Hoert, Ryan. and Toth, Evelin., COP 26 Explained: What to Know About the UN Climate Change Conference, https://unfoundation.org/blog/post/cop- 26-explained-what-to-know-about-the-un-climate- change-conference/?gclid=CjwKCAjwur- SBhB6EiwA5sKtjt2dXpoX6ZeBUAO1agBejU_JBx 9QCa5gdxvm97YyxV_AClRTh5nuMBoCP7sQAvD _BwE UNF, 2021. 21. de Sépibus, J. (2015) Green Climate Fund: How Attractive Is It to Donor Countries?. Carbon & Climate Law Review, 9: 298-313. 22. Schneider, Lambert, and Stephanie La Hoz Theuer. "Environmental integrity of international carbon market mechanisms under the Paris Agreement." Climate Policy 19.3 (2019): 386-400. 7. Unfccc.Int, 2022, https://unfccc.int/parties-observers. 23. Oladi, Reza, et al. “Sequestration and the Engagement of Developing Economies in a Global Carbon Market.” Resource and Energy Economics, vol. 52, 2018, pp. 50–63. , , p p 8. Islam, S. Nazrul and John Winkel (2017). “Climate Change and Social Inequality”. Department of Economic and Social Affairs. Working Paper No. 152: 1. 8. Islam, S. Nazrul and John Winkel (2017). “Climate Change and Social Inequality”. Department of Economic and Social Affairs. Working Paper No. 152: 24. Steinebach, Yves, and Julian Limberg. “Implementing Market Mechanisms in the Paris Era: The Importance 8 SHS Web of Conferences 148, 01026 (2022) https://doi.org/10.1051/shsconf/202214801026 of Bureaucratic Capacity Building for International Climate Policy.” Journal of European Public Policy, 2021, pp. 1-16. 25. Zhongming, Z., Liu W. References "Climate Finance Provided and Mobilised by Developed Countries: Aggregate Trends Updated with 2019 Data." (2021). 26. Carbon Brief. 2022. Analysis: Which countries met the UN’s 2020 deadline to raise ‘climate ambition’? - Carbon Brief. [online] Available at: <https://www.carbonbrief.org/analysis-which- countries-met-the-uns-2020-deadline-to-raise- climate-ambition> [Accessed 29 April 2022]. 27. Carbon Brief. 2022. Analysis: Which countries met the UN’s 2020 deadline to raise ‘climate ambition’? - Carbon Brief. [online] Available at: <https://www.carbonbrief.org/analysis-which- countries-met-the-uns-2020-deadline-to-raise- climate-ambition> [Accessed 29 April 2022]. 28. Hof, A., den Elzen, M., Admiraal, A., Roelfsema, M., Gernaat, D. and van Vuuren, D., 2017. Global and regional abatement costs of Nationally Determined Contributions (NDCs) and of enhanced action to levels well below 2 °C and 1.5 °C. Environmental Science &amp; Policy, 71, pp.30-40. 29. Thwaites, J. and Bos, J., 2021. A Breakdown of Developed Countries’ Public Climate Finance Contributions Towards the $100 Billion Goal. World Resources Institute,. 30. Ambrosi, P.. “How to Keep Momentum Up in Carbon Markets.” (2011). 31. Rocket Boosters to Accelerate Climate Action: Why Article 6 of the Paris Agreement Matters, https://www.edf.org/sites/default/files/documents/Cli matePolicy_Article6_Layout_04_0.pdf. Accessed 28 April 2022, pp.1-4. 32. Silbert, Nicola. “Making International Law, Making Carbon Markets.” Alternative Law Journal, vol. 46, no. 4, Dec. 2021, pp. 263-267. 33. Verret-Hamelin, Antoine. "Carbon Markets: Rehabilitating the Egalitarian Objection." ethic@-An international Journal for Moral Philosophy 17.3 (2018): 389-408. 34. Unfccc.Int, 2022, https://unfccc.int/process-and- meetings/the-convention/history-of-the- convention/proposal-to-amend-annexes-i-and-ii-to- remove-the-name-of-turkey-and-to-amend-annex-i- to-add-the-name 34. Unfccc.Int, 2022, https://unfccc.int/process-and- meetings/the-convention/history-of-the- convention/proposal-to-amend-annexes-i-and-ii-to- remove-the-name-of-turkey-and-to-amend-annex-i- to-add-the-name 9 9

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WONG MERAZ, Víctor Alejandro, CABANAS VEIGA, Manuel, &amp; ALDRETE ACUÑA, Christian Yair, Las ciencias constitucionales y su relevancia en el siglo XXI: estudios en homenaje a Javier Ruipérez Alamillo, Editorial Colex, A Coruña, 2023, 610 págs.

Anuario da Facultade de Dereito da Universidade da Coruña/Anuario da Facultade de Dereito da Universidade da Coruña

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Anuario da Facultade de Dereito da Universidade da Coruña Vol. 27 (2023), pp. 173-182 ISSNe: 2530-6324 || ISSN: 1138-039X DOI: https://doi.org/10.17979/afdudc.2023.27.0.9922 WONG MERAZ, Víctor Alejandro, CABANAS VEIGA, Manuel, & ALDRETE ACUÑA, Christian Yair, Las ciencias constitucionales y su relevancia en el siglo XXI: estudios en homenaje a Javier Ruipérez Alamillo, Editorial Colex, A Coruña, 2023, 610 págs. EDUARDO LAMPÓN SÁNCHEZ Anuario da Facultade de Dereito da Universidade da Coruña Vol. 27 (2023), pp. 173-182 ISSNe: 2530-6324 || ISSN: 1138-039X DOI: https://doi.org/10.17979/afdudc.2023.27.0.9922 WONG MERAZ, Víctor Alejandro, CABANAS VEIGA, Manuel, & ALDRETE ACUÑA, Christian Yair, Las ciencias constitucionales y su relevancia en el siglo XXI: estudios en homenaje a Javier Ruipérez Alamillo, Editorial Colex, A Coruña, 2023, 610 págs. EDUARDO LAMPÓN SÁNCHEZ Anuario da Facultade de Dereito da Universidade da Coruña Vol. 27 (2023), pp. 173-182 ISSNe: 2530-6324 || ISSN: 1138-039X DOI: https://doi.org/10.17979/afdudc.2023.27.0.9922 WONG MERAZ, Víctor Alejandro, CABANAS VEIGA, Manuel, & ALDRETE ACUÑA, Christian Yair, Las ciencias constitucionales y su relevancia en el siglo XXI: estudios en homenaje a Javier Ruipérez Alamillo, Editorial Colex, A Coruña, 2023, 610 págs. WONG MERAZ, Víctor Alejandro, CABANAS VEIGA, Manuel, & ALDRETE ACUÑA, Christian Yair, Las ciencias constitucionales y su relevancia en el siglo XXI: estudios en homenaje a Javier Ruipérez Alamillo, Editorial Colex, A Coruña, 2023, 610 págs. EDUARDO LAMPÓN SÁNCHEZ Investigador predoctoral Universidad de A Coruña Recibido: 14/09/2023 Aceptado: 25/09/2023 Recibido: 14/09/2023 Aceptado: 25/09/2023 La mirada retrospectiva de la presente obra supone el reconocimiento a una labor especialmente fructífera. Los estudiantes que hemos pasado a lo largo de los años por las clases de Javier Ruipérez Alamillo podemos testimoniar la pasión de este veterano educador con la divulgación de su materia de estudio. El enfoque de su prolífica obra académica es un exponente del compromiso del autor con la realidad social en la que tenemos que vivir y que el ordenamiento jurídico nace para regular. Por ello, el estudio publicado por la Colección Iberoamericana de Ciencias Constitucionales de la Editorial Colex supone una oportunidad para recapitular y reflexionar el pensamiento jurídico-filosófico de una figura reconocida entre los académicos y estudiantes que han tenido oportunidad de acercarse a este profesor de la Universidad de A Coruña. Es muy significativo que el libro, coordinado por Víctor Alejandro Wong Meraz (Universidad Autónoma del Estado de México), Manuel Cabanas Veiga (Universidad de Lleida) y Christian Yair Aldrete Acuña (Tribunal Electoral del Estado de México), reúna a autores de diversas regiones, como Argentina, Colombia, España, Italia, México o Perú, convirtiendo este trabajo en un encuentro de representantes de culturas y tradiciones jurídicas diversas, pero deudores de las enseñanzas del homenajeado. Anuario da Facultade de Dereito da Universidade da Coruña Vol. 27 (2023), pp. 173-182 ISSNe: 2530-6324 || ISSN: 1138-039X DOI: https://doi.org/10.17979/afdudc.2023.27.0.9922 WONG MERAZ, Víctor Alejandro, CABANAS VEIGA, Manuel, & ALDRETE ACUÑA, Christian Yair, Las ciencias constitucionales y su relevancia en el siglo XXI: estudios en homenaje a Javier Ruipérez Alamillo, Editorial Colex, A Coruña, 2023, 610 págs. EDUARDO LAMPÓN SÁNCHEZ Wong Meraz, en su estudio introductorio, señala: «Es por todo lo anterior, junto con Jorge Carpizo y Pedro De Vega, Ruipérez es de los profesores que más nos han formado, en virtud de que sus enseñanzas se complementan y ayudan a que podamos entender la relevancia del Derecho Constitucional» (p. 34). Anu Fac Der UDC, 2023, 27: 173-182 174 Veintisiete juristas se reúnen en esta iniciativa para explorar las enseñanzas del autor. El libro se estructura en siete partes que exploran los asuntos que han sido estudiados por el catedrático, lo que supone un amplio repaso por los grandes problemas del Derecho Constitucional, desde sus albores históricos hasta los grandes debates jurídicos de los últimos tiempos. Entre los múltiples estudios jurídicos que componen la obra, encontramos discusiones sobre la naturaleza del poder, la relación entre lo jurídico y lo político, la división de poderes, la democracia constitucional, los derechos fundamentales, la reforma constitucional o la descentralización política. Miguel Ángel Alegre Martínez destaca: «El hilo conductor no es otro que su incansable defensa de los valores constitucionales, y la Constitución entendida como instrumento que conjuga libertad y democracia para los ciudadanos» (p. 273). En su dilatada su trayectoria, Javier Ruipérez ha ejercido magisterio en distintas instituciones académicas, especialmente las universidades de Salamanca, Santiago de Compostela y A Coruña, en la que actualmente ocupa la cátedra de Derecho Constitucional. En su desarrollo académico e intelectual fueron determinantes su formación salamantina y su relación con el catedrático Pedro De Vega García. En el año 1976, un período marcado por el soplo de vientos de cambio, pero aún bajo la vigencia del Código Penal de la dictadura, que contemplaba la ominosa pena de muerte, el futuro docente no pensaba inicialmente en estudiar la carrera de Derecho. Sin embargo, en el epicentro de la transición política, terminó tomando la decisión de matricularse (1977). Así, el profesor de A Coruña inició su singladura en la Facultad de Derecho de la mítica Universidad de Salamanca. Como estudiante, el influjo de académicos como Francisco Tomás y Valiente, Alfredo Calonge, Gloria Begué o, de un modo muy destacado, Enrique Rivero, fue relevante en sus años de formación como jurista. Pero especialmente alumbradora fue la interacción con el profesor Pedro De Vega, quien, desde la primera clase, se convirtió en su mentor en el campo del Derecho Político. Anuario da Facultade de Dereito da Universidade da Coruña Vol. 27 (2023), pp. 173-182 ISSNe: 2530-6324 || ISSN: 1138-039X DOI: https://doi.org/10.17979/afdudc.2023.27.0.9922 WONG MERAZ, Víctor Alejandro, CABANAS VEIGA, Manuel, & ALDRETE ACUÑA, Christian Yair, Las ciencias constitucionales y su relevancia en el siglo XXI: estudios en homenaje a Javier Ruipérez Alamillo, Editorial Colex, A Coruña, 2023, 610 págs. EDUARDO LAMPÓN SÁNCHEZ La capacidad de integrar diferentes corrientes ideológicas, desde el aperturismo franquista hasta el Partido Comunista de España, simboliza la voluntad expresa del poder constituyente de unificar a una sociedad diversa y establecer una democracia avanzada y pluralista. De esta manera, el texto aprobado en 1978 supuso un hito emblemático en la sociedad española, un armisticio que puso fin a siglos de enfrentamientos y conflictos en la nación. Al tiempo que, tras décadas de oscurantismo y aislamiento, el país por fin avanzaba por la senda de la Constitución y la apertura al mundo, el estudiante, bajo la tutela y guía de De Vega, completó su doctorado en la Universidad de Salamanca (1987). Durante estos años de investigación, compartió la odisea con su condiscípula, amiga y confidente, Rogelia Calzada Conde. Juntos enfrentaron desafíos académicos, participaron en debates apasionados y se brindaron apoyo mutuo. Tristemente, la trágica partida prematura de la joven constitucionalista, dejó un vacío que nunca pudo ser colmado, por muchos años que pasen, y una enseñanza sobre la importancia de la amistad y la fraternidad. Desde entonces, como profesor titular en la Universidad de Santiago de Compostela (1990) y, posteriormente, con la obtención de la cátedra en la Universidad de A Coruña (1997), la extensa obra de Ruipérez ha abarcado una globalidad de temas relevantes para el Derecho Constitucional. Sus monografías han sido de necesaria lectura en el estudio del constitucionalismo democrático («Proceso constituyente, soberanía y autodeterminación», 2003), la reforma constitucional («Reforma Versus Revolución», 2014), las autonomías («La protección constitucional de la autonomía», 1994), los derechos sociales («De los derechos fundamentales sociales y su eficacia jurídica: Entre la jurisdicción y la voluntad constitucional», 2019) y la ciudadanía europea («La “Constitución Europea” y la Teoría del Poder Constituyente», 2000). Alfonso Guerra, constituyente de 1978 y negociador entre partidos durante el proceso, enfatiza aún más: «Es de una enorme satisfacción encontrar una concepción del texto constitucional como la del profesor Ruipérez que pone en contacto las diferentes disposiciones para completar una visión que ayude a mejorar la realidad de los ciudadanos a los que protege y obliga el Código Constitucional» (p. 24). La orientación constitucional del autor homenajeado se caracteriza por un enfoque transformador. Anuario da Facultade de Dereito da Universidade da Coruña Vol. 27 (2023), pp. 173-182 ISSNe: 2530-6324 || ISSN: 1138-039X DOI: https://doi.org/10.17979/afdudc.2023.27.0.9922 WONG MERAZ, Víctor Alejandro, CABANAS VEIGA, Manuel, & ALDRETE ACUÑA, Christian Yair, Las ciencias constitucionales y su relevancia en el siglo XXI: estudios en homenaje a Javier Ruipérez Alamillo, Editorial Colex, A Coruña, 2023, 610 págs. EDUARDO LAMPÓN SÁNCHEZ De la mano de su admirado maestro, iniciaba un camino del que ya no se iba a separar. La explicación del profesor De Vega del concepto de libertad, desde la antigua Grecia a nuestros días, fue el punto de partida en la visión del mundo jurídico del estudiante. En los últimos estertores de la universidad del franquismo, la técnica del cripticismo, la simulación y el doble leguaje, así como el estudio de los clásicos, eran la manera de escapar del férreo control ideológico de la dictadura para explicar y denunciar las injusticias del momento vivido. Esta metodología de análisis se mantendría, incluso una vez llegada la democracia, como una de las señales de identidad de la escuela De Vega. Sonia García Vázquez subraya: «Recogiendo su testigo, el Profesor Ruipérez Alamillo, firme defensor del Estado de Derecho y los valores del constitucionalismo, siempre nos ha mostrado la relevancia de estudiar los clásicos y conocer el pasado con la finalidad de poder comprender el presente y prepararnos para el futuro» (p. 170). Pese a la erudición de su exposición, el enfoque de maestro y discípulo no se basa en elucubraciones teóricas, sino que pone todo el estudio de la disciplina al servicio de la construcción y consolidación de una democracia basada en la convivencia pacífica y en la protección de los derechos y libertades de los ciudadanos. Anu Fac Der UDC, 2023, 27: 173-182 175 Alfonso Guerra González, en el prólogo, destaca la importancia que la obra de Pedro De Vega y Javier Ruipérez ha tenido en la formación de una teoría del Derecho Público democrática en un país caracterizado por una historia convulsa, que la Constitución Española de 1978 venía a superar. El vicepresidente del gobierno del PSOE refleja con profundidad: «Se hizo lo que necesitaban los españoles. Un poco más lejos hubiese supuesto el rechazo de la mitad de la Cámara, y de los españoles representados por ella; un poco menos lejos hubiese cosechado la negativa de la otra mitad y de los españoles representados por ella. Se trataba de alcanzar una convivencia pacífica que enviara al desván de la historia las guerras civiles, las asonadas, los pronunciamientos y los golpes de Estado» (p. 29). Anuario da Facultade de Dereito da Universidade da Coruña Vol. 27 (2023), pp. 173-182 ISSNe: 2530-6324 || ISSN: 1138-039X DOI: https://doi.org/10.17979/afdudc.2023.27.0.9922 WONG MERAZ, Víctor Alejandro, CABANAS VEIGA, Manuel, & ALDRETE ACUÑA, Christian Yair, Las ciencias constitucionales y su relevancia en el siglo XXI: estudios en homenaje a Javier Ruipérez Alamillo, Editorial Colex, A Coruña, 2023, 610 págs. EDUARDO LAMPÓN SÁNCHEZ Su interpretación jacobina del Derecho contrasta con las perspectivas conservadoras que buscan mantener el estatus quo, así como con las visiones marxistas que sostienen que el Derecho es una superestructura utilizada por la burguesía para dominar a la sociedad. Por el contrario, la perspectiva progresista sostenida por Ruipérez ve al Derecho como una herramienta excepcional para transformar la realidad en beneficio de los individuos. Según esta interpretación, el Derecho debe adelantarse a la realidad misma, preparando reformas que conduzcan a una revolución política que haga innecesaria una revolución social. Anu Fac Der UDC, 2023, 27: 173-182 176 Su recorrido profesional, de ya casi 40 años, demuestra que la defensa de sus valores es siempre la brújula que guía las exposiciones y publicaciones académicas en que ha participado. Esta constancia, en la teórica y la práctica, ha dejado una huella en el ámbito de su asignatura reconocida por compañeros en los estudios de este homenaje. Las palabras de Diego Valadés, pronunciadas en la presentación del libro el 13 de junio de 2023 en la Universidad Nacional Autónoma de México, lo sitúan como: «El más valioso exponente del constitucionalismo de la democracia social contemporánea». Pero este reconocimiento académico no sólo conmemora a Javier Ruipérez, sino también a otros importantes juristas, como Hermann Heller. Pues como destaca Víctor Alejandro Wong Meraz en el estudio introductorio: «La amistad histórica no es la que surge en el momento en que unas personas se conocen, sino que nos debemos remontar a cuando los ideales y postulados que nos unen, surgen a la superficie y se plasman como teorías para los años venideros, en este sentido, es que podemos trasladarnos hace más de 100 años, cuando Heller inicia su carrera académica, para poder establecer esa fraternidad con el profesor Javier Ruipérez Alamillo» (p. 51). Cabe destacar que la llegada de Heller a España (1933), escapando de la Alemania nazi, para dar clase en la Universidad Complutense de Madrid, marca un punto de partida en la historia de esta disciplina. La amistad histórica entre Heller y Manuel Pedroso, junto con Fernando de los Ríos y Carlos Ollero, es la fuente de la enseñanza de las Ciencias Constitucionales. Estos autores defendieron que, para abordar los desafíos contemporáneos, resultaba esencial analizar los problemas desde una perspectiva constitucional. Anuario da Facultade de Dereito da Universidade da Coruña Vol. 27 (2023), pp. 173-182 ISSNe: 2530-6324 || ISSN: 1138-039X DOI: https://doi.org/10.17979/afdudc.2023.27.0.9922 WONG MERAZ, Víctor Alejandro, CABANAS VEIGA, Manuel, & ALDRETE ACUÑA, Christian Yair, Las ciencias constitucionales y su relevancia en el siglo XXI: estudios en homenaje a Javier Ruipérez Alamillo, Editorial Colex, A Coruña, 2023, 610 págs. EDUARDO LAMPÓN SÁNCHEZ Siguiendo su estela, son varios los estudiosos que reivindican los debates de los grandes constitucionalistas en la República de Weimar como base para enfrentar los dilemas actuales. Javier Tajadura Tejada sostiene: «El estudio del momento constitucional de Weimar nos pone de manifiesto la fragilidad de la democracia y nos permite extraer lecciones para un presente en que el irracionalismo y el populismo han alcanzado nuevamente un protagonismo destacado» (p. 212). Y agrega: «Weimar es un ejemplo claro de cómo una sociedad que abraza el irracionalismo acaba asumiendo toda una serie de mitos y falsedades que le conducen finalmente al desastre» (p. 229). Analizando la tensión entre positivistas y realistas, Wong Meraz resalta la preferencia por Heller, destacando: «la importancia de este autor para el entendimiento de los límites del poder de revisión constitucional» (p. 40-41). Este constitucionalista se diferencia al no limitarse al estudio de la norma por sí sola, ni centrarse exclusivamente en la Constitución, como hace Kelsen, ni politizarla completamente, como hace Schmitt. Más bien, contextualiza la norma. Esta amistad histórica continúa a través de la participación de académicos contemporáneos. Cuando la pandemia del COVID-19 se desató, tomaron la iniciativa de impulsar la creación un seminarito virtual de Ciencias Constitucionales (2020), emulando lo hecho por Tierno Galván (1953) y De Vega (1971) en la Facultad de Derecho de Salamanca. Este seminario se ha convertido en una escuela, semana tras semana, para un grupo emergente de investigadores. En este lugar se discuten y se retoman a los clásicos constitucionales, brindando un espacio de intercambio de ideas en un mundo cada vez más conectado. Anu Fac Der UDC, 2023, 27: 173-182 177 En última instancia, la conclusión a la que se llega es la afirmación de De Vega, en su obra «Estudios Constitucionales» (1987): «La convicción profunda de que el único régimen éticamente defendible, políticamente coherente y científicamente demostrable es el régimen democrático» (p. 8). Esta afirmación sintetiza el enfoque académico que guía a los miembros del seminario. De esta forma, las influencias históricas de figuras como Heller, junto con la defensa de la democracia enunciada por De Vega y Carpizo, sigue iluminando el campo de las Ciencias Constitucionales, y nos insta a continuar explorando los debates jurídicos del pasado para entender los problemas de nuestro tiempo y prepararnos para los desafíos del futuro. Anuario da Facultade de Dereito da Universidade da Coruña Vol. 27 (2023), pp. 173-182 ISSNe: 2530-6324 || ISSN: 1138-039X DOI: https://doi.org/10.17979/afdudc.2023.27.0.9922 WONG MERAZ, Víctor Alejandro, CABANAS VEIGA, Manuel, & ALDRETE ACUÑA, Christian Yair, Las ciencias constitucionales y su relevancia en el siglo XXI: estudios en homenaje a Javier Ruipérez Alamillo, Editorial Colex, A Coruña, 2023, 610 págs. EDUARDO LAMPÓN SÁNCHEZ Entrando en materia, un aspecto central de los trabajos de Javier Ruipérez es el enfoque en relación con los derechos sociales, considerándolos como parte integral de los derechos fundamentales. Para el autor, la protección de estos derechos es fundamental en la construcción de una sociedad avanzada, como la que instaura nuestro Preámbulo constitucional. El Estado Social se consagra en el artículo 1 de la Constitución. En el mismo marco normativo, se reconoce una economía mixta de mercado, en el artículo 38. Junto al reconocimiento de la igualdad formal liberal, en el artículo 14, se recoge también la igualdad sustantiva, a través de la cláusula de transformación social del artículo 9. El artículo 128 subordina toda la riqueza nacional a la consecución del interés general. Por su parte, el artículo 33 reconoce la función social y, por tanto, los límites del derecho de propiedad. Finalmente, el artículo 35 establece el derecho al trabajo. Es decir, la Constitución Española adopta el modelo de Weimar y Querétaro. Sin embargo, pese al tono aparentemente transformador del texto constitucional, su elaboración aparece marcada por un momento histórico de crisis del Estado Social, lo que se reflejó en el tratamiento degradado de los derechos sociales, especialmente en cuanto a sus garantías jurisdiccionales. En vista de lo anterior, el artículo 53 de la Constitución establece grados distintos de protección en función de las tres categorías de derechos que reconoce. En primer lugar, los «Derechos Fundamentales» (artículos 15-29), mayoritariamente derechos tradicionales de libertad, gozan de aplicación directa, el recurso preferente y sumario ante la jurisdicción ordinaria y la posibilidad (aunque cada vez más teórica) de acceder al recurso de amparo ante el Tribunal Constitucional. En una posición intermedia están los «Derechos» (artículos 30- 38), dotados de protección ordinaria. Por último, los «Principio Rectores de la Política Social y Económica» (artículos 39-52), donde se encuentra la mayoría de los derechos sociales, no aparecen configurados como derechos subjetivos, sino como, en la práctica, una vocación de libre configuración para el legislador. Desde esta perspectiva, Ruipérez ha estudiado cómo evoluciona un derecho social básico como el acceso a la vivienda, reconocido en el artículo 47 como un principio rector, y se convierte en un derecho fundamental. El autor aboga por la reforma constitucional, proponiendo una modificación al artículo 53. Anuario da Facultade de Dereito da Universidade da Coruña Vol. 27 (2023), pp. 173-182 ISSNe: 2530-6324 || ISSN: 1138-039X DOI: https://doi.org/10.17979/afdudc.2023.27.0.9922 WONG MERAZ, Víctor Alejandro, CABANAS VEIGA, Manuel, & ALDRETE ACUÑA, Christian Yair, Las ciencias constitucionales y su relevancia en el siglo XXI: estudios en homenaje a Javier Ruipérez Alamillo, Editorial Colex, A Coruña, 2023, 610 págs. EDUARDO LAMPÓN SÁNCHEZ Su propuesta abarca una protección de los derechos sociales, donde no solo el Legislativo y el Ejecutivo, sino también el Poder Judicial, asuman la responsabilidad de garantizar su efectividad. Pero el análisis trasciende las disposiciones legales, abordando preguntas sobre la naturaleza de los derechos en la sociedad actual. En un contexto marcado por continuas crisis económicas esta perspectiva se vuelve aún más necesaria. Anu Fac Der UDC, 2023, 27: 173-182 178 En su estudio sobre la constitucionalización del Derecho Privado, Rafael Colina Garea apunta: «desde los postulados del Estado Social proclamados por la CE de 1978, no es sólo lícito, sino conveniente, un relativo sacrificio de la libertad individual del propietario, siempre y cuando así lo aconseje la realización de un interés social relevante y específico constitucionalmente promovido… Quiere ello decir que, dentro de aquellos objetivos que presiden el modo de actuar del propietario, ya no se halla únicamente presente la satisfacción del interés individual, sino que simultáneamente aparece la realización del interés social de la colectividad… Ambos tipos de interés aparecen íntimamente ligados en el seno del derecho de propiedad» (p. 407). Pero la situación actual demuestra la insatisfacción en torno a la respuesta estatal. La problemática de los desahucios y las ocupaciones de viviendas resalta el delicado equilibrio entre la necesidad de abordar situaciones de vulnerabilidad y la inexcusable tutela de la propiedad privada (artículo 33). Para Luis Jimena Quesada: «El problema reside en una concepción sesgada de la economía (y de la globalización económica) como fin en sí misma, que genera una mediatización o instrumentalización de los derechos sociales» (p. 424). En sentido parecido, Miguel Ángel Alegre Martínez destaca: «El profesor Ruipérez insiste incansablemente en que tan solo en el constitucionalismo democrático y social la eficacia de los derechos fundamentales se ha hecho verdaderamente real; y ello por cuanto que ha sido en él donde las garantías normativas y jurisdiccionales establecidas por los textos constitucionales han podido funcionar... Frente a ello, la lógica de la globalización demuestra un superior interés por eliminar la teoría democrática del poder constituyente del pueblo, resucitando la falacia de la contraposición entre Estado y sociedad, y propugnando un absoluto sometimiento de la política a los dictados de la razón tecnocrática e instrumental...» (p. 277). Anuario da Facultade de Dereito da Universidade da Coruña Vol. 27 (2023), pp. 173-182 ISSNe: 2530-6324 || ISSN: 1138-039X DOI: https://doi.org/10.17979/afdudc.2023.27.0.9922 WONG MERAZ, Víctor Alejandro, CABANAS VEIGA, Manuel, & ALDRETE ACUÑA, Christian Yair, Las ciencias constitucionales y su relevancia en el siglo XXI: estudios en homenaje a Javier Ruipérez Alamillo, Editorial Colex, A Coruña, 2023, 610 págs. EDUARDO LAMPÓN SÁNCHEZ Esta aproximación a los derechos sociales saca a relucir la esencia de un Estado Social y Democrático de Derecho, tal como lo consagran los artículos 1 y 9 de la Constitución Española, generando cuestionamientos sobre la exigibilidad de derechos como la salud (artículo 43) y la vivienda (artículo 47), que carecen de la protección adecuada en nuestro ordenamiento jurídico, y confluye con la construcción de un sistema de protección de los derechos sociales más real y efectivo. Por ende, no consiste solo en una explicación académica de cómo se regulan los derechos en el sistema legal, sino que incita a la acción por una sociedad más respetuosa de los derechos fundamentales sociales de todos sus miembros. Este libro, que surge como un obsequio de respeto y cariño hacia Javier Ruipérez, se origina en el contexto de México, una tierra arraigada en su amor por la libertad y la independencia. Las imágenes de los murales de Diego Rivera y las palabras de Chavela Vargas, «los mexicanos nacemos donde nos da la gana», fueron esgrimidos por el homenajeado en la presentación de la obra como fundamento del lazo que ha tejido con la identidad mexicana. Con su vinculación académica con universidades mexicanas, demuestra ser un gran conocedor de la Constitución de Querétaro de 1917, tal como señala Alegre Martínez (p. 287). Además, la contribución de Diego Valadés resalta que la obra del catedrático ha dejado una huella significativa en Iberoamérica, especialmente en México (p. 111). Anu Fac Der UDC, 2023, 27: 173-182 179 Estos estudios jurídicos exploran cómo la norma y la realidad a menudo interactúan y se desafían en el ámbito constitucional. Wong Meraz, entre otros, resalta la importancia de entender las relaciones entre norma y realidad en el estudio del Derecho Constitucional y la necesidad de abordar los problemas recientes que enfrenta la democracia constitucional en la región (pp. 35-36). De hecho, las crisis en Chile (2019), Perú (2022) o Ecuador (2023) han cuestionado la estabilidad de sus sistemas constitucionales. Pero la brecha entre la norma y la realidad puede llevar a invertir los términos, y convertir la Constitución en un reflejo distorsionado de la realidad. Anuario da Facultade de Dereito da Universidade da Coruña Vol. 27 (2023), pp. 173-182 ISSNe: 2530-6324 || ISSN: 1138-039X DOI: https://doi.org/10.17979/afdudc.2023.27.0.9922 WONG MERAZ, Víctor Alejandro, CABANAS VEIGA, Manuel, & ALDRETE ACUÑA, Christian Yair, Las ciencias constitucionales y su relevancia en el siglo XXI: estudios en homenaje a Javier Ruipérez Alamillo, Editorial Colex, A Coruña, 2023, 610 págs. EDUARDO LAMPÓN SÁNCHEZ Raúl Canosa Usera explora el concepto de Constitución en Iberoamérica y resalta las diferencias con Europa: «Si la plasmación del Estado social en las constituciones europeas es modesta en comparación con las iberoamericanas, la realidad europea demuestra un grado de Justicia social mucho mayor y menos desigualdades materiales. Frente a las desigualdades materiales que no cesan y aún parecen aumentar, los constituyentes en Iberoamérica han doblado una y otra vez su apuesta socializante... Se ceba una pulsión caudillista, una tendencia monárquica en el sentido etimológico del término, que propende a convertir los deseos del presidente en disposiciones constitucionales y ello con más frecuencia de lo deseable debilita la división de poderes y por lo tanto mina la identidad misma del Estado de derecho y acerca su naturaleza a la del autoritarismo» (pp. 244-245). Estas constituciones, pretendidamente transformadoras, se han terminado convirtiendo en un pergamino de sueños incumplidos, sin trascendencia real en la vida de los ciudadanos. Por ello, Canosa Usera habla de "constitucionalismo mágico" para describir esta tendencia. Siguiendo la analogía literaria, y ante las frustraciones que las promesas rotas han generado en gran parte de la sociedad, podríamos llegar a más, calificándolo de "constitucionalismo quijotesco". Las acciones del hidalgo Don Quijote de la Mancha, debido a su falta de comprensión de la realidad, terminan causando más mal que bien a aquellos que pretende ayudar. Como en el caso del personaje creado por Miguel de Cervantes, el mundo imaginario concebido por su idealismo no coincide con el real y las aspiraciones de estos textos constitucionales han culminado en una profunda desazón, al tratar de implementar cambios históricos sin considerar los contextos políticos, sociales y económicos. En su examen sobre la democracia latinoamericana y sus desafíos, Enoc Francisco Morán Torres advierte que el desarrollo de la democracia en América Latina, al menos en los últimos cien años, no ha estado exento de experimentar períodos y regímenes que se autodenominan democráticos en el discurso, pero cuyas acciones cotidianas distan mucho de la postura proclamada: «Ello, sin obviar que dicho desarrollo va adminiculado con factores de índole económica, social y cultural —tan inestables y característicos de la referida región— por lo que, a pesar de prevalecer la democracia como forma de gobierno, ésta sigue generando insatisfacción» (p. 444). Como un ejemplo de esta insatisfacción, Domingo García Belaunde analiza el juicio político en el Perú y su relación con la vacancia presidencial (p. 134). Anuario da Facultade de Dereito da Universidade da Coruña Vol. 27 (2023), pp. 173-182 ISSNe: 2530-6324 || ISSN: 1138-039X DOI: https://doi.org/10.17979/afdudc.2023.27.0.9922 WONG MERAZ, Víctor Alejandro, CABANAS VEIGA, Manuel, & ALDRETE ACUÑA, Christian Yair, Las ciencias constitucionales y su relevancia en el siglo XXI: estudios en homenaje a Javier Ruipérez Alamillo, Editorial Colex, A Coruña, 2023, 610 págs. EDUARDO LAMPÓN SÁNCHEZ Este mecanismo, importado de la Constitución Estadounidense de 1787, se utilizó consecutivamente en los casos de los presidentes Kuczynski, Vizcarra y Castillo en Perú. La terminación del mandato se vuelve la excepción y no la norma, síntoma inequívoco de una grave patología política. Anu Fac Der UDC, 2023, 27: 173-182 180 El ensayo de Lucio Pegoraro muestra cómo los poderes excepcionales de suspensión de derechos y libertades, reglados en el texto constitucional español en su artículo 55 (en relación con el 116), en América Latina a menudo se han utilizado para justificar golpes militares y concentración de poder, erosionando la Constitución en lugar de defenderla (p. 88). Resucitando la visión hobbesiana, se plantea que «La seguridad puede justificar cualquier medida» (p. 96), un discurso que voces autoritarias enuncian de forma elocuente, con la aprobación creciente de una población desesperada por la desigualdad y, su inevitable corolario, la inestabilidad política y social. Sergio Díaz Ricci, en su estudio «La reforma constitucional como institución y procedimiento», nos presenta la reforma constitucional como una institución que reúne in nuce toda la problemática del constitucionalismo (p. 341). Este mecanismo jurídico, regulado en el Título X de nuestra Constitución (artículos 166-168), es imprescindible para adaptarla a los nuevos dictados de la política y la sociedad. Pero Wong advierte que «Al no aceptar que existen límites materiales a la reforma constitucional, estamos dando pie, a que todo se puede modificar e incluso las ideas de libertad y de democracia» (p. 47). Con esta transformación, aparece el peligro del fraude a la Constitución. En su trabajo "¿Quién defiende a la Constitución de la Constitución?", Armando Ramírez Castañeda destaca una conclusión que compartimos: «El Poder de Reforma al ser un órgano constituido tiene límites, y estos son aquellos que hemos aludido y a los que Schmitt señalaba como decisiones políticas fundamentales» (p. 264). Corresponde a juristas y académicos la responsabilidad de velar porque la Constitución no sea, como sucedía en el siglo XIX, un mero ornamento legal, sino un reflejo auténtico de la voluntad de la sociedad y un límite para el poder de los gobernantes. Los debates académicos presentados (solo nos hemos hecho eco de algunos de ellos) revelan la complejidad del Derecho Constitucional. Los autores, desde una perspectiva comparada, nos enseñan como el pensamiento constitucional puede trascender las limitaciones del positivismo y abrazar la realidad. Anuario da Facultade de Dereito da Universidade da Coruña Vol. 27 (2023), pp. 173-182 ISSNe: 2530-6324 || ISSN: 1138-039X DOI: https://doi.org/10.17979/afdudc.2023.27.0.9922 WONG MERAZ, Víctor Alejandro, CABANAS VEIGA, Manuel, & ALDRETE ACUÑA, Christian Yair, Las ciencias constitucionales y su relevancia en el siglo XXI: estudios en homenaje a Javier Ruipérez Alamillo, Editorial Colex, A Coruña, 2023, 610 págs. EDUARDO LAMPÓN SÁNCHEZ Alegre Martínez comparte la visión de huir del tecnicismo constitucional para abordar problemas reales (p. 271). En este mismo sentido, Jimena Quesada defiende: «Cabalmente, el Derecho Constitucional tiene que ofrecer una comprensión global del conjunto del sistema constitucional, lo que para Javier Ruipérez solamente puede lograrse explicando, en términos jurídicos, las relaciones de poder reales existentes en una determinada Comunidad Política y en un concreto momento histórico, tratando de averiguar en lo sucedido lo que está por devenir, con la finalidad de conocer su evolución e influir en el desarrollo de la realidad jurídica y política» (p. 423). Este homenaje es un recordatorio de que el estudio del Derecho Constitucional, siguiendo el ejemplo de Heller, debe ser una búsqueda continua de equilibrio entre las aspiraciones de la norma y las exigencias de la realidad. Wong, concluye: «En el Derecho constitucional, es el lugar donde confluye lo jurídico con lo político, lo normativo con la normalidad. En este sentido, la Constitución no puede ser estudiada solamente desde una perspectiva o uno de sus elementos, el jurídico o político, ya que se eliminaría uno de sus componentes. Por tal motivo, su método debe ser el de las ciencias constitucionales, donde debes tomar todos los elementos que confluyen en la sociedad, desde lo político, jurídico, sociológico, económico, e incluso hasta lo histórico, para poder soluciones a los problemas constitucionales» (p. 51). Por esa razón, las discusiones presentadas en las páginas del libro pretenden preservar los valores Anu Fac Der UDC, 2023, 27: 173-182 181 superiores de la justicia, la libertad, la igualdad, el pluralismo y la dignidad humana como corazón de nuestras constituciones y sistemas políticos. En esta línea, Javier Ruipérez ha asumido la defensa de la democracia constitucional como un componente inherente a su vida. El análisis «Responsabilidad social y militancia a favor de la democracia constitucional en la obra del profesor Ruipérez Alamillo» de Luis Jimena Quesada, destaca su capacidad para transmitir su formación y devoción como constitucionalista comprometido con la Democracia Constitucional tanto a sus estudiantes como a quienes buscan su orientación en el ámbito académico (p. 414). Este autor hace hincapié en la relevancia de tres pensadores en la obra de Ruipérez: Rousseau, Heller y De Vega. Anuario da Facultade de Dereito da Universidade da Coruña Vol. 27 (2023), pp. 173-182 ISSNe: 2530-6324 || ISSN: 1138-039X DOI: https://doi.org/10.17979/afdudc.2023.27.0.9922 WONG MERAZ, Víctor Alejandro, CABANAS VEIGA, Manuel, & ALDRETE ACUÑA, Christian Yair, Las ciencias constitucionales y su relevancia en el siglo XXI: estudios en homenaje a Javier Ruipérez Alamillo, Editorial Colex, A Coruña, 2023, 610 págs. EDUARDO LAMPÓN SÁNCHEZ Rousseau formuló la idea democrática, Heller tradujo este ideal en la formulación jurídica del Estado Social, mientras que De Vega enfatizó la importancia del Estado Social para la supervivencia de la democracia (p. 418). Miguel Ángel Alegre Martínez, en su artículo «Supremacía y defensa de la Constitución según el profesor Ruipérez Alamillo: Revisitando lo clásico cuando todo parece cambiar», examina la creciente desconexión entre los ciudadanos y la toma de decisiones políticas fundamentales, que se ha intensificado debido a la globalización. Alegre Martínez sugiere que, en lugar de abordar estas decisiones desde una perspectiva democrática, a menudo se atienden a criterios económicos, lo que aleja aún más a los ciudadanos del proceso (pp. 273- 274). El texto destaca cómo la defensa de la Constitución se ha transformado en una defensa rígida de un "fetiche mágico-mítico", en lugar de ser un medio para hacer efectivos los principios y valores que fundamentan el constitucionalismo moderno: «Se ha operado, de este modo, la sustitución de la “ideología del constitucionalismo” por la “ideología de la Constitución”» (p. 276). Precisamente por ello, la obra de Ruipérez lo caracteriza, según el autor, «por ser la voz que clama en el desierto de un positivismo jurisprudencial cada vez más sumiso y resignado» (p. 296). En definitiva, la trayectoria del profesor de A Coruña en la teoría constitucional es un ejemplo de la defensa incansablemente de la democracia y sus valores, recordándonos que, a pesar de los desafíos, esta lucha siempre vale la pena. En el estudio «El momento constitucional de Weimar: La fragilidad de la democracia» por Javier Tajadura Tejada, se destaca cómo el homenajeado ha sido un jurista comprometido con los valores de libertad y democracia que caracterizaron el constitucionalismo surgido en la época de Weimar (p. 211). Esta recopilación rinde homenaje a su largo camino universitario. De todo lo apuntado podemos concluir que el agasajado ha contribuido, con su docencia e investigaciones, al florecimiento académico de una disciplina que tuvo su germen en el exilio de Heller a España en 1933, huyendo de la persecución del nazismo. Aunque falleció poco después, sus lecciones han perdurado como una fuente de inspiración para generaciones futuras de constitucionalistas en Europa e Iberoamérica, como Tierno, De Vega, Carpizo, Ruipérez o Wong. Continuándose el estudio de las Ciencias Constitucionales en el siglo XXI y, quizás, más allá. Anuario da Facultade de Dereito da Universidade da Coruña Vol. 27 (2023), pp. 173-182 ISSNe: 2530-6324 || ISSN: 1138-039X DOI: https://doi.org/10.17979/afdudc.2023.27.0.9922 WONG MERAZ, Víctor Alejandro, CABANAS VEIGA, Manuel, & ALDRETE ACUÑA, Christian Yair, Las ciencias constitucionales y su relevancia en el siglo XXI: estudios en homenaje a Javier Ruipérez Alamillo, Editorial Colex, A Coruña, 2023, 610 págs. EDUARDO LAMPÓN SÁNCHEZ La obra no sólo es un reconocimiento a una querida personalidad del mundo académico, sino también un recorrido por el desarrollo del Derecho Constitucional de la mano de los clásicos, cuyo legado ha quedado plasmado, en cada página y en cada reflexión, como tributo a su influencia prolongada. Anu Fac Der UDC, 2023, 27: 173-182 182 La presentación del libro tuvo lugar el Instituto de Investigaciones Jurídicas de la Universidad Nacional Autónoma de México. La grabación del evento puede ser visualizada en el siguiente enlace: https://fb.watch/mAoFv60lEc/. Anu Fac Der UDC, 2023, 27: 173-182

https://openalex.org/W2027957948

https://europepmc.org/articles/pmc3707777?pdf=render

English

Paclitaxel-resistant advanced recurrent breast cancer: a case of partial response due to addition of bevacizumab to paclitaxel therapy: a case report

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* Correspondence: [email protected] 1Breast Center, Dokkyo Medical University Koshigaya Hospital, 2-1-50 Minami- Koshigaya, Saitama 343–8555, Koshigaya, Japan Full list of author information is available at the end of the article Ishizuna et al. BMC Research Notes 2013, 6:254 http://www.biomedcentral.com/1756-0500/6/254 Ishizuna et al. BMC Research Notes 2013, 6:254 http://www.biomedcentral.com/1756-0500/6/254 CASE REPORT Open Access © 2013 Ishizuna 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. Paclitaxel-resistant advanced recurrent breast cancer: a case of partial response due to addition of bevacizumab to paclitaxel therapy: a case report Kazuo Ishizuna1*, Jun Ninomiya1,2, Makoto Kojima1, Miho Kawashima3, Miwako Nozaki3, Hidetsugu Yamagishi4, Yoshihiko Ueda4 and Masatoshi Oya1 © 2013 Ishizuna 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: [email protected] 1Breast Center, Dokkyo Medical University Koshigaya Hospital, 2-1-50 Minami- Koshigaya, Saitama 343–8555, Koshigaya, Japan Full list of author information is available at the end of the article © 2013 Ishizuna 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. Case presentation The patient was a 68-year-old postmenopausal woman with a non-contributory history. In September 2004, she underwent a pectoral muscle-conserving mastectomy with axillary dissection for right-sided breast cancer. Patho- logical diagnosis was papillotubular carcinoma, n = 0/12, nuclear grade 1, ly+, v-, estrogen receptor positive, proges- terone receptor negative, and human epidermal growth factor receptor type 2 negative (UICC classification: pT3N0M0-stage IIB). Adjuvant therapy consisted of 6 cy- cles of CEF (cyclophosphamide 75 mg/m2 (days 1–14), epirubicin 60 mg/m2 (days 1 and 8), and fluorouracil 500 mg/m2 (days 1 and 8), every 4 weeks) and subsequent oral anastrozole (1 mg/day). In August 2007, the patient developed a recurrence in the left axillary lymph node. The chemotherapy was changed to high-dose toremifene (120 mg/day), and radiation therapy was also performed (left axilla: 63 Gy). The patient achieved a complete re- sponse (CR) in March 2008. In April 2009, CT showed left axillary lymph node enlargement once again and multiple lung metastases. Hormone therapy was changed to exemestane (25 mg/day) and long-term stable disease was achieved. In March 2011, the lung and left axillary lymph node metastases were enlarged and progressive disease was noted. Thus, the tumors were determined to be resist- ant to hormone therapy. In May, weekly paclitaxel was begun (80 mg/m2, 3 weeks on and 1 week off). Since par- tial response was achieved, this therapy was continued. In December 2011, CT showed that lung and axillary lymph node metastases were enlarged and progressive disease was observed. Therefore, the tumors were determined to be resistant to PTX (tumor markers, CEA: 2.8 ng/ml, CA 15–3: 27.7 U/ml) (Figure 1a and b). Since the patient be- came resistant to PTX and developed progressive disease, a change to other drugs was considered. However, metas- tases enlarged only slightly. Thus, PTX was continued in combination with bevacizumab whose indications had Figure 1 a and b (CT from December 2011). CT showed a left axillary lymph node that had enlarged to 2.5 cm and lung metastases that had also enlarged. been newly expanded to include breast cancer. PTX was continued to exhaust the therapeutic options based on taxanes – a key drug for metastatic and recurrent breast cancer. In January 2012, bevacizumab and weekly PTX were begun (paclitaxel 90 mg/m2, 3 weeks on and 1 week off; bevacizumab 10 mg/kg, once every 2 weeks). Abstract Background: Paclitaxel plus bevacizumab have shown a high response rate and prolonged progression-free survival in metastatic breast cancer patients. However, overall survival was not prolonged. Thus, no conclusion has been made on the effectiveness of bevacizumab. In our report, taxane plus bevacizumab were used to treat a metastatic breast cancer patient with taxane resistance, and a good therapeutic result was obtained. Case presentation: The patient was a 68-year-old woman with a non-contributory history. In September 2004, she underwent a pectoral muscle-conserving mastectomy with axillary dissection for right-sided breast cancer (pT3N0M0-stage IIB, estrogen receptor positive, progesterone receptor negative, and human epidermal growth factor receptor type 2 negative). Adjuvant therapy consisted of 6 cycles of cyclophosphamide, epirubicin and fluorouracil, and subsequent oral anastrozole. In August 2007, the patient developed a recurrence in the left axillary lymph node. The chemotherapy was changed to high-dose toremifene, and radiation therapy was also performed. The patient achieved a complete response. In April 2009, CT showed left axillary lymph node enlargement once again and multiple lung metastases. Hormone therapy was changed to exemestane and long-term stable disease was achieved. In March 2011, the lung and left axillary lymph node metastases were enlarged and progressive disease was noted. Thus, the tumors were determined to be resistant to hormone therapy, and weekly paclitaxel was begun in May. Since partial response was achieved, this therapy was continued. In December, CT showed that lung and axillary lymph node metastases were enlarged and progressive disease was observed. Therefore, the tumors were determined to be resistant to paclitaxel. In January 2012, bevacizumab and weekly paclitaxel were begun. In April, lung and axillary lymph node metastases were reduced in size, and partial response was achieved. Thereafter the same treatment has been continued, and the patient has been followed up without clinical exacerbation as of January 2013. Conclusion: Taxane plus bevacizumab were used to treat a metastatic breast cancer patient with taxane resistance, and a good therapeutic result was obtained. This result is considered important in increasing treatment options for patients with taxane resistance or patients using adjuvant taxane-based therapy and in examining the effectiveness of bevacizumab in metastatic breast cancer patients. Keyword: Breast cancer, Bevacizumab, Paclitaxel Ishizuna et al. BMC Research Notes 2013, 6:254 http://www.biomedcentral.com/1756-0500/6/254 Ishizuna et al. BMC Research Notes 2013, 6:254 http://www.biomedcentral.com/1756-0500/6/254 Page 2 of 4 Figure 1 a and b (CT from December 2011). Background It is often difficult to cure metastatic and recurrent breast cancer, except for some local recurrences. Im- provement of QOL and extension of survival are the ob- jectives of treatment for metastatic and recurrent breast cancer. In recent years, various new drugs have been used clinically in an effort to achieve these objectives. Bevacizumab is a humanized monoclonal antibody that targets vascular endothelial growth factor (VEGF), which is a major regulator of angiogenesis. In Japan, its indications are colon cancer and lung cancer and have expanded to include breast cancer in September 2011. In this report, we describe a case of paclitaxel (PTX) resistant advanced recurrent breast cancer that achieved partial response due to addition of bevacizumab to pacli- taxel therapy. We also include a brief literature review. Figure 1 a and b (CT from December 2011). CT showed a left axillary lymph node that had enlarged to 2.5 cm and lung metastases that had also enlarged. Case presentation In April, CT showed reduction of size of lung and axillary lymph node metastases, and partial response was achieved (tumor markers, CEA: 2.0 ng/ml, CA 15–3: 20.9 U/ml) (Figure 2a and b). Thereafter the same treatment has been continued, and the patient has been followed up without clinical exacerbation as of January 2013. Abstract CT showed a left axillary lymph node that had enlarged to 2.5 cm and lung metastases that had also enlarged. Discussion Tumor growth requires oxygen and nutrients. Tumors vessels are needed to supply sufficient oxygen and nutri- ents as the tumor size increases. Therefore, angiogenesis Page 3 of 4 Ishizuna et al. BMC Research Notes 2013, 6:254 http://www.biomedcentral.com/1756-0500/6/254 Figure 2 a and b (CT from April 2012). Three months after bevacizumab and weekly PTX were begun, the left axillary lymph node reduced in size to 1.4 cm and the lung metastases also became smaller. Our patient developed resistance to PTX and subse- quently achieved a partial response when bevacizumab was added to PTX therapy. The reason is that bevacizumab in- hibits angiogenesis and improves paclitaxel delivery into tumor tissue. In 2007, there was a report on a phase III randomized trial (E2100) which compared paclitaxel plus bevacizumab and paclitaxel alone as initial chemotherapy for patients with untreated advanced and recurrent breast cancer (n = 722). Although the overall survival was not significantly prolonged (median: 26.7 months vs 25.2 months, respect- ively, HR: 0.88, p = 0.16), the median progression-free sur- vival was prolonged (11.8 months vs 5.9 months, HR: 0.60, p < 0.001) and the response rate increased (36.9% vs 21.2%, p < 0.001) [7]. In the U.S., bevacizumab was promptly ap- proved for metastatic breast cancer in February 2008 based on these results. In Japan, its indications were expanded to include breast cancer patients in September 2011. Figure 2 a and b (CT from April 2012). Three months after bevacizumab and weekly PTX were begun, the left axillary lymph node reduced in size to 1.4 cm and the lung metastases also became smaller. When additional trials were conducted (AVADO and RIBBON-1 trials), the results showed that the progression-free survival was prolonged and the re- sponse rate increased. However, the overall survival was not prolonged. In addition, the results suggested that the risks for adverse events could outweigh the benefits [7-9]. In November 2011, the FDA revoked the approval of bevacizumab for breast cancer. At the present time, it is necessary to thoroughly examine the risks and ben- efits of bevacizumab. When bevacizumab is used in combination with an- other chemotherapeutic agent, a high response rate can be obtained. Thus, bevacizumab is currently considered a drug with major benefits, particularly in patients with life-threatening metastasis. Discussion Dedes KJ, Matter-Walstra K, Schwenkglenks M, Pestolozzi BC, Fink D, et al: Bevacizumab in combination with paclitaxel for HER-2 negative metastatic breast cancer: An economic evaluation. Eur J Cancer 2009, 45(8):1397–1406. doi:10.1186/1756-0500-6-254 Cite this article as: Ishizuna et al.: Paclitaxel-resistant advanced recurrent breast cancer: a case of partial response due to addition of bevacizumab to paclitaxel therapy: a case report. BMC Research Notes 2013 6:254. Competing interests The authors declare that they have no competing interests. Competing interests The authors declare that they have no competing interests. Author details 1 1Breast Center, Dokkyo Medical University Koshigaya Hospital, 2-1-50 Minami- Koshigaya, Saitama 343–8555, Koshigaya, Japan. 2Ninomiya Hospital, 491–6 Shin-eicho, Soka, Saitama 340–0056, Koshigaya, Japan. 3Department of Radiology, Dokkyo Medical University Koshigaya Hospital, 2-1-50 Minami- Koshigaya, Saitama 343–8555, Koshigaya, Japan. 4Department of Pathology, Dokkyo Medical University Koshigaya Hospital, 2-1-50 Minami-Koshigaya, Saitama 343–8555, Koshigaya, Japan. Conclusion We reported a case of paclitaxel-resistant metastatic re- current breast cancer that achieved partial response due to addition of bevacizumab to paclitaxel therapy. The clinical value of bevacizumab has not yet been established in treating advanced and recurrent cancer. It will be neces- sary to accumulate more cases. doi:10.1186/1756-0500-6-254 Cite this article as: Ishizuna et al.: Paclitaxel-resistant advanced recurrent breast cancer: a case of partial response due to addition of bevacizumab to paclitaxel therapy: a case report. BMC Research Notes 2013 6:254. Authors’ contributions KI, JN, MK, MK and MN participated in the clinical diagnosis. HY and YU performed the histological examination. KI and JN contributed to drafting of the manuscript. MO supervised the concept and design of the manuscript. All authors read and approved the final manuscript. Discussion When patients develop re- sistance to taxane during adjuvant therapy (as in our pa- tient), our results suggest that the combination therapy of taxane and bevacizumab can be considered for treat- ment. Thus, our finding can have major significance. In the E2100 trial, metastatic breast cancer patients who had a history of adjuvant taxane chemotherapy were examined. Paclitaxel plus bevacizumab significantly pro- longed progression-free survival as compared with pacli- taxel alone (12 months vs 3 months, respectively; HR: 0.46, P < 0.001) [7]. Figure 2 a and b (CT from April 2012). Three months after bevacizumab and weekly PTX were begun, the left axillary lymph node reduced in size to 1.4 cm and the lung metastases also became smaller. plays an important role in tumor growth and metastasis. VEGF is a major regulator involved in tumor angiogen- esis, growth, and metastasis. It acts as a ligand and binds to VEGF receptors on the endothelial cell surface [1,2]. The molecularly targeted drug bevacizumab (Avastin; Genentech, South San Francisco, CA) selectively binds to a VEGF family member, VEGF-A. Bevacizumab in- hibits binding of VEGF-A to VEGF receptors (VEGFR-1, VEGFR-2, and neuropilin 1) expressed on endothelial cells, thereby blocking the signal transduction pathway. It inhibits angiogenesis in tumor tissue and suppresses tumor growth as a result [3]. In addition, it has been shown to normalize the vascular structure, decrease vas- cular permeability, and lower increased tumor interstitial pressure [4,5]. When the tumor interstitial pressure is reduced by bevacizumab, paclitaxel delivery to the tumor tissue is improved. Thus, the drug concentration in- creases in the tumor tissue [6]. In Japan, it has not been very long since bevacizumab has been indicated for metastatic and recurrent breast cancer. However, since it has not been shown to prolong survival, its blind use is also not recommended from the aspect of health economics [10]. However, bevacizumab has been shown to achieve a high response rate and to prolong progression-free survival. Thus, it could be very beneficial depending on the case. In the future, more cases should be accumulated, and it would be necessary to identify a subset of patients in which bevacizumab will be more effective. Ishizuna et al. BMC Research Notes 2013, 6:254 http://www.biomedcentral.com/1756-0500/6/254 Ishizuna et al. BMC Research Notes 2013, 6:254 http://www.biomedcentral.com/1756-0500/6/254 Page 4 of 4 human epidermal growth factor receptor 2-negative, locally recurrent or metastatic breast cancer. J Clin Oncol 2011, 29(10):1252–1260. 10. References 1. Bossung V, Harbeck N: Angiogenesis inhibitors in the management of breast cancer. Curr Opin Obstet Gynecol 2010, 22(1):79–86. 1. Bossung V, Harbeck N: Angiogenesis inhibitors in the management of breast cancer. Curr Opin Obstet Gynecol 2010, 22(1):79–86. 1. Bossung V, Harbeck N: Angiogenesis inhibitors in the management of breast cancer. Curr Opin Obstet Gynecol 2010, 22(1):79–86. 2. Ferrara N: The role of vascular endothelial growth factor in pathological angiogenesis. Breast Cancer Res Treat 1995, 36(2):127–137. 2. Ferrara N: The role of vascular endothelial growth factor in pathological angiogenesis. Breast Cancer Res Treat 1995, 36(2):127–137. g g 3. Kim KJ, Li B, Winer J, Armanini M, Gillett N, et al: Inhibition of vascular endothelial growth factor-induced angiogenesis suppresses tumor growth in vivo. Nature 1993, 362(6423):841–844. 3. Kim KJ, Li B, Winer J, Armanini M, Gillett N, et al: Inhibition of vascular endothelial growth factor-induced angiogenesis suppresses tumor growth in vivo. Nature 1993, 362(6423):841–844. 4. Willett CG, Boucher Y, Di Tomaso E, Duda DG, Munn LL, et al: Direct evidence that the VEGF-specific antibody bevacizumab has antivascular effects in human rectal cancer. Nat Med 2004, 10(2):145–147. 5. Presta LG, Chen H, O’Connor SJ, Chisholm V, Meng YG, et al: Humanization of an anti-vascular endothelial growth factor monoclonal antibody for the therapy of solid tumors and other disorders. Cancer Res 1997, 57(20):4593–4599. Competing interests Competing interests The authors declare that they have no competing interests. Consent Written informed consent was obtained from the patient for publication of this manuscript and any accompany- ing images. A copy of the written consent is available for review by the Editor-in-Chief of this journal. Abbreviations Abbreviations VEGF: Vascular endothelial growth factor; PTX: Paclitaxel. References 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: • 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: 6. Yanagisawa M, Yorozu K, Kurasawa M, Nakano K, Furugiki K, et al: Bevacizumab improves the delivery and efficacy of paclitaxel. Anticancer Drugs 2010, 21(7):687–694. • Convenient online submission g 7. Miller K, Wang M, Gralow J, Dickler M, Cobleigh M, et al: Paclitaxel plus bevacizumab versus paclitaxel alone for metastatic breast cancer. N Engl J Med 2007, 357(26):2666–2676. 8. Miles DW, Chan A, Dirix LY, Cortes J, Pivot X, et al: Phase III study of bevacizumab plus docetaxel compared with placebo plus docetaxel for the first-line treatment of human epidermal growth factor receptor 2- negative metastatic breast cancer. J Clin Oncol 2010, 28(20):3239–3247. g , ( ) 9. Robert NJ, Dieras V, Glaspy J, Brufsky AM, Bondarenko I, et al: RIBBON-1: randomized, double-blind, placebo-controlled, phase III trial of chemotherapy with or without bevacizumab for first-line treatment of

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Improved Structural Equation Models Using Factor Analysis

Pakistan journal of statistics and operation research

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Abstract We develop an agricultural adaptive structural equation model (SEM) that incorporates a large number of factors. These factors simultaneously account for food production while uncompromising food quality and safety. Using the principal component analysis (PCA), we obtain provisional factors, which we rotate using factor analysis, thus leading to reduced number of variables. To decide on the form of the covariance structure in the estimation of the parameters of the regression model, we conduct analysis of covariance. The generated principal components are incorporated into the SEMs where testing of different inter-associations among latent variables (LV) is conducted. For simplicity of the model, we utilise J𝑜̈reskog linear structural equation (LSE) system throughout the investigation process. Using a comprehensive real-life example, we illustrate the concepts and effects of the outcomes. The results show that factors such as energy, transport, labour and fertilizer make a positive contribution in the increase of the quantity and quality food. In addition, we demonstrate how to determine the key factors that influence food production where some factors are not directly measured. Keywords: Structural Equation Model, Path Analysis, Factor Analysis , J𝑜̈reskog Linear Structural Equation. Improved Structural Equation Models Using Factor Analysis Busanga Jerome Kanyama Department of Statistics, School of Science, College of Science, Engineering and Technology, University of South Africa [email protected] Busanga Jerome Kanyama Department of Statistics, School of Science, College of Science, Engineering and Technology, University of South Africa [email protected] Peter Njuho Department of Statistics, School of Science, College of Science, Engineering and Technology, University of South Africa [email protected] Peter Njuho Department of Statistics, School of Science, College of Science, Engineering and Technology, University of South Africa [email protected] Jean-Claude Malela-Majika Department of Statistics, School of Science, College of Science, Engineering and Technology, University of South Africa [email protected] Jean-Claude Malela-Majika Department of Statistics, School of Science, College of Science, Engineering and Technology, University of South Africa [email protected] j Department of Statistics, School of Science, College of Science, Engineering and Technology, University of South Africa [email protected] 1. Introduction Structural equation models (SEMs) is a popular tool in the field of social sciences (Bentler and Chou, 1986; Bielby and Hausser, 1977) and across many other fields. This technique has been used in different fields of science to analyse cause-effect relationship between latent variables. Steenkamp and Baumgartner (2000) and Babin et al. (2008) applied SEMs techniques in marketing field to examine unobservable phenomena such as consumer attitudes, perception, and intentions. Wang and Staver (2001) examined relationships between factors of science education and student career aspiration. Singh et al. (2002) studied the achievement in mathematics and sciences. Lee et al. (2011) and Nitzl (2016) used SEMs and partial least squares (PLS) techniques in accounting. SEMs technique has been also used in other discipline such as hospitality management, international management, operations management, management informatics system, supply chain management, etc. (Sosik et al., 2009; Peng and Lai, 2012; Kaufmann and Graeckler, 2015; Richter et al. 2016; Ali et al., 2017). More recently, Hair et al. (2017) used a series of Pak.j.stat.oper.res. Vol.XIV No.4 2018 pp995-1012 Busanga Jerome Kanyama, Peter Njuho, Jean-Claude Malela-Majika ordinary least squares regressions to estimate partial model structures of composite-based SEM models. Henseler (2017) developed a variance-based SEM. A statistical and practical concern with published research featuring SEM was presented by Goodboy and Kline (2017). Bolt et al. (2018) used SEM approaches in medical science to empirically derive networks from region of interest (ROI) activity, and to assess the association of ROIs and their respective whole-brain activities networks with task performance using three large samples. The SEM(s) is a powerful tool that can be used to solve complex problems involving diverse factors. In particular, the tool can provide efficient results in the evaluation of the relations among variables and in testing theoretical models. The SEM(s) and path analysis are introduced in agricultural science as powerful tools to solve complex problems encountered. Worldwide, agricultural studies play a significant role in the life of human being and particularly in sub-Saharan Africa where countries are dominated by a high number of hungry people (Mwichabe, 2013). SEM comprises: (i) A set of linear equations identifying or detailing the causal relationship between the variables in the model, and (ii) Several supporting assumptions. Similarly, to linear equations, SEM establishes a direct relationship between any cause and any effect that is generally specified by the coefficients connecting or associating two variables in the equation. 1. Introduction As a result, the coefficient is the variation in effect generated by a one-unit variation in the level of the cause holding the other causes constant. Generally, the value of the coefficient is unknown. Noticing the great need for the development and improvement of new analytical methods in the field of agricultural science, this paper introduces SEM and path analysis by developing appropriate structural equations and path diagrams. The linear relationship in a system of equations models can be represented in different ways, but in this paper, these equations are offered as given in equation 1(a – c). Section 2 presents the basic characteristics of SEMs and path analysis. Their contributions to the field of agricultural science is illustrated through a practical example. In Section 3, we develop a model of observable fact of interesting SEM. The developed model is tested by means of the variance-covariance technique based on Factor analysis in the SEM structure. Conclusion and useful recommendations are given in Section 4. Pak.j.stat.oper.res. Vol.XIV No.4 2018 pp995-1012 2. Structural Equation Model Associated to empirical patterns, the concept of causality has always been an alarming issue in various field of sciences, like in social sciences. In the same way, SEMs come across causality hypothesis that is normally tested in non-empirical studies models. Wright (1921) was the first to suggest SEM in a complete approach with regression analysis as a foundation to test the relationship between observed and implicit variables (Pedhazur, 1997; Raykov and Marcoulides, 2000). In addition, SEM can perform multiple regression test with two or more indirect or hidden variables subjecting to a number of display variables associated with error terms. In general, SEM remains completely subjected to theoretical suggestion that SEM model will demonstrate whether the previously defined connection pattern could be supported or not, by the collected data. In other words, we use SEM in prediction of unknown parameters on linear structure of equation. The variables in a set of SEM equations are directly and indirect observed. In SEM, we assume existence Pak.j.stat.oper.res. Vol.XIV No.4 2018 pp995-1012 996 Improved Structural Equation Models Using Factor Analysis of a causality (or interconnection) structure between the directly observed variables and the indirect measured variables. of a causality (or interconnection) structure between the directly observed variables and the indirect measured variables. Technically, SEMs hold one or more linear regressions that explain how endogenous structures are determined upon exogenous structure. That means, in SEM the focus is in terms of measurement of variables that define just how theoretical (indirect) structures depend on observed variables when assuming causality relationship between indirect variables. Path analysis (PA) and confirmatory factor analysis (CFA) are special types of SEM. PA examines how independent variables are statistically related to a dependent variable. Moreover, PA can allow causal interpretation of statistical dependencies and most importantly, PA allows for the examination of how the data fits to a theoretical model. PA enables us both to draw a path diagram based on the theory and to conduct one or more regression analysis (see Figure 1 and 2). The estimation process in SEM involves different techniques, which include maximum likelihood commonly used by software. It assumes either multivariate normality or generalized least square of robust estimators. Pak.j.stat.oper.res. Vol.XIV No.4 2018 pp995-1012 Busanga Jerome Kanyama, Peter Njuho, Jean-Claude Malela-Majika Figure 1. Path diagram Figure 1 presents a path diagram for a linear SEM that provides solutions to the problem of hunger in Sub-Saharan Africa (SSA) by increasing food production when using the relationship between the exogenous and endogenous variables. Figure 1. Path diagram Figure 1. Path diagram g g Figure 1 presents a path diagram for a linear SEM that provides solutions to the problem of hunger in Sub-Saharan Africa (SSA) by increasing food production when using the relationship between the exogenous and endogenous variables. Figure 1 presents a path diagram for a linear SEM that provides solutions to the problem of hunger in Sub-Saharan Africa (SSA) by increasing food production when using the relationship between the exogenous and endogenous variables. Most often, PA provides a diversity of set of relationships that can be developed among the variables. However, some of these variables are similar. Therefore, there is a need of a more advanced technique (or method) that allows us to reduce a large number of variables into a small number. Factor analysis (FA) serves this purpose. FA is a multivariate statistical method for reducing large numbers of variables to fewer underlying dimensions. This method involves grouping of similar variables into dimensions. This process is used to identify latent variables or constructs. Most often, factors are rotated after extraction. FA has several different rotation methods, and some of them ensure that the factors are orthogonal (i.e. uncorrelated), which eliminates problems of multicollinearity in regression analysis. There are many techniques of FA with principal component analysis (PCA) being the most used followed by the exploratory factor analysis (EFA). PCA is used if the components can be derived or/and summarized. It has been used by many researchers in medical science, education, social science and many other related fields (Wang and Staver, 2001; Bolt et al., 2018). However, EFA is used if the variables have unmeasured variables. It is not as popular as PCA. In this paper, we integrate FA into SEM in order to provide an optimal and cost effective model that explains better the key factors in the food production system. 3.1 The improved Structural Equation Models using Factor Analysis 2. Structural Equation Model SEM using J𝑜̈reson in linear structural relations (LISREL) notations as presented by Bentler and Weeks (1980) follows: 𝜂 = Β 𝜂 + Γ 𝜉 + 𝜁 (1 a) Y = Λ(𝑦) 𝜂 + 𝜀 (1 b) Χ = Λ(𝑥) 𝜉 + 𝛿, (1 c) 𝜂 = Β 𝜂 + Γ 𝜉 + 𝜁 (1 a) Y = Λ(𝑦) 𝜂 + 𝜀 (1 b) Χ = Λ(𝑥) 𝜉 + 𝛿, (1 c) 𝜂 = Β 𝜂 + Γ 𝜉 + 𝜁 Y = Λ(𝑦) 𝜂 + 𝜀 Χ = Λ(𝑥) 𝜉 + 𝛿, (1 a) (1 b) (1 c) (1 c) where, 𝜂 represents the random vector latent dependent variables, Β indicates the weights (parameter matrices) for predicting dependent variables from each other. Γ represents weights (parameter matrices) for predicting dependent variable from independent variables (Β and Γ are coefficient matrices for linear relations of all variables involved in SEM), 𝜉 denotes the random vector latent independent variables, 𝑌 indicates the observed indicator for latent dependent variable, 𝑋 denotes the observed indicator for latent independent variable Λ(𝑥) and Λ(𝑦) are parameter matrices and 𝜀 and 𝛿 are random vectors. Path diagrams represent the models graphically and enable researchers to visualize the conceptual models behind the research and to show statistical results. Path diagrams represent functional relationships among the multiple regression models that are special case of structural equation model. From the output given by the path diagram, when the p- value is greater than 5% level of significance, we conclude that the theoretical model is not a good one for the data. To illustrate this process we use an example. Consider four dependent and four independent latent variables that we want to establish the system of equations of the observed (𝑌 and 𝑋) and theoretical model (𝜂) using (1a), (1b) and (1c) as shown in Figure 1. Pak.j.stat.oper.res. Vol.XIV No.4 2018 pp995-1012 997 Busanga Jerome Kanyama, Peter Njuho, Jean-Claude Malela-Majika Improved Structural Equation Models Using Factor Analysis We outline necessary steps to take in producing SEM using factor analysis after obtaining provisional factors via principal component analysis (PCA) as follow: We outline necessary steps to take in producing SEM using factor analysis after obtaining provisional factors via principal component analysis (PCA) as follow: (i) Screen the data for suitability through testing; creen the data for suitability through testing; (ii) Apply PCA on correlation matrix to obtain provisional factors when the test in Step (i) is statistically significant. Using the Factor analysis (FA), calculate the communalities accounting for pre-set proportion of total variation; (ii) Apply PCA on correlation matrix to obtain provisional factors when the test in Step (i) is statistically significant. Using the Factor analysis (FA), calculate the communalities accounting for pre-set proportion of total variation; (iii) Determine the number of principal components to retain and rotate to obtain orthogonality; (iv) Interpret the new variables (FAs) based on factor loading for each variable; (v) Consider rotating the factors to attain orthogonality. Thus, final factors are orthogonal; (vi) Determine the component score coefficient matrix for the possible models. Estimation of parameters in SEM is by maximum likelihood method. It provides estimates for the linear equations that reduce the deviation between the observed and the proposed model. We incorporate the selected Factors to a number of SEMs and then test for different inter-associations among the latent variables. The correlations between the latent (unobserved) variables and latent (observed) variables were equivalent to factor loading in principal component analysis. The general structural equation model as given in Equation (1a) is equivalent to Equation (2) summarized as 𝜂= 𝛽+𝜂+ Σ𝜉+ 𝜁, (2) (2) where 𝜂= ( 𝜂1 𝜂2 𝜂3 𝜂4 ), 𝛽+ = ( 0 0 𝛽12 0 𝛽13 𝛽14 𝛽23 𝛽24 0 0 0 𝛽34 0 0 0 0 ), Σ = Γ = ( 𝛿11 𝛿21 𝛿12 𝛿22 𝛿13 𝛿14 𝛿23 𝛿24 𝛿31 𝛿32 𝛿33 𝛿34 𝛿41 𝛿42 𝛿43 𝛿44 ) , 𝜉= ( 𝜉1 𝜉2 𝜉3 𝜉4 ) and 𝜁= ( 𝜁1 𝜁2 𝜁3 𝜁4 ) where These structures of random vectors and parameter matrices are used in the data analysis. These structures of random vectors and parameter matrices are used in the data analysis. These structures of random vectors and parameter matrices are used in the data analysis. 3. Methodology The current approach of SEM is more restrictive since it specifies the latent variables that are involved in the analysis and creates the theoretical relations between the variables. There is a huge diversity of set of relationships that could be developed among the variables. The variability of set of relationships point to inconsistent conclusions about the level at which a model truly is equivalent to the observed data. Therefore, a variety of the path diagram are oftentimes utilized. We present a more reliable approach that provides a guideline on how to evaluate the suitability of a given SEM. Researchers in agriculture sector use all possible variables that might be identified for a set data, but using factor analysis through the PCA, researchers will be able to use the most important variables in the model. SEM, commonly applied in many fields is introduced in the agriculture field. 3.1 The improved Structural Equation Models using Factor Analysis Pak.j.stat.oper.res. Vol.XIV No.4 2018 pp995-1012 998 Improved Structural Equation Models Using Factor Analysis Pak.j.stat.oper.res. Vol.XIV No.4 2018 pp995-1012 3.2 Data analysis In the past few decades, a number of researchers have contributed in the development of agricultural models to improve food production in sub-Saharan Africa (SSA). Lamb et al. (2010) developed a model illustrating the application of SEM in plant sciences. When solving the problem of food insufficiency caused by environmental conditions, individual’s solutions exist, but permanent results remain an issue to be addressed, since the model is still unknown and needs to be discovered. Therefore, the true parameters of the model can Pak.j.stat.oper.res. Vol.XIV No.4 2018 pp995-1012 999 Busanga Jerome Kanyama, Peter Njuho, Jean-Claude Malela-Majika only be estimated from the observed core factors. These factors are identified through the PCA and FA procedures. only be estimated from the observed core factors. These factors are identified through the PCA and FA procedures. Consider data from the FAO database http://faostat3.org/home/E from 2015 across 45 African countries. The variables are given in Table 1 with the LISREL notations according to J𝑜̈reskog (2000). Table 1: Crop components classified into three vital factors (crop, livestock and contributors) with various factor levels and denoted by LISREL Components Description of variables LISREL notations Crop Banana 𝑌1 Beans 𝑌2 Cassava 𝑌3 Rice 𝑌4 Groundnut 𝑌5 Maize 𝑌6 Sugar cane 𝑌7 Vegetables 𝑌8 Cereals 𝑌9 Fruits 𝑌10 Livestock Cattle and Buffaloes 𝑌11 Pigs 𝑌12 Poultry 𝑌13 Sheep and Goats 𝑌14 Contributors Fertilizer (Factor 1) Nitrogen 𝑋1 Phosphate 𝑋2 Trade (Factor 2) Export values 𝑋3 Import values 𝑋4 Labour (Factor 3) Rural 𝑋5 Urban 𝑋6 Land (Factor 4) Arable 𝑋7 Permanent 𝑋8 Water (Factor 5) Rainfall 𝑋9 Irrigated land 𝑋10 Energy used (Factor 6) Electricity 𝑋11 Diesel 𝑋12 Transport 𝑋13 Suppose we denote crop components Y1, Y2, . . .,Y10, livestock components Y11, Y12, . . .,Y14 and contributors components X1, X2, . . .,X13. Crop components classified into three vital factors (crop, livestock and contributors) with various factor levels and denoted by LISREL Suppose we denote crop components Y1, Y2, . . .,Y10, livestock components Y11, Y12, . . .,Y14 and contributors components X1, X2, . . .,X13. Suppose we denote crop components Y1, Y2, . . .,Y10, livestock components Y11, Y12, . . .,Y14 and contributors components X1, X2, . . .,X13. In the complexity of these variables and data, PCA is used to determine the direct observed variables in order to decide about the number of factors to be retained in the model. 3.2 Data analysis Table 2: Screening of different variables through PCA based on the total variance explained Component Initial Eigenvalues Rotation Sums of Squared loadings Total % of Variance Cumulative % Total % of Variance Cumulative % 1 4.746 18.985 18.985 3.077 12.310 12.310 2 3.254 13.017 32.002 2.950 11.799 24.109 3 2.742 10.969 42.971 2.930 11.720 35.829 4 2.435 9.741 52.712 2.681 10.725 46.554 5 1.885 7.542 60.254 2.123 8.492 55.046 6 1.545 6.181 66.435 2.052 8.207 63.253 7 1511 6.044 72.479 1.706 6.825 70.078 8 1.402 5.606 78.085 1.532 6.127 76.205 9 1.154 4.615 82.700 1.415 5.661 81.866 10 1.028 4.111 86.811 1.236 4.945 86.811 11 0.804 3.217 90.028 2: Screening of different variables through PCA based on the total variance explained Table 2: Screening of different variables through PCA based on the to explained Extraction Method: Principal Component Analysis From Table 2, about 87% of the total variation is accounted for by 10 out of 25 original variables. Thus, we rotate the 10 principal components using FA to attain orthogonality. 3.2 Data analysis PCA is strongly related to factor analysis by indicating the correlations or associations between the variables and determining the small number of latent variables. Countries were grouped Pak.j.stat.oper.res. Vol.XIV No.4 2018 pp995-1012 1000 Improved Structural Equation Models Using Factor Analysis into crop production, livestock and contributors factors dimensions and make inference about stable estimate parameters for the solutions to the problem of hunger and life of human being at the extreme menace. We used the PCA approach to determine direct and indirect variables. into crop production, livestock and contributors factors dimensions and make inference about stable estimate parameters for the solutions to the problem of hunger and life of human being at the extreme menace. We used the PCA approach to determine direct and indirect variables. The correlation matrix is used to determine the variables that were the most strongly correlated with each component. This screening of variables reduced the number of highly correlated variables from 25 to 10 new independent variables as indicated in Table 2. The retained variables explain much of the total variation in the variable of interest is explained by each component, as this cannot be performed in multiple regressions. The results of PCA determined the levels at which the variables were measured. The variables with the highest sample variances were among the few components taken as each variable received its particular weight in the analysis. To receive equal weight in the analysis we have then standardized variables before carrying out the PCA (performing PCA on a correlated matrix). Table 2 shows the number of components and the eigenvalues (initial and rotation eigenvalues). 3.3 Illustrative Example on Agricultural Data Analysis using SEMs In SSA countries, agriculture is one of the most dominant activities providing jobs to the population. Productivity in this part of the world remains low because of many challenges that go beyond weather, pests and lack of fertilizer. For instance, in the northern part of the African continent, less than thirty percent of land is irrigated and Africa is far behind in the Pak.j.stat.oper.res. Vol.XIV No.4 2018 pp995-1012 1001 Busanga Jerome Kanyama, Peter Njuho, Jean-Claude Malela-Majika use of more advanced agricultural technology. We have used food production to display the values of this modelling method. use of more advanced agricultural technology. We have used food production to display the values of this modelling method. In this section, the proposed technique is implemented using a real-life example based on food production in order to show how the newly proposed model prevails on the existing models. In this illustrative example, most valuable crops, livestock’s products and the contributor’s factors in the SSA are given in column 1 of Table 1. PCA procedure allows for reduction of dimension of the original variables into a few number of the principal components as variables explaining most of the variation in the data set. These principal components are represented by component 1 to 10 as given in Table 3. The bold values are the highest correlations between the original variables and the components in the array. Pak.j.stat.oper.res. Vol.XIV No.4 2018 pp995-1012 3.3 Illustrative Example on Agricultural Data Analysis using SEMs P k j t t V l XIV N 4 2018 995 1012 1002 Table 3: The rotate component matrix Original variables Factor components 1 2 3 4 5 6 7 8 9 10 Bananas .100 -.045 .049 -.099 -.009 -.191 -.105 .802 -.038 -.039 Beans -.050 -.029 .057 -.076 .114 .139 .075 .821 -.001 .073 Cassava -.017 .040 .872 -.072 .089 .012 -.019 .114 -.044 .053 Rice -.072 -.024 .090 .000 -.038 -.117 -.005 .050 -.031 .886 Groundnut .033 .960 .208 -.041 -.009 -.017 .002 -.095 -.050 -.031 Maize .074 .990 -.064 .034 .005 -.004 -.034 .003 -.027 .004 Sugar cane .804 .083 .013 .421 .093 -.050 .203 .061 -.017 .062 Vegetables .023 .993 .068 -.037 .001 -.005 -.032 .004 -.030 .013 Cereals .382 .116 .843 .095 .008 -.088 -.022 .068 .081 .146 Fruit .041 .059 .933 -.114 .023 -.056 .007 -.069 -.024 -.060 Export .707 .016 .124 .254 .029 -.054 -.088 .243 .216 -.124 Import .659 .071 .558 .255 .059 -.077 -.010 .126 .005 -.111 Irrigated .775 .030 .200 .109 .144 -.102 -.065 -.169 -.078 -.051 Rainfall -.131 -.073 -.009 -.020 .274 .070 .868 -.045 .026 .191 Nitrogen -.111 -.019 -.044 -.049 .024 .919 -.008 .062 -.105 -.076 Phosphate -.011 -.012 -.055 -.017 .174 .922 -.010 -.098 .069 -.027 Rural .031 .019 .092 .010 .961 .070 .088 .112 -.054 -.040 Urban .221 -.018 .029 .112 .934 .142 -.025 .002 .029 -.010 Table 3: The rotate component matrix Table 3: The rotate component matrix 1002 Improved Structural Equation Models Using Factor Analysis Electricity .335 -.044 -.047 .860 -.007 .090 -.149 -.067 .058 -.025 Diesel .409 -.060 -.012 .855 -.015 -.010 .032 -.053 -.049 .000 Transport -.016 .029 -.023 .895 .130 -.144 .037 -.106 .044 -.006 Cattle- Buffaloes .120 .000 -.023 -.026 -.158 -.082 .907 .009 .031 -.189 Pigs .120 -.042 -.077 .026 -.173 .076 -.028 -.080 .889 .115 Poultry .128 .102 -.111 -.033 -.304 .222 -.154 -.073 -.726 .361 Sheep - goats -.565 -.064 .348 .162 -.034 -.367 .013 .125 .006 -.396 The dominance variables explaining each of the 10 factors accounting for 87% of the total variation are outlined below: Factor 1 --- Sugar cane, Import, Irrigated and Sheep - Goat Factor 2 --- Groundnut, Maize, and Vegetables Factor 3 --- Cassava, Cereals, and Fruits Factor 4 --- Electricity, Diesel, and Transport Factor 5 --- Rural and Urban Factor 6 --- Nitrogen and Phosphate Factor 7 --- Rainfall and Cattle - Buffalos Factor 8 --- Bananas and Beans Factor 9 --- Pigs and Poultry Factor 10 ---Rice. 3.3 Illustrative Example on Agricultural Data Analysis using SEMs Improved Structural Equation Models Using Factor Analysis Electricity .335 -.044 -.047 .860 -.007 .090 -.149 -.067 .058 -.025 Diesel .409 -.060 -.012 .855 -.015 -.010 .032 -.053 -.049 .000 Transport -.016 .029 -.023 .895 .130 -.144 .037 -.106 .044 -.006 Cattle- Buffaloes .120 .000 -.023 -.026 -.158 -.082 .907 .009 .031 -.189 Pigs .120 -.042 -.077 .026 -.173 .076 -.028 -.080 .889 .115 Poultry .128 .102 -.111 -.033 -.304 .222 -.154 -.073 -.726 .361 Sheep - goats -.565 -.064 .348 .162 -.034 -.367 .013 .125 .006 -.396 The dominance variables explaining each of the 10 factors accounting for 87% of the total variation are outlined below: Improved Structural Equation Models Using Factor Analysis Factor 1 --- Sugar cane, Import, Irrigated and Sheep - Goat Factor 2 --- Groundnut, Maize, and Vegetables Factor 3 --- Cassava, Cereals, and Fruits Factor 4 --- Electricity, Diesel, and Transport Factor 5 --- Rural and Urban Factor 6 --- Nitrogen and Phosphate Factor 7 --- Rainfall and Cattle - Buffalos Factor 8 --- Bananas and Beans Factor 9 --- Pigs and Poultry Factor 10 ---Rice. The test for normality of the variables in each of the observed indicator for endogenous and exogenous variables is validated as shown in Tables 4 and 5. Pak.j.stat.oper.res. Vol.XIV No.4 2018 pp995-1012 1003 Table 4: Test for normality for endogenous variable Observation Factor 1 Factor 2 Factor 3 Factor 4 Factor 5 Factor 6 Chi-square 88.942 263.882 113.417 18.676 8.068 6.940 Degrees of freedom 10 3 6 1 1 1 p-value 0.000 0.000 0.000 0.000 0.005 0.008 Table 5: Test for normality for exogenous variables Observation Factor 1 Factor 2 Factor 3 Chi-square 105.636 69.642 48.157 Table 4: Test for normality for endogenous variable Observation Factor 1 Factor 2 Factor 3 Factor 4 Factor 5 Factor 6 Chi-square 88.942 263.882 113.417 18.676 8.068 6.940 Degrees of freedom 10 3 6 1 1 1 p-value 0.000 0.000 0.000 0.000 0.005 0.008 Table 4: Test for normality for endogenous variable 1003 Busanga Jerome Kanyama, Peter Njuho, Jean-Claude Malela-Majika The variables were normally distributed since 𝑝−𝑣𝑎𝑙𝑢𝑒 is less than 0.05. Therefore, the maximum likelihood estimation can be used. The general linear SEM is given in Equations (1a), (1b) and (1c) (See Tables 6 and 7). The latent endogenous and exogenous models are the highly correlated of the factors load in which the measurement model is obtained by the maximum likelihood. Pak.j.stat.oper.res. Vol.XIV No.4 2018 pp995-1012 Improved Structural Equation Models Using Factor Analysis Improved Structural Equation Models Using Factor Analysis Figure 2. Conceptual path diagram for the structural model Figure 2. Conceptual path diagram for the structural model Table 6 presents the endogenous variables under different models based on the factor loadings obtained from rotated provisional factors. The model equations, measurement model parameters and associated score components, in addition to goodness of fit test statistics are also included Pak.j.stat.oper.res. Vol.XIV No.4 2018 pp995-1012 3.3 Illustrative Example on Agricultural Data Analysis using SEMs The model fit was the result for the goodness-of-fit statistical tests that explain the discrepancy between latent and unobserved variables. In this practice, the model fits well the data as this indicated that no important paths have been omitted from the model. After estimating the endogenous and exogenous latent measurement model separately, a joint model that includes altogether latent model can now be estimated (Figure 2). After estimating the endogenous and exogenous latent measurement model separately, a joint model that includes altogether latent model can now be estimated (Figure 2). Since latent variables are observed, the measurement is obtained indirectly through the latent endogenous and exogenous variables. The latent unobserved variables are represented as ellipses and the latent observed variables are represented as rectangles and because we cannot measure or estimate perfectly the unknown factors or parameters, we can only measure with error and therefore, the errors terms were associated with each of the latent observed variables as they form part of the overall model. The error terms are also represented as ellipse (Figure 2). Based on the type of regression and relationship indicated in the diagram, the SEMs are potentially complex interplay between a large number of observed and unobserved variables including error terms. Using the variables in the data and corresponding identifier notations, we illustrate inter-relationship using the path diagram. The path diagram represented the model in line of the overall outcome of this paper. Using Equation (2) and results in Table 3, the maximum likelihood estimates were obtained. We illustrates the path diagram using these estimates as follows: 1004 Pak.j.stat.oper.res. Vol.XIV No.4 2018 pp995-1012 Pak.j.stat.oper.res. Vol.XIV No.4 2018 1006 Table 6: The endogenous descriptions model 1005 Pak.j.stat.oper.res. Vol.XIV No.4 2018 pp995-1012 Busanga Jerome Kanyama, Peter Njuho, Jean-Claude Malela-Majika Busanga Jerome Kanyama, Peter Njuho, Jean Claude Malela Majika Pak j stat oper res Vol XIV No 4 2018 pp995-1012 1006 Model Factor load Correlation Model equation Measurement model Component score coefficient Goodness- fit test 1 Sugar Export Import Irrigation Sheep & goats 0.804 0.707 0.659 0.775 0.565 𝑌1 = Λ𝑦 𝜂1 + 𝜀 ( 𝑦7 𝑦14 𝑥3 𝑥4 𝑥10) = ( λ71 𝑦 λ14,1 𝑦 λ31 𝑦 λ41 𝑦 λ10,1 𝑦) 𝜂1 + ( 𝜀1 𝜀2 𝜀3 𝜀4 𝜀5) ( 𝑦7 𝑥3 𝑥4 𝑥10 𝑦14) = ( 0 0.649 0.445 0 0 ) 𝜂1 + ( 𝜀1 𝜀2 𝜀3 𝜀4 𝜀5) Chi-square= 8.018 𝑑𝑓 = 4 p-value= 0.005 2 Groundnut Maize Vegetable 0.960 0.990 0.993 𝑌2 = Λ𝑦 𝜂2 + 𝜀 ( 𝑦5 𝑦6 𝑦8 ) = ( λ52 𝑦 λ62 𝑦 λ82 𝑦 ) 𝜂2 + ( 𝜀1 𝜀2 𝜀3 ) ( 𝑦5 𝑦6 𝑦8 ) = ( 0.055 0.936 0.013 ) 𝜂2 + ( 𝜀1 𝜀2 𝜀3 ) Chi-square= 0.637 𝑑𝑓 = 2 p-value= 0.000 3 Cassava Cereals Fruits 0.872 0.843 0.933 𝑌3 = Λ𝑦 𝜂3 + 𝜀 ( 𝑦3 𝑦9 𝑦10 ) = ( λ33 𝑦 λ93 𝑦 λ10,3 𝑦 ) 𝜂3 + ( 𝜀1 𝜀2 𝜀3 ) ( 𝑦3 𝑦9 𝑦10 ) = ( 0.152 0.186 0.688 ) 𝜂3 + ( 𝜀1 𝜀2 𝜀3 ) Chi-square= 15.49 𝑑𝑓 = 2 p-value= 0.000 4 Rainfall Cattle 0.868 0.907 𝑌4 = Λ𝑦 𝜂4 + 𝜀 ( 𝑋9 𝑦11) = ( λ94 𝑦 λ11,4 𝑦) 𝜂4 + ( 𝜀1 𝜀2) ( 𝑋9 𝑦11) = (0.560 0.560) 𝜂4 + ( 𝜀1 𝜀2) Chi-square= 16.68 𝑑𝑓 = 1 p-value= 0.000 5 Banana Beans 0.802 0.821 𝑌5 = Λ𝑦 𝜂5 + 𝜀 ( 𝑦1 𝑦2) = (λ15 𝑦 λ25 𝑦) 𝜂5 + ( 𝜀1 𝜀2) ( 𝑦1 𝑦2) = (0.594 0.594) 𝜂5 + ( 𝜀1 𝜀2) Chi-square= 8.068 𝑑𝑓= 1 p-value= 0.005 Pak.j.stat.oper.res. Table 6: The endogenous descriptions model Vol.XIV No.4 2018 pp995-1012 mproved Structural Equation Models Using Factor Analysis Improved Structural Equation Models Using Factor Analysis Model Factor load Correlation Model equation Measurement model Component score coefficient Goodness- fit test 6 Pigs Poultry 0.889 -0.776 𝑌6 = Λ𝑦 𝜂6 + 𝜀 ( 𝑦12 𝑦13) = (λ12,6 𝑦 λ13,6 𝑦) 𝜂6 + ( 𝜀1 𝜀2) ( 𝑦12 𝑦13) = ( 0.600 − 0.600) 𝜂6 + ( 𝜀1 𝜀2) Chi-square= 6.940 𝑑𝑓 = 1 p-value= 0.008 Model Factor load Correlation Model equation Measurement model Component score coefficient Goodness- fit test 6 Pigs Poultry 0.889 -0.776 𝑌6 = Λ𝑦 𝜂6 + 𝜀 ( 𝑦12 𝑦13) = (λ12,6 𝑦 λ13,6 𝑦) 𝜂6 + ( 𝜀1 𝜀2) ( 𝑦12 𝑦13) = ( 0.600 − 0.600) 𝜂6 + ( 𝜀1 𝜀2) Chi-square= 6.940 𝑑𝑓 = 1 p-value= 0.008 Table 7: The exogenous descriptions model Mode l Factor load Correlatio n Model equatio n Measuremen t model Componen t score coefficient Goodness -fit test 1 Electricit y Diesel Transport 0.860 0.855 0.895 𝑋1 = Λ𝑥 𝜉1 + 𝛿 ( 𝑋11 𝑋12 𝑋13 ) = ( λ33 𝑥 λ93 𝑦 λ10,3 𝑥 ) 𝜉1 + ( 𝛿 1 𝛿 2 𝛿 3 ) ( 𝑋11 𝑋12 𝑋13 ) = ( 0.376 0.582 0.057 ) 𝜉1 + ( 𝛿 1 𝛿 2 𝛿 3 ) Chi-square= 15.49 𝑑𝑓 = 2 p-value= 0.000 2 Rural Urban 0.961 0.934 𝑋2 = Λ𝑥 𝜉2 + 𝛿 ( 𝑋5 𝑋6) = (λ52 𝑥 λ62 𝑥) 𝜉2 + ( 𝛿 1 𝛿 2) ( 𝑋5 𝑋6) = (0.513 0.513) 𝜉2 + ( 𝛿 1 𝛿 2) Chi-square= 69.64 𝑑𝑓 = 1 p-value= 0.000 3 Nitrogen Phosphat e 0.919 0.922 𝑋3 = Λ𝑥 𝜉3 + 𝛿 (𝑋1 𝑋2) = (λ1,3 𝑥 λ2,3 𝑥) 𝜉3 + (𝛿 1 𝛿 3) (𝑋1 𝑋2) = (0.524 0.057) 𝜉3 + (𝛿 1 𝛿 3) Chi-square= 48.16 𝑑𝑓 = 1 p-value= 0.000 In this model three major components were causes for the performance and improvement y ( 𝜀1 𝜀2) 0.600 + ( 𝜀1 𝜀2) p 0.008 Table 7: The exogenous descriptions model Mode l Factor load Correlatio n Model equatio n Measuremen t model Componen t score coefficient Goodness -fit test 1 Electricit y Diesel Transport 0.860 0.855 0.895 𝑋1 = Λ𝑥 𝜉1 + 𝛿 ( 𝑋11 𝑋12 𝑋13 ) = ( λ33 𝑥 λ93 𝑦 λ10,3 𝑥 ) 𝜉1 + ( 𝛿 1 𝛿 2 𝛿 3 ) ( 𝑋11 𝑋12 𝑋13 ) = ( 0.376 0.582 0.057 ) 𝜉1 + ( 𝛿 1 𝛿 2 𝛿 3 ) Chi-square= 15.49 𝑑𝑓 = 2 p-value= 0.000 2 Rural Urban 0.961 0.934 𝑋2 = Λ𝑥 𝜉2 + 𝛿 ( 𝑋5 𝑋6) = (λ52 𝑥 λ62 𝑥) 𝜉2 + ( 𝛿 1 𝛿 2) ( 𝑋5 𝑋6) = (0.513 0.513) 𝜉2 + ( 𝛿 1 𝛿 2) Chi-square= 69.64 𝑑𝑓 = 1 p-value= 0.000 3 Nitrogen Phosphat e 0.919 0.922 𝑋3 = Λ𝑥 𝜉3 + 𝛿 (𝑋1 𝑋2) = (λ1,3 𝑥 λ2,3 𝑥) 𝜉3 + (𝛿 1 𝛿 3) (𝑋1 𝑋2) = (0.524 0.057) 𝜉3 + (𝛿 1 𝛿 3) Chi-square= 48.16 𝑑𝑓 = 1 p-value= 0.000 In this model, three major components were causes for the performance and improvement of food production in SSA. Table 6: The endogenous descriptions model As these components are obtained through the analysis of the data when using PCA approach. The three causes derived from the data were energy (𝜉1), labour (𝜉2), and fertilizer (𝜉3) as indicated in the path diagram (Figure 2). These variables are called “exogenous variables” in this experience. This is because they were governed by the outside factors to the food products. In addition, these variables appear to be random. In other illustration, the exogenous variables maybe fixed by the researcher (Sobel, 1986). On the other hand, we had six effects that were derived from the data: 𝜂1, variable “Sugar cane and sheep - goat”, 𝜂2, variable “Groundnut, maize and vegetable”, 𝜂3, variable “Cassava, cereals and fruit”, 𝜂4, Variable “castle - buffaloes”, 𝜂5, Variable “bananas and beans”, and 𝜂6, Variable “pigs and poultry”. These variables are called “endogenous Table 7: The exogenous descriptions model Mode l Factor load Correlatio n Model equatio n Measuremen t model Componen t score coefficient Goodness -fit test 1 Electricit y Diesel Transport 0.860 0.855 0.895 𝑋1 = Λ𝑥 𝜉1 + 𝛿 ( 𝑋11 𝑋12 𝑋13 ) = ( λ33 𝑥 λ93 𝑦 λ10,3 𝑥 ) 𝜉1 + ( 𝛿 1 𝛿 2 𝛿 3 ) ( 𝑋11 𝑋12 𝑋13 ) = ( 0.376 0.582 0.057 ) 𝜉1 + ( 𝛿 1 𝛿 2 𝛿 3 ) Chi-square= 15.49 𝑑𝑓 = 2 p-value= 0.000 2 Rural Urban 0.961 0.934 𝑋2 = Λ𝑥 𝜉2 + 𝛿 ( 𝑋5 𝑋6) = (λ52 𝑥 λ62 𝑥) 𝜉2 + ( 𝛿 1 𝛿 2) ( 𝑋5 𝑋6) = (0.513 0.513) 𝜉2 + ( 𝛿 1 𝛿 2) Chi-square= 69.64 𝑑𝑓 = 1 p-value= 0.000 3 Nitrogen Phosphat e 0.919 0.922 𝑋3 = Λ𝑥 𝜉3 + 𝛿 (𝑋1 𝑋2) = (λ1,3 𝑥 λ2,3 𝑥) 𝜉3 + (𝛿 1 𝛿 3) (𝑋1 𝑋2) = (0.524 0.057) 𝜉3 + (𝛿 1 𝛿 3) Chi-square= 48.16 𝑑𝑓 = 1 p-value= 0.000 Table 7: The exogenous descriptions model In this model, three major components were causes for the performance and improvement of food production in SSA. As these components are obtained through the analysis of the data when using PCA approach. The three causes derived from the data were energy (𝜉1), labour (𝜉2), and fertilizer (𝜉3) as indicated in the path diagram (Figure 2). These variables are called “exogenous variables” in this experience. Pak.j.stat.oper.res. Vol.XIV No.4 2018 pp995-1012 The SEM given by J𝑜̈reskog given by the Equation (2) is shown in the Table 8. Pak.j.stat.oper.res. Vol.XIV No.4 2018 1008 Table 6: The endogenous descriptions model This is because they were governed by the outside factors to the food products. In addition, these variables appear to be random. In other illustration, the exogenous variables maybe fixed by the researcher (Sobel, 1986). On the other hand, we had six effects that were derived from the data: 𝜂1, variable “Sugar cane and sheep - goat”, 𝜂2, variable “Groundnut, maize and vegetable”, 𝜂3, variable “Cassava, cereals and fruit”, 𝜂4, Variable “castle - buffaloes”, 𝜂5, Variable “bananas and beans”, and 𝜂6, Variable “pigs and poultry”. These variables are called “endogenous In this model, three major components were causes for the performance and improvement of food production in SSA. As these components are obtained through the analysis of the data when using PCA approach. The three causes derived from the data were energy (𝜉1), labour (𝜉2), and fertilizer (𝜉3) as indicated in the path diagram (Figure 2). These variables are called “exogenous variables” in this experience. This is because they were governed by the outside factors to the food products. In addition, these variables appear to be random. In other illustration, the exogenous variables maybe fixed by the researcher (Sobel, 1986). On the other hand, we had six effects that were derived from the data: 𝜂1, variable “Sugar cane and sheep - goat”, 𝜂2, variable “Groundnut, maize and vegetable”, 𝜂3, variable “Cassava, cereals and fruit”, 𝜂4, Variable “castle - buffaloes”, 𝜂5, Variable “bananas and beans”, and 𝜂6, Variable “pigs and poultry”. These variables are called “endogenous Pak.j.stat.oper.res. Vol.XIV No.4 2018 pp995-1012 1007 Busanga Jerome Kanyama, Peter Njuho, Jean-Claude Malela-Majika variables” given that their impact depends stochastically on the operation system of food that solves the problem of hunger in the SSA. The arrows between these variables indicate that one variable was a cause of the other variable and 𝜀𝑖(𝑖= 1, 2, …, 6) and 𝛿𝑖 (𝑖 = 1, 2, and 3) are random variables that are assumed to be multivariate normal distribution. This means the expectation of the vector 𝜀 or 𝛿 is assumed to be equal to zero. For instance, the variance-covariance matrix of 𝜀 or 𝛿 was assumed to be zero and the 𝐶𝑜𝑣 ( 𝜀1, 𝜀2 ) = 𝐶𝑜𝑣 ( 𝜀2, 𝜀3 ) = ... = 𝐶𝑜𝑣 ( 𝜀𝑖, 𝜀𝑗 ) = 0, where 𝑖 = 1, 2, ..., n and j = 1, 2, ..., m. Table 6: The endogenous descriptions model Using the path diagram, the absence of curved arrows between the variables in 𝜀 or 𝛿 indicated that the covariance matrix equals to zero as assumed above. Using the path diagram, the absence of curved arrows between the variables in 𝜀 or 𝛿 indicated that the covariance matrix equals to zero as assumed above. This is the result of the power of the exploratory properties of factor analysis by showing strong indication against orthogonality solutions in this complexity of data. Therefore, the six-measurement model in matrix notation for the exogenous model equivalent to the path diagram 2 represented by Equation (1b) is then given by ( 𝑦1 𝑦2 𝑦3 𝑦4 𝑦5 𝑦6 𝑦7 𝑦8 𝑦9 𝑦10 𝑦11 𝑦12 𝑦13 𝑦14) = ( 0.000 0.000 0.000 0.000 0.594 0.000 0.000 0.000 0.000 0.000 0.594 0.000 0.445 0.000 0.152 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.055 0.000 0.000 0.000 0.000 0.000 0.936 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.013 0.000 0.000 0.000 0.000 0.000 0.000 0.186 0.000 0.000 0.000 0.000 0.000 0.688 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.600 0.000 0.000 0.000 0.000 0.000 −0.60 0.649 0.000 0.000 0.000 0.000 0.000) ( 𝜂1 𝜂2 𝜂3 𝜂4 𝜂5 𝜂6) + ( 𝜀1 𝜀2 𝜀3 𝜀4 𝜀5 𝜀6 𝜀7 𝜀8 𝜀9 𝜀10 𝜀11 𝜀12 𝜀13 𝜀14) (3) (3) In the same way, the exogenous measurement model represented by Equation (1c) is given by In the same way, the exogenous measurement model represented by Equation (1c) is given by ( 𝑋1 𝑋2 𝑋3 𝑋4 𝑋5 𝑋6 𝑋7 𝑋8 𝑋9 𝑋10 𝑋11 𝑋12 𝑋13) = ( 0.376 0 0 0 0 0 0 0 0 0 0 0.582 0 0 0 0.513 0.513 0 0 0 0 0 0.057 0 0 0 0 0 0 0 0 0 0.524 0 0 0 0 0 0.057) ( 𝜉1 𝜉2 𝜉3 ) + ( 𝛿 1 𝛿 2 𝛿 3 𝛿 4 𝛿 5 𝛿 6 𝛿 7 𝛿 8 𝛿 9 𝛿 10 𝛿 11 𝛿 12 𝛿 13) (4) (4) The SEM given by J𝑜̈reskog given by the Equation (2) is shown in the Table 8. Table 6: The endogenous descriptions model 1008 Improved Structural Equation Models Using Factor Analysis Table 8: The parameters estimates and measurement model matrices Β = ( 0 0 0 0 0.527 0 0 0 0.098 0.008 0.094 −0.025 −0.196 0.265 −0.102 0.066 0 0.217 0.548 −0.634 0 0 −0.454 0.207 0 0 0 0 0 0.244 0 0 0 0 0 0 ) 𝜂= ( 𝜂1 𝜂2 𝜂3 𝜂4 𝜂5 𝜂6) Γ = ( 0.707 −0.087 0.384 0 0.673 −0.036 −0.141 0 0.193 0 0 0 0.761 0 0 0 −0.567 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 ) 𝜁= ( 𝜁1 𝜁2 𝜁3 𝜁4 𝜁5 𝜁6) 𝜉= ( 𝜉1 𝜉2 𝜉3 𝜉4 𝜉5 𝜉6) The structural model estimated with the class of the linear model as given in Equation (2) is equivalent to Table 8: The parameters estimates and measurement model matrices The structural model estimated with the class of the linear model as given in Equation (2) is equivalent to The structural model estimated with the class of the linear model as given in Equation (2) is equivalent to ( 𝜂1 𝜂2 𝜂3 𝜂4 𝜂5 𝜂6) = ( 0 0 0 0 0.527 0 0 0 0.098 0.008 0.094 −0.025 −0.196 0.265 −0.102 0.066 0 0.217 0.548 −0.634 0 0 −0.454 0.207 0 0 0 0 0 0.244 0 0 0 0 0 0 ) ( 𝜂1 𝜂2 𝜂3 𝜂4 𝜂5 𝜂6) + ( 0.707 −0.087 0.384 0 0.673 −0.036 −0.141 0 0.193 0 0 0 0.761 0 0 0 −0.567 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 ) ( 𝜉1 𝜉2 𝜉3 𝜉4 𝜉5 𝜉6) + ( 𝜁1 𝜁2 𝜁3 𝜁4 𝜁5 𝜁6) Having the latent scores for 𝜂1,𝜂2, 𝜂3, 𝜂4, 𝜂5 and 𝜂6, and 𝜉1, 𝜉2 and 𝜉3, we can use the information from the model to compare the productivity level for all the identified components. Based on this information, Figure 2 entails that a primary crop production level was simultaneously controlled by the support of livestock (using manure) and the contributor’s factors. The SEMs obtained extract more information about the food production then when using a single linear model for instance maize. Table 6: The endogenous descriptions model In so doing with latent scores, we were able to estimate a single linear equation by using ordinary least squared (OLS) through 𝜂1 as endogenous variable. This procedure generates the equation 𝜂1 = - 0.0479𝜉1 – 0.0182 𝜉2 + 0.404 𝜉3 . For illustration of the model, this suggested that 𝜂1 was a linear function of 𝜉1 , 𝜉2 and 𝜉3 and as a result, the components units can be ranked either on the basis of 𝜂1 or - 0.0479 𝜉1 – 0.0182 𝜉2 + 0.404 𝜉3. As indicated earlier, the approach adopted by SEM was based on the variance-covariate matrix between the variables in the data and the initial path diagrams that hypothesizes the causal relationships among the variables. These path diagrams were later translated into a Pak.j.stat.oper.res. Vol.XIV No.4 2018 pp995-1012 1009 Busanga Jerome Kanyama, Peter Njuho, Jean-Claude Malela-Majika diverse set of linear equations describing the relationships that define a certain pattern when using variance-covariance matrix. diverse set of linear equations describing the relationships that define a certain pattern when using variance-covariance matrix. The results were as amazingly natural as the correlations between latent (unobserved) variables and observed variables were found highly correlated (all above 0.80) and in positive direction except Y (poultry) that was negatively strong (- 0.73) and Y representing sheep and goats (− 0.57) that was acceptable relationship. By contrast, the relationship between the latent (unknown) variables was positively weak but statistically significant. Given these patterns, it appears both a direct and indirect effect between exogenous and endogenous variables. The six endogenous variables derived from the diverse type of crop and kind of livestock affect mutually the three direct cause-factors exogenous: energy, labour and fertilizer as this is likely to maintain claims by revealing how well it is organized. Conversely, the energy used as a factor, labour and fertilizer types were likely to be exceptionally confident, as these factors were key feature to create more productivity of crop and conserve healthy livestock. Acknowledgements The authors thank the University of South Africa and the Master and Doctoral Research Support Programme (MDSP) for their support. 4. Conclusion and Recommendations SEM and path analysis have been used in many fields of science to solve complex problems. This paper introduced the use and application of SEM in agricultural field in an explicit and illustrative manner. Path analysis is a technique to be used in agricultural studies since it helps to focus on the key activities of food production and how they all fit together. SEM and path analysis being statistical techniques of making decision, they also have their own strength and limitations. The best method should be the one addressing the purpose of the research. We have assumed that there is a causal structure among a set of latent variables, therefore SEM technique applied to food production has validated that the livestock’s products and crop in its diversities are likely to be integrated. The results also revealed that factors such as energy, labour and fertilizer are anticipated to make positive contributions to the increase of food production in SSA. 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Bentler, P.M. and Chou, C.P. (1986). Practical Issues in Structural Modelling. Sage journals. Los Angeles. 4. Bentler, P.M. and Weeks, D.G. (1980). Linear Structural Equations with Latent Variables. Psychometrika, 45(3), 289-308. 5. Bolt, T., Prince, E.B., Nomi, J.S., Messinger D., llabre, M.M. and Uddin, L.Q. (2018). Combining region-and network-level brain-behavior relationships in a Structural equation model. NeuroImage, 1656 158-16. 6. Bielby, W.T. and Hauser, R.M. (1977), Structural Equation Models. Annual Review of Sociology, 3, 137-161. 7. Goodboy, A.K., and Kline, R B. (2017). Statistical and Practical Concerns with Published Communication Research featuring Structural Equation Modeling. Communication Research Reports, 34, 1, 68-77. 8. Hair, J. F., Hult, G.T.M., Ringle, C.M., Sarstedt, M. and Thiele, K.O. (2017). Mirror, Mirror on the wall: a Comparative Evaluation of Composite-based Structural Equation Modeling Methods. 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BK Channel-Mediated Microglial Phagocytosis Alleviates Neurological Deficit After Ischemic Stroke

Frontiers in cellular neuroscience

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ORIGINAL RESEARCH published: 01 July 2021 doi: 10.3389/fncel.2021.683769 Edited by: Kai Zhou, Karolinska Institutet, Sweden Edited by: Kai Zhou, Karolinska Institutet, Sweden Karolinska Institutet, Sweden Reviewed by: Burak Yulug, Alanya Alaaddin Keykubat University, Turkey Dennis Qing Wang, Southern Medical University, China *Correspondence: Zhijun Zhang [email protected] Reviewed by: Burak Yulug, Alanya Alaaddin Keykubat University, Turkey Reviewed by: Burak Yulug, Alanya Alaaddin Keykubat University, Turkey Dennis Qing Wang, Southern Medical University, China Dennis Qing Wang, Southern Medical University, China *Correspondence: Zhijun Zhang [email protected] Specialty section: This article was submitted to Cellular Neuropathology, a section of the journal Frontiers in Cellular Neuroscience Keywords: ischemic stroke, BK channels, microglia, phagocytosis, ERK INTRODUCTION Received: 22 March 2021 Accepted: 25 May 2021 Published: 01 July 2021 Stroke is the major cause of death and long-term neurological disability, seriously threatening quality of life in China and across the world (Wu et al., 2019). Ischemic stroke, comprising 80% of all stroke cases, annually affects about 4 million people in China (Wang et al., 2019). Current therapeutic approaches for ischemic stroke (recombinant tissue plasminogen activator and thrombolysis) are limited by short time window and the risk of hemorrhage transformation (Xiong et al., 2019; Chang et al., 2021). Timely treatment in the acute stage of ischemic stroke could effectively limit infarct volume and rescue neuronal death in the peri-focal region (Chamorro et al., 2020). BK Channel-Mediated Microglial Phagocytosis Alleviates Neurological Deficit After Ischemic Stroke Shuxian Huang1, Tingting Chen1, Qian Suo1, Rubing Shi1, Haroon Khan1, Yuanyuan Ma1,2, Yaohui Tang1, Guo-Yuan Yang1,2 and Zhijun Zhang1* 1 Shanghai Jiao Tong University Affiliated Sixth People’s Hospital, School of Biomedical Engineering, Shanghai Jiao Tong University, Shanghai, China, 2 Department of Neurology, Ruijin Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China Microglial phagocytosis benefits neurological recovery after stroke. Large-conductance Ca2+-activated K+ currents are expressed in activated microglia, and BK channel knockout aggravates cerebral ischemic injury. However, the effect of BK channels on microglial phagocytosis after ischemic stroke remains unknown. Here, we explored whether BK channel activation is beneficial for neurological outcomes through microglial phagocytosis after ischemic stroke. ICR mice after transient middle cerebral artery occlusion (tMCAO) were treated with dimethyl sulfoxide (DMSO), BK channel activator NS19504, and inhibitor Paxilline. The results showed a decrease in BK channel expression after tMCAO. BK channel activator NS19504 alleviates neurological deficit after experimental modeling of tMCAO in mice compared to the control. Furthermore, we treated primary microglia with DMSO, NS19504, and Paxilline after oxygen glucose deprivation (OGD). NS19504 promoted primary microglial phagocytosing fluorescent beads and neuronal debris, which reduced neuronal apoptosis after stroke. These effects could be reversed by BK channel inhibitor Paxilline. Finally, NS19504 increased relative phosphorylated extracellular signal-regulated kinase 1/2 expression compared to the Paxilline group at the third day after stroke. Our findings indicate that microglial BK channels are a potential target for acute stage of ischemic stroke therapy. Animal Experiments All animal experiments were conducted in accordance with the guidelines of the Institutional Animal Care and Committee (IACUC) of Shanghai Jiao Tong University, Shanghai, China. Adult male ICR mice, weighting 28–32 g, were kept in a 12- h light/dark cycle with free access to standard food and water. Mice were randomly divided into four groups: (1) sham group; (2) saline plus dimethyl sulfoxide (DMSO) group; (3) Paxilline group; and (4) NS19504 group. Drugs were administered via intraperitoneal injection once a day for three consecutive days after tMCAO. The modified neurological severity score (mNSS) was assessed at 1, 3, 7, and 14 days after tMCAO to evaluate neurological function. Hinging wire tests were assessed at 3, 7, and 14 days to evaluate motor function. At day 3 after tMCAO, three mice were sacrificed for RNA and Western blot analysis while the remaining mice were used for immunostaining examination. y g Ischemic stroke leads to shortage of oxygen and brain nutrients, triggering cell death at the ischemic core (Adav and Sze, 2020; Xu et al., 2020). Timely clearance of apoptotic cells and cellular debris ameliorates ischemic injury and promotes neuroregeneration and tissue repair leading to improved stroke outcomes (Taylor and Sansing, 2013; Zhang et al., 2019). During the acute stage of stroke, microglia engulf granulocytes within 24 h after damage to alleviate apoptosis of neurons (Neumann et al., 2008). Microglial phagocytosis mediated by TREM2 improved neurological outcomes following experimental stroke (Kurisu et al., 2019), indicating the beneficial role of microglial phagocytosis for neuronal survival after ischemic brain injury. However, knockout of phagocytotic protein mer receptor tyrosine kinase (MERTK) and milk fat globule- epidermal growth factor 8 (MFG-E8) depressed microglial phagocytosis, exhibiting a significant reduction in mouse brain atrophy and improvements in neurological behavior (Neher et al., 2013). These studies suggest that microglial phagocytosis plays controversial roles at different stages of ischemic stroke. Citation: Huang S, Chen T, Suo Q, Shi R, Khan H, Ma Y, Tang Y, Yang G-Y and Zhang Z (2021) BK Channel-Mediated Microglial Phagocytosis Alleviates Neurological Deficit After Ischemic Stroke. Huang S, Chen T, Suo Q, Shi R, Khan H, Ma Y, Tang Y, Yang G-Y and Zhang Z (2021) BK Channel-Mediated Microglial Phagocytosis Alleviates Neurological Deficit After Ischemic Stroke. Microglial phagocytosis plays an important role in maintaining CNS homeostasis. During mammalian brain development, microglial pruning overproduced synapses and myelin along Front. Cell. Neurosci. 15:683769. doi: 10.3389/fncel.2021.683769 Front. Cell. Neurosci. 15:683769. doi: 10.3389/fncel.2021.683769 July 2021 | Volume 15 | Article 683769 1 Frontiers in Cellular Neuroscience | www.frontiersin.org Huang et al. BK Channels in Ischemic Stroke with neurons (Cobley, 2018). Microglial phagocytosis is required to eliminate myelin debris and protein aggregates in the aging process (Pluvinage et al., 2019; Zhu et al., 2019). However, under pathological conditions, microglial phagocytosis has shown a biphasic role. Microglial abnormal synapse pruning may lead to developmental disorders such as autism (Paolicelli et al., 2011). Knockout of TREM2 in an Alzheimer’s disease (AD) mouse model inhibited microglial phagocytotic effects on removing Aβ plaques and cell debris as well as induced extracellular lipid accumulation and secondary inflammation (Nugent et al., 2020). Depletion of TDP-43 enhanced microglial phagocytosis of amyloid protein in AD mice, while inducing excessive phagocytosis of synapses, consequently aggravating cognitive disorders (Paolicelli et al., 2017). Hence, the effect of microglial phagocytosis on disease progression and retrogression is worth studying. with neurons (Cobley, 2018). Microglial phagocytosis is required to eliminate myelin debris and protein aggregates in the aging process (Pluvinage et al., 2019; Zhu et al., 2019). However, under pathological conditions, microglial phagocytosis has shown a biphasic role. Microglial abnormal synapse pruning may lead to developmental disorders such as autism (Paolicelli et al., 2011). Knockout of TREM2 in an Alzheimer’s disease (AD) mouse model inhibited microglial phagocytotic effects on removing Aβ plaques and cell debris as well as induced extracellular lipid accumulation and secondary inflammation (Nugent et al., 2020). Depletion of TDP-43 enhanced microglial phagocytosis of amyloid protein in AD mice, while inducing excessive phagocytosis of synapses, consequently aggravating cognitive disorders (Paolicelli et al., 2017). Hence, the effect of microglial phagocytosis on disease progression and retrogression is worth studying. Transient Middle Cerebral Artery Occlusion Model The transient middle cerebral artery occlusion (tMCAO) model was performed as previously described (Tang et al., 2014). Briefly, after mice were anesthetized with isoflurane (1.5–2%), a midline neck incision was made, and the right common carotid artery (CCA), internal carotid artery (ICA), and external carotid artery (ECA) were isolated carefully. Then, the CCA and ICA were temporarily ligated with 4–0 silk suture. The distal end of ECA was permanently ligated, and a small incision was made for suture insertion. A silicone-coated 6–0 nylon monofilament suture was carefully inserted from the ECA into the ICA to block the MCA. The insertion depth was 9.5 ± 0.5 mm. After 90 min of occlusion, the suture was gently withdrawn for reperfusion. The blood flow of MCA was measured prior to and after the surgery by laser Doppler flowmetry (Moor Instruments, Devon, United Kingdom). About 80% decline in blood flow indicated successful occlusion of the MCA. g BK channels are highly expressed in the central nervous system (Contet et al., 2016). It consists of channel-forming alpha subunits and four accessory beta subunits that can be activated via both intracellular calcium and magnesium levels and membrane potential (Wu et al., 2018, in Chinese). The protective effect of activating BK channels has been proven in many disease models (Soltysinska et al., 2014; Dai et al., 2017; Jacobsen et al., 2018). BK channel activator pretreatment limited intestinal ischemia and reperfusion injury via an oxidant- dependent mechanism, reduced tumor necrosis factor-α (TNF- α), and protected mucosal permeability (Dai et al., 2017). Activation of BK channels during acute spinal cord injury improved motor functional recovery (Jacobsen et al., 2018). BK channels were demonstrated as a molecular target of several drugs for ischemic stroke therapy including vitamin C (Li L. et al., 2019), chlorpromazine (Li et al., 2014), and Baifuzi (Chi et al., 2010). BK currents were exclusively recorded in activated microglia (Schilling and Eder, 2007). BK channels in microglia played an indispensable role in morphine-induced hyperalgesia (Hayashi et al., 2016); it could be a molecular target of S-ketamine on relieving neuropathic pain (Hayashi et al., 2011). However, the role of microglial BK channels in acute stage of stroke is obscure. Citation: It has been demonstrated that lipopolysaccharide (LPS)- induced macrophage phagocytosis of myelin was inhibited after BK channel blocker Paxilline treatment (Vanheel et al., 2012); microglia are macrophages located in the central nervous system. Thus, we hypothesized that activation of BK channels during the acute stage of ischemic stroke may promote microglial phagocytosis, which improves neurological outcomes after experimental tMCAO. Real-Time PCR The selective BK channel antagonist Paxilline (Zhou and Lingle, 2014) and activator NS19504 (Nausch et al., 2014) powder (Alomone Labs, Jerusalem, Israel) were, respectively, dissolved in DMSO to the final storage concentrations of 2 and 50 mM. Mice received a single dose of 14 µg/kg Paxilline or 14 mg/kg NS19504 via intraperitoneal injection. The injection concentrations of Paxilline and NS19504 were 500 µM and 5 mM, respectively, having the same concentration of DMSO [10% (Li and Zhao, 2007)]. One µM Paxilline and 10 µM NS19504 were added into primary microglial culture in vitro. Mice were sacrificed and then transcardially perfused with PBS. Total RNA was isolated from the striatum of the infarcted area using TRIzol reagent (Invitrogen, Carlsbad, CA, United States). After reverse transcription, quantitative real-time PCR was performed using primers specific for the genes encoding BK channels and inflammatory mediators. All procedures were performed following the manufacturer’s protocol (Yisheng, Shanghai, China). Neurological and Motor Function Assessment The 14-score mNSS was used to evaluate mouse neurological function, including motor test (six scores), sensory tests (two scores), and bean balance tests (six scores). Higher scores indicate severe neurological deficits. The format of mNSS was described in a previous study (Jiang et al., 2017). July 2021 | Volume 15 | Article 683769 Frontiers in Cellular Neuroscience | www.frontiersin.org Frontiers in Cellular Neuroscience | www.frontiersin.org 2 BK Channels in Ischemic Stroke Huang et al. dilution, CST, Massachusetts, United States), total-ERK (1:1000 dilution, CST), and β-actin (1:1000 dilution, Invitrogen, Carlsbad, CA, United States) for more than 16 h at 4◦C. After washing three times with TBST buffer, membranes were incubated with a horseradish peroxidase (HRP)-conjugated secondary antibody for 1 h at room temperature. Protein bands were detected with a chemiluminescent HRP substrate (MeilunBio, Dalian, China). The results were analyzed by ImageJ software. The hinging wire test was used to investigate the motor function, especially forelimbs, of mice. Mice were placed at the middle of a horizontal wire about 20 cm above the table, and only their forelimbs were allowed to grab the wire at the beginning. The initial score was kept as 10 points; one point was given when mice arrived at either end, while one point was deducted when mice fall off. We recorded the time and score every time mice fall down or arrive at the end during 180 s. We ended up with a graph of scores per second. Frozen Section of Mouse Brain Gene name Forward primer (5′–3′) Reverse primer (5′–3′) BK GCGGCTTGAAGATGAGCAG TGCCAGGAATTAACAAGGGG IL-1α TCGGCAAAGAAATCAAGATG ATGGTCAATGGCAGAACTGTA IL-1β TACATCAGCACCTCACAAGC AGAAACAGTCCAGCCCATAC IL-6 TGATGCACTTGCAGAAAACAA GGTCTTGGTCCTTAGCCACT IL-10 GCGCTGTCATCGATTTCTCCC TGGCCTTGTAGACACCTTGG TNF-α ACCCTCACACTCAGATCATCTT GGTTGTCTTTGAGATCCATGC TGF-β CACCGGAGAGCCCTGGATA TGTACAGCTGCCGCACACA GAPDH AAATGGTGAAGGTCGGTGTG AGGTCAATGAAGGGGTCGTT Gene name Forward primer (5′–3′) BK GCGGCTTGAAGATGAGCAG IL-1α TCGGCAAAGAAATCAAGATG IL-1β TACATCAGCACCTCACAAGC IL-6 TGATGCACTTGCAGAAAACAA IL-10 GCGCTGTCATCGATTTCTCCC TNF-α ACCCTCACACTCAGATCATCTT TGF-β CACCGGAGAGCCCTGGATA GAPDH AAATGGTGAAGGTCGGTGTG Reverse primer (5′–3′) Mice were sacrificed and then transcardially perfused with phosphate-buffered saline (PBS), followed by 4% paraformaldehyde (PFA). Then, the brain was carefully removed from the endocast and rapidly put in isopentane (chilling at −80◦C) for 1 min. Finally, the brain was taken out and wrapped in tin foil, stored at −80◦C. Before slicing, frozen brain, metal pallet, and blades were placed in the freezing microtome (Leica CM1860, Leica, Nussloch, Germany) at −20◦C for 20 min, then the brain was fixed in an optimal cutting temperature compound (OCT, Leica, Nussloch, Germany) on the metal pallet. After OCT solidification, the brain was sliced into 20 µm and the slices were stored at −80◦C. TGCCAGGAATTAACAAGGGGT ATGGTCAATGGCAGAACTGTAG AGAAACAGTCCAGCCCATACT GGTCTTGGTCCTTAGCCACTC TGGCCTTGTAGACACCTTGG GGTTGTCTTTGAGATCCATGC TGTACAGCTGCCGCACACA AGGTCAATGAAGGGGTCGTT Western Blotting Analysis g Brain sections were fixed with 100% methanol (chilled at −20◦C) for 2 min and incubated with 0.3% Triton X-100 solution for 10 min, then subjected to sodium citrate buffer microwave antigen retrieval and blocked with 1% bovine serum albumin (BSA) for 1 h. Then, sections were incubated with primary antibodies anti-BK α-subunit (1:50, Santa Cruz, TX, United States), GFAP (1:200, EMD Millipore Corp., Billerica MA, United States), IBA1 (1:200, Novusbio, Littleton, CO, United States), CD11b (1:200, Bio-Rad, Hercules, CA, United States), NeuN (1:200, EMD Millipore Corp., Billerica, MA, United States), and MAP2 (1:200, EMD Millipore Corp., Billerica, MA, United States) overnight at 4◦C. After washing three times with PBS, sections were incubated with fluorescent conjugated secondary antibodies for 1 h at 37◦C. Sections were washed again three times with PBS and air dried in a dark room, then covered by an anti-fluorescence attenuation sealant (with DAPI, Meilun, Dalian, China) and coverslip. The fluorescent images were taken by confocal microscope (Leica, Solms, Germany). g y Mice were sacrificed and then transcardially perfused with PBS. The striatum of stroke and the contralateral hemispheres were lysed in extraction buffer, which comprised the following components: 10× radioimmunoprecipitation lysis buffer (RIPA, EMD Millipore Corp., Billerica, MA, United States); 10× phosphatase inhibitor PhoSTOP (Merck KGaA, Darmstadt, Germany); 100× protease inhibitor cocktail (Beyotime, Shanghai, China); 100× phenylmethanesulfonyl fluoride (PMSF, Thermo Scientific, Waltham, United Kingdom); and ddH2O = 10:10:1:1:78. After ultrasonic homogenization, samples were centrifuged at 12,000 rpm (4◦C) for 20 min, and a liquid supernatant was taken for subsequent steps. The concentration of each protein sample was determined using the BCA kit (Thermo Scientific, Waltham, United Kingdom), and each amount of protein (30 µg per line) was loaded for SDS-PAGE. Then, proteins were transferred to polyvinylidene fluoride membrane (Merck KGaA, Darmstadt, Germany). Membranes were blocked with 5% non-fat milk for 1 h and incubated with the primary antibodies of BK channels (1:1000 dilution, Abcam, Cambridge, United States), p-ERK (1:1000 Cell coverslips were carefully taken out and put in a 24-well plate, and washed by PBS, then treated the same as July 2021 | Volume 15 | Article 683769 Frontiers in Cellular Neuroscience | www.frontiersin.org 3 BK Channels in Ischemic Stroke Huang et al. the brain sections. Antigen retrieval was not needed for the cell slide staining. the brain sections. Antigen retrieval was not needed for the cell slide staining. twice to obtain neuronal debris. CCK8 Assay Microglia were seeded in a 96-well plate at a density of 2 × 105 cells/well with 100 µl conditional media, respectively, containing 0.1, 1, 2, 5, and 10 µM Paxilline and 1, 5, 10, and 100 µM NS19504. Five duplicate wells were set for each concentration. After a 12-h treatment, 10 µl CCK8 was added into each well. The plates were incubated for 1–4 h in the incubator. The absorbance at 450 nm was measured by a microplate reader. Oxygen-Glucose Deprivation (OGD) and Reoxygenation Model In order to determine which kind of cell types expressed BK channels after stroke, we sacrificed mice at 3 days after tMCAO and performed immunofluorescent staining. The results showed that BK channels were mainly expressed in microglia and neurons, not in astrocytes (Figure 2A). Similar results were observed in vitro (Figure 2B). At 3 days after tMCAO, we found that most BK channels were located in microglia, while few with neurons and astrocytes (Figure 2C), which suggested that BK channels on microglia play a major role after stroke. Microglia were seeded in 24-well plates at a density of 15 × 105– 20 × 105 cells/well with 500 µl conditional media. Cells were washed twice with sterile water and once with PBS, then cultured in glucose-free DMEM. Cultures were placed into a round anaerobic chamber filled with 95% N2 and 5% CO2 and kept at 37◦C for 1 h of oxygen-glucose deprivation (OGD). An oxygen meter was placed in the chamber to monitor the oxygen concentration. Then, the medium was replaced by DMEM with 10% FBS. Cells were maintained under normoxic conditions for 11 h at 37◦C for reoxygenation. Neurons were subjected to OGD for 8 h and reoxygenation for 12 h to get debris. Western Blotting Analysis Debris then were stained using the PKH26 kit (Merck, Kenilworth, United States). After OGD, reoxygenation, and 12 h treatment with normal microglia medium containing 1 µM Paxilline or 10 µM NS19504, microglia were incubated in normal medium with beads or debris for 2 h. Cultures were washed with PBS about five times and fixed with 4% PFA. All experiments were repeated at least three times independently, and each experimental group had three parallel wells. Three areas per well were photographed and statistically analyzed. BK Channel Expression Decreased After tMCAO To investigate the expression of BK channels after stroke in vivo, we examined the mRNA and protein levels of BK channels in the peri-infarct area of mice brain after tMCAO. The RNA level of BK channels decreased during 3–14 days after tMCAO (Figure 1A). The protein level of BK channels also reduced after tMCAO and reached the bottom at 7 dpi (Figures 1B,C). These results suggested that BK channels were downregulated in the brain after ischemic stroke. Cell Culture Primary microglia and neurons were isolated from cerebral cortices of Sprague Dawley rats (SD, JSJ, Shanghai, China) born within 24 h. Briefly, for primary microglia, isolated cells were plated into poly-D-lysine (PDL, Sigma-Aldrich, St. Louis, MO, United States)-coated 75-cm2 flasks at the density of 1 × 105– 1.4 × 105 cells/cm2. Cells were cultured in Dulbecco’s modified Eagle medium (DMEM, Gibco Laboratories, Grand Island, NY, United States) with 10% fetal bovine serum (FBS, Gibco Laboratories, Grand Island, NY, United States). Microglia were collected by shaking flasks in ∼10 days after seeding then plated into a 24-well plate and cultured for 24 h for phagocytosis assay and coculture. Statistical Analysis Results were presented as the mean ± SEM, and the differences of two groups were analyzed by Students’ t-test, three groups analyzed by one-way ANOVA followed by the Bonferroni corrections. A value of p < 0.05 was taken as statistically significant. These analyses were done using GraphPad 5.0 software. For neurons, isolated cells were plated into six-well plates coated with PDL at the density of 6 × 105–8 × 105 cells/well and cultured in DMEM with 10% FBS. After 4 h, the medium was changed to Neurobasal medium (Gibco, Carlsbad, CA, United States) with B27 (Gibco, Carlsbad, CA, United States). Neurons were ready to use in ∼5 days after seeding. All cells were kept in a humidified incubator containing 5% CO2/95% O2 at 37◦C. Phagocytosis Assay Data are mean ± SE, n = 3 per group. *p < 0.05, **p < 0.01, tMCAO vs. control. FIGURE 2 | BK channels are mainly expressed in microglia and neurons, but not in astrocytes. (A) Representative photomicrographs show BK (green), GFAP (red), MAP2 (red), and IBA1 (red) double staining in primary cultured cells. Scale bar = 25 µm. Right-side image indicates that BK is expressed in the IBA1+ microglia by 3D two-photo microscope image. Scale bar = 10 µm. (B) Representative photomicrographs show BK (green), GFAP (red), MAP2 (red), and IBA1 (red) double staining in the cortex of the normal mouse brain. Scale bar = 25 µm. Right-side image indicates that BK was expressed within the IBA1+ microglia, scale bar = 30 µm. (C) Representative photomicrographs of BK/GFAP, BK/NeuN, and BK/IBA1 double staining at 3 days after tMCAO. Scale bar = 50 µm. Right-side image indicated that BK was expressed in the IBA1+ microglia by 3D two-photo microscope image. Scale bar = 20 µm. FIGURE 2 | BK channels are mainly expressed in microglia and neurons, but not in astrocytes. (A) Representative photomicrographs show BK (green), GFAP (red), MAP2 (red), and IBA1 (red) double staining in primary cultured cells. Scale bar = 25 µm. Right-side image indicates that BK is expressed in the IBA1+ microglia by 3D two-photo microscope image. Scale bar = 10 µm. (B) Representative photomicrographs show BK (green), GFAP (red), MAP2 (red), and IBA1 (red) double staining in the cortex of the normal mouse brain. Scale bar = 25 µm. Right-side image indicates that BK was expressed within the IBA1+ microglia, scale bar = 30 µm. (C) Representative photomicrographs of BK/GFAP, BK/NeuN, and BK/IBA1 double staining at 3 days after tMCAO. Scale bar = 50 µm. Right-side image indicated that BK was expressed in the IBA1+ microglia by 3D two-photo microscope image. Scale bar = 20 µm. FIGURE 2 | BK channels are mainly expressed in microglia and neurons, but not in astrocytes. (A) Representative photomicrographs show BK (green), GFAP (red), MAP2 (red), and IBA1 (red) double staining in primary cultured cells. Scale bar = 25 µm. Right-side image indicates that BK is expressed in the IBA1+ microglia by 3D two-photo microscope image. Scale bar = 10 µm. (B) Representative photomicrographs show BK (green), GFAP (red), MAP2 (red), and IBA1 (red) double staining in the cortex of the normal mouse brain. Scale bar = 25 µm. Phagocytosis Assay Previous study has indicated that inhibiting BK channels could block LPS-induced phagocytosis of macrophages (Vanheel et al., 2012). To investigate the effect of BK channel activator NS19504 on microglial phagocytosis, we cultured primary microglia and performed OGD to mimic the condition of stroke in vivo. First, we examined the effects of NS19504 and Paxilline concentration gradient on cell viability to determine the optimum dosing concentration. The results showed that 1 µM Paxilline and 10 µM The phagocytosis activity of microglia was assessed by measuring the uptake of fluorescent beads or neuronal debris under a confocal microscope (Leica, Solms, Germany). Fluorescent latex beads (diameter 0.7 µm, CellMeter, United States) showed enhanced red fluorescence when phagocytosed by microglia. After 8 h of OGD and 12 h of reoxygenation, neuron media were centrifuged at 10,000 g for 5 min, resuspended in DMEM July 2021 | Volume 15 | Article 683769 Frontiers in Cellular Neuroscience | www.frontiersin.org BK Channels in Ischemic Stroke Huang et al. FIGURE 1 | BK expression in the ipsilateral hemisphere decreased after mouse tMCAO. (A) Bar graph shows the BK mRNA expression in the mouse ipsilateral hemisphere at 3, 7, and 14 days after 90-min tMCAO. Data are mean ± SE, n = 3–7 per group. **p < 0.05. tMCAO vs. control. (B) Representative image of Western blot shows BK expression in the mouse ipsilateral hemisphere at 3, 7, and 14 days after 90-min tMCAO. (C) Bar graph shows that the semiquantitative data of BK protein levels at 3, 7, and 14 days after tMCAO. Data are mean ± SE, n = 3 per group. *p < 0.05, **p < 0.01, tMCAO vs. control. FIGURE 1 | BK expression in the ipsilateral hemisphere decreased after mouse tMCAO. (A) Bar graph shows the BK mRNA expression in the mouse ipsilateral hemisphere at 3, 7, and 14 days after 90-min tMCAO. Data are mean ± SE, n = 3–7 per group. **p < 0.05. tMCAO vs. control. (B) Representative image of Western blot shows BK expression in the mouse ipsilateral hemisphere at 3, 7, and 14 days after 90-min tMCAO. (C) Bar graph shows that the semiquantitative data of BK protein levels at 3, 7, and 14 days after tMCAO. Data are mean ± SE, n = 3 per group. *p < 0.05, **p < 0.01, tMCAO vs. control. protein levels at 3, 7, and 14 days after tMCAO. Phagocytosis Assay Primary microglia were divided into the control group (basic microglia medium); LPS group (200 ng/ml LPS); LPS + NS19504 group (200 ng/ml LPS and 10 µM NS19504); and LPS + Paxilline group (200 ng/ml LPS and 1 µM Paxilline). (F) Bar graph shows semiquantitative analysis of the proportion of microglia that phagocytose beads. Data are mean + SE, n = 3 per group. **p < 0.01, compared with the control group. #p < 0.05, compared with the Paxilline group. (G) Bar graph shows the result of CCK-8 assay in the primary microglia without or with different concentrations of Paxilline and NS19504 for 12 h of OGD. Data are mean ± SE, n = 3 per group. #p < 0.05, ##p < 0.01, compared with the control group. *p < 0.05, **p < 0.01; NS represents for NS19504, and Pax represents for Paxilline. FIGURE 3 | BK channel activator NS19504 promoted microglial phagocytosis after OGD in primary culture. (A) Representative images of beads (red) phagocytosed by CD11b+ microglia (green) after 1 h of OGD. Control group: primary microglia cultured with basic microglia medium; NS19504 group: primary microglia cultured with 10 µM NS19504 in basic microglia medium; Paxilline group: primary microglia cultured with 1 µM Paxilline in basic microglia medium. Scale bar = 25 µm. (B) Bar graph showed that semiquantitative data of the relative bead density versus microglia. Data are mean + SE, n = 3 per group. *p < 0.05, control vs. NS19504 or Paxilline vs. NS19504 group. (C) Representative photomicrographs showed that neuronal debris (red) phagocytosed by microglia (green) in the NS19504- and Paxilline-treated mice following tMCAO. Scale bar = 25 µm. (D) Bar graph shows semiquantitative data of the proportion of microglia that phagocytose neuronal debris. Data are mean + SE, n = 3 per group. *p < 0.05, NS19504 vs. control or Paxilline vs. NS19504 group. (E) Representative photomicrographs show FITC and PE-positive cells. FITC-labeled beads were phagocytosed by PE-labeled microglia. Primary microglia were divided into the control group (basic microglia medium); LPS group (200 ng/ml LPS); LPS + NS19504 group (200 ng/ml LPS and 10 µM NS19504); and LPS + Paxilline group (200 ng/ml LPS and 1 µM Paxilline). (F) Bar graph shows semiquantitative analysis of the proportion of microglia that phagocytose beads. Data are mean + SE, n = 3 per group. **p < 0.01, compared with the control group. Phagocytosis Assay Right-side image indicates that BK was expressed within the IBA1+ microglia, scale bar = 30 µm. (C) Representative photomicrographs of BK/GFAP, BK/NeuN, and BK/IBA1 double staining at 3 days after tMCAO. Scale bar = 50 µm. Right-side image indicated that BK was expressed in the IBA1+ microglia by 3D two-photo microscope image. Scale bar = 20 µm. NS19504 were not cytotoxic within effective concentrations (Figure 3G). After OGD and reoxygenation, microglia were treated with NS19504 or Paxilline for 12 h. Then, microglia were incubated with fluorescent beads, which are PH sensitive, so the fluorescence would be enhanced after being phagocytosed. We measured the gray value of fluorescent beads versus CD11b+ microglia. The NS19504 group showed increased the relative beads/microglia gray value compared to the control group (Figures 3A,B). Paxilline treatment significantly reduced microglial phagocytosis after OGD compared with the NS19504 treatment. In order to mimic the condition in vivo, we substituted neuronal debris for fluorescent beads. After the same treatment, microglia of three groups were incubated with neuronal debris which were stained with PKH26. The results showed that more July 2021 | Volume 15 | Article 683769 Frontiers in Cellular Neuroscience | www.frontiersin.org 5 Huang et al. BK Channels in Ischemic Stroke FIGURE 3 | BK channel activator NS19504 promoted microglial phagocytosis after OGD in primary culture. (A) Representative images of beads (red) phagocytosed by CD11b+ microglia (green) after 1 h of OGD. Control group: primary microglia cultured with basic microglia medium; NS19504 group: primary microglia cultured with 10 µM NS19504 in basic microglia medium; Paxilline group: primary microglia cultured with 1 µM Paxilline in basic microglia medium. Scale bar = 25 µm. (B) Bar graph showed that semiquantitative data of the relative bead density versus microglia. Data are mean + SE, n = 3 per group. *p < 0.05, control vs. NS19504 or Paxilline vs. NS19504 group. (C) Representative photomicrographs showed that neuronal debris (red) phagocytosed by microglia (green) in the NS19504- and Paxilline-treated mice following tMCAO. Scale bar = 25 µm. (D) Bar graph shows semiquantitative data of the proportion of microglia that phagocytose neuronal debris. Data are mean + SE, n = 3 per group. *p < 0.05, NS19504 vs. control or Paxilline vs. NS19504 group. (E) Representative photomicrographs show FITC and PE-positive cells. FITC-labeled beads were phagocytosed by PE-labeled microglia. Activation of BK Channels Reduced Neuronal Apoptosis After tMCAO We used mNSS to evaluate the neurological deficit at 1, 3, 7, and 14 days after tMCAO. Hinging wire tests were conducted at 3 days to test motor function. The results showed that the neurological score was lower at 3 dpi in the NS19504 group than in the DMSO and Paxilline groups (Figure 5C). Results of the hinging wire test demonstrated that the score of the NS19504 group was higher compared to that of the DMSO and Paxilline groups (Figure 5D). The Paxilline group gained the lowest end score at 180 s among Next, we examined the effect of microglial phagocytosis on apoptotic cells after BK channel activation in ischemic mice. We quantified the number of DAPI+/Iba1+ microglial phagocytosis TUNEL-positive cells, which represented that microglia phagocytes apoptotic cells. The number of phagocytic microglia increased in the NS19504 group compared to DMSO and Paxilline groups, which indicated that microglia FIGURE 4 | Activation of BK channels promoted the phagocytic function of microglia after tMCAO and decreased neuronal apoptosis. (A) Representative photomicrograms of IBA1+ microglia (green) and TUNEL+ apoptotic cells (red) in the peri-infarct region of brain slice in the DMSO, NS19504, and Paxilline groups at 3 days after tMCAO. Scale bar = 50 µm. (B) Bar graph shows quantitative analysis of the number of IBA1+ TUNEL+ cells per field. Data are mean + SE, n = 3 per group. *vs. DMSO group, **p < 0.01. (C) Representative photomicrograms of NeuN+ neurons (green) and TUNEL+ apoptotic cells (red) in the peri-infarct region of brain slice in the DMSO, NS19504, and Paxilline groups at 3 days after tMCAO. Scale bar = 50 µm. (D) Bar graph shows quantitative analysis of the number of NeuN+/TUNEL+ cells per vision. Data are mean + SE, n = 3 per group. *vs. DMSO group, **p < 0.01. FIGURE 4 | Activation of BK channels promoted the phagocytic function of microglia after tMCAO and decreased neuronal apoptosis. (A) Representative photomicrograms of IBA1+ microglia (green) and TUNEL+ apoptotic cells (red) in the peri-infarct region of brain slice in the DMSO, NS19504, and Paxilline groups at 3 days after tMCAO. Scale bar = 50 µm. (B) Bar graph shows quantitative analysis of the number of IBA1+ TUNEL+ cells per field. Data are mean + SE, n = 3 per group. *vs. DMSO group, **p < 0.01. use Outcomes After Ischemic Stroke Since microglial M2 polarization could promote clearance of apoptotic cells and improve tissue repair after ischemic stroke (Yang et al., 2017; Wen et al., 2020), we explored whether stimulation of BK channels could change the polarization of microglia. APC-CD206 was used to label M2 microglia; flow cytometry was used to determine the number of M2 microglia of different groups. Figure 3F shows that the four groups of APC+ cell number had no significant difference, demonstrating that regulation of BK channels had no effect on the polarization of M2 microglia. Phagocytosis Assay #p < 0.05, compared with the Paxilline group. (G) Bar graph shows the result of CCK-8 assay in the primary microglia without or with different concentrations of Paxilline and NS19504 for 12 h of OGD. Data are mean ± SE, n = 3 per group. #p < 0.05, ##p < 0.01, compared with the control group. *p < 0.05, **p < 0.01; NS represents for NS19504, and Pax represents for Paxilline. July 2021 | Volume 15 | Article 683769 Frontiers in Cellular Neuroscience | www.frontiersin.org Frontiers in Cellular Neuroscience | www.frontiersin.org 6 BK Channels in Ischemic Stroke Huang et al. of the NS19504 group displayed higher phagocytic activity (Figures 4A,B). cells in the NS19504 group engulfed neuronal debris, while microglia of the Paxilline group showed a similar level of phagocytosis with the control group (Figures 3C,D). Then, we performed immunostaining to measure the number of apoptotic neurons. Results showed that the number of NeuN+/TUNEL+ neurons decreased in the NS19504 group compared to the DMSO and Paxilline groups (Figures 4C,D). To further examine microglial phagocytosis after Paxilline and NS19504 treatment, we also used flow cytometry. We detected PE-labeled CX3CR1+ microglia engulfing FITC-labeled beads. After LPS stimulation, microglia were treated with Paxilline or NS19504 or normal microglial medium. The results showed that the LPS group had a higher percentage of PE+FITC+ cells compared to other groups. Paxilline significantly inhibited LPS- induced microglial phagocytosis (Figures 3E,F). BK Channels Activation Promoted Mouse Outcomes After Ischemic Stroke BK channel-knocked out mice show a larger infarct area after ischemic stroke (Liao et al., 2010). To confirm the effect of BK channel activation on stroke outcomes, we treated mice with a BK channel-specific activator (NS19504) or inhibitor (Paxilline) for 3 days after tMCAO. Figure 5A shows the timeline of animal experiments. First, we recorded weight changes of three groups. As shown in Figure 5B, mice lost the most weight at 3 days and there was no significant difference among groups during 14 days after tMCAO. Then, we investigated the number of surviving mice and found that about more than 80% of mice of the Paxilline group failed to live at 14 dpi (Figure 5E), which suggests that inhibition of BK channels at the acute phase affected the survival rate after stroke. Activation of BK Channels Reduced Neuronal Apoptosis After tMCAO (C) Representative photomicrograms of NeuN+ neurons (green) and TUNEL+ apoptotic cells (red) in the peri-infarct region of brain slice in the DMSO, NS19504, and Paxilline groups at 3 days after tMCAO. Scale bar = 50 µm. (D) Bar graph shows quantitative analysis of the number of NeuN+/TUNEL+ cells per vision. Data are mean + SE, n = 3 per group. *vs. DMSO group, **p < 0.01. July 2021 | Volume 15 | Article 683769 7 Frontiers in Cellular Neuroscience | www.frontiersin.org BK Channels in Ischemic Stroke Huang et al. FIGURE 5 | Activation of BK channels ameliorated stroke outcomes. (A) Experiment design of the animal experiment. Three days before tMCAO, mice were subjected behavior training. After a 90-min tMCAO surgery, mice were divided randomly into four groups (sham, DMSO, NS19504, and Paxilline). At 1, 3, 7, and 14 days, behavior tests were performed, and protein and RNA samples were collected. (B) Weight changes of mice in three groups at 1, 2, 3, 7, and 14 days after tMCAO. (C) Modified Neurological Severity Score of three groups at 1, 3, 7, and 14 days after tMCAO. (D) Average score per second of three groups of hinging wire at 3 days after tMCAO. (E) Survivorship curve during 14 days of three groups after tMCAO. Data are mean + SE, n = 21–23 per group at 1 day, n = 17–23 per group at 3 days, n = 8–13 per group at 7 days, n = 3–9 per group at 14 days. *NS19504 vs. Paxilline group, **p < 0.01. #NS19504 vs. DMSO, #p < 0.05. FIGURE 5 | Activation of BK channels ameliorated stroke outcomes. (A) Experiment design of the animal experiment. Three days before tMCAO, mice were subjected behavior training. After a 90-min tMCAO surgery, mice were divided randomly into four groups (sham, DMSO, NS19504, and Paxilline). At 1, 3, 7, and 14 days, behavior tests were performed, and protein and RNA samples were collected. (B) Weight changes of mice in three groups at 1, 2, 3, 7, and 14 days after tMCAO. (C) Modified Neurological Severity Score of three groups at 1, 3, 7, and 14 days after tMCAO. (D) Average score per second of three groups of hinging wire at 3 days after tMCAO. (E) Survivorship curve during 14 days of three groups after tMCAO. DISCUSSION three groups, which suggests that inhibition of BK channels aggravates motor function deficit. These results demonstrate that activating BK channels could promote mouse motor functions after ischemic stroke. In this study, we explored the role of BK channels in microglial phagocytosis after tMCAO and demonstrated that activation of BK channels during the first 3 days after tMCAO promoted microglial phagocytosis and alleviated neurological deficit after ischemic stroke; ERK was involved in the activation of BK channel-mediated microglial phagocytosis. Activation of BK Channels Reduced Neuronal Apoptosis After tMCAO Data are mean + SE, n = 21–23 per group at 1 day, n = 17–23 per group at 3 days, n = 8–13 per group at 7 days, n = 3–9 per group at 14 days. *NS19504 vs. Paxilline group, **p < 0.01. #NS19504 vs. DMSO, #p < 0.05. Activation of BK Channels Promoted ERK Expression After tMCAO BK Channels in Ischemic Stroke FIGURE 6 | Inflammatory factors and phosphorylated-ERK expressions under different treatments at 3 days after tMCAO. (A) Bar graph represents the relative mRNA levels of inflammatory factors including TNF-α, TGF-β, IL-10, IL-6, IL-1β, and IL-1α. Data are mean + SE, n = 3 per group. (B) Representative image of Western blot of phosphorylated-ERK1/2 (p-ERK), total-ERK1/2 (t-ERK), and β-actin at 3 days after tMCAO. (C) Bar graph showing the relative protein levels of p-ERK vs. t-ERK at 3 day after tMCAO. Data are mean + SE, n = 9 per group. *NS19504 vs. Paxilline group, *p < 0.05; ns, no significance. FIGURE 6 | Inflammatory factors and phosphorylated-ERK expressions under different treatments at 3 days after tMCAO. (A) Bar graph represents the relative mRNA levels of inflammatory factors including TNF-α, TGF-β, IL-10, IL-6, IL-1β, and IL-1α. Data are mean + SE, n = 3 per group. (B) Representative image of Western blot of phosphorylated-ERK1/2 (p-ERK), total-ERK1/2 (t-ERK), and β-actin at 3 days after tMCAO. (C) Bar graph showing the relative protein levels of p-ERK vs. t-ERK at 3 day after tMCAO. Data are mean + SE, n = 9 per group. *NS19504 vs. Paxilline group, *p < 0.05; ns, no significance. Paxilline is a classic BK channel inhibitor, which is the most effective and selective non-endogenous inhibitor of BK channels (Yu et al., 2016). Besides, NS19504 and Paxilline can both cross the blood–brain barrier. In this study, we found that NS19504 has a protective effect against ischemic stroke while activating the BK channel. This effect is accomplished by promoting the phagocytosis of microglia and can be reversed by Paxilline. of BK channels promoted microglial phagocytosis in vitro and in vivo and alleviated neuronal loss at 3 day after stroke, which was accompanied by better neurobehavioral score and less co- location of TUNEL and NeuN. The phenotype of microglia changes dynamically during several disease progression, therefore exhibiting different effects on injury (Ma et al., 2017). In the acute stage of ischemic stroke, microglia tend to polarize into M2 (Zhao et al., 2017), which participated in clearance of dead cells and release of anti- inflammatory factors (Wang et al., 2018), meanwhile reducing the level of pro-inflammatory factors (Lambertsen et al., 2019). Activation of BK Channels Promoted ERK Expression After tMCAO Inflammation response is a general feature in nervous system diseases and is the initial defense mechanism against injury (Chung et al., 2019; Li K. et al., 2019; Sarkar et al., 2019). Since NS19504 treatment promoted the outcome of stroke, we detected whether inflammation affects the significant difference between groups, and the mRNA levels of inflammation factors TNF-α, IL-1β, IL-6, IL-10, and TGF-β were examined. The results of RT-PCR showed that there was no difference between three groups, but the Paxilline group had the trend to severer inflammation (Figure 6A). Previous studies suggest that BK channels have neurovascular protection in cerebrovascular diseases. Knockout of the BK channel shows more severe behavioral defects and higher mortality (Liao et al., 2010). However, the expression pattern and the mechanism of neuroprotection after ischemic stroke of BK channels are still unclear. In this study, our results showed that the BK channel expression of the mouse ipsilateral hemisphere was continuously lower than that of the sham group from 1 to 14 days after ischemic stroke; BK channels were mainly expressed on microglia and neurons, but almost not expressed on astrocytes in the sham group. At 3 days after ischemic stroke, BK channels are mainly located in microglia but few in neurons. Therefore, the BK channel on microglia may play a key role in post-stroke injury. ERK has been reported to play an important role in TREM2- mediated microglial phagocytosis (Fu et al., 2014) and participate in the regulation of WNK1 to BK channels (Liu et al., 2015). We investigated that ERK1/2 function in BK channels mediated microglial phagocytosis. Western blot was used to detect phosphorylated ERK expression versus total ERK expression at 3 days after tMCAO. The results showed that activation of BK channels upregulated the pERK1/2/tERK1/2 level compared with Paxilline, which suggested that ERK was involved in BK channel- mediated microglial phagocytosis in the early stage of ischemic stroke (Figures 6B,C). Then we used drugs that can efficiently and specifically regulate BK channels, including activators and inhibitors. NS19504 is a novel BK channel activator having a higher specificity than the commonly used BK channel activator NS1619, NS19504, which does not activate the small- conductance potassium channel (IK channels) while activating the BK channels (Nausch et al., 2014; Catacuzzeno et al., 2015). July 2021 | Volume 15 | Article 683769 Frontiers in Cellular Neuroscience | www.frontiersin.org 8 Huang et al. SUPPLEMENTARY MATERIAL The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fncel. 2021.683769/full#supplementary-material ETHICS STATEMENT The animal study was reviewed and approved by the Institutional Animal Care and Committee (IACUC) of Shanghai Jiao Tong University, Shanghai, China. FUNDING This study was supported by grants from the National Natural Science Foundation of China projects 81974179 (ZZ), 81901185 (YM), 81771244 (ZZ), 81771251 (G-YY), 81801170 (YT), 82071284 (YT), and 81870921 (YW); the National Key R&D Program of China 2019YFA0112000 (YT); the Scientific Research and Innovation Program of Shanghai Education Commission 2019-01-07-00-02-E00064 (G-YY); the Scientific and Technological Innovation Act Program of Shanghai Science and Technology Commission, 20JC1411900 (G-YY); the Science and Technology Opening Program of the Education Ministry of Henan Province (182106000061, G-YY); and K. C. Wong Education Foundation (G-YY). This study was supported by grants from the National Natural Science Foundation of China projects 81974179 (ZZ), 81901185 (YM), 81771244 (ZZ), 81771251 (G-YY), 81801170 (YT), 82071284 (YT), and 81870921 (YW); the National Key R&D Program of China 2019YFA0112000 (YT); the Scientific Research and Innovation Program of Shanghai Education Commission 2019-01-07-00-02-E00064 (G-YY); the Scientific and Technological Innovation Act Program of Shanghai Science and Technology Commission, 20JC1411900 (G-YY); the Science and Technology Opening Program of the Education Ministry of Henan Province (182106000061, G-YY); and K. C. Wong Education Foundation (G-YY). In summary, our results demonstrate that BK channels are mainly expressed in microglia and neurons. Activation of BK channels promotes microglial phagocytosis both in vivo and in vitro, which reduces neuronal loss and alleviates neurobehavior deficit; ERK1/2 was involved in this process. BK channel- mediated microglial phagocytosis may be a potential target for stroke therapy. Activation of BK Channels Promoted ERK Expression After tMCAO Our results showed that the regulation of BK channels had no impact on microglial polarization, which explained why there was no significant difference of the inflammatory factor levels among DMSO, NS19504, and Paxilline groups (Figure 6A). Clearance of apoptotic cell debris is vital to the improvement of stroke outcomes (Yew et al., 2019). Several studies suggest that inhibiting microglial activation results in improved neurogenesis (Hoehn et al., 2005; Liu et al., 2007). Four-week minocycline treatment at 4 days after tMCAO decreased the number of activated microglia and increased the number of BrdU+/NeuN+ cells (Liu et al., 2007). Overexpressing miR-98 inhibited microglial phagocytosis to attenuate neuronal death in the penumbra area at 3 days after tMCAO (Yang et al., 2021). However, microglial phagocytotic receptor TREM2 knockout mice showed less tissue resorption and a larger lesion area, along with severe limb bias, which suggested that microglial phagocytosis improved synapse and axon regeneration after injury (Werneburg et al., 2020). Neuronal and synapse regeneration contributes to brain neural network remodeling, which improved cognitive and motor function after injury (Wu et al., 2020). Our evidence supported the notion that activation Extracellular signal-regulated kinase 1/2 (ERK1/2) is a key member of the mitogen-activated protein kinase (MAPK) family, which can be stimulated by cellular growth factors. Activated ERK1 and ERK2 are transferred into the nucleus and activate downstream transcription factors and modulate cell proliferation and differentiation (Zhang and Liu, 2002). In the nervous system, ERK1/2 participates in the neuroprotection effect of several components. Plasmalogens (PIs) alleviated neuronal apoptosis after serum deprivation via activating the MAPK/ERK July 2021 | Volume 15 | Article 683769 Frontiers in Cellular Neuroscience | www.frontiersin.org Frontiers in Cellular Neuroscience | www.frontiersin.org 9 Huang et al. BK Channels in Ischemic Stroke DATA AVAILABILITY STATEMENT pathway (Sun et al., 2015). The brain-derived neurotrophic factor (BDNF) stimulated the differentiation and survival of human umbilical cord blood mesenchymal stem cells (HUCB- MSCS) into neurons (Lim et al., 2008) through the activation of MAPK/ERK. Inhibition of ERK promoted HMGB1-mediated neuronal death in vitro (Kim et al., 2011). ERK1/2 was involved in promoting neuronal survival and differentiation (Tejeda and Diaz-Guerra, 2017; Xie et al., 2020). After ischemic stroke, cell debris of injury induce microglial phagocytosis via the TREM-2/DAP12/ERK/PKC pathway (Fu et al., 2014). TREM2 participates in microglial phagocytosis of apoptotic neurons (Wu et al., 2017) and displays anti-inflammation effects after cerebral ischemic and reperfusion injury (Takahashi et al., 2005). Stimulation of TREM2 on microglia increased ERK/MAPK phosphorylation and promoted microglial phagocytosis, which was reversed after ERK inhibitor treatment (Takahashi et al., 2005). Our results supported that significantly upregulated p-ERK1/2 protein levels after BK activation were accompanied by the increase of microglial phagocytosis, which suggests that ERK1/2 is involved in the neuroprotection of BK channel- mediated microglial phagocytosis. The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation. AUTHOR CONTRIBUTIONS ZZ was responsible for the generation of the project idea. SH and ZZ were responsible for the experiment design. SH was responsible for the most of the experiment operation, analysis of the results, and generation of the manuscript. TC and QS were responsible for animal behavior test, western blot, primary microglia culture. RS was responsible for the mNSS test. YM and HK were responsible for the polish of the manuscript and experiment operation. G-YY, ZZ, and YT supervised the project. All authors contributed to the article and approved the submitted version. Our results found that mice of the NS19504 group showed a significantly lower score of the mNSS test, higher average score of the hinging wire test, and decreased number of TUNEL+/NeuN+ cells in the NS19504 group at 3 days after tMCAO. During 1–14 days after tMCAO, the NS19504 group had decreased mortality compared with the Paxilline group. We speculated that there may be two reasons for the significant neurological improvement only on 3 days after tMCAO. 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Ventriculoperitoneal shunt insertion in human immunodeficiency virus infected adults: a systematic review and meta-analysis

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Loan et al. BMC Neurology (2020) 20:141 https://doi.org/10.1186/s12883-020-01713-4 Loan et al. BMC Neurology (2020) 20:141 https://doi.org/10.1186/s12883-020-01713-4 Open Access © The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Ventriculoperitoneal shunt insertion in human immunodeficiency virus infected adults: a systematic review and meta- analysis James J. M. Loan1,2,3*, Michael T. C. Poon1,2, Steven Tominey2, Ncedile Mankahla3, Graeme Meintjes4 and A. Graham Fieggen3 Abstract Background: Hydrocephalus is a common, life threatening complication of human immunodeficiency virus (HIV)- related central nervous system opportunistic infection which can be treated by insertion of a ventriculoperitoneal shunt (VPS). In HIV-infected patients there is concern that VPS might be associated with unacceptably high mortality. To identify prognostic indicators, we aimed to compare survival and clinical outcome following VPS placement between all studied causes of hydrocephalus in HIV infected patients. Methods: The following electronic databases were searched: The Cochrane Central Register of Controlled Trials, MEDLINE (PubMed), EMBASE, CINAHL Plus, LILACS, Research Registry, the metaRegister of Controlled Trials, ClinicalTrials.gov, African Journals Online, and the OpenGrey database. We included observational studies of HIV- infected patients treated with VPS which reported of survival or clinical outcome. Data was extracted using standardised proformas. Risk of bias was assessed using validated domain-based tools. Results: Seven Hunderd twenty-three unique study records were screened. Nine observational studies were included. Three included a total of 75 patients with tuberculous meningitis (TBM) and six included a total of 49 patients with cryptococcal meningitis (CM). All of the CM and two of the TBM studies were of weak quality. One of the TBM studies was of moderate quality. One-month mortality ranged from 62.5–100% for CM and 33.3–61.9% for TBM. These pooled data were of low to very-low quality and was inadequate to support meta-analysis between aetiologies. Pooling of results from two studies with a total of 77 participants indicated that HIV-infected patients with TBM had higher risk of one-month mortality compared with HIV non-infected controls (odds ratio 3.03; 95% confidence-interval 1.13–8.12; p = 0.03). Conclusions: The evidence base is currently inadequate to inform prognostication in VPS insertion in HIV-infected patients. A population-based prospective cohort study is required to address this, in the first instance. Keywords: Systematic review, Meta-analysis, Ventriculoperitoneal shunt, Cerebrospinal fluid, Hydrocephalus, Human immunodeficiency virus, Acquired immunodeficiency syndrome * Correspondence: [email protected] 1Centre for Clinical Brain Sciences and Centre for Discovery Brain Sciences, Chancellor’s Building, 49 Little France Crescent, Edinburgh EH16 4SB, UK 2Edinburgh Medical School, Chancellor’s Building, 49 Little France Crescent, Edinburgh EH16 4SB, UK 1Centre for Clinical Brain Sciences and Centre for Discovery Brain Sciences, Chancellor’s Building, 49 Little France Crescent, Edinburgh EH16 4SB, UK 2Edinburgh Medical School, Chancellor’s Building, 49 Little France Crescent, Edinburgh EH16 4SB, UK g , Full list of author information is available at the end of the article g , Full list of author information is available at the end of the article Description of the intervention A ventriculoperitoneal shunt (VPS) consists of a surgi- cally implanted catheter running from the cerebral ven- tricles into the peritoneal cavity. This permits drainage of excess CSF according to prespecified parameters de- termined by a pressure or flow-regulated valve [29]. To reduce VPS associated infection, the catheter may be im- pregnated with the antibiotics rifampicin and clindamy- cin [30]. Catheter impregnation with silver has also been trialled [30]. In the absence of permanent CSF diversion therapy, the identification and treatment underlying causative factors will often result in normalisation of CSF hydro- dynamics. Sometimes however, a state of chronically raised CSF pressure develops [9]. Age-related cerebral tissue loss as well as HIV associated neurocognitive dis- order are associated with enlargement of CSF spaces due to generalised brain atrophy and may result in the radio- logical appearance of hydrocephalus, but are associated with normal CSF pressure and flow, so called “hydro- cephalus ex vacuo” [10, 11]. This is not an indication for surgical treatment and, consequently, is not discussed in this study [10]. Complications of VPS insertion include infection, catheter occlusion, migration or fracture, and over- or under-drainage [31]. In a United States adult cohort, ap- proximately 20% of patients suffered a complication in the first year post-insertion, whereas in a Kenyan, mostly paediatric, cohort this rate was recorded at 35% [32, 33]. This latter cohort possibly underestimates complication rates in this high-HIV prevalence, lower-middle income country because of high risk of attrition bias [33–35]. Alternatives to VPS include ventriculoatrial and ventri- culopleural shunting. Where acute, temporary cranial CSF diversion is required an external ventricular drain (EVD) may be sited as a primary procedure [29]. For some patients with communicating hydrocephalus it may be possible to avoid cranial surgery by draining CSF through serial lumbar puncture, a lumbar drain or a lumboperitoneal shunt [29]. Endoscopic third ventricu- lostomy may be used to treat some patients with non- communicating hydrocephalus, but is skill and resource intensive and may not be available in emergency or re- source limited settings [14]. y Meningeal infections including TBM, cryptococcal meningitis and bacterial meningitis, may impair CSF re- sorption at arachnoid granulations, resulting in commu- nicating hydrocephalus [9, 12, 13]. Additionally, in TBM, where thick basal exudate may occlude ventricular out- flow pathways, non-communicating hydrocephalus can develop [14]. Description of the condition Human immunodeficiency virus (HIV) is the causative pathogen for the acquired immunodeficiency syndrome (AIDS). Without treatment, AIDS will usually occur within 10 years of HIV seroconversion [1–3]. AIDS may predispose to raised cerebrospinal fluid (CSF) pressures and hydrocephalus by various aetiologies. These include tuberculous meningitis (TBM), cryptococcal meningitis (CM), primary central nervous system (CNS) lymphoma, coccoidal meningitis and immune reconstitution inflam- matory syndrome (IRIS) [4–8]. Background might be required to acutely control extremely high ini- tial CSF pressures and, where raised pressure persists, a permanent CSF shunt may be required [28]. Loan et al. BMC Neurology (2020) 20:141 Loan et al. BMC Neurology (2020) 20:141 Page 2 of 16 Description of the intervention Without appropriate antimicrobial treat- ment, TBM with associated hydrocephalus is almost uni- versally fatal, although this is believed to be principally a consequence of basal cerebral vasculitis and infarction, rather than hydrocephalus [15, 16]. Even with optimal medical management, death occurs in 30% of HIV non- infected patients with TBM, and many of those in whom antimicrobial management is successful suffer from chronic hydrocephalus [17, 18]. In the context of HIV infection, medically managed TBM has been reported to be associated with over 4 times higher mortality than HIV non-infected patients [18–20]. This excess mortality might be a consequence of synergistic virulence mecha- nisms of HIV and Mycobacterium tuberculosis, depletion of physiological reserve consequent to chronic HIV in- fection and demographic differences between HIV- infected and non-HIV infected populations [21–26]. Types of studies Reports of randomised or non-randomised studies, ex- cepting < 2-person case series, were eligible for inclusion. Aims and hypotheses not causing symptoms and has not become infected or undergone surgical revision or removal at any point. We aimed to identify all published studies of survival and outcome in HIV-infected patients undergoing VPS using a systematic review process and synthesise these data using quantitative and narrative meta-analyses to determine the risks and benefits of VPS insertion in the context of HIV infection. We hypothesised that the evidence base would be insufficient to reliably inform clin- ical practice or to conduct quantitative meta-analysis. Why it was important to conduct this review Why it was important to conduct this review HIV-infected patients are at high risk of developing con- ditions which predispose to hydrocephalus, such as TBM or CM [36, 37]. However, VPS insertion which is a mainstay of treatment for hydrocephalus in HIV non- infected individuals might be particularly high risk in the context of HIV: HIV infection might predispose to shunt infection and patients with hydrocephalus in the context of advanced HIV infection, might have such poor overall predicted survival as to render neurosurgical interven- tion futile or inappropriate [20, 38]. The literature from HIV non-infected patient populations therefore cannot be generalised to those with HIV infection. Clinicians therefore require a dedicated synthesis of high-quality evidence to appropriately counsel patients and their rela- tives of the risks and benefits of VPS insertion in the context of HIV. Other HIV-associated meningeal infections, such as CM more typically result in communicating hydroceph- alus, which usually resolves with successful antimicrobial treatment and intermittent therapeutic lumbar puncture to treat initially raised CSF pressures [9, 13, 27]. How- ever, insertion of a temporary CSF drainage system Loan et al. BMC Neurology (2020) 20:141 Page 3 of 16 Page 3 of 16 Loan et al. BMC Neurology (2020) 20:141 Data collection and analysis Selection of studies Each abstract was independently screened against eligi- bility criteria by two of three reviewers (JL, NM, MP, ST). Where eligibility criteria were met, or possibly met, the full manuscript was obtained and reviewed by two reviewers to determine final inclusion. Any disagree- ments were resolved by consensus discussion. Search methods for the identification of studies Electronic searches Our objectives were to: 1) Compare post-VPS mortality at 12, 6 and 1 months between the different aetiologies of HIV-associated hydrocephalus; 2) Produce a narrative review of risks of VPS infection and malfunction and other complications in HIV-infected patients and; 3) Identify baseline patient characteristics predictive of good outcome following VPS in HIV infection. The following electronic online databases were systematic- ally searched using terms for ventriculoperitoneal shunting or CSF diversion and HIV infection (see supplementary materials for full search strategies): The Cochrane Central Register of Controlled Trials (CENTRAL), MEDLINE (PubMed), EMBASE, CINAHL Plus (EBSCOhost), LILACS (BIREME), Research Registry (www.researchregistry.com), the metaRegister of Controlled Trials (mRCT) (www.con- trolled-trials.com), ClinicalTrials.gov (www.clinicaltrials. gov) and African Journals Online (AJOL). Grey Literature searching will be performed using the OpenGREY database. These were last updated on 17 October 2019. Our search sensitivity was validated confirming that candidate studies identified during scoping searches are included in the search yields [20, 44–46]. Data extraction and management We included studies which described VPS insertion using any technique, catheter or valve. Data was extracted using standardised proformas (supple- mentary materials) and entered into Review Manager [47]. Reference management was done using EndNote [48]. Outcomes Our primary outcome was one-year survival post-VPS in- sertion with comparison between all identified aetiologies of hydrocephalus. Secondary outcomes were overall sur- vival, AIDS specific mortality, VPS survival, risk of peri- operative complications and clinical outcome score [41–43] at 1 month, 6 months and one-year post-VPS insertion. Study design and protocol registration We conducted a systematic review of all published lit- erature stored in 10 electronic online databases, with no language or date restrictions. Our study protocol has been published in a peer-reviewed open access format and is registered on PROSPERO (CRD42016052239) [39, 40]. The methods used are summarised here. Types of participants We included studies of HIV-infected patients aged 16 years or older who underwent a VPS procedure for any indication. This included infectious and non-infectious illness as well as illness unrelated to HIV status. Searching other resources To ensure that no relevant studies were arbitrarily ex- cluded from this work, we included all studies which met all of the below criteria: The reference lists of included studies were scrutinized to identify any additional studies for inclusion. Two ex- perts in relevant fields (GM and GF) were consulted to identify key studies not identified by electronic searches. No hand searching was employed. Data synthesis Included studies were synthesised in a narrative review addressing each of our pre-specified outcomes in turn. The strength of the evidence available to inform each outcome was assessed using the GRADE method and summarised in summary of finding (SOF) tables using GRADEpro [52]. Where substantial clinical, methodo- logical or statistical heterogeneity was detected meta- analysis was not attempted. Comparative meta-analysis of homogenous studies was planned using the Peto fixed-effects method [40]. However, as none of the stud- ies were of high quality, study size varied, and as between-studies variation in control matching was sig- nificant, it was not clear that fixed-effect assumptions were met. We therefore elected to use the Mantel- Haenszel method, with random effects, and Review Manager v5.3 [47]. As effect sizes from just two studies could be determined it was not possible to generate meaningful funnel plots. As criteria for further meta- analysis were not met, only limited meta-analysis was conducted. Our planned extended meta-analysis has been detailed previously [39]. The reasons for exclusion from this review are as fol- lows: Two studies included patients with multiple, un- documented modalities of CSF diversion [54, 55]; one study provided a just single case report [56]; three stud- ies reported just one or two cases of surgical interven- tion and no survival/outcome data [57, 60, 61]; one study included 44 patients who underwent VPS in the context of CM but no outcome data [80]; two studies were review articles [58, 66]; eight reported no HIV- infected patients who underwent VPS [20, 59, 62–65, 71, 76–78]; in one study reporting < 50% HIV-infected pa- tients it was not possible to associate outcome with HIV status [8] and; no relevant measures of survival/outcome were studied in four [67–70]. Measures of effect Our narrative review summarises each included study’s reported measures of effect [39]. For quantitative meta- analysis, we report pooled odds ratios [51]. Studies of cryptococcal meningitis: characteristics and risk of bias As no comparative studies were included in this group it was impossible to calculate size of effect. Characteristics of included studies are summarised in Table 1 and risk of bias in Table 2. Description of studies Our literature search yielded 793 reports of studies, 721 of which were unique. Two further reports were identified by review of reference lists of included studies and were considered for inclusion [53, 54]. yielding a total of 723 unique records for screening (Fig. 1). No further studies were identified by expert opinion. 624 records were ex- cluded by title and abstract screening. 33 records were se- lected for full text review [8, 20, 44–46, 53–78]. Nine studies met criteria for inclusion [44–46, 53, 72–75, 79] and 24 were excluded [8, 20, 54–71, 80]. All included studies were observational studies. Three studies included a total of 75 patients with TBM [44, 46, 79] and six in- cluded a total of 49 patients with CM [45, 53, 72–75]. Assessment of heterogeneity On the basis of extracted data, included studies were compared for clinical and methodological heterogeneity. Clinically and methodologically studies reporting similar outcome measures at similar time points, were assessed for statistical heterogeneity by calculation of the I2 statis- tic (%) [47, 51]. We defined substantial heterogeneity as I2 greater than 50%. Two studies were published only in abstract form and did not present sufficient outcome data for inclusion [67, 68]. The corresponding authors of these records were therefore contacted to request further details to as- sess eligibility [8, 67, 68]. The authors one of these responded but were unable to provide further informa- tion [8, 68]. This study was therefore excluded. Excluded studies f h l d d Of the excluded studies, 16 concerned CM [54–57, 59– 63, 66–69, 76, 78, 80], four TBM [20, 58, 64, 77], two bacterial meningitis [70, 71], one coccoidal meningitis [8], and one included only cases of diagnostic uncer- tainty [65]. Assessment of risk of bias in included studies We included studied which reported overall survival, AIDS specific mortality, VPS survival or measures of functional outcome/disability. We defined VPS survival as the proportion of patients who have a VPS which is Each included study was initially assessed independently for risk of bias by two reviewers (JL, MP, ST) using the Canadian National Collaborating Centre for Methods Page 4 of 16 Page 4 of 16 Loan et al. BMC Neurology (2020) 20:141 and Tools domain based “Quality Assessment Tool for Quantitative Studies” [49, 50]. Disagreement was re- solved by consensus discussion. and Tools domain based “Quality Assessment Tool for Quantitative Studies” [49, 50]. Disagreement was re- solved by consensus discussion. Dealing with missing data The corresponding authors of all published abstracts of studies that were potentially eligible for inclusion, but for which a full study report was not published, were contacted electronically to request data not provided by the abstract. All authors contacted were allowed a mini- mum of 18 months to respond, prior to submission of our completed article. *median (range); ** mean (range); a) HIV infected cohort, b) d = days, m = months, y = years Calvo, et al. 2003 [75] This is an interrupted time series that presents five HIV- infected patients who were treated with VPS whilst ad- mitted to a single Intensive Care Unit (ICU) in Montevideo, Uruguay. It is unclear if the data was col- lected in a prospective or retrospective fashion or whether patients were recruited consecutively. Median initial GCS was 12 (range 9–15). CD4+ cell count was not reported for any patient, nor was the make or model of VPS system. The VPS was inserted as a primary pro- cedure for four patients with CSF pressures >23cmH2O. One patient initially received an EVD with intraventricu- lar pressure monitoring before having a VPS inserted. All patients were managed with antifungal agents and hyperventilation to a target pCO2 of 30 mmHg. Mannitol boluses were used in an unclear number of patients. At discharge from ICU all five patients were alive. Time to discharge from ICU was not reported. At 6 months two patients had been lost to follow-up and three survived. Outcome in this study is described as “good recovery”, “moderately disabled” or “dead” but the criteria for as- signment to these descriptors are not described. At ICU discharge, 4 patients had made a “good recovery” and one was “moderately disabled”. At 3 months, two had a “good recovery” and one was “moderately disabled”. No complications or shunt failure are reported. At 1 month, five of the eight VPS patients survived. Three deaths had occurred, one due to post-extubation aspiration pneumonia, one due to septic shock of un- documented aetiology and the other due to a gram- negative bacterial pneumonia. At 6 months, five patients were still alive, and no new deaths were recorded. At 12 months, three patients were alive, four were dead and one was lost to follow-up. The additional death was sec- ondary to an unspecified respiratory complication. Of the three patients initially treated for CM IRIS, at both one and 6 months, two were alive and one dead. At 12 months, two were confirmed dead and the other lost to follow-up. One instance of shunt infection was recorded at 12 months. Two other patients, who were followed up for 1512 and 1485 days, suffered a shunt occlusion and a shunt infection, respectively, at some point during their follow-up period. However, the time to these complica- tions was not reported. Bach, et al. 1997 [45] BMC Neurology Table 2 Quality assessment summary for cryptococcal meningitis studies Using the Canadian national collaborating centre for methods and tools effective public health practice project Quality Assessment Tool for Quantitative Studies [49] Table 2 Quality assessment summary for cryptococcal meningitis studies outcome measure. 40% of patients were lost to follow-up at 6 months. outcomes, rates of shunt failure and complication were not presented. Although this study included consecutive patients and therefore minimised selection bias, it was classified as being globally weak. An interrupted time series is a weak study design. Coupled with small sample size, this ren- dered meaningful multivariable analysis impossible and it was not attempted. The time until loss to follow up of one patient was unclear, and the reasons for this are not documented. Mortality is a robust outcome measure. Cherian, et al. 2016 [74] This two-cohort analysis reported all patients with CM and CM IRIS presenting to a single hospital in Texas, USA. 49 of 50 patients had associated HIV infection. Fourty patients had communicating hydrocephalus, and it is unclear if the patient with non-communicating hydrocephalus was HIV-infected or not. CD4+ cell count was not documented for any patient, nor was ini- tial neurological status. All further demographic and outcome data are specific for the HIV-infected group. Patients who went on to receive CSF diversion had ini- tial lumbar puncture opening pressure > 25 cm H2O. They were initially managed with antifungals, antiretro- viral therapy and a variable number of therapeutic lum- bar punctures (median 7; range 2–40) for a variable period (median 27 days; range 4–281 days) before pro- ceeding to VPS. Eight patients underwent VPS. Bach, et al. 1997 [45] Lists of citations for each stage of review and risk of bias assessment tables are available from the corresponding author. This was a retrospective consecutive interrupted time series of all patients admitted with severe intracranial Loan et al. BMC Neurology (2020) 20:141 Page 5 of 16 Loan et al. BMC Neurology Fig. 1 PRISMA Flow diagram of screened and included study reports Fig. 1 PRISMA Flow diagram of screened and included study reports was not reported. Neither makes nor models of the VPS systems used were described. was not reported. Neither makes nor models of the VPS systems used were described. hypertension (lumbar puncture opening pressure > 35cmCSF) secondary to CM who received CSF diver- sion. Four patients received a VP shunt. Three patients were initially managed with serial lumbar puncture. One received primary VPS. All patients presented with visual disturbance, one had seizures and one had bilateral ab- ducens nerve palsies. Initial Glasgow Coma Score (GCS) One patient was lost to follow-up at an unclear time point. At 1 month, all three remaining patients were alive. One death at 6 months was attributed to “wasting syndrome”. At 10 months a further death was attributed to Pneumocystis jiroveci pneumonia. Functional Table 1 Characteristics of included studies Aetiology Number of patients with HIV and VPS Mean age (years) (SD)a Mean CD4 count (cells/μL; ±SD)a Included outcome Follow- upb Number of non-HIV infected controls (mean age [±SD]) Country Bach 1997 [45] CM 4 39.25 (±6.1) 28.75(±14.4) Survival 3d-12y N/A USA Calvo 2003 [75] CM 5 28.2 (±7.7) unknown Survival 6 m N/A Uruguay Cherian 2016 [74] CM 8 38.9 (±8.2) Unknown Survival 6 m-5.7y N/A USA Corti 2014 [73] CM 14 33 (18–53)* 50 (6–511)* Survival 1y N/A Argentina Liu 2014 [72] CM 9 32.2(±6.0) 10.75(±9.4) Survival 1 m-1y N/A China Vidal 2012 [53] CM 9 Unknown Unknown Survival Discharge N/A Brazil Nadvi 2000 [46] TBM 15 26.1 (±16.4) 171.7(±161.5) GOS 1 m 15 patients (age 10.7[±9.6]) South Africa Sharma 2015 [44] TBM 30 31.3(±7.8) 143 (26–445)** GOS 3 m 30 patients (age 31[±9.9]) India Harrichandparsad 2019 [79] TBM 30 28.4(±13.4) 227(±163.9) GOS 1 m N/A South Africa *median (range); ** mean (range); a) HIV infected cohort, b) d = days, m = months, y = years Table 1 Characteristics of included studies Loan et al. BMC Neurology (2020) 20:141 Page 6 of 16 Loan et al. Corti, et al. 2014 [73] This retrospective interrupted time series included 15 patients with CM who presented to medical services in Buenos Aires, Argentina. 14 of these patients were HIV- infected, however it was not possible to identify which demographic or outcome data pertained to the HIV- non-infected patient. CSF diversion was undertaken in patients who had lumbar puncture opening pressure persistently > 25 cm H20 after 2 weeks of therapeutic lumbar puncture and antifungal treatment. 14 of the 15 patients were managed with VP shunt, and one with lumboperitoneal shunting. Again, it was not possible to isolate demographic and outcome data from this patient. Medtronic® systems were used for all patients but com- ponent models are not described. This study was classified as being globally weak due to a weak study design and unknown risk of selection bias. No confounders were identified, and the primary out- come was survival – which is a robust outcome measure. Three patients suffered complications including sepsis, multiorgan failure, two cases of meningitis and abdom- inal pseudocyst formation related to the distal catheter tip and requiring catheter revision. These occurred at unclear time points and the patients were excluded from survival analysis by the study authors. However, the au- thors did report that at least one of the patients with meningitis and multiorgan failure died. Of the remaining patients, all survived to 12 months. No other complica- tions or outcomes were reported in this group. Vidal, et al. 2012 [53] This retrospective cohort study investigated associations between various parameters derived from quantitative CSF microscopy with mortality in a cohort of patients who presented to hospital in São Paulo, Brazil, with HIV-associated CM. Included patients were admitted between January 2006 and June 2008, but it is unclear what rate of case acquisition this represents. A median CD4+ cell count of 36 cells/μL, with interquartile range of 17–87 cells/μL was reported across all patients. Neurosurgical intervention was considered in patients with lumbar puncture opening pressure persistently >20cmH2O after 14 days therapeutic lumbar puncture and antifungal treatment. This study was classified as being globally weak with high risk of selection bias, a weak study design and small sample size which did not permit adjustment for factors potentially confounding outcome, and poorly docu- mented exclusion from follow up causing high risk of at- trition bias. Survival is a validated outcome measure but was unreliably reported in the group with complications. Although survival was presented in the series of 9 pa- tients undergoing VPS, no other dependent or independ- ent variables were reported in this subgroup to allow comparisons and therefore, for our purposes, this study provided case-series data. Initial neurological status and type of shunt valve or catheter were not described. Of the 9 patients who underwent VPS, 6 survived until hos- pital discharge. However, time to discharge and length of stay were not reported. No other outcomes were reported. Calvo, et al. 2003 [75] One other patient who was followed up for 383 days suffered a subdural haematoma, which was attributed to VPS over-drainage. Again, the time point at which this occurred was not reported. Functional outcomes were not reported. This study was classified as being globally weak with a high risk of selection bias and weak study design which, coupled with small sample size, did not permit adjust- ment for modulators of risk of mortality or poor out- come. Outcome assessment did not use a validated Page 7 of 16 Page 7 of 16 Loan et al. BMC Neurology (2020) 20:141 Loan et al. BMC Neurology (2020) 20:141 This study was classified as being globally weak with no attempt to undertake multivariable survival analysis, undocumented reasons for loss to follow-up and lack of reporting of times of complications. Survival is a vali- dated outcome measure but was unreliably reported in patients discharged to hospice care. Rates of complica- tion were not measured using a validated method. Pa- tients were consecutively recruited, and the selection process was clearly documented. A two-cohort analysis study has moderate risk of bias. puncture and mannitol administration. VPS was under- taken in all patients using a Medtronic® system, but the valve and catheter model are not documented. The au- thors define raised opening pressure as “> 400 cm H20” which, presumably, is a typographic error (raised CSF opening pressure defined as > 20-35 cm H2O in other in- cluded studies [45, 53, 73–75]). Two patients were de- scribed as having loss of consciousness at baseline, but no objective measures were provided. Eight of the nine patients studied were alive at 1 month. Three patients were lost to follow up at 6 months and no new deaths were reported. At 12 months, one more patient had died, yielding a survival of four out of six patients for whom follow-up was available. Given the low initial CD4 counts, it is likely that deaths would have occurred in those lost to follow-up by this time point and therefore there is a risk of attrition bias at this time point. The causes of death were VPS ob- struction at 1 month and an unspecified reaction to anti- retroviral drugs at 12 months. No other outcome measures, shunt failures or complications were reported. Included studies of tuberculous meningitis: characteristics and risk of bias This prospective cohort analysis compared outcome and mortality at 1 month following VPS between HIV- infected and HIV non-infected patients. Patients who underwent VPS for TBM, over an undefined recruitment period prior to commencement of the national South African antiretroviral treatment (ART) programme in 2004, were recruited from a single hospital in KwaZulu- Natal, South Africa. Of the 30 patients recruited, 15 were HIV-infected and 15 HIV non-infected. The two cohorts differed widely in age range with the HIV- infected group being mostly adult and the HIV non- infected group being mostly paediatric. Patients were di- agnosed with hydrocephalus on the basis of CT, trans- cranial doppler or clinical observation in keeping with hydrocephalus and intraoperative ventricular pressure > 20cmCSF. Median initial GCS was 14 (range 9–15) in the HIV-infected group and 12 (range 9–15) in the HIV non-infected group. Use of EVD or therapeutic lumbar puncture was not documented for any patient. VPS was undertaken using a 102 cm catheter and low-profile, medium pressure Codman / Unishunt valve (Codman / Johnson & Johnson®, Raynham, MA). p p At 1 month, 13/21 HIV-infected and 20/26 HIV non- infected patients were alive. At 6 months, 9 HIV- infected and 18 HIV non-infected patients were alive. In the HIV-infected cohort, unfavourable initial GCS was associated with higher rates of mortality. GOS at 3 months was also dichotomized as unfavourable (1–3) or favourable (4, 5). 16/21 HIV-infected, and 9/26 HIV- non-infected patients had an unfavourable outcome at 3 months. Binomial logistic regression analysis confirmed that HIV infection (p = 0.038) and low Palur grade (p = 0.024) [82] were significantly associated with unfavour- able outcome. No measure of effect was presented for these analyses. Anaemia (haemoglobin < 10 mg/dL) was associated with unfavourable outcome in the HIV- infected group (Exp.[β] = 25.6; p = 0.011). y At 1 month, 5/15 and 11/15 HIV-infected, and HIV non-infected patients were alive, respectively. 11/15 and 6/15 patients in the HIV-infected and HIV non-infected cohorts had an unfavourable Glasgow Outcome Score [43] (GOS 1–3) dichotomised GOS at 1 month. Multi- variable analysis was not attempted. Univariate analysis revealed a significant association of CD4+ cell count and outcome (p = 0.031), but the magnitude of this associ- ation was not reported. This study was classified as being globally weak. A prospective cohort study provides a moderate study de- sign. Liu, et al. 2014 [72] This retrospective single cohort study included nine pa- tients with HIV-associated CM managed with VPS over a five-year period at a Shanghai hospital, China. Al- though exclusion criteria are documented, the number of exclusions is not. Included patients had a mean CD4+ cell count of 11 cells/μL. Neurosurgical intervention was considered in patients with raised lumbar puncture opening pressure and deteriorating neurological status despite antifungal therapy, repeated therapeutic lumbar This study was classified as being globally weak with an unknown risk of selection bias and weak study design and size which did not permit adjustment for Page 8 of 16 Page 8 of 16 Loan et al. BMC Neurology (2020) 20:141 Loan et al. BMC Neurology (2020) 20:141 Loan et al. BMC Neurology in Bangalore, India, between June 2002 and October 2012 were included. These patients were compared against 30 HIV non-infected control patients that were matched for age, sex and clinical grade of TBM. Patients were included who “had symptoms of raised intracranial pressure, with clinical, radiological and CSF findings characteristic of TBM”. However, specific inclusion cri- teria were not documented. Initial GCS was dichoto- mized as 3–8 (unfavourable) and this was present in 13.3 and 3.3% in the HIV-infected and non-infected pa- tients, respectively. The rest had GCS 9–15, which was classified as favourable. 23 and 22 patients in the HIV- infected and non-infected cohorts, respectively, had communicating hydrocephalus. Patients underwent VPS as a primary procedure unless extensive basal infarcts were evident on computed tomography (CT) scan of the brain. In these patients, a frontal EVD was placed and converted to VPS if improvement in “sensorium” was noted. The proportion of those who underwent primary VPS is not reported. VPS was with a medium-pressure Chhabra shunt (Surgiwear® Inc.). 9 HIV-infected patients and 4 HIV non-infected patients were lost to follow-up. confounding variables regards our research question. Survival is a strong primary outcome measure. confounding variables regards our research question. Survival is a strong primary outcome measure. Included studies of tuberculous meningitis: characteristics and risk of bias The study included representative patients, but the control group was poorly age matched and this was not adjusted for. It is not clear that patients were enrolled consecutively, yielding an unknown risk of selection bias. The authors were not blinded to HIV status. Mortality is a robust primary outcome measure. As this study was undertaken prior to ART being made widely available in South Africa, its findings may not be generalisable to settings where ART is available [81]. This study was classified as being globally of moderate quality. A case-control study provides a moderate quality of study design. The study included representative con- secutive cases with appropriate matching of controls. Po- tential confounders were adjusted for in the multivariable analysis. The authors were not blinded to HIV status. Just under a third of initially recruited pa- tients were lost to follow-up at later time points, but this was appropriately documented. GCS and dichotomised GOS are robust outcome measures but not validated in the context of TBM. Mortality is a robust outcome measure. Harrichandparsad, et al. 2019 [79] This retrospective case-control study compared outcome and mortality between HIV-infected and HIV non- infected patients following VPS. All 30 eligible HIV- infected patients who underwent VPS at a single hospital This case control study compared 15 HIV-infected pa- tients who were using ART prior to presentation who consecutively underwent VPS for TBM in 2017 with 15 Page 9 of 16 Loan et al. BMC Neurology (2020) 20:141 Page 9 of 16 Loan et al. BMC Neurology (2020) 20:141 Loan et al. BMC Neurology (2020) 20:141 and follow-up varied greatly. Further, all of the studies of CM were of weak design, and two of the three studies of TBM also being graded as weak. There was significant methodological heterogeneity between CM and TBM studies and it was therefore not possible to conduct meaningful meta-analytic comparison of outcomes be- tween these groups. We therefore present a narrative analysis. The results of pooling of reported outcomes within each group are presented in SOF tables, with the purpose of summarising reported outcomes to date. These are of very low to low GRADE of evidence (see risk of bias, above), indicating that the true outcomes for each pathology may be, or are likely to be, substantially different from the pooled measure [83].. historical retrospectively identified controls who under- went the same procedure but were not using ART. All patients receiving ART were prescribed single pill for- mulation of tenofovir and emtricitabine as well as efavir- enz. The diagnosis of TBM was made on the basis of typical clinical, radiological and CSF findings. Not all pa- tients had positive CSF cultures. All patients had radio- logical features of hydrocephalus with CSF pressures higher than 20 cm H2O on ventriculostomy and under- went VPS as a primary procedure. Both cohorts had similar initial GCS and Palur grade on admission. All patients were followed up as inpatient for a minimum of 1 month post-operatively. Outcome was assessed at discharge using the dichotomised GOS (GOS 4–5 good outcome; 1–3 poor outcome). At discharge overall 15 patients achieved good outcome, one poor outcome and four died. Eleven patients receiving anti- retroviral therapy achieved a good outcome with no in- stances of poor outcome and four deaths. This was better than the historical control group only four of which achieved a good outcome, one poor outcome and ten deaths. This difference was statistically significant in unadjusted analysis (p = 0.027). Risk of bias between studies: tuberculous meningitis Risk of bias between studies: tuberculous meningitis As two comparative cohort studies were included asses- sing the effect of HIV infection on outcome, it was pos- sible to pool reported measures of effect of HIV- infection on survival at one-month post-VPS. Risk of bias is summarised in Table 3. Four studies reported 12-month survival [45, 72–74]. Liu, et al. reported a further death. Cherian, et al. re- ported a death in one of their patients who had been di- agnosed with CM IRIS, contributing to AIDS-specific mortality. Another of their patients was lost to follow- up. One of Bach et al.’s patients had died of Pneumocyc- stis jiroveci pneumonia, also contributing to AIDS- specific mortality. Corti, et al. report that all of their 12 patients followed-up for 1 year were alive. In total 20 of 29 patients with 12-month follow-up survived. Five of the 17 (29%) patients reported by studies describing Cryptococcal meningitis Cryptococcal meningitis Pooled outcome measures are reported in Table 4. Harrichandparsad, et al. 2019 [79] Patients in the ART treated group were more likely to have a higher GCS at 1 month than at presentation than the ART untreated group (p = 0.042). Survival Three studies reported survival in CM at one-month post VPS [45, 72, 74]. Cherian, et al. reported three deaths in eight patients. Three of these patients had a diagnosis of CM IRIS, one of which died (AIDS-specific mortality). Liu, et al. report one death in nine patients at 1 month. This was due to shunt obstruction. Bach, et al.’s four patients with documented follow up were all alive at 1 month. In total, 17 of 21 (81%) patients de- scribed in these studies survived 1 month, with one epi- sode of shunt failure documented. This study was classified as being globally weak. A case-control study provides a moderate quality of study design. Consecutive patients were enrolled, reducing chances of selection bias and there was no attrition. Al- though matching of cases and controls by severity was attempted, differences in spread of age and gender were present. These were not statistically significantly differ- ent between groups at p < 0.05, but this may reflect high variation in these characteristics and the multivariable analysis that would be required to adjust for these was not possible. The study describes no blinding. GCS and dichotomised GOS are robust measures but not vali- dated in the context of TBM. Mortality is a robust out- come measure. At 6 months post-VPS, four studies reported survival [45, 72, 74, 75]. Three of Liu, et al’s patients had been lost to follow-up. However, no new deaths were con- firmed. Bach, et al. reported one death due to HIV- wasting syndrome. Another had died of cytomegalovirus pancreatitis. These both therefore contributed to AIDS- specific mortality [84]. Calvo, et al. obtained 3 months follow up for three patients, all of whom survived. Cher- ian et al. followed up all of their eight patients and re- ported no new deaths. With loss to follow-up and addition of three surviving patients from Calvo, et al’s cohort, overall survival remained stable: 17 of 21 (81%) patients that were followed up at 6 months were alive. No new cases of shunt failure were documented. Synthesis of results Effect of pathology Nine studies of survival and/or outcome in TBM and CM were identified [44–46, 53, 72–75, 79]. However, none of these compared survival or outcome between TBM and CM. Study populations, outcome measures Page 10 of 16 Loan et al. BMC Neurology (2020) 20:141 Table 3 Quality assessment summary for tuberculous meningitis studies Using the Canadian national collaborating center for methods and tools effective public health practice project Quality Assessment Tool for Quantitative Studies [49] Using the Canadian national collaborating center for methods and tools effective public health practice project Quality Assessment Tool for Quantitative Studies [49] Table 4 Summary of findings – cryptococcal meningitis Outcomes Pooled outcome №of participants (studies) Quality of the evidence (GRADE) Comments Survival Survival follow up: 1 months 17 (81.0%; range 62.5–100%) 21 (3 observational studies) [45, 72, 74] ⨁◯◯◯ VERY LOW a,b,d,f,g,h,k Survival follow up: 6 months 16 (76.2%; range 62.5–100%) 21 (4 observational studies) [45, 72, 74, 75] ⨁◯◯◯ VERY LOW a,b,d,f,g,h Survival follow up: 12 months 20 (69.0%; range 33–100%) 29 (4 observational studies) [45, 72–74] ⨁◯◯◯ VERY LOW a,b,d,f,g,h,i AIDS specific mortality AIDS specific mortality follow up: 1 months 1 (4.8%; range 0–12.5%) 21 (3 observational studies) [45, 72, 74] ⨁◯◯◯ VERY LOW a,b,d,f,g,h,k AIDS specific mortality follow up: 6 months 3 (16.7%; range 12.5–33%) 18 (3 observational studies) [45, 72, 74] ⨁◯◯◯ VERY LOW a,b,d,f,g,h,k AIDS specific mortality follow up: 12 months 5 (29.4%; range 25–66%) 17 (3 observational studies) [45, 72, 74] ⨁◯◯◯ VERY LOW a,b,d,f,g,h,k VPS survival VPS survival follow up: 1 months 8 (88.9%) 9 (1 observational study) [72] ⨁◯◯◯ VERY LOW a,d,e,f,g Cherian, et al. [74] reported two shunt failures at unspecified timepoints. Corti, et al. [73] reported three shunt failures at unspecified timepoints. VPS survival follow up: 6 months 5 (83.3%) 6 (1 observational study) [72] ⨁◯◯◯ VERY LOW a,d,e,f,g Cherian, et al. [74] reported two shunt failures at unspecified timepoints. Corti, et al. [73] reported three shunt failures at unspecified timepoints. VPS survival follow up: 12 months 5 (83.3%) 6 (1 observational study) [72] ⨁◯◯◯ VERY LOW a,d,e,f,g Cherian, et al. [74] reported two shunt failures at unspecified timepoints and one at 12 months. Corti, et al. [73] reported three shunt failures at unspecified timepoints. Operative complication 4 (23.5%; range 0–50% 17 (2 observational studies) [72, 74] ⨁◯◯◯ VERY LOW a,b,c,d,e,f,g Corti,et al. [73] reported one case of multiorgan failure or unspecified aetiology. Synthesis of results Effect of pathology Validated outcome score No studies reported validated outcome measures at any time point Calvo, et al. [75] reported unvalidated outcome measures at 6 months Table 4 Summary of findings – cryptococcal meningitis Page 11 of 16 Page 11 of 16 Loan et al. BMC Neurology (2020) 20:141 cause of death had AIDS-defining illness as a primary or major contributing cause of death by 12 months. Survival Three studies reported 1-month survival following VPS for HIV-associated TBM [44, 46, 79]. Sharma et al. re- ported 13 of 21 patients surviving for one-month post- VPS. Nadvi, et al. reported just 5 of 15 patients surviving for one-month. Harrichandparsad, et al. reported 14 out of 30 patients surviving for 1 month, although 10 of these deaths were in the ART untreated group. Liu et al., reported one shunt failure at 1 month. Cher- ian and colleagues reported one shunt failure at 12 months, but as they also reported two other instances of shunt failure at unclear timepoints, their shunt failure data was not included in the pooled analysis. Operative complications Sharma et al. reported 9 of 21 patients surviving 6 months. Sharma et al. also reported “long-term” survival of 7 of 21 with mean follow-up of 130 days, range 91– 760 days. No studies reported causes of death or mea- sures of shunt survival. Complications of treatment were reported by two studies [72, 74]. In Liu, et al.’s series, none of these were related to the surgical management of their pa- tients at 12 months. In addition to their three in- stances of shunt failure, Cherian and colleagues reported one instance of over drainage subdural haematoma at an unspecified time. Follow-up in this series ranged from 28 to 1512 days. Functional outcome Two studies reported GOS in HIV-infected patients 1 month following VPS [46, 79]. 11 of 15 patients in Nadvi, et al.’s study had an unfavourable functional out- come, with 10 of these being dead. Therefore four of the five surviving patients attained GOS 4–5, signifying moderate to low levels of disability at one month [43]. Harrichandparsad, et al. reported 15 of 30 patients achieving an unfavourable outcome, including death at No studies reported any measures of functional outcome at any time point. Calvo, et al. reported that two of three patients made a “good recovery” at 6 months but this measure is not validated [75]. Tuberculous meningitis Pooled outcome measures are reported in Table 5. Table 5 Summary of findings – tuberculous meningitis Outcomes Pooled outcome №of participants (studies) Quality of the evidence (GRADE) Comments Survival Survival follow up: 1 months 34 (51.5%; range 33.3– 61.9%) 66 (3 observational studies) [44, 46, 79] ⨁⨁◯◯ LOW a Survival follow up: 6 months 9 (42.9%) 21 (1 observational study) [44] ⨁◯◯◯ VERY LOW a,b Survival follow up: 12 months 7 (33.3%) 21 (1 observational study) [44] ⨁◯◯◯ VERY LOW a,b AIDS specific mortality No studies reported AIDS specific mortality at any point VPS survival No studies reported VPS failure or survival rates at any time point Operative complication No studies reported rates of operative complication at any time point Validated outcome score Glasgow outcome score (GOS) follow up: 1 month GOS 1–3: 26 (57.8%) GOS 4–5: 19 (42.2%) 45 (2 observational studies) [46, 79] ⨁⨁◯◯ LOW a GOS 1–3 signifies death to severe disability GOS 4–5 signifies moderate to low disability Glasgow outcome score (GOS) follow up: 6 months GOS 1–3: 16 (76.2%) GOS 4–5: 5 (23.8%) 21 (1 observational study) [46] ⨁◯◯◯ VERY LOW a,c GOS 1–3 signifies death to severe disability GOS 4–5 signifies moderate to low disability Glasgow outcome score (GOS) follow up: 12 months No studies reported a validated outcome score at 12 months a Possibly selected observational case series level data b Incomplete long-term follow-up c Mean follow up 130 days. Range of 3–25 months Table 5 Summary of findings – tuberculous meningitis Page 12 of 16 Page 12 of 16 Loan et al. BMC Neurology (2020) 20:141 one month, 11 of which were in the ART untreated group [79]. puncture and drainage of CSF [86]. These guidelines emphasise that repeat lumbar puncture should be under- taken to control symptomatic hydrocephalus until reso- lution and do not describe any role for permanent CSF diversion in CM. The Infectious Diseases Society of America 2010 guidelines for the management of crypto- coccal disease recommended early ventricular CSF diver- sion for non-communicating hydrocephalus in CM [87]. For chronic communicating hydrocephalus or communi- cating hydrocephalus causing significant neurological impairment, permanent CSF diversion may be under- taken [87]. Effect of human immunodeficiency virus infection As two studies comparing HIV-infected and HIV non- infected patients with TBM were identified, it was pos- sible to examine the impact of HIV-infection on survival [44, 46]. At 1 month, the odds ratio for mortality in Sharma, et al.’s HIV-infected group was 2.02 (95% confidence interval [CI]: 0.58–7.01). In Nadvi’s group it was 4.73 (95% CI: 0.58–7.01). The pooled odds ratio (Mantel- Haenszel) for one-month mortality in the HIV-infected group was 3.03 (95% CI: 1.13–8.12; p = 0.03; chi2 = 0.92; df = 2; I2 = 0%; Fig. 2), indicating significantly increased risk of mortality at one-month post-VPS in HIV infection. Summary of evidence Only studies of patients with who underwent ventriculo- peritoneal shunting because of HIV-associated hydro- cephalus secondary to TBM or CM were identified by our study. Six series of patients with CM [45, 53, 72–75] and two controlled studies of patients with TBM [44, 46] were included. Tuberculous meningitis Three cohort studies were included. These provide a low level of evidence to inform prognostication at one- month, in terms of survival and functional outcome, for patients undergoing VPS for HIV-associated TBM. One- month survival varied from 33 to 61.9% between these studies, yielding a very cautious pooled estimate of one- month survival of 51.5% and a pooled estimate of 42.2% good outcome at 1 month. We anticipate that this esti- mate differs substantially from the true outcome. The in- cluded studies provide a very low level of evidence to inform later prognostication in terms of survival and functional outcome. For all other measures of outcome – including AIDS-specific mortality, shunt failure risk and perioperative complication – the included studies provided no data to inform clinical decision-making. Nadvi, et al. and Sharma, et al’s studies demonstrate that patients who underwent VPS for HIV-associated TBM had poorer outcomes than their HIV non-infected coun- terparts. Meta-analysis of these findings yielded in- creased risk of mortality (odds ratio 2.93) in the HIV- infected group. However, one of these studies compared a largely paediatric HIV non-infected population with a largely adult HIV-infected population [46]. The clinical phenotypes and pathophysiological responses to brain injury in these groups differ widely and so comparisons should be interpreted with caution [88, 89]. It is also im- portant to note that whilst one of the studies employed ventricular CSF diversion as a primary measure for man- agement of hydrocephalus [44], it is unclear if this was undertaken primarily, or following failure of lumbar Sharma et al. reported six-month survival, but Nadvi, et al. did not. Cryptococcal meningitis The included studies of CM provided a very low level of evidence on which to inform clinical practice. Reported sur- vival by included studies of CM varied extremely widely – from 33 to 100% at 1 year – and this is likely a consequence of study heterogeneity. The relatively high pooled 12- month survival of 69% might represent selection and attri- tion biases, which many included studies were considered to be at a high risk of. For comparison, 12-month survival in a study of 549 patients with HIV-associated CM who did not receive shunting varies from 55 to 93%, contingent on patients’ antiretroviral status [85]. The 2018 World Health Organisation (WHO) guide- lines on CM recommend that CM-associated hydro- cephalus be initially managed by therapeutic lumbar Fig. 2 Forest Plot - One-month mortality HIV-infected vs. HIV non-infected tuberculous meningitis Fig. 2 Forest Plot - One-month mortality HIV-infected vs. HIV non-infected tuberculous meningitis Page 13 of 16 Loan et al. BMC Neurology (2020) 20:141 in the context of TBM (ART 73% vs. no ART 33% 1 month survival) [79]. It is striking that 1 month survival in this small ART treated cohort is similar to that of the pooled data from HIV uninfected patients in Sharma et al., and Nadvi, et al’s studies (75.6%). Two studies of CM published in 2003 [75] and 1997 [45] reported 0– 50% 6 month mortality, respectively. However, these contributed small numbers of cases to our analysis of CM and so the effect of lack of availability of ART for these cases on our analysis is likely less than the effect of other factors such as incomplete follow-up. drainage/medical management in the other study [46]. This heterogeneity limits the interpretation of compari- sons between reported outcomes in HIV-infected and non-infected cohorts. The British Infection Society published guidelines in 2009 for the management of TBM [90]. These guidelines did not recommend altering the medical management of patients with TBM on the basis of their HIV status but did not comment on their surgical management with re- spect to HIV. On the basis of observational studies of HIV non-infected patients, early VPS is recommended by these guidelines for non-communicating hydroceph- alus [90–92]. It is noted that response to external ven- tricular drainage poorly predicts response to VPS [91]. Review limitations This review is limited in certain respects. As just two studies reporting effect sizes could be included in our meta-analysis, it was not possible to meaningfully assess for publication bias. Publication bias in case series tends to over-represent good outcomes and therefore our pooled results are likely to indicate better survival and outcomes than is the truly the case [93]. Included stud- ies in our review were of low-moderate quality study de- sign. The decision to utilise designs such as single cohort studes or small case series is often a pragmatic choice on behalf of the study authors: conduct of larger standardised observational studies, with blinding of out- come assessment and multivariable adjustment of factors influencing clinical outcome may not have been justified by available resources. Unfortunately, observer bias and the effect of unmeasured confounding factors in these types of study can substantially impact on study out- come and this therefore reduces our confidence in our pooled estimates of outcome following VPS insertion in HIV-infected individuals [94, 95]. Furthermore, our review has provided evidence that available data is of inadequate quality to reliably inform clinical decision-making related to VPS insertion for HIV-associated CM and TBM. The burden of HIV- associated CM and TBM in low-middle income settings in the era of ART are unclear yet believed to remain sig- nificant, with one study estimating over 600,000 HIV- associated CM deaths occurring in 2006 [37] and TBM and CM accounting for approximately 10% of AIDS re- lated deaths in a Ghanaian study from 2007 [36]. As hydrocephalus or raised CSF pressure is present in ap- proximately 50% of cases of HIV-associated TBM [100, 101] and 60% of HIV-associated CM [102], the lack of evidence to guide use of VPS in this setting is concerning. Four of our included studies were published 1997– 2003, prior to the widespread availability of ART [81]. Use of ART is an important predictor of survival and this could potentially impact on the generalisability of these studies outcomes to contemporary practice [86, 90]. These included Nadvi et al’s study of TBM, which reported lower one-month survival (at 33%) than the more recent study by Sharma, et al. (61.9%) [44, 46, 81]. Harrichandparsad, et al. Suggestions for future research Our review has included studies of patients with either TBM or CM. Although other causes of hydrocephalus in HIV have been reported, none of these reports met our criteria for inclusion [5–8, 96, 97]. Understanding how outcomes differ following VPS insertion in HIV-infected and non-infected patients with causes of hydrocephalus not directly attributable to HIV infection (e.g. subarach- noid haemorrhage, Chiari malformation, etc) would allow determination of the relative impacts of undertak- ing VPS in the context of active CSF infection and HIV- infection. Unfortunately, to date, most large studies of VPS insertion for “all cause” hydrocephalus have ex- cluded HIV-infected patients, have been conducted in populations with low HIV-infection prevalence, or do not report HIV-infection rates within their cohort [30, 98, 99]. Cryptococcal meningitis Our study’s findings would indicate that HIV status might impact significantly on surgical outcome and therefore caution should be employed by future guide- line working groups before generalizing the results of studies conducted of HIV non-infected populations to inform the surgical management of HIV-infected patients. Consent for publication All h h i d All authors have reviewed the final manuscript and consent for publication. Authors’ contributions JL, NM, GM and GF conceived and designed the study. JL, ST, MP and NM reviewed studies and extracted data. JL analysed data and drafted the manuscript. All authors critically reviewed and approved the completed manuscript. Abbreviations 9. Graybill JR, Sobel J, Saag M, et al. Diagnosis and management of increased intracranial pressure in patients with AIDS and cryptococcal meningitis. The NIAID mycoses study group and AIDS cooperative treatment groups. Clin Infect Dis. 2000;30(1):47–54. HIV: Human immunodeficiency virus; AIDS: Acquired immune deficiency syndrome; CSF: Cerebrospinal fluid; CNS: Central nervous system; TBM: Tuberculous meningitis; CM: Cryptococcal meningitis; IRIS: Immune reconstitution inflammatory syndrome; ART: Antiretroviral therapy; VPS: Ventriculoperitoneal shunt; EVD: External ventricular drain; GCS: Glasgow coma score; ICU: Intensive care unit; GOS: Glasgow outcome score; CT: Computed tomography 10. Greenberg MS. Hydrocephalus - general aspects. In: Greenberg MS, editor. Handbook of neurosurgery. 8th ed. New York: Thieme; 2016. p. 394–413. 11. Filippi CG, Ulug AM, Ryan E, Ferrando SJ, van Gorp W. Diffusion tensor imaging of patients with HIV and normal-appearing white matter on MR images of the brain. AJNR Am J Neuroradiol. 2001;22(2):277–83. 12. Vinnard C, Macgregor RR. Tuberculous meningitis in HIV-infected individuals. Curr HIV/AIDS Rep. 2009;6(3):139–45. 12. Vinnard C, Macgregor RR. Tuberculous meningitis individuals. Curr HIV/AIDS Rep. 2009;6(3):139–45. References 1. Buonaguro L, Tornesello ML, Buonaguro FM. Human immunodeficiency virus type 1 subtype distribution in the worldwide epidemic: pathogenetic and therapeutic implications. J Virol. 2007;81(19):10209–19. 2. Sabin CA, Lundgren JD. The natural history of HIV infection. Curr Opin HIV AIDS. 2013;8(4):311–7. 3. Kumar P. Long term non-progressor (LTNP) HIV infection. Indian J Med Res. 2013;138(3):291–3. 4. Sacktor N. The epidemiology of human immunodeficiency virus-associated neurological disease in the era of highly active antiretroviral therapy. J Neuro-Oncol. 2002;8(Suppl 2):115–21. Supplementary information Supplementary information accompanies this paper at https://doi.org/10. 1186/s12883-020-01713-4. Supplementary information Supplementary information accompanies this paper at https://doi.org/10. 1186/s12883-020-01713-4. Supplementary information Supplementary information accompanies this paper at https://doi.org/10. 1186/s12883-020-01713-4. 5. McArthur JC, Brew BJ, Nath A. Neurological complications of HIV infection. Lancet Neurol. 2005;4(9):543–55. 6. Croucher A, . Winston, A. Neurological complications of HIV. HIV and AIDS 2013;41(8):450–455. 7. Price RW. Neurological complications of HIV infection. Lancet. 1996; 348(9025):445–52. Additional file 1. Supplementary Material: search terms 8. Drake KW, Adam RD. Coccidioidal meningitis and brain abscesses: analysis of 71 cases at a referral center. Neurology. 2009;73(21):1780–6. Competing interests All h f h It is not possible to reliably determine the risks and bene- fits of VPS surgery in patients with HIV on the basis of the currently available literature. All studies of outcomes following VPS for HIV-associated CM are weak, with only case-series data available. Studies of survival and/or out- come following VPS for HIV-associated CM and TBM are either weak or of moderate quality. This included three cohort studies of patients with TBM, at differing risk of bias. Included studies indicate that only a very low to low quality evidence base exists on which to inform clinical decision making regarding VPS insertion for HIV-infected patients. This is reflected in weak recommendations in existing clinical guidelines for neurosurgical practice in HIV-infected patients [87, 90, 104, 105]. In the first in- stance, a rigorously conducted prospective, population based observational study of outcome and survival in HIV-associated hydrocephalus should be conducted. This is an urgent research priority and would inform design of future randomised clinical trials. All authors confirm that they have no competing interests and have received no financial support in the production of this research. Received: 27 March 2019 Accepted: 31 March 2020 Received: 27 March 2019 Accepted: 31 March 2020 Funding f d No funding was obtained for the conduct of this study. Publication charges were funded by the University of Cape Town. Author details 1 1Centre for Clinical Brain Sciences and Centre for Discovery Brain Sciences, Chancellor’s Building, 49 Little France Crescent, Edinburgh EH16 4SB, UK. 2Edinburgh Medical School, Chancellor’s Building, 49 Little France Crescent, Edinburgh EH16 4SB, UK. 3Division of Neurosurgery, University of Cape Town, H53 Old Main Building, Groote Schuur Hospital, Main Road, Observatory, Cape Town 7925, South Africa. 4Institute of Infectious Disease and Molecular Medicine, Faculty of Health Sciences, University of Cape Town, Anzio Road, Observatory, Cape Town 7925, South Africa. Review limitations directly assessed the impact of ART provision and showed a dramatic, yet expected, as- sociation between ART provision and better outcomes We therefore propose that a large, multi-centre, pro- spective, population-based survival analysis of all pa- tients with HIV-infection and hydrocephalus be urgently conducted. This should include both surgically and non- surgically managed patients with hydrocephalus occur- ring in association of HIV, either as a consequence of HIV-infection or any other cause, not directly related to HIV-infection. Such a study should collect baseline clin- ical data – such as pre-admission ART use, neurological grade, CD4+ cell count, haemoglobin and CSF cell Page 14 of 16 Loan et al. BMC Neurology (2020) 20:141 Loan et al. BMC Neurology (2020) 20:141 Loan et al. BMC Neurology (2020) 20:141 count, protein and glucose, microbiological results and opening pressure – which are currently used to prognos- ticate and guide selection of surgical candidates. Ideally, all included patients would be matched to HIV non- infected controls, however identification of appropriate control subjects might be difficult for conditions such as CM, which infrequently affect HIV non-infected individ- uals [103]. This study would allow validation of these potential markers of prognosis and would allow identifi- cation of patient groups for which there may be equi- poise regarding the benefit and harms of VPS. Definition of such populations would provide a strong rationale for undertaking an appropriately powered randomised clin- ical trial, conducted in low-middle income settings. Ethics approval and consent to participate Ethics approval and consent to participate Ethical approval and consent were not sought for this study as no primary patient data was collected. pp p p Ethical approval and consent were not sought for this study as no primary patient data was collected. Availability of data and materials All study data and search strategy outputs will be made available on reasonable request to the corresponding author. All study data and search strategy outputs will be made available on reasonable request to the corresponding author. Acknowledgements Th h f ll 13. Loyse A, Wainwright H, Jarvis JN, et al. Histopathology of the arachnoid granulations and brain in HIV-associated cryptococcal meningitis: correlation with cerebrospinal fluid pressure. AIDS. 2010;24(3):405–10. The authors gratefully acknowledge for the assistance of Dr. Ann Shien Loo in translation of titles, abstracts and manuscripts written in Chinese languages. 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Local and global even-odd effects in prompt emission in fission

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1. Introduction distributions p(Z,A) are provided by the Zp model of Wahl [11–13]. Lately [1–5] our attention was turned to a subject, not yet investigated, namely proton (Z) and neutron (N) even- odd effects in prompt emission in fission. We have studied even-Z nuclei fissioning spontaneously or induced by thermal neutrons, like 233,235U(n,f), 234U(n,f), 239Pu(nth,f), 236,238,240,242,244Pu(SF), 252Cf(SF), because the extensive studies (e.g., [6–9]) regarding the Z even-odd effect in fragment distributions showed that the effect is most pronounced for this type of nuclei. We started to study the basic features of the even-odd effect in prompt emission quantities, e.g., the behaviour of different average quantities like q(Z), q(A) of even-Z and odd-Z fragmentations, the behaviour of the global Z even- odd effect in different total average quantities, defined as [1,2]: δ<q> = <q>even−Z −<q>odd−Z <q> (1) (1) This topic is of major importance for a more profound understanding of the nuclear fission process, for the determination of the fragment distributions (which depends on knowing with high accuracy the prompt emission data, including even-odd effects). where <q> even-Z, <q> odd-Z and <q> are any quantities corresponding to even-Z, to odd-Z and to all- Z fragmentations, respectively. Recently, we have investigated some particular aspects related to the even-odd effects in prompt emission [5], like: the periodicity of five mass units in average quantities as a function of fragment mass, the intrinsic even-odd effect of prompt emission, the local Z even-odd effect in prompt neutron multiplicity and TXE. The prompt emission calculations were done in the frame of the Point-by-Point (PbP) model (described in [10] and references therein). The primary results of the PbP model are the multi-parametric matrices of different quantities characterizing the fission fragments and the prompt emission, generally labelled as q(A,Z,TKE) (e.g., prompt neutron multiplicity ν(A,Z,TKE), prompt γ -ray energy Eγ (A,Z,TKE)). Average quantities as a function of Z (q(Z)), of A (q(A)), of TKE (q(TKE)) and total average <q> are obtained by averaging the PbP matrices over the fragment distributions Y(A,Z,TKE) in different ways (details are given in Refs. [1,2,10] and references therein). These distributions are constructed as Y(A,Z,TKE) = Y(A,TKE) p(Z,A) in which Y(A,TKE) are experimental distributions (usually reconstructed from the single ones Y(A), TKE(A) and σTKE(A)) and the isobaric charge Georgiana Giubega1,2,a, Anabella Tudora1, and Franz-Josef Hambsch3 1 University of Bucharest, Faculty of Physics, Bucharest Magurele POB MG-11, 077125, Romania 2 National Institute for Laser, Plasma and Radiation Physics, Bucharest Magurele POB MG-36, 077125, Romania 3 EC-JRC Directorate G – Nuclear Safety and Security, Retieseweg 111, 2440, Geel, Belgium 1 University of Bucharest, Faculty of Physics, Bucharest Magurele POB MG-11, 077125, Romania 2 National Institute for Laser, Plasma and Radiation Physics, Bucharest Magurele POB MG-36, 077125, Romania 3 EC-JRC Directorate G – Nuclear Safety and Security, Retieseweg 111, 2440, Geel, Belgium Abstract. The investigation of the proton even-odd effects in prompt emission in fission for even-Z actinides revealed basic features of the global even-odd effect in prompt emission similar with those in fission fragment yields and some particular aspects, such as: (1) the even-odd effects in prompt emission are the result of two contributions: a dominant intrinsic even-odd effect due to the even-odd nuclear character of fragments reflected in their properties and a weak even-odd effect caused by the fragment distributions (over which the multi-parametric matrices are averaged); (2) oscillations with a periodicity of about 5 mass units are present in different prompt emission quantities corresponding to even-Z and odd-Z fragmentations independent on the size of the even-odd effect in the charge yield Y(Z). These oscillations are due to the periodicity of nuclear properties of fragments; (3) a local even-odd effect in prompt emission quantities has been recently investigated. Similarities between prompt emission quantities and fragment yields were found in the case of the local even-odd effect, too. The local even-odd effect in both fragment charge yields and prompt emission quantities exhibit a pronounced increase at asymmetry values corresponding to fragmentations in which the heavy fragment (Z = 50 and/or N = 82) or the light one (Z = 28) is magic. c⃝The 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/). a e-mail: giubega [email protected] EPJ Web of Conferences 146, 04039 (2017) ND2016 EPJ Web of Conferences 146, 04039 (2017) ND2016 EPJ Web of Conferences 146, 04039 (2017) ND2016 DOI: 10.1051/epjconf/201714604039 c⃝The 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/) 2. Basic features of the global Z even-odd effect in prompt emission 80 90 100 110 120 130 140 150 160 170 0 1 2 3 4 5 6 7 80 90 100 110 120 130 140 150 160 0 1 2 3 4 5 6 7 IRMM Hambsch et al. all Z even Z odd Z 252Cf(SF) Y (%) A IRMM Straede et al. all Z even Z odd Z 235U(nth,f) Y (%) 30 32 34 36 38 40 42 44 46 48 50 52 54 56 58 60 62 0,0 0,5 1,0 1,5 2,0 2,5 3,00 5 10 15 20 25 PbP results Prompt neutron multiplicity Z 235U(nth,f) δY(Z) = 21.89% Y (%) Figure 1. 235U(nth,f): Y(Z) projection (upper part) and prompt neutron multiplicity as a function of Z (lower part). 30 32 34 36 38 40 42 44 46 48 50 52 54 56 58 60 62 0,0 0,5 1,0 1,5 2,0 2,5 3,00 5 10 15 20 25 PbP results Prompt neutron multiplicity Z 235U(nth,f) δY(Z) = 21.89% Y (%) Z Figure 1. 235U(nth,f): Y(Z) projection (upper part) and prompt neutron multiplicity as a function of Z (lower part). A Figure 3. Y(A) of even-Z fragmentations (red circles) and odd-Z fragmentations (blue diamonds), Y(A) of all fragmenta- tions (open green squares) and experimental Y(A) data (full black squares) for the fissioning systems 235U(nth,f) (upper part) and 252Cf(SF) (lower part). 120 130 140 150 160 170 180 190 0 5 10 15 20 25110 120 130 140 150 160 170 180 190 200 210 0 5 10 15 20 25 242Pu(SF) 244Pu(SF) 236Pu(SF) 238Pu(SF) 240Pu(SF) Z even-odd effect of <ν>(TKE) (%) TKE (MeV) 235U(nth,f) 233U(nth,f) 239Pu(nth,f) 252Cf(SF) Figure 2. Z even-odd effect in <ν>(TKE) for: 235,233U(nth,f), 239Pu(nth,f), 252Cf(SF) (uppert part) [1] and 236Pu(SF), 238Pu(SF), 240Pu(SF), 242Pu(SF) and 244Pu(SF) (lower part) [2]. 120 130 140 150 160 170 180 190 0 5 10 15 20 25110 120 130 140 150 160 170 180 190 200 210 0 5 10 15 20 25 242Pu(SF) 244Pu(SF) 236Pu(SF) 238Pu(SF) 240Pu(SF) Z even-odd effect of <ν>(TKE) (%) TKE (MeV) 235U(nth,f) 233U(nth,f) 239Pu(nth,f) 252Cf(SF) rms(A) of the isobaric charge distribution p(Z,A), well described by the Zp model of Wahl [11–13], in the asymmetric fission region [6]. G¨onnenwein has made a connection between the oscillations of Z(A) and rms(A) with a period A ≈5 and the presence of even-odd effects in charge yield Y(Z) [6]. 2. Basic features of the global Z even-odd effect in prompt emission (3) the Z even-odd effect increases with increasing kinetic energy of the fission fragments. In the case of prompt emission, this fact is emphasized by the following function: δ<q>(T K E) = (<q> e(T K E)- <q> o(T K E))/<q>(T K E) introduced in Ref. [1]. An example of this function is given in Fig. 2 for <ν>(TKE) of 233,235U(nth,f), 239Pu(nth,f) and 252Cf(SF) (upper part) and 236,238,240,242,244Pu(SF) (lower part). In the case of Z(A) and rms(A) the magnitude of their oscillation amplitudes is proportional with the size of the even-odd effect in Y(Z). At limit, zero amplitude, i.e., no oscillation of Z(A) and rms(A), means no even-odd effect in Y(Z) (details are given in Ref. [5]) 2. Basic features of the global Z even-odd effect in prompt emission The main features of the global Z even-odd effect in prompt emission [1–3] are similar with those in fission fragment charge yields Y(Z) [6–9]: (1) the global Z even-odd effect in prompt emission decreases with increasing mass of the fissioning nucleus, e.g., from 9% (233,235U(nth,f)) to about 6% (252Cf(SF)). The global Z even-odd effect in Y(Z) decreases from about 21% (236U) to 4% (252Cf(SF)). (1) the global Z even-odd effect in prompt emission decreases with increasing mass of the fissioning nucleus, e.g., from 9% (233,235U(nth,f)) to about 6% (252Cf(SF)). The global Z even-odd effect in Y(Z) decreases from about 21% (236U) to 4% (252Cf(SF)). EPJ Web of Conferences 146, 04039 (2017) ND2016 EPJ Web of Conferences 146, 04039 (2017) ND2016 DOI: 10.1051/epjconf/201714604039 30 32 34 36 38 40 42 44 46 48 50 52 54 56 58 60 62 0,0 0,5 1,0 1,5 2,0 2,5 3,00 5 10 15 20 25 PbP results Prompt neutron multiplicity Z 235U(nth,f) δY(Z) = 21.89% Y (%) Figure 1. 235U(nth,f): Y(Z) projection (upper part) and prompt neutron multiplicity as a function of Z (lower part). 15 20 25 ) (%) 235U(nth,f) 233U(nth,f) 239Pu(nth,f) 252 80 90 100 110 120 130 140 150 160 170 0 1 2 3 4 5 6 7 80 90 100 110 120 130 140 150 160 0 1 2 3 4 5 6 7 IRMM Hambsch et al. all Z even Z odd Z 252Cf(SF) Y (%) A IRMM Straede et al. all Z even Z odd Z 235U(nth,f) Y (%) Figure 3. Y(A) of even-Z fragmentations (red circles) and odd-Z fragmentations (blue diamonds), Y(A) of all fragmenta- tions (open green squares) and experimental Y(A) data (full black squares) for the fissioning systems 235U(nth,f) (upper part) and 252Cf(SF) (lower part). 80 90 100 110 120 130 140 150 160 170 0 1 2 3 4 5 6 7 80 90 100 110 120 130 140 150 160 0 1 2 3 4 5 6 7 IRMM Hambsch et al. all Z even Z odd Z 252Cf(SF) Y (%) A IRMM Straede et al. all Z even Z odd Z 235U(nth,f) Y (%) Figure 3. Y(A) of even-Z fragmentations (red circles) and odd-Z fragmentations (blue diamonds), Y(A) of all fragmenta- tions (open green squares) and experimental Y(A) data (full black squares) for the fissioning systems 235U(nth,f) (upper part) and 252Cf(SF) (lower part). 2. Basic features of the global Z even-odd effect in prompt emission g y ( ) In the case of Z(A), rms(A) and Y(A) of even-Z and odd-Z fragments, only the magnitude of the oscillations amplitudes is related to the size of the even-odd effect in Y(Z). This fact can be seen in the example given in Fig. 3 where Y(A) is plotted separately for even-Z (red circles), odd-Z (blue diamonds) and all-Z fragmentations (black squares) for two fisioning nuclei (the extreme fissioning systems in terms of the size of the even-odd effect in Y(Z)): 235U(nth,f) (upper part) and 252Cf(SF) (lower part). It can be observed that Y(A) of even-Z and odd-Z fragmentations oscillate in anti-phase with a period A ≈5. It can be also seen that in the case of 235U(nth,f) (for which the global even-odd effect in Y(Z) is high, of about 22%) the amplitudes of the oscillations are visibly higher for even-Z fragmentations than for odd-Z ones while for 252Cf(SF) (with a lower δY(Z), of about 4%) the amplitudes of the oscillations are almost equal. Thus, higher amplitudes of Y(A) of even-Z fragmentations compared to those of odd-Z fragmentations means the presence of the even-odd effect in Y(Z). At limit, equal amplitudes in anti- phase cancel the even-odd effect in Y(Z). TKE (MeV) Figure 2. Z even-odd effect in <ν>(TKE) for: 235,233U(nth,f), 239Pu(nth,f), 252Cf(SF) (uppert part) [1] and 236Pu(SF), 238Pu(SF), 240Pu(SF), 242Pu(SF) and 244Pu(SF) (lower part) [2]. (2) prompt neutron multiplicity as a function of Z, ν(Z), exhibit a visible staggering in the asymmetric fission region as Y(Z) does. This fact can be seen in the examples given in Fig. 1. (2) prompt neutron multiplicity as a function of Z, ν(Z), exhibit a visible staggering in the asymmetric fission region as Y(Z) does. This fact can be seen in the examples given in Fig. 1. (2) prompt neutron multiplicity as a function of Z, ν(Z), exhibit a visible staggering in the asymmetric fission region as Y(Z) does. This fact can be seen in the examples given in Fig. 1. (3) the Z even-odd effect increases with increasing kinetic energy of the fission fragments. In the case of prompt emission, this fact is emphasized by the following function: δ<q>(T K E) = (<q> e(T K E)- <q> o(T K E))/<q>(T K E) introduced in Ref. [1]. An example of this function is given in Fig. 2 for <ν>(TKE) of 233,235U(nth,f), 239Pu(nth,f) and 252Cf(SF) (upper part) and 236,238,240,242,244Pu(SF) (lower part). 3. Particular aspects related to even-odd effects in prompt emission Regarding the prompt emission quantities, the os- cillations with the period A ≈5 persists even when fragment distributions without even-odd effects are used to obtain different average quantities. This fact was shown in Ref. [5], where relevant quantities for prompt emission, such as the energy release (Q-value) and the total excitation energy of fully accelerated fragments We have seen in our studies that average quantities as a function of A corresponding to even-Z and to odd-Z fragmentations exhibit oscillations with a periodicity of about 5 mass units [1–5]. The same periodicity was seen in experimental data of charge polarization Z(A) and the root-mean-square 2 DOI: 10.1051/epjconf/201714604039 EPJ Web of Conferences 146, 04039 (2017) 2016 EPJ Web of Conferences 146, 04039 (2017) ND2016 28 32 36 40 44 48 52 56 60 64 68 0 5 10 15 20 28 32 36 40 44 48 52 56 60 64 0 5 10 15 20 25 ∆Z=|0.5|, rms=0.6 δY(Z) = (0.069+-0.005)% 252Cf(SF) ∆Z(A), rms(A) δY(Z) = (4.775+-0.005)% Y (%) Z ∆Z=|0.5|, rms=0.6 δY(Z) = (0.33+-0.04)% 235U(nth,f) ∆Z(A), rms(A) δY(Z) = (22.32+-0.04)% Y (%) Figure 4. Y(Z) projections obtained in two cases of using Z and rms: Z = |0.5| and rms = 0.6 (navy squares), Z(A) and rms(A) (red stars) [5]. 120 125 130 135 140 145 150 155 160 150 160 170 180 190 200 150 160 170 180 190 200 235U(nth,f) without Z even-odd effect (∆Z=|0.5|, rms=0.6) all Z even Z odd Z δ = 1.26 % Q (MeV) AH 235U(nth,f) with Z even-odd effect (∆Z(A), rms(A)) all Z even Z odd Z δ = 1.28 % Q (MeV) Figure 5. 235U(nth,f): average Q(A) obtained by averaging Q(A,Z) over Y(A,Z,TKE) with Z even-odd effect (upper part) and another one without Z even-odd effects (lower part) [5]. 40 45 50 55 252Cf(SF) with Z even-odd effect (∆Z(A), rms(A)) all Z even Z odd Z MeV) 28 32 36 40 44 48 52 56 60 64 68 0 5 10 15 20 28 32 36 40 44 48 52 56 60 64 0 5 10 15 20 25 ∆Z=|0.5|, rms=0.6 δY(Z) = (0.069+-0.005)% 252Cf(SF) ∆Z(A), rms(A) δY(Z) = (4.775+-0.005)% Y (%) Z ∆Z=|0.5|, rms=0.6 δY(Z) = (0.33+-0.04)% 235U(nth,f) ∆Z(A), rms(A) δY(Z) = (22.32+-0.04)% Y (%) Figure 4. 3. Particular aspects related to even-odd effects in prompt emission 130 135 140 145 150 155 160 165 170 25 30 35 40 45 50 25 30 35 40 45 50 55 252Cf(SF) without Z even-odd effect (∆Z=|0.5|, rms=0.6) all Z even Z odd Z δ = 5.7% TXE (MeV) AH 252Cf(SF) with Z even-odd effect (∆Z(A), rms(A)) all Z even Z odd Z δ = 6% TXE (MeV) Figure 4. Y(Z) projections obtained in two cases of using Z and rms: Z = |0.5| and rms = 0.6 (navy squares), Z(A) and rms(A) (red stars) [5]. (TXE), were averaged over two types of Y(A,Z,TKE) distributions: one with even-odd effects (constructed by taking in the Gaussian expression of p(Z,A) oscillating Z(A) and rms(A)) and one without even-odd effects (constructed by considering for all fragments Z = |0.5| and the same value of 0.6 for the rms of p(Z,A)). In Fig. 4 are given examples of Y(Z) projections for 235U(nth,f) (upper part) and 252Cf(SF) (lower part), obtained in the two cases of Y(A,Z,TKE) distributions. As it can be seen, the even-odd effect in Y(Z) disappears when constant Z and rms are used, being reflected in the lack of Y(Z) staggering and in almost equal to zero global Z even-odd effect (given in the legend) [5]. Figure 6. 252Cf(SF): average TXE(A) obtained by averaging TXE(A,Z,TKE) over Y(A,Z,TKE) with Z even-odd effect (upper part) and without Z even-odd effects (lower part) [5]. Examples of Q(A) and TXE(A), obtained by averaging over the two types of distributions, are plotted in Fig. 5 (235U(nth,f)) and 6 252Cf(SF)), respectively. and (ii) a weak even-odd effect caused by the fragment distributions. For both fissioning systems Q(A) and TXE(A) of even-Z and odd-Z fragmentations exhibit oscillations with a periodicity of about 5 mass units in both cases of Y(A,Z,TKE) (with or without even-odd effect). This fact proves that the periodicity of these oscillations is independent of the existence or not of an even-odd effect in charge distributions, being a consequence of the periodicity in the nuclear properties of the fragments (e.g., mass excesses, binding energies, pairing energies). The dominance of the intrinsic even-odd effect was also demonstrated by the even-odd nucleus 234U(n,f) at 14 incident neutron energies ranging from 0.2 MeV to 5 MeV (see Ref. [4]). 3. Particular aspects related to even-odd effects in prompt emission Y(Z) projections obtained in two cases of using Z and rms: Z = |0.5| and rms = 0.6 (navy squares), Z(A) and rms(A) (red stars) [5]. 120 125 130 135 140 145 150 155 160 150 160 170 180 190 200 150 160 170 180 190 200 235U(nth,f) without Z even-odd effect (∆Z=|0.5|, rms=0.6) all Z even Z odd Z δ = 1.26 % Q (MeV) AH 235U(nth,f) with Z even-odd effect (∆Z(A), rms(A)) all Z even Z odd Z δ = 1.28 % Q (MeV) Figure 5. 235U(nth,f): average Q(A) obtained by averaging Q(A,Z) over Y(A,Z,TKE) with Z even-odd effect (upper part) and another one without Z even-odd effects (lower part) [5]. 28 32 36 40 44 48 52 56 60 64 68 0 5 10 15 20 28 32 36 40 44 48 52 56 60 64 0 5 10 15 20 25 ∆Z=|0.5|, rms=0.6 δY(Z) = (0.069+-0.005)% 252Cf(SF) ∆Z(A), rms(A) δY(Z) = (4.775+-0.005)% Y (%) Z ∆Z=|0.5|, rms=0.6 δY(Z) = (0.33+-0.04)% 235U(nth,f) ∆Z(A), rms(A) δY(Z) = (22.32+-0.04)% Y (%) 120 125 130 135 140 145 150 155 160 150 160 170 180 190 200 150 160 170 180 190 200 235U(nth,f) without Z even-odd effect (∆Z=|0.5|, rms=0.6) all Z even Z odd Z δ = 1.26 % Q (MeV) AH 235U(nth,f) with Z even-odd effect (∆Z(A), rms(A)) all Z even Z odd Z δ = 1.28 % Q (MeV) Figure 5. 235U(nth,f): average Q(A) obtained by averaging Q(A,Z) over Y(A,Z,TKE) with Z even-odd effect (upper part) and another one without Z even-odd effects (lower part) [5]. Z 130 135 140 145 150 155 160 165 170 25 30 35 40 45 50 25 30 35 40 45 50 55 252Cf(SF) without Z even-odd effect (∆Z=|0.5|, rms=0.6) all Z even Z odd Z δ = 5.7% TXE (MeV) AH 252Cf(SF) with Z even-odd effect (∆Z(A), rms(A)) all Z even Z odd Z δ = 6% TXE (MeV) Figure 6. 252Cf(SF): average TXE(A) obtained by averaging TXE(A,Z,TKE) over Y(A,Z,TKE) with Z even-odd effect (upper part) and without Z even-odd effects (lower part) [5]. 4. Local even-odd effect in prompt emission in fission The values of the global Z even-odd effect in <TXE>, (and <Q>), given in the legends of Figs. 5 and 6, are almost the same in both cases of fragment distributions (with and without Z even-odd effect). This fact together with the important role played by TXE in the prompt emission has demonstrated that the even-odd effects in different quantities related to the prompt emission are mainly due to the nuclear properties of fission fragments. Consequently, the even-odd effects in prompt emission are the result of two contributions [4,5]: (i) a dominant intrinsic even-odd effect due to the even-odd nuclear character of fragments reflected in their properties (and consequently in the emitted prompt neutrons and γ -rays) The behavior of different quantities corresponding to the four possible types of fragmentation of a fissioning nucleus (i.e., even-even, even-odd, odd-even and odd-odd for an even-even fissioning nucleus) [1–5] suggested the possibility of defining, for the first time, a local even-odd effect in prompt emission quantities (generally labelled “q”), as [5]: δp <q> = 1 2 <q>even−Z −<q>odd−Z <q>even−Z + <q>odd−Z (2) (2) where <q>even-Z and <q>odd-Z are normalized quantities corresponding to even-Z and to odd-Z fragmentations. 3 EPJ Web of Conferences 146, 04039 (2017) 6 EPJ Web of Conferences 146, 04039 (2017) ND2016 DOI: 10.1051/epjconf/201714604039 0,00 0,05 0,10 0,15 0,20 0,25 0,30 0,35 0,40 0,45 0,0 0,2 0,4 0,6 0,8 local even-odd effect in TXE in νpair PbP calculation 5 Z per A, Y(A,TKE) Straede excluding TXE < 9 MeV 235U(nth,f) exp. data Caamano et al. local even-odd effect asymmetry Figure 7. Local even-odd effect in TXE (red circles) and prompt neutron multiplicity of fragment pair (blue squares) for 235U(nth,f) in comparison with the experimental data of the local even-odd effect in fragment distributions of Caamano et al. [9] (black squares) plotted as a function of asymmetry parameter [5]. 0,00 0,05 0,10 0,15 0,20 0,25 0,30 0,35 0,40 0,45 0,0 0,2 0,4 0,6 0,8 local even-odd effect in TXE in νpair PbP calculation 5 Z per A, Y(A,TKE) Straede excluding TXE < 9 MeV 235U(nth,f) exp. data Caamano et al. local even-odd effect asymmetry to the periodicity of nuclear properties of fragments, being independent of the presence or not of the even-odd effect in the charge yield Y(Z). References [1] A. Tudora, F.-J. Hambsch, G. Giubega, I. Visan, Nucl. Phys. A. 929, 260–292 (2014) In Fig. 7 are given examples of local even-odd effect in TXE (red circles) and prompt neutron multiplicity of fragment pair (blue squares) as a function of asymmetry parameter, defined as as = (Z H −ZL)
Z0, for 235U(nth,f). δp<νpair> is a little bit higher than δp<TXE> fact confirmed by the slightly higher global even-odd effect for <ν> compared to <TXE> [1]. An interesting observation is that the local even-odd effect in both TXE and νpair exhibits a similar behaviour as the experimental data of Caama˜no et al. [14] (black squares) concerning the local even-odd effect in fragment distributions [5]. y [2] A. Tudora, F.-J. Hambsch, G. Giubega, I. Visan, Nucl. Phys. A 933, 165–188 (2015) [3] A. Tudora, F.-J. Hambsch, G. Giubega, I. Visan, Phys. Proc. 64, 62–72 (2015) [4] A. Tudora, F.-J. Hambsch, G. Giubega, I. Visan, Rom. Rep. Phys. 68(2), 571–581 (2016) [5] A. Tudora, F.-J. Hambsch, G. Giubega, Eur. Phys. J. A 52, 182–193 (2016) [6] C. Wagemans The Nuclear Fission Process (CRC Press, BocaRaton, chapter 8, 1991) The pronounced increase of the local even-odd effect at asymmetry values corresponding to fragmentations in which one of the fragment is magic or double magic (seen in Fig. 7 at asymmetry values of about 0.7 corresponding to fragmentations with magic heavy fragment (NH = 82) and around 0.4 corresponding to very asymmetric fragmentations in which the light fragment is magic (ZL = 28)) is a consequence of the important role played by the fragment properties reflecting the even-odd nuclear character of fragments (i.e., the contribution of the intrinsic even-odd effect). [7] F. G¨onnenwein, Physics Procedia 47, 107–114 (2013) [8] B.L. Tracy, J. Chaumont, R. Klapisch, J.M. Nitschke, A.M. Paskanzer, E. Raeckl, C. Thibault, Phys. Rev. C 5(1), 222–234 (1972) [9] M. Caama˜no, F. Rejmund, K.H. Schmidt, J. Phys. G. Nucl: Part. Phys. 38, 035101 (2011) [10] R. Capote, Chen Y.J., F.-J. Hambsch, N. Kornilov, J.P. Lestone, O. Litaize, B. Morillon, D. Neudecker, S. Oberstedt, N. Otuka, V.G. Pronyaev, A. Saxena, O. Serot, O.A. Scherbakov, Shu N.C., D.L. Smith, P. Talou, A. Trkov, A.C. Tudora, R. Vogt and A.S. Vorobyev, Nucl. Data Sheets 131, 1 (2016) O. Serot, O.A. Scherbakov, Shu N.C., D.L. Smith, 4. Local even-odd effect in prompt emission in fission The even-odd effect in prompt emission quantities is the result of two contributions: a dominant intrinsic even- odd effect due to the nuclear properties of fragments and a weaker even-odd effect brought by the fragment distributions (over which the multi-parametric matrices are averaged). The local even-odd effect in TXE and prompt neutron multiplicity exhibits the same behavior as the local even- odd effect in fragment yields. The feature of the local even-odd effect, consisting in a pronounced increase at asymmetry values corresponding to fragmentations in which the heavy fragment (Z = 50 and/or N = 82) or the light one (Z = 28) is magic, is present in both the charge yield and prompt emission quantities. Figure 7. Local even-odd effect in TXE (red circles) and prompt neutron multiplicity of fragment pair (blue squares) for 235U(nth,f) in comparison with the experimental data of the local even-odd effect in fragment distributions of Caamano et al. [9] (black squares) plotted as a function of asymmetry parameter [5]. 5. Conclusions The basic features of the even-odd effect in prompt emission are similar with those in fragment yields. [11] A.C. Wahl, Atomic Data and Nuclear Data Tables 39, 1–156 (1988) [12] A.C. Wahl, Compilation and evaluation of fission yield nuclear data (Final report of CRP 1991–1996) The periodicity A ≈5 of the oscillations in the charge polarization Z(A), rms(A) of the isobaric charge distribution, as well as in the fragment mass yields Y(A) and different quantities related to the prompt emission corresponding to even-Z and odd-Z fragmentations are due [13] A.C. Wahl, Fission Product Yield Data for the Transmutation of Minor Actinide Nuclear Waste (IAEA STI/PUB/1286, 2008) 4

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Neovascular glaucoma: a review

International journal of retina and vitreous

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© The Author(s) 2016. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Abstract Neovascular glaucoma (NVG) is a secondary glaucoma generally associated with poor visual prognosis. The devel‑ opment of new vessels over the iris and the iridocorneal angle can obstruct aqueous humor outflow and lead to increased intraocular pressure. The underlying pathogenesis in most cases is posterior segment ischemia, which is most commonly secondary to proliferative diabetic retinopathy or central vein retinal occlusion. The neovasculariza‑ tion process in the eye is driven by the events that alter the homeostatic balance between pro-angiogenic factors, such as the vascular endothelial growth factor and anti-angiogenic factors, such as the pigment-epithelium-derived factor. Early diagnosis of this condition through slit lamp examination of the iris, iridocorneal angle and retina can help to avoid the development of goniosynechia and obstruction of aqueous humor outflow, with consequent intraocular pressure elevation. Historically, NVG treatment was focused on reducing the posterior segment ischemic process that caused the formation of new vessels, through panretinal photocoagulation. Recently, several studies have investi‑ gated the application of intravitreal anti-VEGF therapies in NVG. If clinical treatment with the use of hypotensive topi‑ cal drops is not sufficient, laser and/or surgical procedures are required for intraocular pressure control. Keywords:  Neovascular glaucoma, Refractory, Anti-VEGF, Diabetes, Central retinal vein occlusion light perception in a tertiary hospital in Brazil. The inci- dence of NVG was similar between genders, with slight higher prevalence of men. It more commonly affects the elderly. It was observed that 46.16 % of the patients were between 60 and 79 years of age at onset and 30.68 % were over the age of 80. NVG usually requires not only medi- cation, but also surgical procedures in order to control IOP. The cost of this treatment, both clinical and surgical is often high. In fact, a study in a tertiary hospital in Bra- zil showed that glaucoma treatment may consume up to 30 % of a family income [3]. Neovascular glaucoma: a review Gustavo B. Rodrigues1*  , Ricardo Y. Abe1, Camila Zangalli1, Savio L. Sodre1, Flavia A. Donini1, Danilo C. Costa1, Andre Leite1, Joao P. Felix1, Marcelo Torigoe1, Alberto Diniz‑Filho2 and Homero Gusmão de Almeida2 Introduction Neovascular glaucoma (NVG) is a potentially blinding secondary glaucoma, characterized by the development of neovascularization of the iris, elevated intraocular pressure (IOP) and, in many instances, poor visual prog- nosis. In the past, it used was referred to as congestive glaucoma, rubeotic glaucoma or diabetic hemorrhagic glaucoma. In 1963 Weiss and colleagues, proposed the term NVG [1]. Coats first described the histological find- ings of new vessels on the iris on a patient with central retinal vein occlusion. With the introduction of clinical gonioscopy, the visualization of new vessels in the angle was possible and the origin of elevated IOP was explained by the closure of the iridocorneal angle [1]. There is a high rate of severe visual loss associated with the dis- ease and final visual acuities of hand movements or light perception is not uncommon [1]. Vasconcellos et al. [2] found around 70 % of NVG patients had visual acuity of *Correspondence: [email protected] 1 Department of Ophthalmology, Faculdade de Ciências Médicas ‑ UNICAMP, University of Campinas, Caixa Postal ‑ 6111, Campinas, SP 13083‑970, Brazil Full list of author information is available at the end of the article Rodrigues et al. Int J Retin Vitr (2016) 2:26 DOI 10.1186/s40942-016-0051-x Rodrigues et al. Int J Retin Vitr (2016) 2:26 DOI 10.1186/s40942-016-0051-x International Journal of Retina and Vitreous International Journal of Retina and Vitreous Open Access © The Author(s) 2016. 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. Etiology and diagnosish invariably develops before the intraocular pressure rises [5]. The most common causes of NVG are central vein retinal occlusion, proliferative diabetic retinopathy and ocular ischemic syndrome, and central retinal artery obstruc- tion [21]. Neovascularization is a multi-step process that involves complex interactions of a variety of angiogenic actors. New vessel formation in the eye is affected to a large extent by an unbalance between pro-angiogenic factors (such as, vascular endothelial growth factor-VEGF) and other anti-angiogenic factors (such as pigment-epithe- lium-derived factor) [6]. In Table  1, the conditions that can lead to NVG are summarized and divided by common causes [22–25], uncommon causes, such as those related to ocular tumors [26–30], systemic diseases [31–37] and other rare diseases that can lead to NVG [38–41]. The diagno- sis of NVG is clinical and requires detailed patient’s his- tory and a complete ophthalmological examination. Case history is important to determine the origin of ischemia. Patients may be asymptomatic, especially when the IOP rise occurs slowly, or they can present with symptoms such as low vision, ocular pain and photophobia. In the early stages, exam findings can be subtle, requiring the ophthalmologist to maintain high index of suspicion in face of conditions that are commonly associated with NVG such as diabetic retinopathy, central retina vein occlusion or ocular ischemic syndrome [4, 5]. VEGF plays a major part in mediating active intraocu- lar neovascularization in patients with ischemic retinal diseases [7]. VEGF and insulina growth-1 factors are pro- duced locally in the human eye by a variety of cells includ- ing Mueller cells, retinal pigment epithelial cells, retinal capillary pericytes, endothelial cells and ganglion cells [8]. VEGF is sufficient to produce iris neovasculariza- tion in a nonhuman primate [9]. Neovascularization was consistent with increased of insulin growth-1 factor and induction of VEGF expression in retinal glial cells. Insu- lin growth-1 factor accumulated in aqueous humor may cause rubeosis iridis and subsequently adhesions between the cornea and iris may block aqueous humor drainage [10]. Concentration of VEGF can decline after the regres- sion of iris neo-vessels [11]. The non-pigmented cili- ary epithelium is an important site of VEGF synthesis in patients with NVG. In fact, a recent study considered the ciliary epithelium as an additional focus of treatment in the management of NVG, especially in eyes that were not responsive to panretinal photocoagulation (PRP) [12]. Pathogenesis N G NVG is a severe form of glaucoma attributed to new blood vessels obstructing aqueous humor outflow, sec- ondary to posterior segment ischemia [4]. It is associ- ated with the development of a fibrovascular membrane on the anterior surface of the iris and iridocorneal angle of anterior chamber [5]. Invasion of the anterior cham- ber by a fibrovascular membrane initially obstructs aque- ous outflow in an open-angle fashion and later contracts to produce secondary synechial angle-closure glaucoma with high IOP [4]. Iris and angle neo-vessels almost *Correspondence: [email protected] 1 Department of Ophthalmology, Faculdade de Ciências Médicas ‑ UNICAMP, University of Campinas, Caixa Postal ‑ 6111, Campinas, SP 13083‑970, Brazil Full list of author information is available at the end of the article Rodrigues et al. Int J Retin Vitr (2016) 2:26 Page 2 of 10 Etiology and diagnosish y In diabetic patients, the onset of NVG is generally correlated with poor glycemic control, leading to prolif- erative diabetic retinopathy and consequently, neovas- cularization of the anterior segment. Sudden painless visual loss occurring months before, in turn, would be typical related to NVG associated to central retina vein occlusion. NVG may appear between 2 weeks to 2 years after occlusion of the central retinal vein, but most com- monly appears after 3 months. History of occlusion of the carotid artery with elevated IOP in ipsilateral eye raises the suspicion of ocular ischemic syndrome. In cases of NVG after CRAO, the onset can be as early as 2 weeks after the onset of artery obstruction [42]. Others potential pro-angiogenic initiating factors have been investigated in previous studies. The inflamma- tory cytokine IL-6 concentration in aqueous humor was increased spatially and temporally correlated with the grade of neovascularization of the iris in patients of NVG secondary to central retina vein occlusion [13]. It was also found a possible involvement of basic fibroblast growth factor (bFGF) in the pathogenesis of anterior-segment dis- orders, such as NVG [14]. Furthermore, increased levels of transforming growth factor-beta 1 and -beta 2 [15], nitric oxide [16] and endothelin-1 [17] in the aqueous humor of patients with NVG was observed. Previous study also sug- gested a strong correlation with free-radicals such as the superoxide in the aqueous humor of NVG patients [18]. Etiology and diagnosish Table 1  Summary of conditions that can lead to neovascu- lar glaucoma Common causes Ocular tumors Central retinal vein occlusion Retinoplastoma Branch retinal vein occlusion Uveal melanomas Proliferative diabetic retinopathy Ciliary body medulloepithelioma Caroid arterial obstruction Vasoproliferative tumors of the retina Central retinal artery occlusion Ocular metastasis Systemic diseases Other causes Juvenile myelomonocytic leucemia Uveitis Systemic lupus erythematosus Purtscher’s retinopathy Juvenile xanthogranuloma Altered expression of aquaporins Cryoglobulinemia type 1 Familial amyloid polyneuropathy Neurofibromatosis type 1 Arteritis from cytomegalovirus retinitis Table 1  Summary of conditions that can lead to neovascu- lar glaucoma Common causes Ocular tumors Central retinal vein occlusion Retinoplastoma Branch retinal vein occlusion Uveal melanomas Proliferative diabetic retinopathy Ciliary body medulloepithelioma Caroid arterial obstruction Vasoproliferative tumors of the retina Central retinal artery occlusion Ocular metastasis Systemic diseases Other causes Juvenile myelomonocytic leucemia Uveitis Systemic lupus erythematosus Purtscher’s retinopathy Juvenile xanthogranuloma Altered expression of aquaporins Cryoglobulinemia type 1 Familial amyloid polyneuropathy Neurofibromatosis type 1 Arteritis from cytomegalovirus retinitis Table 1  Summary of conditions that can lead to neovascu- lar glaucoma Table 1  Summary of conditions that can lead to neovascu- lar glaucoma In  poorly controlled diabetic patients, with wide- spread posterior segment ischemia that goes un- recognized and untreated, progression from iris neovascularization to NVG is frequent and can occur after 12-month following the development of iris neo- vascularization [19] NVD is some diabetic eyes can take a more indolent course, and not immediately result in NVG. In patients with  ischemic central reti- nal vein occlusion, NVG occurs typically between 1.5 to 6 months after ischemic event [20]. Rodrigues et al. Int J Retin Vitr (2016) 2:26 Rodrigues et al. Int J Retin Vitr (2016) 2:26 Page 3 of 10 On physical examination, a careful examination of iris and anterior chamber angle is essential before pupil dila- tion for fundus evaluation. Anterior biomicroscopy can reveal: rubeosis iridis (neovessels are vessels that do not follow an organized growth pattern, while iris vessels usually grow radially symmetric), mild anterior chamber reaction, corneal edema due to sharp increase of IOP, cil- iary injection and uveal ectropion by contraction of the fibrovascular membrane over the iris (Fig. 1) [4, 5]. Rube- osis starts from the pupillary border with the appearance of tiny tufts of dilated capillaries (Fig. 2) or red spots that can’t be seen unless the iris is examined under high mag- nification. Etiology and diagnosish Rubeosis iridis is usually present, though not always, before neovascularization of the angle. In rare cases, there may be neovascularization of angle without neovascularization of the pupillary border, especially after ischemic central retinal vein occlusion. Therefore, it is important to perform gonioscopy even when the bor- der of the pupil is not involved. Initially, the iridocorneal angle appears open on goni- oscopy but with the progression of the disease, neoves- sels can appear over the angle structures (Fig. 3). In the final stages, peripheral anterior synechiae can occur and lead to complete angle closure (Fig. 4) [4, 5]. The IOP may be normal in the early stages of the disease, but usually goes to high levels in advanced stages of the disease when the angle is closed by the contraction of the fibrovascular membrane. On fundus examination, glaucomatous optic nerve damage may already be present depending on the duration of elevated IOP and its levels. Despite the clinical diagnosis, in some cases, a func- tional test such as the electroretinography can be used to differentiate between ischemic and non-ischemic forms of central retinal vein occlusion, helping to detect patients more prone to the development of neovasculari- zation of the iris [5]. Both interocular amplitude differ- ence of −23 microV and interocular amplitude ratio of 60  % were good cutoff points to differentiate ischemic from nonischemic central vein retinal occlusion [41] Iris angiography can also be useful in some borderline cases because it shows fluorescein leakage, which is not normally seen [5]. Although these tools can aid in early detection of neovascularization, they are expensive and not always available. In contrast, gonioscopy is a widely available e, fast and low cost procedure that can detect neovascularization of the angle. Retinal angiography may also help diagnosis elucidation, especially in cases of reti- nal vascular disorders and it can also guide the treatment with retinal photocoagulation. A Doppler ultrasound may be necessary to identify carotid stenosis if obvious retinal ischemia causes are not found [5]. Fig. 1  a Rubeosis iridis, b Anterior chamber in NVG ig 1 a Rubeosis iridis b Anterior chamber in NVG retinal ischemia causes are not found [5]. Fig. 1  a Rubeosis iridis, b Anterior chamber in NVG Fig. 2  Iris neovessels Fig. 3  a Iridocorneal angle neovessels, b Iridocorneal angle neoves‑ sels Fig. 3  a Iridocorneal angle neovessels, b Iridocorneal angle neoves‑ sels Fig. Medical treatmentshi This treatment has variable outcome depending on the underlying cause of NVG and also the stage in which the disease was diagnosed. For example, in diabetic retinopa- thy, after panretinal photocoagulation, complete regres- sion of retinal neovascularization can be reached in 67–77 % of cases, visual loss can be prevented in 59–73 % and IOP reduction can be achieved in 42 % of the cases [48]. If neovascularization persists, additional laser treat- ment can be performed until complete regression of the neovascularization. In such successfully treated cases of posterior segment neovascularization, anterior segment neovascularization almost never occurs. In central reti- nal vein occlusion patients, panretinal photocoagulation is indicated in the ischemic form of central retinal vein occlusion due to the high risk of NVG development [49]. Panretinal photocoagulation is also indicated in cases of iris, angle and retinal neovascularization.h The first step to prevent visual loss and relieve pain or discomfort associated with NVG is to lower the high IOP levels. One of the strategies of medical management of NVG consists of IOP-lowering agents, such as topi- cal β-adrenergic antagonists, α-2 agonists and topical or oral carbonic anhydrase inhibitors. These pharmacologic agents work by suppressing aqueous production and pos- sibly increasing uveoscleral outflow [5, 43]. Prostaglandin analogs should be avoided in order to prevent further breakdown of the blood-aqueous barrier with worsen- ing of the intraocular inflammation [44]. Pilocarpine and other anticholinergic agents are generally contrain- dicated, because they may increase inflammation, cause miosis, worsen synechial angle closure and decrease uve- oscleral outflow. Topical atropine may be used for cyclo- plegia and might even lower the pressure by increasing the uveoscleral outflow. Atropine also reduces the inci- dence of hyphema. Since some patients with NVG have some degree of intraocular inflammation, it may helpful to give topical corticosteroids to reduce any inflamma- tory component that may be present [45]. Oral carbonic anhydrase inhibitors, such as acetazolamide and met- hazolamide, can be prescribed when topical treatment is not enough to lower IOP [46, 47]. The treatment of NVG secondary to ocular ischemic syndrome should be multidisciplinary with the involve- ment of a cardiologist and/or vascular surgeon for carotid arteries imaging and possible carotid endarterec- tomy if indicated [24]. Photocoagulation is indicated in ocular ischemic syndrome patients with iris and poste- rior segment neovascularization to prevent development of secondary NVG. Medical treatmentshi However, is noteworthy to mention that uveal ischemia alone can be responsible for neo- vascularization and panretinal photocoagulation should be performed if fluorescein fundus angiography shows retinal ischemia due to retinal capillary obliteration [50]. Previous report has suggested that panretinal photoco- agulation alone can increase IOP and may compromise Etiology and diagnosish 3  a Iridocorneal angle neovessels, b Iridocorneal angle neoves‑ sels Fig. 1  a Rubeosis iridis, b Anterior chamber in NVG Fig. 2  Iris neovessels Fig. 2  Iris neovessels Fig. 2  Iris neovessels Page 4 of 10 Rodrigues et al. Int J Retin Vitr (2016) 2:26 Fig. 4  a Peripheral anterior synechiae, b peripheral anterior synechiae pigment-epithelium-derived factor [6]. Panretinal pho- tocoagulation remains the mainstay in controlling the neovascular drive and should be considered in all cases of NVG when retinal ischemia is present [5]. It is still believed to be helpful in eyes whose angles are already occluded by the new vessels. The procedure is character- ized by photocoagulation of the peripheral retina using a slit lamp or indirect laser with 1200–1600 burns and approximately 500 microns spot size. Panretinal photo- coagulation is commonl performed over 1–3 sessions. In cases of NVG, the sessions should be peformed as fast as possible. The procedure is usually performed under topi- cal anesthesia. If t topical anesthesia is insufficient, sub- conjunctival anesthesia or even peribulbar anesthesia can be performed. Panretinal photocoagulation is indicated not only in initial rubeosis, but also in late stages of NVG with goniosynechiae. In cases of cloudy media precluding transpupillary laser, consideration for PRP performed in the operating room during pars plana vitrectomy can be performed. Historically, pan retinal peripheral cryother- apy was done is such cases but this procedure is rarely done now. Fig. 4  a Peripheral anterior synechiae, b peripheral anterior synechiae Fig. 4  a Peripheral anterior synechiae, b peripheral anterior synechiae Vascular endothelial growth factor inhibitors Recently, use of anti-VEGF in the management of NVG has been extensively investigated [52]. Since 1996, sev- eral studies have been reporting VEGF as an important and predominant factor in the pathogenesis of neovascu- larization [9, 53]. VEGF inhibitors can stifle the neovas- cularization process secondary to retinal ischemia [54]. The administration of anti-VEGF is currently becoming established, supported by several studies suggesting bet- ter visual prognosis and IOP control following anti-VEGF injections [6, 55]. Anti-VEGF injections can lead to regression of both iris and angle neovascularization, and intraocular pressure control when the angle remains open [56]. However, the effects of anti-VEGF agents seemed to induce only a tem- porary regression of new vessels in the anterior chamber angle as well as IOP reduction, generally during between four to six weeks [6]. In the current review, we report some of the main results of some studies about use of anti-VEGF in the treatment of NVG. Yazdani et al. [57] investigated the effect of intravitreal bevacizumab on NVG in a randomized controlled trial with 26 eyes from 26 patients. All eyes received conven- tional treatment for NVG and were randomly allocated to three 2.5  mg intravitreal bevacizumab injections at 4-week intervals or a sham procedure. Authors concluded that intravitreal injections of bevacizumab reduced iris neovascularization and IOP in NVG and may be consid- ered as an adjunct to more definitive surgical procedures for NVG. In addition, Wittstrom et  al. investigated the effect of a single intravitreal injection of bevacizumab for NVG after ischemic central retinal vein occlusion [58]. In this study 19 eyes from 19 patients were randomly allo- cated to either an intravitreal bevacizumab injection and panretinal photocoagulation (10 eyes) or panretinal pho- tocoagulation alone (9 eyes). Their results suggested that intravitreal injection of bevacizumab might be valuable in the treatment of NVG by improving the resolution of neovascularization.fi i A systematic review by Simha et  al. [61] found that there is no evidence to evaluate statistically the effec- tiveness of anti-VEGF treatments, even as an adjunct to conventional treatment in reducing the IOP in NVG. More recently, Tang et al. [62] performed a prospective non-randomized study with 43 eyes of 43 neovascular glaucoma patients. Photocoagulation Th b f h The basis for the treatment of NVG is to reduce pos- terior segment ischemia and recover the homeo- static balance between pro-angiogenic factors such as VEGF and anti-angiogenic factor, such as the Rodrigues et al. Int J Retin Vitr (2016) 2:26 Page 5 of 10 addition there was a significant, though modest, best- corrected visual acuity improvement in intravitreal ranibizumab injection group. They also had less post- operative complications and lower failure ratio than Ahmed surgery. However, in a recent study conducted by Olmos et al. [60] intravitreal injection of bevacizumab was not superior than panretinal photocoagulation. The study was a retrospective, comparative, case series of 163 eyes of 151 patients with NVG, including 99 treated without and 64 treated with intravitreal bevacizumab. Medical and surgical treatments for NVG were assessed. They found that IOP decreased to 18.3  ±  13.8 mmHg in the non-bevacizumab group and 15.3  ±  8.0 mm Hg in the bevacizumab group. Panretinal photocoagula- tion substantially reduced the need for glaucoma sur- gery (P  <  0.001) in bevacizumab treated NVG eyes. Therefore, although bevacizumab delayed the need for glaucoma surgery, panretinal photocoagulation was the most important factor that reduced the need for surgery. Vision and IOP in eyes with NVG treated with bevaci- zumab showed no long-term differences when compared with eyes that were not treated with bevacizumab. Thus, intravitreal bevacizumab serves as an effective temporiz- ing treatment, but is not a replacement for close moni- toring and definitive treatment of NVG. optic nerve head circulation. Therefore surgical carotid endarterectomy would be the best treatment in these cases [51]. Trabeculectomy NVG h b Zhou et al. [64] conducted a systematic review to eval- uate the efficacy and tolerability of Ahmed glaucoma valve implantation with intravitreal bevacizumab injec- tion pretreatment in the treatment of NVG.h y NVG has been associated with high rates of failure after trabeculectomy [67, 68] but the adjunct use of antimetab- olites has improved the success rate of the surgery [69]. Sisto et al. [69] showed 55 % of success rate in a mean follow-up of 35  months with the use of postoperative 5-fluorouracil and 54 % of success rate in a mean follow- up 18  months with intraoperative mitomycin C. Still, compared to other types of glaucoma, NVG is a known risk factor for surgical failure [70]. Moreover, it has been suggested that a postoperative hyphema, a common complication in patients with NVG, may be associated with higher rates of trabeculectomy failure in NVG [71]. They found that the intravitreal bevacizumab group was associated with significant greater complete success rates compared with the control group. However, it did not show a significant difference for the qualified success rate between them. In addition, the intravitreal bevaci- zumab group was associated with a significantly lower frequency of hyphema than the control group.l More recently, newer anti-VEGF agents such as afliber- cept have also been used in the treatment of NVG [65]. Soohoo et al. reported a case series study with 4 newly diagnosed stage 1 or 2 NVG patients. They were treated with intravitreal aflibercept at the time of diagnosis, and repeated injections at 4, 8 and then every 8 weeks there- after up until 52 weeks after study initiation. They found that intravitreal aflibercept resulted in rapid regression of iris and angle neovascularization. IOP was stable or reduced in all patients at the 52-week study visit, sug- gesting that intravitreal aflibercept may be an effective treatment for stage 1 and 2 NVG, even though further research is needed to determine the full duration of effect and the optimal dose and timing of administration.f Glaucoma drainage devices Gl d d Glaucoma drainage devices are usually considered the first treatment option for refractory glaucoma. However, NVG patients are at greater risk for surgical failure after Ahmed glaucoma valve surgery compared with con- trols. Yalvac reported 63.2 and 56.2 % of success rates at 1 and 2 years after Ahmed glaucoma valve implantation, respectively [72]. Hernandez-Oteyza recently reported a success rate of 60 % at 1 year of follow-up and found that a hypertensive phase in the postoperative period and a worse preoperative BCVA to be risk factors for Ahmed valve surgical failure in patients with NVG [73]. Net- land et  al. found that the success rate was significantly lower over time in eyes with NVG compared with con- trols. They reported success rates at 5 years of 81.8 % for control and 20.6 % for patients with NVG [74]. Similar results have been reported with other types of glaucoma drainage devices [75–78]. Furthermore, there is no evi- dence of improved surgical outcomes with glaucoma drainage devices as opposed to augmented trabeculec- tomy. Similar results have been reported when treat- ment with Ahmed Glaucoma valve was compared to trabeculectomy with mitomycin C. Shen et al. reported success rates of 70 and 65 % at 1 year and 60 and 55 % at 2 years after Ahmed glaucoma valve and trabeculectomy with mitomycin C, respectively [79]. Therefore, proper control of retinal neovascularization in addition to either trabeculectomy with mitomycin C or glaucoma drainage device implantation seem appropriate treatment options for IOP control in NVG patients. In conclusion, there still a debate about the real effec- tiveness of anti-VEGF in the management of NVG. There is evidence showing that a pre-treatment with anti-VEGF before definitive IOP lowering glaucoma surgeries can significantly lower the frequency of hyphema. But further research is still needed to evaluate the impact on long- term IOP control, visual acuity and cost-effectiveness of the anti-VEGF injections in the management of NVG. It is also important to remember that continuous intravit- real anti-VEGF injections may cause both transient and sustained elevation in IOP [66]. Vascular endothelial growth factor inhibitors In this study, patients were assigned to receive either 0.5 mg intravitreal ranibizumab for three to 14 days before a Ahmed glaucoma valve implantation (n  =  21) or Ahmed glaucoma valve implantation alone (n = 22). They found a success rate of 73.7 vs. 71.4 % at 6 months and 72.2 vs. 68.4 % at 12 months in the injec- tion group and the control group, respectively. There were no significant differences in the two groups with respect to intraocular pressure, best corrected visual acu- ity, anti-glaucoma medications or postoperative compli- cations at 6 or 12 months. They concluded, therefore that a single intravitreal ranibizumab before surgery has no significant effect on the medium- or long-term outcomes of neovascular glaucoma treated with Ahmed glaucoma valve implantation. Liu et  al. [59] investigated the efficacy and safety of intravitreal ranibizumab injection combined with trab- eculectomy compared it with Ahmed valve surgeries. In this prospective study, they have included 37 eyes from 36 NVG patients, in which 18 NVG eyes were given intravitreal ranibizumab injection one week before tra- beculectomy. Ahmed valve implantation surgery was performed in 19 eyes. Their results showed that IOP was significantly decreased following intravitreal ranibizumab injection combined with trabeculectomy treatment. In Sahyoun et al. [63] also evaluated the long-term results of the Ahmed glaucoma valve implantation in association with bevacizumab in NVG patients in a retrospective study.h Their study included 39 eyes of 34 patients, which were divided in two groups. The first group consisted of 19 eyes that received an injection of intravitreal Rodrigues et al. Int J Retin Vitr (2016) 2:26 Page 6 of 10 formation and angle closure have occurred. Surgical interventions for NVG include: trabeculectomy with anti- metabolites, glaucoma drainage devices, cyclophoto- coagulation, among others. NVG is a refractory type of glaucoma that poses a challenge for proper IOP control and is often associated with increased risk for postopera- tive complications including hyphema and vision loss. bevacizumab 7  days preoperatively, whereas the sec- ond group without the injection, included 20 eyes. Even though, preoperative intravitreal bevacizumab before Ahmed glaucoma valve surgery was not associated with a better surgical success, IOP control, or best-corrected visual acuity. Its administration significantly decreased postoperative hyphema and number of last visit’s antiglaucoma medications. Cyclodestructive procedures Transcleral application of diode laser cyclophotoco- agulation consists of the destruction the ciliary body epithelium and stroma with consequent reduction of aqueous humor production and IOP levels [90–92]. Transcleral cyclophotocoagulation with and without the use anti-VEGF has been shown to be effective in lowering IOP and relieving pain in advanced cases of NVG [70, 93–95]. When compared to Ahmed valve implantation in a randomized controlled trial, no sig- nificant difference was found in the success rate at 24  months between the diode cyclophotocoagulation (61.18  %) and Ahmed glaucoma valve implantation (59.26  %) in NVG treatment [91]. It is important to note, however, that the underlying diagnosis of NVG poses an increased risk for hypotony after transcleral- cyclophotocoagulation [94–97]. Endo-cyclophotoco- agulation was also shown to be effective in NVG. A study showed success rates at 24 months of 70.59 and 73.53  % for the Ahmed and endo-cyclophotocoagula- tion groups, respectively [98]. Since NVG and proliferative diabetic retinopathy are usually co-existing conditions, it is not uncommon for patients with NVG to have a positive history of prior vit- rectomy. Studies that evaluated implantation of Ahmed glaucoma valve for IOP control in vitrectomized eyes, showed the safety and efficacy of the procedure [82, 83], with success rates of 62.5 % after 3 years for vitrec- tomized eyes, which was not statistically different from the 68.5 % success rate for the nonvitrectomized group. Ahmed glaucoma valve can control the IOP in the major- ity of eyes after pars plana vitrectomy and silicone oil injection, when implanted in the anterior chamber or inferonasal or inferotemporal quadrant, preventing oil to clogging the tube. [84]. If this surgery is selected, intra- silicone injection of anti-VEGF in posterior segment for regressing iris neovascularization is considered safe and effective [85]. However, intraocular silicone oil tam- ponade was found to be a risk factor for surgical failure [83]. The combination of 23-gauge pars plana vitrectomy and Ahmed valve implantation in the same procedure is also a treatment option for these cases and has been shown to be safe and effective in patients with prolifera- tive diabetic retinopathy and refractory NVG [86, 87]. Wallsh et al. confirmed these findings in a retrospective study with a 22 patients, in which 95.8 % of eyes had IOP below 21 mmHg in the final follow-up (mean follow-up of 7.39 ± 1.11 months). Best-corrected visual acuity also improved significantly [88]. Surgical treatment Although the mainstay of therapy of NVG is the treat- ment of retinal ischemia with panretinal photocoagu- lation, surgical interventions to control IOP are often necessary since the use of eye-drops may not lower IOP enough to prevent optic nerve damage. Especially in those cases in which peripheral anterior synechia Rodrigues et al. Int J Retin Vitr (2016) 2:26 Page 7 of 10 [89] However, prospective and comparative studies with longer follow-up are still needed. A randomized clinical trial by Arcieri et  al. investi- gated the efficacy and safety of intravitreal bevacizumab in eyes with NVG undergoing Ahmed glaucoma valve implantation. They enrolled 40 patients who were ran- domized to receive intravitreal bevacizumab (1.25 mg) or not during Ahmed valve implant surgery. Injections were administered intra-operatively, 4 and 8 weeks after surgery. Their results suggest a trend that using with intravitreal bevacizumab as an adjunct can lower IOP levels and the number of post operative medications in NVG patients who underwent Ahmed glaucoma valve implantation. It is important to note, however, that patients with NVG are at a higher risk for certain post- operative complications and poor visual outcomes, pos- sibly due to progression of underlying disease. Loss of light perception is not rare among NVG patients after surgical procedures [74, 75, 77] and hyphema is often encountered [80]. Compared to other types of glau- coma, NVG eyes also seem to be at higher risk for tube shunt exposure [81]. Cyclodestructive procedures Finally, a retrospective study evaluated the results of combined pars plana vitrectomy and pars plana Baerveldt tube placement. A significant IOP decrease was achieved with the procedure while visual acuity remained unchanged. However, it is impor- tant to note that 38 % experienced a decrease in vision Other surgical options Due to the relatively low long-term success rates of the existing treatment options for NVG, new surgical approaches have been proposed for IOP control. For example, manual and bimanual maneuvers to remove the fibrovascular membrane from the anterior chamber angle have been described [99]. The use of drainage devices made of porous material such as the Ahmed M4 [100]— and the Express shunt [101] has also been attempted. However, more studies and randomized clinical trials are needed to assess the efficacy of such procedures. Abbreviations NVG: Neovascular glaucoma; IOP: Intraocular pressure; VEGF: Vascular endothelial growth factor. Conclusion NVG is an important secondary glaucoma associated with poor visual prognosis, due to the optic nerve dam- age from high IOP and also complications from retinal vascular diseases. Even though treatment options with panretinal photocoagulation and anti-VEGF might be used in attempt to control the neovascularization pro- cess, in some cases surgical procedures are necessary in order to achieve normal levels of IOP and avoid optic nerve damage. Proper management and early diagnosis of this condition is crucial to reduce the chances of visual impairment. 1028). 32. Kiratli H, Tarlan B, Soylemezoglu F. 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https://strathprints.strath.ac.uk/63229/1/Danzerl_etal_RPG_2017_Maximising_wind_generation_through_optimised_operation_of_on_load_tap_changing_transformers.pdf

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Maximising wind generation through optimised operation of on‐load tap changing transformers in active distribution networks

Journal of engineering

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1 Introduction are highly susceptible to voltage rise problems when various DG resources are connected [6]. Power generations from intermittent re- newable resources such as wind and solar compound the challenge and undermine the performance of the OLTCs to efficiently regulate the voltage. These complexities have rendered conventional OLTC transformers alone, inefficient and unreliable to control the voltage, limiting DG connection capacities on the network. Centralised voltage control methods in distribution networks have traditionally relied on on-load tap changing (OLTC) transformers as the most common control device to regulate and maintain network voltages within required limits [1]. In the UK, the statutory voltage limits are defined in the Electricity Safety, Quality and Continuity (ESQC) Regulations 2002 [2] and it specifies that, low- voltage (LV) customers’ be supplied at 400/230 V with tolerance of +10/−6%, whereas high-voltage (HV) customers, at tolerance of ±6%. The OLTC operates by moving their tap positions to select appropriate transformer turns ratio that suits a range of power flow conditions on the network. To simplify and automate voltage control, automatic voltage control (AVC) relays are used in conjunction with line drop compensation (LDC) equipment. The AVC relay continually monitors the network to detect voltage variation and initiates a tap change command to the motorised OLTC when the voltages are outside the pre-set limits [3]. The paradigm shift towards an intelligent and advanced distribu- tion management systems (DMS) requires sophisticated control strategies that optimise existing network assets. Addressing the voltage rise problem efficiently will require a well-coordinated DMS that can control transformer tap changers, voltage regulators, power plants, compensators, and loads at the primary substation [7]. The advent of active network management (ANM) techniques offers a feasible solution in this direction in developing an efficient, flexible, and reliable network. It involves real-time monitoring and control of the network and is seen to enhance greater DG connec- tion capacities without reinforcement. Recent ANM schemes and trial projects in the UK have investigated control strategies that address thermal and power flow congestion problems on the network. However, voltage constraint issues are becoming a more complex challenge and one that has not been fully investigated and trailed in current ANM schemes. The AVC relay operation usually incorporates a time delay setting between 10 and 120 s from detecting an out of range voltage and starting a tap-change command. Maximising wind generation through optimised operation of on-load tap changing transformers in active distribution networks Daniel Danzerl1, Simon Gill2, Olimpo Anaya-Lara2 1Wind Energy CDT, Department of Electronic and Electrical Engineering, University of Strathclyde, Glasgow, UK 2Department of Electronic and Electrical Engineering, University of Strathclyde, Glasgow, UK E-mail: [email protected] ublished in The Journal of Engineering; Received on 12th October 2017; Accepted on 3rd November 2017 Abstract: On-load tap changing transformers are the most common control device to regulate and maintain distribution network voltage within required limits. Voltage rise issues on the other hand have become a major factor limiting greater penetration of low carbon generators, par- ticularly in weak distribution networks. Here, the voltage rise problem is addressed through the application of optimised set-point voltage technique that aims to improve network hosting capacity to accommodate high wind penetration. It assesses the effectiveness of the technique on a realistic 289-node UK generic 11 kV distribution network using time-series optimal power flow simulations. The results reveal that when the tap changer is operated at the optimised set-point voltage, it can lead to greater energy yields. It also shows a reduction in the number of tap changing operations when the transformer is operated within the optimised deadband allowing for an improved life-span and minimum main- tenance cost. 1 Introduction The time-delay setting is to avoid unnecessary tap operation during short-term voltage fluctuations on the network. The LDC is used to compen- sate for voltage drop variations on the line between the transformer and loads situated towards the far end of the feeder In this paper, the voltage rise problem is addressed and mitigated. The study investigates an optimised set-point voltage technique that aims to improve network hosting capacity to accommodate high wind penetration levels. It presents a detailed case-study assessment of the benefits and impact on DG capacities using time-series AC optimal power flow (OPF) simulations. It concludes that, when the OLTC is operated at the optimised set-point voltage, it can lead to greater generation levels. Previous passive operation of the network assumed unidirection- al power flows and uncontrollable resources from the transmission system into distribution networks at the grid supply points (GSP) [4]. However, recent proliferation of distributed generator (DG) connections renders such assumptions invalid and as such have initiated significant changes to the previous passive methods to an active approach to network management. Operational challenges such as reverse power flows from the DGs in current active distri- bution systems adversely affect the operation of OLTCs to efficient- ly regulate and maintain the voltages [5]. Weak distribution networks, particularly, rural networks with poor X/R characteristics, The 6th International Conference on Renewable Power Generation (RPG) The 6th International Conference on Renewable Power Generation (RPG) The 6th International Conference on Renewable Power Generation (RPG) 19–20 October 2017 J. Eng., 2017, Vol. 2017, Iss. 13, pp. 2339–2344 doi: 10.1049/joe.2017.0749 2 Optimisation problem The nodal power balance (2) can be expanded as: PGi −PDi −
NG i=1 ViVjYij   cos (uij −di + dj) = 0 (8) QGi −QDi −
NG i=1 ViVjYij   sin (uij −di + dj) = 0 (9) (8) (9) The parameter constraint limits in (3) is expanded as: The parameter constraint limits in (3) is expanded as: l d l d i The proposed control technique in this paper is formulated as an optimisation problem that makes use of mathematical AC OPF ana- lysis tools using time-series simulations. It utilises the standard ACOPF [11, 12] formulation at each time-step and treats the tap position as control variables. The optimisation aims to achieve the following system objectives: Generator constraints: Generator voltages and real and reactive power outputs are constrained by their upper and lower limits as follows: V min Gi ≤VGi ≤V max Gi (10) Pmin Gi ≤PGi ≤Pmax Gi (11) Qmin Gi ≤QGi ≤Qmax Gi (12) (10) (11) † maximise wind generation outputs at minimum cost, † maximise wind generation outputs at minimum cost, † improving network hosting capacity, (12) † minimise the impact of DGs on voltage profiles, † minimise the number of OLTC transformer tap operations. Transformer constraints: The OLTC transformer tap settings are bounded as follows: Transformer constraints: The OLTC transformer tap settings are bounded as follows: The ACOPF-based problem is modelled as a set of non-linear equations consisting equality and inequality constraints and is given as follows: Tmin i ≤Ti ≤Tmax i (13) (13) Slack bus constraint: The slack bus voltage is bounded as: Optimise: f (x, u) (1) Subject to: g(x, u) = 0 (2) h(x, u) ≤0 (3) Optimise: f (x, u) (1) Subject to: g(x, u) = 0 (2) h(x, u) ≤0 (3) (1) vmin s ≤vs ≤vmax s (14) (14) Security constraints: These include the constraints of voltages at load buses and thermal power flow limits Security constraints: These include the constraints of voltages at load buses and thermal power flow limits h(x, u) ≤0 (3) (3) V min i ≤Vi ≤V max i (15) Sij   ≤Smax ij (16) (15) where f represents the objective function, g the physics of the power system enforced through the power flow equations, and h the par- ameter limits of the system. This is an open access article published by the IET under the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0/) J. Eng., 2017, Vol. 2017, Iss. 13, pp. 2339–2344 doi: 10.1049/joe.2017.0749 2 Optimisation problem x represents a vector of state variables and it includes the real power generation of the slack bus PG(slack), load bus voltages Vi, reactive power generation QGi, and line thermal flows Si and can be expressed as: (16) The ACOPF formulation in this paper involves a two-stage opti- misation process that treats the tap positions as discrete and continu- ous control variables similar to the methods discussed in [10]. The initial stage is a discrete method which models the voltage step ratio as discrete variables that can vary between a certain maximum vmax s and minimum vmin s set-point voltage by a fixed step-size Δv. The AVC model manually moves up or down by one step size Δv at a time and locks the transformer tap position to a pre-determined voltage set-point. Each optimisation cycle requires a physical move- ment of the tap position to the desired set-point, where the voltage is held fixed at the slack bus to connect the multiple DG schemes. The control logic can be expressed mathematically as: x = [PG(slack) ViQGiSi] (4) (4) u represents the vector of control variables and includes generator real power PGi, generator voltage VGi, and transformer tap changer Ti and can be expressed as: u = [PGi VGi Ti] (5) (5) vs = vs + Dv if vk −vref , Dm and vs , vmax s ; vmin s = vmax s vs if vk −vref   , Dm; vmin s = vmax s vs −Dv if vk −vref . Dm and vs . vmax s ; vmin s = vmax s ⎧ ⎪⎨ ⎪⎩ (17) To maximise wind generation capacities PGi, the objective function f takes the form in (6): To maximise wind generation capacities PGi, the objective function f takes the form in (6): (17) Maximise
NG G=1 PGi (6) Maximise
NG G=1 PGi (6) ( 7) where vs represents the set-point voltage at the slack bus, vk the regulated voltage quantity, vref is the reference voltage, and Δm where vs represents the set-point voltage at the slack bus, vk the regulated voltage quantity, vref is the reference voltage, and Δm (6) This is an open access article published by the IET under the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0/) J. Eng., 2017, Vol. 2017, Iss. 13, pp. 2 Optimisation problem Previous work reported by Deckmyn et al. [8] involved an area control strategy of OLTC operation using local network measure- ment. The authors used voltage information of remote measurement J. Eng., 2017, Vol. 2017, Iss. 13, pp. 2339–2344 doi: 10.1049/joe.2017.0749 This is an open access article published by the IET under the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0/) The quadratic cost function of the generators is represented as: The quadratic cost function of the generators is represented as: points to determine optimal tap positions that minimises voltage de- viation on the network. This was achieved through heuristic-based optimisation methods which treated the tap changer as discrete vari- ables. A decision-making algorithm is proposed in [9] to find ad- equate set-point voltage generated through simulations or historical performance to control voltage rise using OLTCs. In [3], the authors discuss voltage regulation using a coordination between OLTC and LDC in medium-voltage feeders to improve DG connection capacities. A similar area-based coordinated control strategy that controls the voltage set-points of the AVC relay at the primary substation have been studied in [6] using time- domain simulations. The authors investigated the dynamic oper- ation and responses of the AVC relays and tap changer mechanism to the system load variations. In [10], the authors discuss an interior point (IP) method based on a non-linear complementarity model for OPF problems with load tap changes. The authors initially treat the tap positions as continuous variables to identify the upper and lower bounds of the transformer using OPF. The complementarity con- straints of the transformer taps are then modelled into the OPF and solved using IP optimisation method. min
NG i=1 ai + bi(PGi) + ci(PGi)2 (7) (7) where ai, bi, and ci represent the cost coefficient of active power output PGi. The OPF model assigns high cost values to the swing bus to discourage grid active power imports from the GSP and low cost values to all the DGs to encourage active demands on the network be met by the DGs. 3 UK generic distribution test network The proposed strategy is applied to a realistic 11 kV UK generic distribution system (UKGDS) to assess its effectiveness. Fig. 1 shows a single-line representation of a 289-node radial distribution network developed in IPSA with full network parameters given in [13]. The model comprises a mix of urban and rural sections con- sisting of underground cables followed by overhead lines charac- terised by varied X/R ratios and medium conductor lengths. The primary substation supplies three 11 kV feeders and is linked to a 33 kV distribution system represented as a source of real and react- ive power. The primary substation is equipped with two identical 33/11 kV OLTC transformers, each rated at 22 MVA connected in parallel to regulate the network voltage to a pre-defined target. The proposed strategy is applied to a realistic 11 kV UK generic distribution system (UKGDS) to assess its effectiveness. Fig. 1 shows a single-line representation of a 289-node radial distribution network developed in IPSA with full network parameters given in [13]. The model comprises a mix of urban and rural sections con- sisting of underground cables followed by overhead lines charac- terised by varied X/R ratios and medium conductor lengths. The primary substation supplies three 11 kV feeders and is linked to a 33 kV distribution system represented as a source of real and react- ive power. The primary substation is equipped with two identical 33/11 kV OLTC transformers, each rated at 22 MVA connected in parallel to regulate the network voltage to a pre-defined target. Here, the OLTCs at the 33/11 kV substation controls the second- ary nominal voltage to a reference set-point and has a maximum and minimum tap settings of +4 to −14%, respectively, in 19 steps with a step size of 1.0%. The AVC relay bandwidth is allowed an output voltage deviation of ±3% of the nominal. The secondary bus vol- tages are constrained at current DNO operational limits and allowed to vary within a permissible range of ±3% of the nominal. The medium circuit conductor lengths with varied MVA Fig. 3 Normalised wind profile (2 weeks) ratings are considered as additional thermal constraints on the network. However, thermal limits on the network are deliberately relaxed to enable the voltage limits to operate as binding constraints to control the generators. 2 Optimisation problem 2339–2344 doi: 10.1049/joe.2017.0749 This is an open access article published by the IET under the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0/) J. Eng., 2017, Vol. 2017, Iss. 13, pp. 2339–2344 doi: 10.1049/joe.2017.0749 Fig. 2 Active demand profile (2 weeks) Fig. 3 Normalised wind profile (2 weeks) Fig. 2 Active demand profile (2 weeks) represents the AVC relay deadband. The ACOPF discrete model investigates a narrowed deadband that aims to optimise DG capaci- ties within the specified network constraints. Within this narrowed deadband, lies an optimum set-point voltage. In the second stage, the narrowed AVC deadband is modelled into the OPF and treats the transformer tap positions as continuous variables. The continu- ous method assumes a small tap step ratio Δv and models the dis- crete switches as approximated continuous variation of the set-point voltage vs. In this case, the ACOPF can choose optimum set-point values between the narrowed upper maximum limit vmax s(optimised) and lower minimum limits vmin s(optimised) that maxi- mises the objective function at each iteration. This can be repre- sented mathematically as: Fig. 2 Active demand profile (2 weeks) Fig. 3 Normalised wind profile (2 weeks) Fig. 3 Normalised wind profile (2 weeks) vmin s(optimised) ≤vs(optimised) ≤vmax s(optimised) (18) vmin s(optimised) ≤vs(optimised) ≤vmax s(optimised) (18) (18) vmin s(optimised) ≤vs(optimised) ≤vmax s(optimised) 3 UK generic distribution test network In this rule, the OPF engine optimally shares limited network capacity by assigning greater curtailment in order of generator with the most impact on voltage rise constraint. The model is deployed in Matpower [14] where simulation studies are carried over 1 month period at half- hourly resolution consisting a total of 1440 time-steps. 4 Results To demonstrate and quantify the effectiveness of the proposed strat- egy to mitigate the voltage rise problem, five scenarios for connect- ing the multiple DG schemes have been investigated and are summarised in Table 1. Each scenario configures the set-point Fig. 1 11 kV radial distribution network (HV_UG_OHb) [13] Table 1 Summary of AVC set-point voltages Scenario % kV p.u. 1 +3 11.33 1.03 2 +2 11.22 1.02 3 +1 11.11 1.01 4 0 11.00 1.00 5 −1 10.89 0.99 J. Eng., 2017, Vol. 2017, Iss. 13, pp. 2339–2344 doi: 10.1049/joe.2017.0749 This is an open access article published by the IET under the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0/) Fig. 1 11 kV radial distribution network (HV_UG_OHb) [13] 3 UK generic distribution test network Half-hourly time-series load profiles are connected on all secondary buses and consists a mixture of residen- tial, industrial, and commercial loads. These are aggregated values scaled from a single load profile (peak loads) provided in [13] with minimum demands set at 25% of the peak load. Fig. 2 presents a 2-week long snapshot of half-hourly demand data. Here, the OLTCs at the 33/11 kV substation controls the second- ary nominal voltage to a reference set-point and has a maximum and minimum tap settings of +4 to −14%, respectively, in 19 steps with a step size of 1.0%. The AVC relay bandwidth is allowed an output voltage deviation of ±3% of the nominal. The secondary bus vol- tages are constrained at current DNO operational limits and allowed to vary within a permissible range of ±3% of the nominal. The medium circuit conductor lengths with varied MVA The use of historic wind resource time-series, such as normalised output of a nearby wind farm, is used to estimate potential gener- ation. Fig. 3 shows a 2-week sample of normalised wind generation profile. A suit of eight ‘non-firm’ distributed wind generator (DWG) schemes of varied capacities are connected to the network at nodes 1244, 1144, 1105, 1191, 1120, 1310, 1358, and 1387 and assumed to operate an ANM scheme. The DWGs are modelled and connected to the network as (P, Q) nodes and operated at unity power factor mode with no voltage control capabilities. The principle of access rule for connecting the multiple generators and sharing of curtailment is via a technical best arrangement. Here, all the DG schemes are assumed to have equal priority and access to the network. In this rule, the OPF engine optimally shares limited network capacity by assigning greater curtailment in order of generator with the most impact on voltage rise constraint. The model is deployed in Matpower [14] where simulation studies are carried over 1 month period at half- hourly resolution consisting a total of 1440 time-steps. nodes and operated at unity power factor mode with no voltage control capabilities. The principle of access rule for connecting the multiple generators and sharing of curtailment is via a technical best arrangement. Here, all the DG schemes are assumed to have equal priority and access to the network. 4.3 Scenario 5: set-point voltage fixed at −1% of nominal 4.3 Scenario 5: set-point voltage fixed at −1% of nomina Here, the set-point voltage is further lowered to −1% of the nominal voltage. Simulation results presented in Fig. 6 show a significant improvement in generation capacities, allowing the remotely con- nected generators greater network access and subsequent wind yields. Generator H is now observed to be generating up to a maximum of 36% of its installed capacity. In this study, −2 and −3% set-point voltages are ignored as a result of undervoltage conditions occurring towards the ends of the feeders. The OPF failed to converge due to breach of lower voltage limits at certain time-steps. A sample of the scenarios studied and corresponding simulation results obtained are presented in the following sections. The total energy realised across the investigated set-point vol- tages are presented in Fig. 7. It is seen that, the total generation across the DG schemes increases by lowering the slack bus set- point voltage. Operating the AVC at high set-points significantly reduces generation capacities. For example, at +3% set-point voltage, a total energy yield of 1071 MWh was realised out of 12,851 MWh available energy representing a total generation cap- acity of 8.3% across the DGs. In the case of −1% set point voltage, a total generation of 10,909 MWh was realised out of the total available energy representing an improved generation capacity of 85% across the DG schemes. 4.1 Scenario 1: set-point voltage fixed at +3% of nominal This scenario represents a worse-case operation condition of the AVC set-point voltage. In this study, the set-point voltage at the slack bus is raised and held fixed at the upper maximum limit (+3% of nominal) which serves as a reference point to the rest of the secondary bus voltages. The ACOPF reveals poor DG perfor- mances in terms of generation levels (Fig. 4) with the biggest size generator Gen A (which is electrically closer to the primary sub- station) generating a maximum capacity of 19%. In comparison to Gen H (which is the smallest size and electrically the most fur- thest away), the generator is observed to be generating ∼1% of its rated capacity. All the DGs are observed to experience significant curtailment due to reduced voltage margins and substantial impact on voltage rise constraints from active generations. Generator performance profile across the investigated set-point voltages are presented in Fig. 8. From here, it is observed that, Fig. 7 Total energy generation Fig. 8 Generator performance profile e Commons J. Eng., 2017, Vol. 2017, Iss. 13, pp. 2339–2344 doi: 10.1049/joe.2017.0749 Fig. 7 Total energy generation 4 Results To demonstrate and quantify the effectiveness of the proposed strat- egy to mitigate the voltage rise problem, five scenarios for connect- ing the multiple DG schemes have been investigated and are summarised in Table 1. Each scenario configures the set-point Table 1 Summary of AVC set-point voltages Table 1 Summary of AVC set-point voltages Scenario % kV p.u. 1 +3 11.33 1.03 2 +2 11.22 1.02 3 +1 11.11 1.01 4 0 11.00 1.00 5 −1 10.89 0.99 Fig. 1 11 kV radial distribution network (HV_UG_OHb) [13] J. Eng., 2017, Vol. 2017, Iss. 13, pp. 2339–2344 doi: 10.1049/joe.2017.0749 This is an open access article published by the IET under the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0/) J. Eng., 2017, Vol. 2017, Iss. 13, pp. 2339–2344 doi: 10.1049/joe.2017.0749 This is an open access article published by the IET under the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0/) Fig. 4 Energy generated and curtailed at +3% set-point voltage Fig. 6 Energy generated and curtailed at −1% set-point voltage Fig. 4 Energy generated and curtailed at +3% set-point voltage Fig. 6 Energy generated and curtailed at −1% set-point voltage voltage of the AVC relay to a pre-determined fixed target value to assess the impact on the DGs and adequacy of the technique to miti- gate the problem. Each scenario provides a quantitative assessment of renewable energy yields and curtailment levels required to main- tain the network limits when the strategies are implemented. 6 Conclusion This paper has presented a constraint optimisation strategy that con- figures the set-point voltage of OLTC transformers at distribution primary substation. It investigates an optimum set-point voltage control technique of the tap-changer transformer with an overall aim to improve network hosting capacity to accommodate greater wind connections at weak distribution networks. The results obtained demonstrated that, configuring the AVC relays at lower set-point voltage significantly improves the network hosting capaci- ties. By doing so, the DG voltage margins can be improved and their active power impact on the constraint effectively minimised. It also shows that, controlling the transformer within a narrowed optimum deadband effectively reduces the number of tap changing operations allowing for an improved life-span and reduced mainten- ance cost. 4.2 Scenario 3: set-point voltage fixed at +1% of nominal In this scenario, the set-point voltage is lowered and held fixed at +1% of the nominal. The ACOPF results show improved generation levels of the DGs with generators A, B, and C operating at maximum installed capacities. Subsequently, the OPF allows an improved access for the rest of the DGs to generate onto the network. Generator H is now observed to be generating up to a maximum of 27% of its rated capacity when compared with scen- ario 1 and is shown in Fig. 5 Fig. 7 Total energy generation Fig. 5 Energy generated and curtailed at +1% set-point voltage Fig. 8 Generator performance profile This is an open access article published by the IET under the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0/) J. Eng., 2017, Vol. 2017, Iss. 13, pp. 2339–2344 doi: 10.1049/joe.2017.0749 Fig. 5 Energy generated and curtailed at +1% set-point voltage Fig. 8 Generator performance profile Fig. 5 Energy generated and curtailed at +1% set-point voltage Fig. 8 Generator performance profile This is an open access article published by the IET under the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0/) J. Eng., 2017, Vol. 2017, Iss. 13, pp. 2339–2344 doi: 10.1049/joe.2017.0749 This is an open access article published by the IET under the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0/) This is an open access article published by the IET under the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0/) Fig. 10 Changes in set-point voltage at full range Fig. 11 Changes in set-point voltage at optimised deadband Fig. 10 Changes in set-point voltage at full range the DGs generation capacities decrease as we move further along the feeders and away from the primary substation. Generators located closer to the substation showed greater penetration levels across all voltage set-points when compared with the remotely located DGs. For instance, at +3% set-point, it seen that generators A, B, and C are most favoured with greater network access due to their strong location. Conversely, the rest of the DGs are least favoured (with highly restricted or no access to the network) due to their weak remote location. Comparatively, it is seen that the OPF allows an improved access for all DGs schemes when the slack bus set-point voltages are lowered to levels that reduce their impact on voltage rise constraint. Fig. 10 Changes in set-point voltage at full range 7 Acknowledgment This work was funded by the UK’s Engineering and Physical Science Research Council (EPSRC), project reference number EP/G037728/1. ( ) p j EP/G037728/1. 8 References [1] Lakervi E., Holmes E.J.: ‘Electricity distribution network design’ (2007, 2nd edn.) [2] Department of Trade and Industry: ‘Guidance on the Electricity Safety, Quality and Continuity regulations 2002’. Rep. Number URN 02/1544, October 2002, pp. 1–47 [3] Viawan F.A., Sannino A., Daalder J.: ‘Voltage control with on-load tap changers in medium voltage feeders in presence of distributed generation’, Electr. Power Syst. Res., 2007, 77, (10), pp. 1314–1322 [4] Vovos P.N., Kiprakis A.E., Wallace A.R., ET AL.: ‘Centralized and dis- tributed voltage control: impact on distributed generation penetra- tion’, IEEE Trans. Power Syst., 2007, 22, (1), pp. 476–483 Fig. 9 Maximum voltage profile J. Eng., 2017, Vol. 2017, Iss. 13, pp. 2339–2344 doi: 10.1049/joe.2017.0749 This is an open access article published by the IET under the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0/) Fig. 9 Maximum voltage profile J. Eng., 2017, Vol. 2017, Iss. 13, pp. 2339–2344 doi: 10.1049/joe.2017.0749 This is an ope 5 Discussion The optimisation engine controls and dictates generation capacities according to DGs active power impact on local voltage rise at their point of connection. Setting the AVC at high-voltage set-points ad- versely limits the network’s headroom capacity to cope with high DG penetration. In effect, active generation from the DGs become highly susceptible to pushing the network’s voltage profiles up to the maximum limits forcing the OPF to heavily curtail genera- tions to levels that satisfy the constraint. In such extreme cases, gen- erators located on the stronger sections of the network (with minimum impact on voltage rise) are allowed access, whereas the remotely connected DGs have no access as seen in Fig. 8. Therefore, to influence fairness and greater connection capacities for all DGs across the various network location points, there is a scope to lower the voltage settings at the slack bus to enhance DG voltage margins and allow greater active generation capabil- ities. By doing this, the voltage profiles and network headroom cap- acity can be improved to accommodate high wind penetration levels. Fig. 9 shows single time-step profiles of maximum bus voltage magnitudes recorded during the optimisation. Here, by comparing the voltage profiles of selected set-point voltages (scen- arios 1, 3, and 5), it seen that operating the AVC at −1% set-point voltage improves the voltage profile and mitigates the voltage rise problem. Voltage profile at −1% showed the least number of points at which the voltage magnitudes reach the upper maximum limits implying reduced curtailments when compared with +3 and +1%. To compare the actions of the tap operation with respect to changes in set-point voltages, the tap positions are treated as con- tinuous variables. A base-case is initially investigated where the slack bus set-point voltage is modelled to vary between the full set of minimum and maximum operational limits −3% ≤vs ≤+3%. In this study, the optimised deadband for the OLTC is established at 0% ≤vs (optimised) ≤−1%. Fig. 10 (base- case) shows high variation and changes in set-point voltage of the AVC which controls the OLTC indicating a potential increase in the number of tap operation of the OLTC transformer. Conversely, Fig. 11 shows a significant reduction in changes in voltage variation when the AVC is operated within the narrowed optimised deadband. This indicates a potential reduction in the Fig. 11 Changes in set-point voltage at optimised deadband Fig. [1] Lakervi E., Holmes E.J.: ‘Electricity distribution network design’ (2007, 2nd edn.) [2] Department of Trade and Industry: ‘Guidance on the Electricity Safety, Quality and Continuity regulations 2002’. Rep. Number URN 02/1544, October 2002, pp. 1–47 [3] Viawan F.A., Sannino A., Daalder J.: ‘Voltage control with on-load tap changers in medium voltage feeders in presence of distributed generation’, Electr. Power Syst. Res., 2007, 77, (10), pp. 1314–1322 [4] Vovos P.N., Kiprakis A.E., Wallace A.R., ET AL.: ‘Centralized and dis- tributed voltage control: impact on distributed generation penetra- tion’, IEEE Trans. Power Syst., 2007, 22, (1), pp. 476–483 [2] Department of Trade and Industry: ‘Guidance on the Electricity Safety, Quality and Continuity regulations 2002’. Rep. Number URN 02/1544, October 2002, pp. 1–47 This is an open access article published by the IET under the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0/) [1] Lakervi E., Holmes E.J.: ‘Electricity distribution network design’ (2007, 2nd edn.) 5 Discussion 11 Changes in set-point voltage at optimised deadband number of tap changing operation of the OLTC transformer, enhan- cing the life-span and reducing the cost of maintenance. 8 References [1] Lakervi E., Holmes E.J.: ‘Electricity distribution network design’ (2007, 2nd edn.) [2] Department of Trade and Industry: ‘Guidance on the Electricity Safety, Quality and Continuity regulations 2002’. Rep. Number URN 02/1544, October 2002, pp. 1–47 [3] Viawan F.A., Sannino A., Daalder J.: ‘Voltage control with on-load tap changers in medium voltage feeders in presence of distributed generation’, Electr. Power Syst. Res., 2007, 77, (10), pp. 1314–1322 [4] Vovos P.N., Kiprakis A.E., Wallace A.R., ET AL.: ‘Centralized and dis- tributed voltage control: impact on distributed generation penetra- tion’, IEEE Trans. Power Syst., 2007, 22, (1), pp. 476–483 [1] Lakervi E., Holmes E.J.: ‘Electricity distribution network design’ (2007, 2nd edn.) [2] Department of Trade and Industry: ‘Guidance on the Electricity Safety, Quality and Continuity regulations 2002’. Rep. Number URN 02/1544, October 2002, pp. 1–47 pp [3] Viawan F.A., Sannino A., Daalder J.: ‘Voltage control with on-load tap changers in medium voltage feeders in presence of distributed generation’, Electr. Power Syst. Res., 2007, 77, (10), pp. 1314–1322 [3] Viawan F.A., Sannino A., Daalder J.: ‘Voltage control with on-load tap changers in medium voltage feeders in presence of distributed generation’, Electr. Power Syst. Res., 2007, 77, (10), pp. 1314–1322 g , y , , , ( ), pp [4] Vovos P.N., Kiprakis A.E., Wallace A.R., ET AL.: ‘Centralized and dis- tributed voltage control: impact on distributed generation penetra- tion’, IEEE Trans. Power Syst., 2007, 22, (1), pp. 476–483 Fig. 9 Maximum voltage profile Fig. 9 Maximum voltage profile J. Eng., 2017, Vol. 2017, Iss. 13, pp. 2339–2344 doi: 10.1049/joe.2017.0749 J. Eng., 2017, Vol. 2017, Iss. 13, pp. 2339–2344 doi: 10.1049/joe.2017.0749 This is an open access article published by the IET under the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0/) [5] Kulmala A.: ‘Active voltage control in distribution networks includ- ing distributed energy resources’. PhD thesis, 2014 [9] Xu T., Wade N.S., Davidson E.M., ET AL.: ‘Case-based reasoning for coordinated voltage control on distribution networks’, Electr. Power Syst. Res., 2011, 81, (12), pp. 2088–2098 [6] Kulmala A., Mäki K., Repo S., ET AL.: ‘Active voltage level management of distribution networks with distributed generation using on load tap changing transformers’. PowerTech, 2007, pp. 455–460 [10] Li B., Bai X., Wei H.: ‘An interior point method based on nonlinear complementarity model for OPF problems with load tap changing transformers’. Asia-Pacific Power Energy Eng. Conf. APPEEC, 2010 [11] Carpentier J.: ‘Optimal power flows’, Int. J. Electr. J. Eng., 2017, Vol. 2017, Iss. 13, pp. 2339–2344 doi: 10.1049/joe.2017.0749 This is an open access article published by the IET under the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0/) This is an open access article published by the IET under the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0/) J. Eng., 2017, Vol. 2017, Iss. 13, pp. 2339–2344 doi: 10.1049/joe.2017.0749 8 References Power Energy Syst., 1979, 1, (1), pp. 3–15 [7] Strbac G., Jenkins N., Hird M., ET AL.: ‘Integration of operation of em- bedded generation and distribution networks’. Manchester Centre for Electrical Energy, 2002, pp. 1–95 [12] Dommel H., Tinney W.: ‘Optimal power flow solutions’, IEEE Trans. Power Appar. Syst., 1968, PAS-87, (10), pp. 1866–1876 [8] Deckmyn C., Vandoorn T.L., Meersman B., ET AL.: ‘A coordinated voltage control strategy for on-load tap changing transformers with the utilisation of distributed generators’. 2016 IEEE Int. Energy Conf. ENERGYCON 2016, 2016 [13] UKGDS: ‘UK generic distribution system’, 2015. [Online]. Available at https://github.com/sedg/ukgds [13] UKGDS: ‘UK generic distribution system’, 2015. [Online]. A [13] UKGDS: ‘UK generic distribution at https://github.com/sedg/ukgds at https://github.com/sedg/ukgds [14] Zimmerman R.D., Murillo-Sanchez C.E.: ‘Matpower 6.0b1’, vol. 22203, February 2017 This is an open access article published by the IET under the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0/)

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Samuel Moyn* It is not clear what there is left for a commentator to say once a symposium has unfolded in such a way as to cancel itself out. But in case others read it differently than I do, I am happy to explain how I think this process occurs across the wonderful though self-canceling pages of the American Journal of International Law symposium on the International Criminal Tribunal for the Former Yugoslavia (ICTY) and the International Criminal Tribunal for Rwanda (ICTR) and—through valedictory reflection on those enterprises—on con- temporary international criminal law so far. The self-cancellation process, as I see it, takes place in the move from creation story and doctrinal evolution to impact measurement amidst legacy rhetoric. One might take this result as an index of where things stand (or whether anything stands) in the fascinating emergence of a prestigious enterprise—and what might come next. A sense of fragility has haunted the contemporary revival of international criminal law from the first. In their excellent overview of how the U.S. government has engaged with the enterprise, from the self-interested perspective of two of its representatives in the pivotal 1990s, Michael J. Matheson and David J. Scheffer vigorously deny that the inception of the enterprise was “simply a token alternative to effective action or a mere act of political contrition.”1 They are, of course, right in this contention. But it may not be so much or only, as Matheson and Scheffer themselves contend, because the outrage of ongoing impunity demanded justice. That may have been the motivation of key actors, but, to put it bluntly, states do not have a very good record so far of providing justice for the crimes of world history, and so one has to ask why they started when they did and in the places they did. y p y An obvious answer follows, though it is not in the symposium: there was a cultural frame that made it meaningful and a geopolitical context for specific action. There had always been political evil (defined in different ways), and atrocity wrongs as one of its versions. To the rare extent some response to evil occurs, it is always a matter of which evil the powerful have singled out for attention and which response the powerful have chosen to match—if only because they think the weak want it. ASIL and Samuel Moyn © 2016 * Jeremiah Smith, Jr. Professor of Law and Professor of History, Harvard University. Originally published online 23 November 2016. 1 Michael J. Matheson & David Scheffer, The Creation of the Tribunals, 110 AJIL 173, 173 (2016). 2 Karen L. Engle, Anti-Impunity and the Turn to Criminal Law in Human Rights, 100 CORNELL L. REV. 1069 (2015). See more generally SAMUEL MOYN, THE LAST UTOPIA: HUMAN RIGHTS IN HISTORY (2010). 3 Matheson & Scheffer, supra note 1, at 187. 4 For my own thinking about this history, see Samuel Moyn, From Aggression to Atrocity: Rethinking the History of International Criminal Law, in OXFORD HANDBOOK TO INTERNATIONAL CRIMINAL LAW (Kevin Jon Heller et al. eds., forthcoming). 5 Compare DAVID SCHEFFER, ALL THE MISSING SOULS: A PERSONAL HISTORY OF THE WAR CRIMES TRIBUNALS (2011). For prelimi- nary political accounts of two state agendas, see DAVID BOSCO, ROUGH JUSTICE: THE INTERNATIONAL CRIMINAL COURT IN A WORLD OF POWER POLITICS (2014) or RONEN STEINKE, THE POLITICS OF INTERNATIONAL CRIMINAL JUSTICE: GERMAN PERSPECTIVES FROM NUREMBERG TO THE HAGUE (2012). 6 Darryl Robinson & Gillian MacNeil, The Tribunals and the Renaissance of International Criminal Law: Three Themes, 110 AJIL 191 (2016). Samuel Moyn* By 1992, the frame had been set by a convergence of a human rights revolution and Holocaust memory— and indeed the latter belatedly transforming the former. As Karen Engle has noted, in the early years of the human rights movement, Amnesty International (as its very name implies) aimed at getting people out of jail, not throwing people into it. More broadly, the early human rights movement traded on a newfound skepti- ASIL and Samuel Moyn © 2016 258 https://doi.org/10.1017/S2398772300009120 Published online by Cambridge University Press ON A SELF-DECONSTRUCTING SYMPOSIUM 2016 259 cism about state power, and about its penal authority and methods in particular.2 Early transnational human rights movements had been concerned by political repression and police states, and even their Latin American versions in the 1970s and 1980s were not so much about mass atrocity and genocide as disappearance and terror. But by the 1990s, as anyone who lived through it will remember, the very phrase “human rights” had become so tightly linked to a surge of Holocaust memory across the prior two decades that political evil had now become synonymous with forms of horror that appeared analogous to the World War II events. In the absence of other agents, it seemed critical for states to act to prevent and punish such outrages. It mattered utterly, in this rich and specific context, that the first move towards international criminal ac- countability occurred in response to the shock of atrocity on the European continent—at a moment when European identity depended so much on its sense of having put the Holocaust behind it—even though the subsequent geography of the field has been essentially postcolonial and “southern.” It was in part for this reason, as Matheson and Scheffer nervously admit, that the new international criminal accountability reversed the priorities of the very Nuremberg precedent it has so often claimed to honor, by demoting aggression as “counterproductive”3 (and, for the American state, committing to permanent struggle against its return as a chargeable offense).4 I would not disagree for an instant with Matheson and Scheffer that morality counted for something too, but it was defined in a specific way. And it is remarkable that as state actors Matheson and Scheffer provide next to no information about how state interests (including those of their own state) must have concurred with that new definition. Samuel Moyn* For all the resistance of some states, the new atrocity law emerged at a time of the decline of interstate war, and the persistence of civil war. In claim- ing Nuremberg’s legacy, indeed, the new accountability broke fundamentally from it not simply in its reorientation to atrocity but also in freeing itself from the original context of interstate war (or even war of any kind). As for the closing thought experiment that Robinson and MacNeil offer—to ask what international crimi- nal law might look like without the ad hoc tribunals—I confess that it feels a bit strange. Counterfactual history has experienced a vogue of late, but asking what international criminal law would look like today without the ad hoc tribunals is unlikely to be illuminating because it is not as if the field has had any real life (certainly in recent decades) except through the accident of the ad hoc tribunals in the first place. It is somewhat like asking what someone’s life might look like had her parents never met: she would not have existed at all. Similarly, without the contingent but decisive intervention of the 1990s tribunals, there would not really exist a field of international criminal law to speak about. Especially given the elusive and transformed precedent of Nurem- berg—followed in the 1950s in extant proposals to set up an aggression court—atrocity law was essentially brought about by the ad hoc tribunals, not merely “helped” or “hurt” by them as an intervening factor. It is in turning from creation and doctrine to impact that the symposium makes the self-deconstructing move. In the best book ever written about international criminal law, Judith Shklar long ago indicted exclusive focus on doctrine—that is to say, the focus that Robinson and MacNeil take up—as if “the future of international law” were an important aim regardless of whether Nuremberg improved the world in any way. “To think of either the immediate political needs or the ideological impact of the Trial on Germany would have been to descend to mere politics,” she allowed. “Nevertheless, it was these and these alone that justified the trial.”7 I fully accept the implicit argument of Robinson and MacNeil that the reason doctrinal evolution mattered is that it allowed for just retribution for hitherto unpunished crime. But a wider political lens investigates whether that retribution in fact accrued, compared to imaginable alternative mechanisms. Samuel Moyn* For example, Matheson and Scheffer allude briefly to the fact that the United States might not have ratified a treaty creating a tribunal, so that it was fortunate that the Security Council could act. Scheffer has written movingly of his action in memory of “all the missing souls,” but someday, especially once state archives have been opened, it will be fascinating to learn more about all the present interests—and all the souls that may not have not been missed as much either before or after this pivotal 1990s moment.5 It is already obvious that a unique geopolitical conjuncture obtained, with a post-Cold War glow seducing statesmen and—women into thinking that a discontinuous moralization of global affairs was now possible. It strikes me as highly unlikely that states would have converged in the same way at any other time—even leaving out the most recent controversies over the International Criminal Court and the spike in its unpopu- larity. Once created, the tribunals for Yugoslavia and Rwanda took on a jurisprudential life of their own, as Dar- ryl Robinson and Gillian MacNeil record in their knowledgeable contribution.6 Aside from providing yet more evidence of how fundamentally Nuremberg-era law had to be abandoned or revised to suit the search for accountability in noninternational armed conflict or indeed outside the setting of armed conflict altogeth- er, not to mention to allow pursuit of rape as a war crime, Robinson and MacNeil provide a rich account of how contemporary international criminal law was made. Their reflections on the collectivist turn of the jurisprudence are especially thought-provoking. Voltaire once remarked that he feared to break with the https://doi.org/10.1017/S2398772300009120 Published online by Cambridge University Press Vol. 110 260 AJIL UNBOUND Zeitgeist, for those who lack the spirit of an age may still have all of its defects. Robinson and MacNeil acknowledge contemporary scholarly worries about “progress” narratives, but the real risk is that they portray the conformity of international criminal law with contemporary assumptions, imperatives, and understand- ings about the purposes of such a body of law as more surprising than it in fact is. However interesting it may be to recall that the application of extant war crimes law to noninternational armed conflicts was once controversial, for example, it seems unlikely that a set of doctrines originally crafted for interstate war could have failed for long to be applied to noninternational armed conflict. 7 JUDITH N. SHKLAR, LEGALISM 181, 147 (1963). 8 See Samuel Moyn, Towards Instrumentalism at the International Criminal Court, YALE J. INT’L L. ONLINE 39, 55-65 (Spring 2014). 9 Marko Milanović, The Impact of the ICTY on the Former Yugoslavia: An Anticipatory Postmortem, 110 AJIL 233, 235 (2016). 10 Sara Kendall & Sarah M. H. Nouwen, Speaking of Legacy: Toward an Ethos of Modesty at the International Criminal Tribunal for Rwanda, 110 AJIL 212, 217 (2016) (footnote omitted). Samuel Moyn* And it inquires into what wider effects—both intended and inadvertent—the chosen mechanisms achieved.8 In his superlative “anticipatory postmortem” for the ICTY, Marko Milanović concedes that retribution happened that would not have occurred otherwise. But he rightly indicts the tendency to “continue theorizing about the potential impact” of international criminal law without checking.9 Meanwhile, in their sophisticated piece, Sara Kendall and Sarah M.H. Nouwen willingly acknowledge that the ICTR has struck a blow for international law in some form, but rightly worry that in the absence of “demonstrable findings, many of the claims made about the ICTR’s impact are either hypotheses, setting forth how the Tribunal could have an impact, or assertions of hopes or normative opinions as to what its impact should be.”10 https://doi.org/10.1017/S2398772300009120 Published online by Cambridge University Press ON A SELF-DECONSTRUCTING SYMPOSIUM 2016 261 I read these two fine essays combined as upsetting a current asymmetry in literature on the contemporary “justice cascade” of prosecutions. Like justice, the “empiricism” currently championed in international law scholarship is a specific kind of empiricism, privileging quasi-scientific proof. More than challenging that orthodoxy, the symposium implies how odd it is that “empiricism” is most associated not with critics but with promoters of international criminal processes, who have positioned themselves as cautious fact-finders compared to both conservatives who propose to do nothing and radicals who long for doing something else. These essays suggest that if a promotional empiricism is familiar when it comes to atrocity law, it is because too few empirical questions have been asked.11 Milanović appeals to “objective” evidence of what impact the ICTY has had on how peoples of the region (Bosnia, Croatia, and Serbia) understand what went wrong—and in particular, what responsibility their own people or leaders might bear.12 The results are sobering about how little the proceedings have affected domi- nant narratives, but even more illuminating is Milanović’s observation that one of the ICTY’s smallest acts— the indictment of Vojislav Šešelj—had the biggest consequences for the Serbian people, paving the way for the “soft” authoritarian rule under which they find themselves today. The important lesson is that interna- tional courts are on the hook not just for the little progress they may make towards their avowed or expected aims (from retribution to reconciliation), but also for the whole set of consequences that follow from their deployment. 11 See KATHRYN SIKKINK, THE JUSTICE CASCADE: HOW HUMAN RIGHTS PROSECUTIONS ARE CHANGING WORLD POLITICS (2011) or Hyeran Jo and Beth A. Simmons, Can the International Criminal Court Deter Atrocity?, 70 INT’L ORG. 443 (2016); For counterpoint, see Samuel Moyn, Anti-Impunity as Deflection of Argument, in ANTI-IMPUNITY AND THE HUMAN RIGHTS AGENDA (Karen L. Engle et al. eds., 2016). 12 Milanović, supra note 9, at 235. 13 Kendall & Nouwen, supra note 10, at 213. 14 Id. at 226. 14 Id. at 226. Samuel Moyn* Of course, no one can guess what would have happened had the ICTY or the current wave of international criminal law never come about. But to flee in response to Milanović’s findings into world-weary observations about the opacity and unknowability of things is hardly going to work as justification for trying again. Presumably one would want a better defense of the rise of international criminal processes pursuing atrocity than the speculative guess that doing nothing might have made things even worse. Especially since, as Kendall and Nouwen register in passing, postgenocide Rwanda provides a case in which local justice—not to mention other choices than prosecution whether local or international—has proceeded in tandem with the ICTR.13 Ascending to a brilliant higher-order inquiry into what the desire for a “legacy” and the search for it might involve, Kendall and Nouwen distinguish legacy talk from impact measurement of the sort Milanović attempts. Along the lines of their analysis, indeed, one might suggest that legacy talk is a prime way to distract from an avoidance of impact measurement. Dwelling on the epistemological quandaries involved in assessing a legacy, Kendall and Nouwen persuasively show that it all depends who is asking and from what vantage point, including how distantly from the events: legacy talk, it seems, is not infrequently image management, which does not necessarily mean it gains more plausibility the further away the assess- ment takes place or the less involved those conducting it are. Emphasizing the selectivity of retributive justice and its doubtful contribution to reconciliation, Kendall and Nouwen also worry that the ICTR has played havoc with historical knowledge, and not simply provided the foundation for it. They even cast doubt on how powerful an effect the tribunal had on domestic legal processes and reform, attributing even positive changes to a “confluence of interests.”14 From their survey, Kendall and Nouwen wisely infer that an indefinite sense of fallibility is much more important for the ICTR than a quick bid for immortality. The sheer difficulty of achieving progress, as Ken- dall and Nouwen argue, counsels modesty about this sort of undertaking. Yet the enterprise of international https://doi.org/10.1017/S2398772300009120 Published online by Cambridge University Press Vol. Samuel Moyn* 110 262 AJIL UNBOUND criminal law has had so much invested in it, literally and rhetorically, that it may be difficult to restore it to the usual constraints in which even normalized legal reform must operate: with effort, it may make a bit of difference for the good, while eternally risking failure. The very overhyping lavished on the agenda of atrocity law, up to and including the International Criminal Court, may lead to equally irrational backlash, not so much reasonable moderation. One would not want that result either. criminal law has had so much invested in it, literally and rhetorically, that it may be difficult to restore it to the usual constraints in which even normalized legal reform must operate: with effort, it may make a bit of difference for the good, while eternally risking failure. The very overhyping lavished on the agenda of atrocity law, up to and including the International Criminal Court, may lead to equally irrational backlash, not so much reasonable moderation. One would not want that result either. Not to put words in their mouths, but by the end it seems as if the conclusion Kendall and Nouwen reach—like the self-cancellation process of the overall symposium they complete—feels quite a bit more devastating than they openly state. Kendall and Nouwen (citing Jean-Marie Katamali) call the ICTR a case of “experimental justice,” which is a good label for the entire post-1989 endeavor so far. True, potshots after the fact dishonoring such inevitably imperfect experiments, not to mention those who have committed their lives to them, are frustrating in the extreme, but the same is true of experiments that are never allowed to fail, assuming it made sense to try them to begin with. The symposium forces readers to ask: Has the rise of “atrocity law” in our time been worth it? Would selective retribution alone, to the extent it is being achieved (as all commentators acknowledge), suffice to allow an affirmative response? I do not know the answers to these questions after witnessing the symposium self-deconstruct, but the experience leads to a different outcome than moderation. It feels more paralyzing, but more productive too. It is the intuition that what people need is not exactly a legacy monument for the ICTY or the ICTR so much as a reality check about what it might actually take to improve a recalcitrant and violent world. https://doi.org/10.1017/S2398772300009120 Published online by Cambridge University Press Samuel Moyn* https://doi.org/10.1017/S2398772300009120 Published online by Cambridge University Press

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Air monitoring system of the Krasnoyarsk Science Center SB RAS

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© The Authors, published by EDP Sciences. This is an open access article distributed under the terms of the Creative Commons Attribution License 4.0 (http://creativecommons.org/licenses/by/4.0/). Air monitoring system of the Krasnoyarsk Science Center SB RAS 1Institute of Computational Modeling SB RAS, Krasnoyarsk, Russia 2Federal Research Center Krasnoyarsk Science Center SB RAS, Krasnoyarsk, Russia Abstract. The paper deals with the problem of monitoring the surface layer of the atmosphere of the Siberian city of Krasnoyarsk with a population of one million people in order to study the regularities of the formation of a space-time field of particulate matter concentrations in the air. It is shown that such research can be performed only with the help of a system of scientific monitoring of atmospheric air. The Krasnoyarsk Scientific Center SB RAS air monitoring system is a set of special equipment and software designed to collect and analyze objective data on the state of atmospheric air in Krasnoyarsk. The main task of creating and operating the monitoring system is to form an information and analytical basis for research and development aimed at solving the problems of improving the environmental situation in Krasnoyarsk. The article presents the results of almost two years of experience in the operation of the monitoring system and the main results. * Corresponding author: [email protected] E3S Web of Conferences 223, 03020 (2020) RPERS 2020 E3S Web of Conferences 223, 03020 (2020) RPERS 2020 https://doi.org/10.1051/e3sconf/202022303020 1 Introduction The state air monitoring system is regulated by a variety of regulatory documents. Due to this circumstance, the introduction of advanced and generally recognized in many countries technologies and means of monitoring the state of the air environment is practically not implemented in Russia. For example, Hydrometcenter of Russia (Hydrometeorological Research Center of Russian Federation) still measures suspended matter, while many years of international experience show that it is necessary to control particulate matter. It has been shown that it is particulate matter that have a very negative effect on human health [1, 2]. At the level of the constituent entity of the Russian Federation, a regional environmental monitoring system has been created, the operator of which is a subordinate institution of the Ministry of Ecology of the Territory. As part of this system in Krasnoyarsk and the suburbs, there are nine automated observation posts (AOP), where particulate matter concentrations are measured. However, the information received cannot be interpreted in an accessible and understandable form for the population due to bureaucratic reasons prescribed in the regulatory legal acts of Hydrometcenter of Russia. The residents of Krasnoyarsk are particularly concerned about the smog phenomena, which the people and the media call "black sky". The intensity of the smog is determined by E3S Web of Conferences 223, 03020 (2020) RPERS 2020 https://doi.org/10.1051/e3sconf/202022303020 the concentration of particulate matter. In this regard, it becomes necessary to study the regularities of the formation of the spatio-temporal field of concentrations of particulate matter in the surface layer of the atmosphere of the city of Krasnoyarsk. For such study of the air environment over the territory of one million city, nine AOP equipped with dust meters are clearly not enough. This problem can be solved by installing relatively cheap air monitoring stations that measure PM concentration and some meteorological parameters [3]. the concentration of particulate matter. In this regard, it becomes necessary to study the regularities of the formation of the spatio-temporal field of concentrations of particulate matter in the surface layer of the atmosphere of the city of Krasnoyarsk. For such study of the air environment over the territory of one million city, nine AOP equipped with dust meters are clearly not enough. This problem can be solved by installing relatively cheap air monitoring stations that measure PM concentration and some meteorological parameters [3]. 2.2 Measured indicators The System contains two blocks of information: meteorological characteristics and data on air pollution. Meteorological characteristics are temperature (in degrees Celsius), pressure (mm Hg), relative humidity (percent). Air pollution data is the concentration of particulate matter (PM) with a diameter of less than 10 microns (PM10) and less than 2.5 microns (PM2.5) in the ambient air. 1 Introduction The above circumstances have necessitated the development of a system for scientific monitoring of the air environment. Systems of this type are now being created in many regions, countries and cities of the world [4-8]. 2.1 Air monitoring system The air monitoring system of the Krasnoyarsk Scientific Center of the Siberian Branch of Russian Academy of Sciences (hereinafter referred to as the System) is a set of special equipment and software designed to collect and analyze objective data on the state of atmospheric air in Krasnoyarsk, which, firstly, includes certified and calibrated automatic stations for monitoring atmospheric air pollution (monitoring stations), recording the characteristics of the atmosphere and transmitting this information to a centralized database via a cellular network. Secondly, there are special software tools such as embedded software of monitoring stations and a web interface for their administration, a subsystem for storing registered data and software interfaces for their loading through web services, tools for operational data processing and visualization [9]. The main task of creating and operating the System is to form an information and analytical basis for scientific research and development aimed at solving the problems of improving the environmental situation in Krasnoyarsk. 2.3 Air Quality Index Instant AQI air quality index is calculated based on measurements of suspended particles concentration in the System. The Air Quality Index (AQI) is a widely used worldwide tool for providing information on ambient air pollution in a simple and visual form. In the System, it is currently calculated based on the concentrations of particulate matter (PM10 and PM2.5), which are recalculated (normalized) by piecewise linear interpolation to a single scale of pollution levels (from 0 to 500 units). For each class of this scale, a color designation is also introduced. The main idea of the AQI is to present data on air pollution in terms of human health impact levels. Along with the basic indicator – the air quality index AQI – its modifications are widely used in practice: NowCast AQI, Asian AQI, Instant AQI (InstantCast AQI), etc. They use revised calculation formulas, in which the averaging of suspended solids concentrations is 2 E3S Web of Conferences 223, 03020 (2020) RPERS 2020 https://doi.org/10.1051/e3sconf/202022303020 not performed in 24 hours as in the case of AQI, but in a shorter period – 12 hours (NowCast AQI) or 1 hour (Instant AQI). The result is more objective operational information about air quality; that is why such modifications are recommended for use in public warning systems. The System provides the Instant AQI (instantaneous, operational AQI). not performed in 24 hours as in the case of AQI, but in a shorter period – 12 hours (NowCast AQI) or 1 hour (Instant AQI). The result is more objective operational information about air quality; that is why such modifications are recommended for use in public warning systems. The System provides the Instant AQI (instantaneous, operational AQI). The color-coded AQI index scale shows how air pollution affects human health; on its basis, recommendations were formulated for the population to stay in the open air. 3 Materials and methods The system is based on many years of experience in scientific research and development in the field of solving problems of environmental monitoring and assessment of the state of the natural environment by a team of authors from the Institute of Computational Modeling SB RAS (ICM SB RAS) [10]. The software and technological platform for development is the Geoportal of the ICM SB RAS, including its subsystem "Operational monitoring data". 23 certified CityAir air monitoring stations developed by a group of companies from the Novosibirsk Technopark and the Skolkovo Innovation Center are used as the basic equipment of the air monitoring system of the Krasnoyarsk Scientific Center of the SB RAS. 23 certified CityAir air monitoring stations developed by a group of companies from the Novosibirsk Technopark and the Skolkovo Innovation Center are used as the basic equipment of the air monitoring system of the Krasnoyarsk Scientific Center of the SB RAS. To assess the data coming from the CityAir air monitoring stations, information from four AOP is used on atmospheric air pollution from the Regional Departmental Information and Analytical System of Data on the State of the Environment of the Krasnoyarsk Territory, which is operated by the Regional State Budgetary Institution Center for the Implementation of Environmental Management and Protection Measures the environment of the Krasnoyarsk Territory". This work is carried out within the framework of intensive information and technical interaction and cooperation, with the support of the regional Ministry of Ecology. The developed software for the environmental monitoring system structurally consists of a set of interrelated components. The core of the development is a service-oriented data transmission system and a geospatial database, which receives all the collected information. A set of web interfaces, including those adapted for mobile devices, has been created to present data to end users. Data visualization is carried out in several forms: on a map and using a specialized table (Fig. 1). 3 3 E3S Web of Conferences 223, 03020 (2020) RPERS 2020 https://doi.org/10.1051/e3sconf/202022303020 Fig. 1. Air monitoring system user interface. A tabular view of monitoring posts is shown in Fig. 1 on the left side. All posts are displayed as separate "cards" with the ability to search by title and sort. The card contains up-to-date data on air pollution and meteorological information. The color scheme reflects the degree of hazard according to the AQI scale. By selecting a separate post, the user opens an extended view of data with the dynamics of changes in indicators for the last 2 days or a month. The cartographic presentation of the data (in Fig. 2 on the right) provides display of the hourly averaged pollution values at each observation point, with the ability to scroll through the values for the last 3 days. 4 Results and discussion The system has been operating steadily since October 2018. The advantages of the System include the fact that it stably operates at temperatures of minus 35-40 °C. Under these conditions, measurements on the edge ALP are not carried out for technical reasons. Thus, using the System, for the first time, the concentration of PM2.5 in the atmosphere of the city of Krasnoyarsk was determined during the period of severe frosts [5]. In addition, on the basis of the data obtained using the System, studies are carried out on the regularities of pollution during anticyclones and the effect of forest fire smoke, PM2.5 concentration fields are studied depending on the relief of the city territory and the proximity of the non-freezing Yenisei River. A comparative analysis of the data for 4 winter months was carried out from 01.11.2019 to 29.02.2020. Graphs of daily average values averaged over all 7 posts of the regional system of the Ministry of Ecology of the Territory (dashed red graph) and averaged over all 16 monitoring stations of the Federal Research Center of the KSC SB RAS (solid blue graph) are shown in Fig. 2. Fig. 2. PM2.5 particulate matter concentrations (mg/m3), averaged over monitoring stations in Krasnoyarsk from November 2019 to February 2020. Here are the periods of unfavourable weather conditions announced by state services (gray blocks), the level of average daily maximum allowable concentrations (green line), monitoring data from two independent city monitoring systems (dashed red & solid blue graphs). Fig. 2. PM2.5 particulate matter concentrations (mg/m3), averaged over monitoring stations in Krasnoyarsk from November 2019 to February 2020. Here are the periods of unfavourable weather conditions announced by state services (gray blocks), the level of average daily maximum allowable concentrations (green line), monitoring data from two independent city monitoring systems (dashed red & solid blue graphs). 4 4 E3S Web of Conferences 223, 03020 (2020) RPERS 2020 https://doi.org/10.1051/e3sconf/202022303020 This presented graph also shows the days on which the mode of unfavourable meteorological conditions (UMC) was announced, this was 7 times during the period under consideration, and the standard for the average daily maximum permissible concentration PM2.5 = 0.035 mg/m3. g The mean values of PM2.5 concentrations at the monitoring stations for this period are shown in Fig. 3. 4 Results and discussion Analysis of the presented spatial distribution of PM2.5, taking into account the inhomogeneity of the relief, suggests that the pollution of the city's atmosphere in the winter period is primarily due to autonomous heat supply sources (stove heating). Fig. 3. Average concentrations of particulate matter PM2.5 (μg/m3) in winter (for 4 months, from November 2019 to February 2020) at monitoring stations in Krasnoyarsk. Fig. 3. Average concentrations of particulate matter PM2.5 (μg/m3) in winter (for 4 months, from November 2019 to February 2020) at monitoring stations in Krasnoyarsk. 5 Conclusions Within the framework of the presented interdisciplinary research, a development was designed and implemented based on solving the problems of studying the characteristics of the natural environment of a million-strong city and creating information and computing technologies for the development of an effective service-oriented analytical system. As a result, a network of scientific research monitoring of atmospheric pollution in a large industrial city was formed, based on a large number of spatially distributed operational monitoring stations. This network provides capabilities for detailed spatial and temporal data analysis. For example, it is possible to determine the centers of the initial appearance of pollutants, to trace the dynamics of the spread of pollution across the territory at the level of individual microdistricts of the city. Such data can help to identify sources of pollution, to reveal the characteristic tendencies of its distribution. The preliminary results of processing the obtained information show a significant effect of furnace heating on the pollution of Krasnoyarsk. The unevenness of the relief, along with the speed and direction of the wind, largely determines the characteristics of the spread of pollution in the city. Further research in this area is needed to obtain reliable results, analyze the pollution situation and justify proposals for regional authorities. 5 https://doi.org/10.1051/e3sconf/202022303020 E3S Web of Conferences 223, 03020 (2020) RPERS 2020 E3S Web of Conferences 223, 03020 (2020) References 1. Song C., He J., Wu L., Jin T., Chen X., Li R., Ren P., Zhang L., Mao H. Environ. Pollut. 223, 575 (2017) 2. S. Gulia, S.M. Shiva Nagendra, M. Khare, I. Khanna. Atmospheric Pollution Research, 6 (3), 286-304 (2015) ( ) ( ) 3. Changqing Lin et al. Atmospheric Environment. 227, 117410 (2020) 4. M. Ghodousi, F. Atabi, J. Nouri, A. Gharagozlou. Polish Journal of Environmental Studies, 26 (2), 593-603 (2017) 5. Xuezhen Qiu et al. Frontiers of Environmental Science & Engineering, 9 (6), 1056 (2015) 6. T. Savu, B.A. Jugravu, D. Dunea. REVISTA DE CHIMIE, 68 (4), 796 (2017 7. L. Hu, P. Yue, M. Zhang, J. Gong, L. Jiang, X. Zhang. Earth Science Informatics, 8 (3), 511 (2015) 8. Hua Wang et al. Journal of Environmental Sciences, 29, 178 (2015) 9. Kadochnikov A.A., Tokarev A.V., Zavoruev V.V., Yakubailik O.E. IOP Conference Series: Materials Science and Engineering. 537 (6), 062052 (2019) 10. Yakubailik O.E., Kadochnikov A.A., Tokarev A.V. Optoelectronics, Instrumentation and Data Processing, 54 (3), 243 (2018) 6 6

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Design of modular gellan gum hydrogel functionalized with avidin and biotinylated adhesive ligands for cell culture applications

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RESEARCH ARTICLE Editor: Rachael Ann Oldinski, University of Vermont, UNITED STATES Editor: Rachael Ann Oldinski, University of Vermont, UNITED STATES Received: November 27, 2018 Accepted: August 19, 2019 Published: August 30, 2019 Copyright: © 2019 Gering 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. Received: November 27, 2018 Accepted: August 19, 2019 Published: August 30, 2019 Copyright: © 2019 Gering 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. Design of modular gellan gum hydrogel functionalized with avidin and biotinylated adhesive ligands for cell culture applications Christine GeringID1*, Janne T. KoivistoID1, Jenny Parraga1, Jenni Leppiniemi1, Kaisa Vuornos1,2, Vesa P. Hyto¨nenID1,3, Susanna Miettinen1,2, Minna Kelloma¨ki1 1 Faculty of Medicine and Health Technology, BioMediTech, Tampere University, Tampere, Finland, 2 Research, Development and Innovation Center, Tampere University Hospital, Tampere, Finland, 3 Fimlab Laboratories, Tampere, Finland a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 * [email protected] * [email protected] * [email protected] Abstract This article proposes the coupling of the recombinant protein avidin to the polysaccharide gellan gum to create a modular hydrogel substrate for 3D cell culture and tissue engineering. Avidin is capable of binding biotin, and thus biotinylated compounds can be tethered to the polymer network to improve cell response. The avidin is successfully conjugated to gellan gum and remains functional as shown with fluorescence titration and electrophoresis (SDS- PAGE). Self-standing hydrogels were formed using bioamines and calcium chloride, yield- ing long-term stability and adequate stiffness for 3D cell culture, as confirmed with compres- sion testing. Human fibroblasts were successfully cultured within the hydrogel treated with biotinylated RGD or biotinylated fibronectin. Moreover, human bone marrow stromal cells were cultured with hydrogel treated with biotinylated RGD over 3 weeks. We demonstrate a modular and inexpensive hydrogel scaffold for cell encapsulation that can be equipped with any desired biotinylated cell ligand to accommodate a wide range of cell types. OPEN ACCESS Citation: Gering C, Koivisto JT, Parraga J, Leppiniemi J, Vuornos K, Hyto¨nen VP, et al. (2019) Design of modular gellan gum hydrogel functionalized with avidin and biotinylated adhesive ligands for cell culture applications. PLoS ONE 14 (8): e0221931. https://doi.org/10.1371/journal. pone.0221931 Citation: Gering C, Koivisto JT, Parraga J, Leppiniemi J, Vuornos K, Hyto¨nen VP, et al. (2019) Design of modular gellan gum hydrogel functionalized with avidin and biotinylated adhesive ligands for cell culture applications. PLoS ONE 14 (8): e0221931. https://doi.org/10.1371/journal. pone.0221931 Avidin-functionalized gellan gum hydrogels for cell culture applications no negative cell response.[1,5] However, rather than only providing a passive environment, hydrogels are also required to promote certain cell functions and support cell recognition and response.[5] In practice, the hydrogel should contain cell recognition moieties, such as peptide sequences or proteins like growth factors, i.e., sites that actively guide cell response. In a similar fashion, the mechanical properties and degradation profile of the hydrogel matrix must actively influence the response of the seeded cells. (VPH) and by The Doctoral Programme in Biomedical Sciences and Engineering at Tampere University (CG). 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. Further, the stiffness of the microenvironment is directly conveyed to the cytoskeleton through cell attachment and integrin signaling. This phenomenon, called mechanotransduc- tion, is known to affect the differentiation of cells[8–10], cell attachment, and migration. [5,11,12] In turn, mechanotransduction requires the integrin ligand to be strongly tethered to the polymeric network. This attachment is necessary to promote cell spreading and to prevent the diffusion of the cellular cues.[4] Finally, there are a few more technical issues of hydrogel design that include the manipula- tion and handling as well as the availability and cost-efficiency of the material.[13] Clearly, the material must be affordable to enable its use on a more general basis. The ability to handle, transport, and analyze the final hydrogel product has definite practical advantages over more sensitive constructs. Ultimately, the design of a hydrogel must strike a balance between func- tional complexity and technical simplicity. One approach to the design of hydrogels for tissue engineering applications is the conjuga- tion of bioactive molecules to the polymer. Examples of conjugation techniques include the formation of zero-length bonds, bio-orthogonal coupling, and the use of protein-ligand bind- ing. Zero-length bonds form a short, direct chemical bond between the polymer and the cou- pled compound that are achieved using carbodiimide coupling and thiol-based conjugation techniques.[14] The so-called bio-orthogonal coupling, such as the strain-promoted azide- alkyne cycloaddition[15], do not interfere with compounds found in living organisms. Here, we exploit the protein-ligand binding, which provides a simple and native tool to form sub- strate-ligand complexes. Introduction Biomaterials are essential instruments in the field of tissue engineering and regenerative medi- cine that are required to support cells and mimic natural tissue.[1,2] Hydrogels are a class of biomaterials that can simulate the native, physiological, and three-dimensional (3D) environ- ment of mammalian cells and act as an artificial extracellular matrix.[3–8] It has been well- established that 3D tissue matrices must be considered over planar, two-dimensional (2D) sur- faces for cell culture applications and in vitro disease modeling.[5] The biomaterial should rec- reate all aspects of the natural cell environment, including dimensionality, physical, mechanical, and biochemical properties. These properties are then engineered to control cell attachment and cell fate Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: The project was funded from several sources: the Innovation Funding Agency ’Business Finland’ (formerly TEKES) as the Human Spare Parts program (MK, CG, JK, JP), by the Competitive State Research Financing of the Expert Responsibility area of Tampere University Hospital (KV, SM), by Academy of Finland project 290506 mechanical, and biochemical properties. These properties are then engineered to control cell attachment and cell fate. There are several issues that need to be considered when designing a hydrogel for cell cul- ture applications. Foremost, all components must assert their biocompatibility and the final material, as well as the reagents used in the preparation method, must be non-toxic and elicit PLOS ONE | https://doi.org/10.1371/journal.pone.0221931 August 30, 2019 1 / 22 PLOS ONE | https://doi.org/10.1371/journal.pone.0221931 August 30, 2019 Certain proteins have the ability to bind small, specific molecules with high affinity and selectivity. One of the most in-depth studied protein systems is the avi- din-biotin binding system.[16–18] Avidin is a protein with the capability to bind biotin with outstanding selectivity and specificity. The avidin-biotin interaction is deemed to be the stron- gest non-covalent bond in nature with a dissociation constant of Kd ~10−15 M, and it has often been used in biochemical assays, diagnostics, and tissue engineering.[18] Furthermore, this type of protein-affinity system has been exploited for many different applications in chemistry, biosciences, and tissue engineering. Indeed, modular approaches, such as the use of nanocellu- lose for 3D printing [19], 2-hydroxyethyl methacrylate flat substrates[20], agarose for spatial patterning [21], and porous poly-L-lactic acid scaffolds [22], have been presented and will be discussed in chapter 4.1. The base polymer used in this study is gellan gum (GG), which is an anionic polysaccharide that has previously been investigated for various cell culture applications.[23–26] GG is able to form self-supporting hydrogels, that do not flow and fracture under high stress and also described as “true gels” in contrast to “weak gels”.[27] To form true hydrogels, GG is hereafter ionically crosslinked with the bioamine spermidine. In its native state, GG has been found to be biocompatible, but most cell types do not readily adhere to or favor the material. Previously, our research group established that functionalization of gellan gum with ECM proteins is needed to obtain better cell attachment and migration.[24] Here, we propose the coupling of the avidin protein to gellan gum to create a modular material that can be modified through the addition of biotinylated cell cues. The cues can enable cell attachment, guide differentiation, or even present drug molecules. In this study, we use an avidin analogue called charge-neutralized chimeric avidin, which has been developed PLOS ONE | https://doi.org/10.1371/journal.pone.0221931 August 30, 2019 2 / 22 Avidin-functionalized gellan gum hydrogels for cell culture applications and produced by our group. It has identical affinity for biotin, but increased stability against pH and temperature treatment compared to wild-type avidin.[28] Furthermore, we have sought suitable applications for the charge-neutralized chimeric avidin in the field of tissue engineering.[29] Biotin is a small organic compound that can be chemically coupled to a desired ligand. Before the gelation step, the hydrogel precursor can be modified without any additional chemical functionalization steps. Purification Gellan gum (GelzanTM, low acyl, Mw 1 kg/mol) was purified to remove counterions in the product and to replace them with sodium ions.[30,31] Briefly, a gellan gum solution (0.5% w/ v, 400 mL) in dI water (milliQ) was combined with Dowex cation exchange resin (5 g, H + form, 50–100 mesh, prerinsed in HCl) and stirred for 30 min at 60 ˚C. The exchange resin was removed from the solution through decantation and the pH was adjusted to 7.5 with NaOH. The purified product was then precipitated in i-propanol and lyophilized over 2 days. Gellan gum (GelzanTM, low acyl, Mw 1 kg/mol) was purified to remove counterions in the product and to replace them with sodium ions.[30,31] Briefly, a gellan gum solution (0.5% w/ v, 400 mL) in dI water (milliQ) was combined with Dowex cation exchange resin (5 g, H + form, 50–100 mesh, prerinsed in HCl) and stirred for 30 min at 60 ˚C. The exchange resin was removed from the solution through decantation and the pH was adjusted to 7.5 with NaOH. The purified product was then precipitated in i-propanol and lyophilized over 2 days. The ion concentration of NaGG was determined with inductively coupled plasma optical emission spectrometry (ICP-OES). Hence, a part of the sample was digested in sulfuric acid and hydrogen peroxide following the protocol by Kirchmajer et al. (2014)[31] and measured with Agilent 5110 ICP-OES (Agilent Technologies). Materials and methods All materials were acquired from Sigma-Aldrich, if not otherwise stated. Charge-neutralized chimeric avidin (avd) was kindly donated by the Protein Dynamics group at the Tampere Uni- versity and is commercially available for research use at Ref. [29]. The choice of biotinylated species can range from attachment factors, such as RGD, to drug molecules, and to growth factors (GF), such as vascu- lar endothelial GF, which also affects differentiation. Ultimately, the goal is to design a non- specialized platform that is adaptable to different applications, while still possessing the innate ability to support cell growth and to allow for the convenient analysis of the tissue engineering construct. To underline the suitability of the proposed modular hydrogel, we characterized the mechanical properties and avidin functionality of the gellan gum-avidin material. First, GG was purified to remove excess counter-ions, yielding sodium-purified GG (NaGG).[23,30] Then, charge-neutralized chimeric avidin (avd) was coupled to NaGG via carbodiimide conju- gation using EDC and NHS[14,23], yielding NaGG-avd. True hydrogels can be formed with suitable crosslinker concentration and compression testing revealed that mechanical behavior was not impaired by the functionalization. Avidin coupled to NaGG is functional and its abil- ity to bind biotin is not impaired by the coupling. Additionally, avidin is shown to be cova- lently coupled to the polymer network as it does not diffuse from the gel. Human fibroblasts were cultured in NaGG-avd and their viability was assessed. Similarly, the effect of NaGG-avd on human bone marrow-derived stem cells was studied for a total culture time of 3 weeks, highlighting the long-term stability of the gels. Functionalization and structural characterization The functionalization reaction was based on the publication by Ferris et al. who similarly acti- vated gellan gum with 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) and N-hydro- xysuccinimide (NHS) to couple the peptide sequence RGD.[23] A solution of sodium-purified gellan gum (NaGG, 10 mg/mL, 10 or 20 mL) was dissolved in HEPES buffer (50 mM, pH 6.5) and stirred at 40 ˚C. The gellan gum was then activated with EDC (0.4 M) and sulfo-NHS (1.0 M) for 15 min and consequently quenched with β-mercapthoethanol (28 μL, final PLOS ONE | https://doi.org/10.1371/journal.pone.0221931 August 30, 2019 3 / 22 Avidin-functionalized gellan gum hydrogels for cell culture applications Fig 1. Reaction scheme of NaGG functionalization with avidin via activation with EDC and NHS. EDC activates the carboxyl group of NaGG, and the intermediate is stabilized with NHS to prevent activation of avidin with EDC. The final product is a combination of functionalized [m] and non- functionalized [n] NaGG repeating units. https://doi org/10 1371/journal pone 0221931 g001 Fig 1. Reaction scheme of NaGG functionalization with avidin via activation with EDC and NHS. EDC activates the carboxyl group of NaGG, and the intermediate is stabilized with NHS to prevent activation of avidin with EDC. The final product is a combination of functionalized [m] and non- functionalized [n] NaGG repeating units. Fig 1. Reaction scheme of NaGG functionalization with avidin via activation with EDC and NHS. EDC activates the carboxyl group of NaGG, and the intermediate is stabilized with NHS to prevent activation of avidin with EDC. The final product is a combination of functionalized [m] and non- functionalized [n] NaGG repeating units. https://doi.org/10.1371/journal.pone.0221931.g001 https://doi.org/10.1371/journal.pone.0221931.g001 https://doi.org/10.1371/journal.pone.0221931.g001 https://doi.org/10.1371/journal.pone.0221931.g001 concentration 20 mM). Finally, charge-neutralized chimeric avidin (1 mg/mL, 3.5 mL in HEPES 50 mM, pH 6.5) was added and the mixture was stirred for 5 h at 40 ˚C. The functiona- lized product was dialyzed over 5 days (MWCO 12–14 kDa) and subsequently lyophilized. Two different batches of NaGG-avd were studied. Each batch differed in the amount of avidin used and the final avidin concentration in the material. Here, these modular hydrogels are termed NaGG-avd(L) for low avidin concentration (4 mg avidin/ 1 g NaGG) and NaGG-avd (H) for high avidin concentration (21 mg avidin/ 1 g NaGG). The reaction scheme of the functionalization is shown in Fig 1. The unstable intermediate of the active ester is stabilized through the addition of NHS, which forms an amine-reactive sulfo-NHS ester. PLOS ONE | https://doi.org/10.1371/journal.pone.0221931 August 30, 2019 Functionalization and structural characterization The primary amines of avidin react with the stable intermediary to form a peptide bond. Notable side reactions include the activation of the carboxyl bonds present in the protein structure of avidin. It can, however, be assumed that EDC quickly deactivates through hydrolysis in aqueous media. The resulting structure was investigated by means of avidin-biotin binding and fluores- cence spectroscopy as well as with electrophoresis (SDS-PAGE with urea) and elution analysis. A fluorescence titration curve was prepared from NaGG-avd (1 mg/mL in HEPES pH 7, 10% sucrose) and the biotinylated fluorescent dye b5F (biotin-5-fluorescein, 2 μM in DMSO and PBS). Control curves were prepared with NaGG and NaGG-avd blocked with biotin (3 μL, 0.17 mg/mL in 50 mM sodium-phosphate, 100 mM NaCl buffer, pH 7). Aliquots of 25 μL b5F were added to a 2 mL sample in a cuvette and each measured after 2 min with a QuantaMaster PTI spectrofluorometer (Photon Technology International, Inc., Lawrenceville, NJ, USA) (excitation at 495 nm, emission at 520 nm, slits 2 nm). For elution analysis, hydrogel samples were prepared with 5 mg/mL NaGG-avd and 10 μM b5F (14 μL/mL NaGG-avd) and crosslinked with 0.5 mg/mL spermidine (SPD). The samples were incubated in the mold overnight and then placed into 500 μL PBS for up to 48 h in an incubator under shaking at 37 ˚C. The eluate was taken at time points of 1 h, 6 h, 24 h, and 48 PLOS ONE | https://doi.org/10.1371/journal.pone.0221931 August 30, 2019 4 / 22 Avidin-functionalized gellan gum hydrogels for cell culture applications h, and the fluorescence intensity was measured. As a reference sample, NaGG was mixed with avidin without conjugation and gels were formed as described above. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was used to further examine the covalent nature of the NaGG-avd conjugate through comparison with a non- covalent mixture of NaGG and avidin (avd). To improve the resolution of the SDS-PAGE, urea was added to the samples and the cast gel. The PAGE gel was cast according to standard procedure, but urea (8 M) was added. All samples were incubated with biotin (85 μg/mL final concentration) to stabilize the avidin tetramer. After addition of loading buffer, the samples were heated to 50 ˚C for 15 min. After a short cooling period, 8 M urea was added to each sam- ple. Functionalization and structural characterization The electrophoresis was performed at +4 ˚C for 3 h at 100 V. Finally, the gel was stained with OrioleTM fluorescent gel stain (Bio-Rad) and imaged with a ChemiDoc MP imaging sys- tem (Bio-Rad Laboratories) with Image Lab software. Physical properties Hydrogel samples were prepared with a uniform mixing technique, which has been described earlier30, to yield homogenous, disc-shaped samples crosslinked with the cationic compound SPD. Gellan gum (5 mg/mL in HEPES/sucrose solution, pH 6.5) was mixed with spermidine (0.5 mg/mL) in 5:1 volume ratio. If CaCl2 was used instead of spermidine crosslinker, a con- centration of 10 mM was added in same volume ratio. The hydrogel solution was then warmed in a mixing vial under constant stirring (300 rpm) and the crosslinking solution was added. The solution was then swiftly transferred to the mold before the true gel was formed. Compression behavior was analyzed with a Bose BioDynamic ElectroForce Instrument 5100 using WinTest 4.1 software (TA Instruments, USA). Disc-shaped samples with a diame- ter of 1.2 cm and a height of 4.5 mm were prepared in PP/PE molds, and the number of paral- lel samples (n) was 5. The test was carried out as uniaxial, unconfined compression in air at ambient pressure and temperature. The sample was prevented from sliding with wet cellulose paper and compressed with a speed of 10 mm/min to 65% of the original sample height. From the resulting stress-strain curve, the fracture strain and fracture strength were analyzed. The swelling behavior of the functionalized hydrogel NaGG-avd was monitored over 3 weeks. The chosen hydrogel compositions for the swelling corresponded to the studied to compression samples. The gels were incubated in 500 μL of PBS or DMEM F-12 at 37˚C for up to 3 week. The samples were then weighed for their wet weight and consecutively lyophi- lized and weighed again to determine the dry mass. (Data shown in S4 Appendix). PLOS ONE | https://doi.org/10.1371/journal.pone.0221931 August 30, 2019 Cell culture Human fibroblasts WI-38 (passage 24–26, Sigma-Aldrich/Culture Collections, Public Health England) we expanded for one week in WI-38 medium, consisting of 10 vol% FBS, 25 U/mL penicillin/streptomycin (pen/strep; Lonza, Basel, Switzerland) in DMEM-F12 1:1 (Gibco, Thermo Fisher Scientific, Waltham, MA) until confluent. The hydrogel solutions of NaGG and NaGG-avd were sterilized prior to cell culture through filtration (Whatman1 FP30/0.2 CA-S). The cells were seeded on top of the hydrogel (2D, 63 000 cells/cm2) and encapsulated in the hydrogel (3D, 950 000 cells/mL) using a Greiner Cellstar 48-well plate (Sigma-Aldrich). The samples were prepared similarly as described for the compression test sample, where the NaGG-avd solution was heated and stirred in a mixing vial at 37 ˚C.[32] For cell culture pur- poses, the biotinylated compound was added, followed by the cell suspension and mixed gently (300 rpm) to achieve homogeneous 3D distribution. Finally, the crosslinker spermidine (0.5 mg/mL, 17 vol% of NaGG-avd) was added and the hydrogel mixture was cast into the well- plate. 5 / 22 PLOS ONE | https://doi.org/10.1371/journal.pone.0221931 August 30, 2019 Avidin-functionalized gellan gum hydrogels for cell culture applications The tested material compositions included NaGG-avd with biotin (0.17 mg/mL in 50 mM sodium-phosphate, 100 mM NaCl buffer, pH 7), biotinylated cyclic RGD (cyclo[Arg-Gly-Asp- D-Phe-Lys(Biotin-PEG-PEG)] 0.1 mg/mL in H2O, 0.3 μg/mL in final gel), and biotinylated human fibronectin (bFN, 2.52 mg/mL, 33 μg/mL in final gel; fibronectin was purified by gela- tin affinity chromatography from outdated plasma preparation and chemically biotinylated). The concentration of biotinylated species was set to match the number of avidin binding sites determined with fluorescence titration. As a reference, the fibroblasts were grown on the well- plate (PS) bottom. The cells were then cultured for 3 days and LIVE/DEAD1 stained. 1 The concentration of biotinylated species was set to match the number of avidin binding sites determined with fluorescence titration. As a reference, the fibroblasts were grown on the well- plate (PS) bottom. The cells were then cultured for 3 days and LIVE/DEAD1 stained. The cell culture samples were stained using the LIVE/DEAD1 viability/cytotoxicity assay (Molecular probes, Thermo Fisher Scientific) containing calcein acetoxymethyl ester (Ca-AM) and ethidium homodimer-1 (EthD1). The dyes were diluted in PBS (Lonza) (final solution concentration Ca-AM 0.8 μM and EthD1 1.0 μM) and added on top of the cell culture samples. PLOS ONE | https://doi.org/10.1371/journal.pone.0221931 August 30, 2019 Cell culture The samples were incubated for 30 min at room temperature and imaged with an Olympus IX51 inverted microscope and an Olympus DP30BW digital camera (Olympus, Tokyo, Japan). The images were then analyzed with ImageJ software (U.S. National Institutes of Health, Bethesda, MD)[33] through the particle counting algorithm. The cell viability was determined from the area according to Eq (1), while cell spreading was determined from the same data using Eq (2): Viability ¼ area of live cells area of live cells þ area of deadcells ð1Þ Spreading ¼ area of live cells image area ð2Þ Viability ¼ area of live cells area of live cells þ area of deadcells ð1Þ ð1Þ Spreading ¼ area of live cells image area ð2Þ Spreading ¼ area of live cells image area ð2Þ ð2Þ Primary human bone marrow stromal cells (hBMSC) were previously harvested, isolated, and cryo-preserved in gas phase nitrogen by the Adult Stem Cell Group, BioMediTech, Tam- pere University, in accordance with the Regional Ethics Committee of the Expert Responsibil- ity area of Tampere University Hospital, ethical approval R15174. The hBMSCs were isolated from an anonymous donor (labeled 6/16) with the patient’s written informed consent during surgery at the Department of Orthopaedics and Traumatology at Tampere University Hospi- tal. The isolation of hBMSCs was performed as described previously with slight modifications. [34,35] Briefly, the bone marrow aspirate was rinsed with DPBS (Lonza), resuspended in Ficoll (GE Healthcare, Chicago, IL, USA), centrifuged 800 g for 20 min at room temperature after which mononuclear cells were collected, washed twice with α-MEM (Gibco, Thermo Fisher Scientific) and centrifuged 400 g for 15 min at room temperature. In the following, cells were seeded into PS flasks (Nunclon; Sigma-Aldrich) in basic medium containing 5% HS (Biowest, Nuaille´, France) in α-MEM and 1% 100 U/mL pen/strep and 5 ng/mL hFGF-2 (Miltenyi Bio- tec, Bergisch Gladbach, Germany), and expanded until 80% confluence. The isolated hBMSCs were characterized with flow cytometry (S6 Appendix). The hBMSCs (passage 6) were thawed and expanded for one week in T75 PS flasks (Nun- clon; Sigma-Aldrich) in basic medium until confluent. The cells were then harvested and seeded into the hydrogel via uniform mixing (cell density 950 000 cells/mL), as described for the fibroblast test using spermidine as crosslinker. The studied materials were NaGG (as a ref- erence) and NaGG-avd with the addition of biotinylated cyclic RGD (2.5 μM final solution concentration). The cells were cultured for 21 days in the hydrogels with the addition of osteo- genic medium (5 vol% HS, 0.25 mM ascorbate-2-phosphate, 10 mM β-glycerophosphate, 1% 100 U/mL pen/strep in α-MEM, with the addition of dexamethasone 5 nM), which was replaced every other day. 6 / 22 PLOS ONE | https://doi.org/10.1371/journal.pone.0221931 August 30, 2019 Avidin-functionalized gellan gum hydrogels for cell culture applications Viability assay was carried out on day 3, 14, and 21 similar to the WI-38 experiment. Ca- AM and EtHD-1 were diluted in PBS (final solution concentration Ca-AM 0.5 μM and EthD1 0.25 μM) and added on top of the cell culture samples. On day 21, the samples were stained with phalloidin (0.17 μg/mL in 1% bovine serum albumin BSA) and 4’,6-diamidino-2-pheny- lindole (DAPI; dilution 1:2000 in PBS; Sigma-Aldrich). Results GG was purified to remove counterions (Ca2+, K+, Mg2+) and to replace them with sodium (Na+). ICP-OES data can be found in S1 Appendix. The result of the ion analysis shows that the calcium content was reduced to below 0.1 wt%, closely matching reported literature values 24,29. NaGG was successfully functionalized with the recombinant protein ‘charge-neutralized chimeric avidin’ (avd) through carbodiimide conjugation. NaGG-avd solution was prepared in HEPES buffer with sucrose. Hydrogel samples were prepared through gelation with SPD or CaCl2 overnight in disc-shaped molds with dimensions of about 4.1 mm in height and 11.6 mm in diameter. Success of functionalization Fluorescence analysis was carried out in different ways to prove the binding and functionality of avidin. The biotinylated fluorescence dye biotin-5-fluorescein (b5F) was used in all cases. Fluorescence titration with b5F was carried out in order to confirm that avidin retains its abil- ity to bind biotin after being coupled to gellan gum. Therefore, small amounts of b5F were added to the analyte. This dye shows a quenching effect to approximately 50% of fluorescence strength when bound to an avidin specific binding site, which results in a non-linear curve. The concentration of available biotin binding sites can be derived from the intersection of the quenched curve and the linear region after saturation. Fig 2 shows the curves of coupled NaGG-avd, unmodified NaGG, and coupled NaGG-avd that has been blocked with biotin. The unmodified and blocked samples show a linear increase in fluorescence intensity, whereas the avidin-modified samples show a quenched curve followed by a linear increase. From the titration curves in Fig 2, the avidin concentration was estimated to be 0.075 (L) and 0.375 (H) μM in 1mg/mL NaGG-avd. The degree of functionalization (dfunct) can be cal- culated as the molar ratio between avidin-functionalized GG repeating units [n] and non-func- tionalized repeating units retaining the carboxyl (COOH) group [m]. The indices n and m refer to the reaction scheme in Fig 1, and the functionalization degrees are derived from Eq (3): dfunct ¼ ½nŠ ½mŠ ¼ molðavidinÞ molðGG COOHÞ
100% ð3Þ ð3Þ A functionalization degree of 0.005 mol% (L) and 0.027 mol% (H) was achieved between different functionalization degrees, assuming one avidin is bound to only a single carboxyl group. A functionalization degree of 0.005 mol% (L) and 0.027 mol% (H) was achieved between different functionalization degrees, assuming one avidin is bound to only a single carboxyl group. To ascertain the covalent binding between gellan gum and avidin, hydrogel samples were prepared containing b5F. The samples were incubated in phosphate buffered saline (PBS) at 37 ˚C, with separate samples for each time point of 1 h, 6 h, 24 h, and 48 h, and the fluores- cence intensity was measured. As a reference sample, NaGG hydrogel samples were prepared with the addition of a comparable amount of avidin. Fig 3(A) shows the resulting graph. Spreading ¼ area of live cells image area ð2Þ The samples were then fixed (0.1% Tri- ton x-100 and paraformaldehyde PFA) and blocked (1% BSA) before staining. PLOS ONE | https://doi.org/10.1371/journal.pone.0221931 August 30, 2019 Success of functionalization Initially, the fluorescence intensity of both eluates is very similar, alluding to a leaching of whole polymer chains from the gel sample carrying the b5F. However, after roughly 24 h in PLOS ONE | https://doi.org/10.1371/journal.pone.0221931 August 30, 2019 7 / 22 Avidin-functionalized gellan gum hydrogels for cell culture applications Fig 2. Fluorescence titration curves with step-wise addition of b5F. Non-functionalized NaGG (green) and functionalized NaGG-avd blocked with biotin (red) show a linear curve, whereas functionalized NaGG-avd (yellow and blue) with available biotin-binding sites show quenching behavior at low b5F concentration. From the intersection of quenched and linear parts the biotin binding site concentration can be deduced. From the intersection or linear and polynomial curves, the biotin-binding concentration was calculated. https://doi org/10 1371/journal pone 0221931 g002 Fig 2. Fluorescence titration curves with step-wise addition of b5F. Non-functionalized NaGG (green) and functionalized NaGG-avd blocked with biotin (red) show a linear curve, whereas functionalized NaGG-avd (yellow and blue) with available biotin-binding sites show quenching behavior at low b5F concentration. From the intersection of quenched and linear parts the biotin binding site concentration can be deduced. From the intersection or linear and polynomial curves, the biotin-binding concentration was calculated. https://doi.org/10.1371/journal.pone.0221931.g002 https://doi.org/10.1371/journal.pone.0221931.g002 suspension, the fluorescence intensity of NaGG-avd starts to reach a plateau and virtually no further b5F leaves the gel. In contrast, the reference sample NaGG+avd continues to exude b5F, and after 48 h the fluorescence intensity of the eluate is considerably higher. When choosing methods to investigate the gellan gum-avidin bonding, it was surmised that 1H-NMR does not sufficiently distinguish the newly formed carbodiimide-coupled peptide bond from the already present bonds in the studied biomacromolecule. Similarly, FT-IR spec- troscopy is unable to show the bond characteristics between the two macromolecules. Thus SDS-PAGE was the method of choice to confirm the gellan gum-avidin binding. The urea PAGE shows a strong band for avidin tetramer in the reference sample (NaGG+avd) and pure avidin, but a faint, diffuse band for the conjugated sample (NaGG-avd) in Fig 3(B). The disper- sion of the band may be due to avidin coupled with short GG chain fragments, leading to a wider molar mass range. A very faint band for the avidin monomer can be observed for NaGG-avd, but not for the reference sample NaGG+avd. GG itself has a molecular weight of around 1000 kDa and does not appear as a band on the SDS-PAGE. PLOS ONE | https://doi.org/10.1371/journal.pone.0221931 August 30, 2019 Success of functionalization The full uncropped and un-altered blot and gel image is available in S2 Appendix. A simple washing test was carried out is described in S8 Appendix, demonstrating a 4-fold increase in retained biotinylated fibro- nectin when comparing unfunctionalized (NaGG) and functionalized GG (NaGG-avd). This further underlines the functionality of the proposed modular hydrogel. PLOS ONE | https://doi.org/10.1371/journal.pone.0221931 August 30, 2019 8 / 22 Avidin-functionalized gellan gum hydrogels for cell culture applications Fig 3. Avidin-GG coupling analysis. (A) Elution analysis: Fluorescence intensity of supernatants of hydrogel samples immersed in PBS. Supernatant was collected from separate samples over 2 days and analyzed with fluorescence spectrometer. Error bars represent standard deviation calculated from three independent samples, and logarithmic fit curves were added to guide the eye. (B) SDS-PAGE: Electrophoretic separation and comparison of conjugated (NaGG-avd) and unconjugated (NaGG+avd) samples. Tetrameric avidin band (57 kDa) is very faint and blurry for NaGG-avd, compared with NaGG+avd and pure avd, indicating covalent bonding between avidin and polymer. https://doi.org/10.1371/journal.pone.0221931.g003 Fig 3. Avidin-GG coupling analysis. (A) Elution analysis: Fluorescence intensity of supernatants of hydrogel samples immersed in PBS. Supernatant was collected from separate samples over 2 days and analyzed with fluorescence spectrometer. Error bars represent standard deviation calculated from three independent samples, and logarithmic fit curves were added to guide the eye. (B) SDS-PAGE: Electrophoretic separation and comparison of conjugated (NaGG-avd) and unconjugated (NaGG+avd) samples. Tetrameric avidin band (57 kDa) is very faint and blurry for NaGG-avd, compared with NaGG+avd and pure avd, indicating covalent bonding between avidin and polymer. https://doi.org/10.1371/journal.pone.0221931.g003 https://doi.org/10.1371/journal.pone.0221931.g003 PLOS ONE | https://doi.org/10.1371/journal.pone.0221931 August 30, 2019 Physical properties The gelation time can be estimated through a qualitative tube-tilt test. NaGG-avd forms true gels during a period of about 30 seconds to one minute after the hydrogel has been transferred to the mold. For mechanical testing, the hydrogel samples were prepared as described earlier and incubated overnight at 37 ˚C before testing. The samples were compressed once to 65% of their original height and then discarded. Representative compression curves for each sample type are shown in Fig 4(A). The results of fracture strength and strain are plotted for compari- son in Fig 4(B) and shown in S3 Appendix. From Fig 4, a significant difference in fracture point between SPD and CaCl2 gels can be seen. Hydrogels formed with CaCl2 are more elastic and have a higher fracture strain of ~46%, while gels formed with SPD are more brittle and have fracture occurring already at around 35% strain. However, the fracture strength values for either crosslinking method are similar. The extensive error exhibited by the strain value may be due to a manual error when position- ing the sample on the compression piston. One-way analysis of variance (ANOVA) was per- formed assuming a confidence level of 95% (p < 0.05) with Microsoft Office Excel. The analysis showed that there is a significant difference between all shown samples (p < 0.05), but the samples prepared with SPD showed no significant difference (p = 0.07). Remarkably, it appears that the addition of avidin to the polymer consistently increases both the fracture strength and fracture strain of the hydrogel samples. The samples were pre- pared in a buffer at pH 6.5, whereas during cell culture experiments the pH was at 7.4 deter- mined by the cell culture medium. Although charge neutralized chimeric avidin has an isoelectric point (pI) of 6.92 (theoretical value), it can be assumed that the terminal N-acetyl glucosamine, as well as lysine and arginine groups, will carry a positive charge within the range of the mentioned pH values regardless. These positively charged amino groups aid in PLOS ONE | https://doi.org/10.1371/journal.pone.0221931 August 30, 2019 9 / 22 Avidin-functionalized gellan gum hydrogels for cell culture applications Fig 4. Compression testing results. (A) Representative compression curves of samples with different composition. (B) plot of fracture strength against fracture strain When examining all data, fracture strength (p = 9.28 x10-11) and fracture strain (p = 1.4 x10-4) are statistically different. Physical properties Testing within samples formed with SPD, however, no significant difference is observed between fracture strength (p = 0.06) and fracture strain (p = 0.54). Error bars represent one standard deviation calculated from five independent samples. https://doi.org/10.1371/journal.pone.0221931.g004 Fig 4. Compression testing results. (A) Representative compression curves of samples with different composition. (B) plot of fracture strength against fracture strain When examining all data, fracture strength (p = 9.28 x10-11) and fracture strain (p = 1.4 x10-4) are statistically different. Testing within samples formed with SPD, however, no significant difference is observed between fracture strength (p = 0.06) and fracture strain (p = 0.54). Error bars represent one standard deviation calculated from five independent samples. https://doi.org/10.1371/journal.pone.0221931.g004 https://doi.org/10.1371/journal.pone.0221931.g004 the crosslinking of the anionic gellan gum as additional crosslinker via charge screening. This is beneficial as the net effect of avidin functionalization is increased mechanical toughness. An initial test with cell culture medium (DMEM) was carried out to observe the behavior of the gels in cell culture conditions. The hydrogel was crosslinked with SPD, and DMEM was added on top after the gels were formed. The samples were then incubated overnight before testing. The cationic species in DMEM diffuse into the hydrogel and increase the fracture strength of the gels significantly. The swelling test showed no swelling of the gels, but rather a contraction over time. On average, the gels lost 3% of their original mass over 3 weeks. The results of the swelling test can be found in S4 Appendix. PLOS ONE | https://doi.org/10.1371/journal.pone.0221931 August 30, 2019 Cell culture Human WI-38 fibroblasts were cultured in avidin-modified gellan gum for three days. Three different biotinylated compounds (biotin, RGD, and fibronectin) were added to the hydrogel as separate samples. As control samples, the bare well bottom (TCP, 2D) as well as the unmod- ified gellan gum (3D) were used. The cells were alive when cultured on top of the hydrogel (2D) as well as when encapsulated in the matrix (3D). Fig 5 shows LIVE/DEAD1 fluorescence images after three days in culture (Fig 5A1–5D1 and 5A3–5D3). The images show aggregation to large cell clusters in 2D and 3D. During encapsulation, the 3D distribution of fibroblasts was homogeneous (S9 Appendix). The most notable difference can be observed in the cell dis- tribution and density between unmodified/unpurified GG (control) and the modified hydro- gel samples (NaGG-avd). Subsequently, the samples were fixed and immuno-stained (DAPI, phalloidin, fibronectin antibody; Fig 5A2–5D2 and 5A4–5D4). Immunostaining shows no meaningful cell spreading, judging from the shape of the actin cytoskeleton (red). Fibroblasts produced their own fibronectin, as revealed by the fibronectin immunostaining in samples without added bFN. To analyze the images statistically, images were taken of each well from random areas and the total area of live (green pixels) and dead (red pixels) cells was compared, as shown in Fig 6. Due to strong clustering of cells, the area was used rather than the number of particles. Simi- larly, the large error, especially for the 2D samples, was caused by aggregation of cells and the PLOS ONE | https://doi.org/10.1371/journal.pone.0221931 August 30, 2019 10 / 22 Avidin-functionalized gellan gum hydrogels for cell culture applications Fig 5. WI-38 fibroblasts after three days in culture. A1-D2 (left side): 2D culture with 63 000 cells/cm2, A3-D4 (right side): 3D culture with 950 000 cells/mL in gel. Images A1-D1 and A3-D3 LIVE/DEAD1 staining with live cells (Ca- AM, green channel) and dead cells (EthD1, red channel); images A2-D2 and A4-D4 actin filaments (red channel, TRITC-phalloidin), fibronectin (green channel, fibronectin antibody), cell nuclei (blue channel, DAPI). https://doi.org/10.1371/journal.pone.0221931.g005 Fig 5. WI-38 fibroblasts after three days in culture. A1-D2 (left side): 2D culture with 63 000 cells/cm2, A3-D4 (right side): 3D culture with 950 000 cells/mL in gel. Images A1-D1 and A3-D3 LIVE/DEAD1 staining with live cells (Ca- AM, green channel) and dead cells (EthD1, red channel); images A2-D2 and A4-D4 actin filaments (red channel, TRITC-phalloidin), fibronectin (green channel, fibronectin antibody), cell nuclei (blue channel, DAPI). Cell culture https://doi.org/10.1371/journal.pone.0221931.g005 https://doi.org/10.1371/journal.pone.0221931.g005 large difference of perceived cell density between different images of the same well. As seen in Fig 6A, all 3D samples show viability above 85% but are ultimately very similar with no statisti- cal difference. ANOVA was performed assuming a confidence level of 95% with the result that there is a significant difference between the cell count of all the shown 2D samples, but no large difference of perceived cell density between different images of the same well. As seen in Fig 6A, all 3D samples show viability above 85% but are ultimately very similar with no statisti- cal difference. ANOVA was performed assuming a confidence level of 95% with the result that there is a significant difference between the cell count of all the shown 2D samples, but no Fig 6. [A] Cell viability and [B] spreading of WI-38 fibroblasts in different materials as calculated from LIVE/DEAD1 stain image analysis. Control materials are tissue culture plastic (TCP, 2D) and unmodified, unpurified GG (3D). Bars represent mean values ± SD, n  10. [A] Cell viability (%live cells vs sum of live and dead cells) demonstrates that all 3D samples (GG, NaGG-avd) are statistically the same (p = 0.09), while the 2D samples (TCP, NaGG-avd) are statistically different (p < 0.05). Similarly, cell spreading (area% of live cells in image) demonstrates no significant difference between 2D GG based samples (p = 0.20), while there is significant difference between 3D samples (p = 3.94 x10-8). https://doi.org/10.1371/journal.pone.0221931.g006 Fig 6. [A] Cell viability and [B] spreading of WI-38 fibroblasts in different materials as calculated from LIVE/DEAD1 stain image analysis. Control materials are tissue culture plastic (TCP, 2D) and unmodified, unpurified GG (3D). Bars represent mean values ± SD, n  10. [A] Cell viability (%live cells vs sum of live and dead cells) demonstrates that all 3D samples (GG, NaGG-avd) are statistically the same (p = 0.09), while the 2D samples (TCP, NaGG-avd) are statistically different (p < 0.05). Similarly, cell spreading (area% of live cells in image) demonstrates no significant difference between 2D GG based samples (p = 0.20), while there is significant difference between 3D samples (p = 3.94 x10-8). https://doi.org/10.1371/journal.pone.0221931.g006 PLOS ONE | https://doi.org/10.1371/journal.pone.0221931 August 30, 2019 11 / 22 Avidin-functionalized gellan gum hydrogels for cell culture applications Fig 7. Cell culture hBMSC encapsulation results (A1-C1 and A2-C2) LIVE/DEAD1 stain of hBMSC encapsulated in NaGG (control) and NaGG-avd with bRGD over 21 days. Red stain = dead cells, Green stain = live cells. (D1-D2) Immunocytochemical cytoskeleton staining on day 21. Blue = cell nuclei (DAPI) and Red = actin filaments (phalloidin-TRITC). https://doi.org/10.1371/journal.pone.0221931.g007 Fig 7. hBMSC encapsulation results (A1-C1 and A2-C2) LIVE/DEAD1 stain of hBMSC encapsulated in NaGG (control) and NaGG-avd with bRGD over 21 days. Red stain = dead cells, Green stain = live cells. (D1-D2) Immunocytochemical cytoskeleton staining on day 21. Blue = cell nuclei (DAPI) and Red = actin filaments (phalloidin-TRITC). https://doi.org/10.1371/journal.pone.0221931.g007 https://doi.org/10.1371/journal.pone.0221931.g007 significant difference between the 3D samples. Full cell viability data from particle counting analysis is shown in S5 Appendix. Using the area% of the live cell data, cell spreading was ana- lyzed as shown in Fig 6B, referring to the area covered by live cells on the investigated image. ANOVA test reveals no significant difference between cell spreading on or in GG-based sam- ples (GG and NaGG-avd), due to the aforementioned large standard deviation and clustering. There is, however, a significant difference between 3D samples of GG formulations. Consequently, human bone marrow derived stromal cells (hBMSC) were encapsulated and cultured in NaGG-avd with added bRGD for up to 21 days. Fluorescence images with LIVE/ DEAD1 stain of the time points 3, 14, and 21 days are shown in Fig 7. After 21 days, the sam- ples were fixed and stained with DAPI (cell nuclei) and phalloidin (actin filaments), referred to as immunocytochemical cytoskeleton staining, as shown in Fig 7D. The LIVE/DEAD1 images demonstrate that the hBMSC are viable in both materials in 3D suspension. Evidently, the image quality degrades the longer the cells are in culture and it becomes difficult to achieve good microscope images due to opaque material. This behavior may indicate a mineralization by the hBMSC differentiating towards bone, but conclusions about differentiation cannot be drawn from actin cytoskeleton alone. In identical culture con- ditions, stained cell-free blank samples showed no background fluorescence after 21 days (the background image is available in S7 Appendix). Thus, the developing opacity is caused by the cells and is not due to the GG material. Immunostaining of hBMSC samples at day 21 confirmed the observations of the LIVE/ DEAD1 images. A majority of the cells appear rounded, but some of them do show elonga- tion. PLOS ONE | https://doi.org/10.1371/journal.pone.0221931 August 30, 2019 Functionalization strategies There is an abundance of approaches to design hydrogels described in the literature. These approaches include the covalent functionalization of passive polymers with bioactive cues, blending with bioactive native polymers or peptides, and the use of native ECM components. [9,36] A broad selection of materials for 3D cell culture is also available commercially. Hydrogels prepared from decellularized ECM or ECM components are a popular choice as promoting scaffolds because they naturally contain cell recognition sites. Commercially avail- able examples include PuraMatrix1[37], a peptide sequence of arginine, alanine, and aspartic acid, and Matrigel1, a reduced growth factor basement membrane matrix.[38] Both materials provide an excellent cell environment and achieve good cell response for a vast range of mam- malian cell types. Their mechanical properties are, however, poor. Bulk hydrogels have low stiffness, degrade quickly, and are rather expensive. On the other hand, there is a plethora of synthetic and natural hydrogel materials that are conjugated with bioactive compounds. These compounds range from small peptide sequences to proteins or other large biological compounds, such as gelatin. An non-exhaustive list of examples includes the conjugation of N-cadherin to alginate[39], conjugation of the peptide sequences RGD and SIKVAV to poly(2-hydroxyethyl methacrylate)[40], or the conjugation of RGD peptide to gellan gum.[23] These attached compounds enhance cell attachment and can guide cell fate. The mechanical properties of the gels are determined by the base polymer and the stiffness can be adjusted to the cell type requirement. However, the synthesis and produc- tion of these cell culture materials are elaborate and expensive, while only a narrow application area is targeted. Usually, specific chemistry development is needed in each case of polymer modification. This means that developing a material for neural cell culture does not benefit the needs for hepatic cell culture, while both are still soft tissues. Even though modular strategies are not as common as single molecule functionalization, avidin-biotin-based approaches have been presented by Kojima (2006), Hobzova et al. (2011), Wylie et al. (2011), and Leppiniemi et al. (2017).[19–22] Kojima et al. (2006) adsorbed avidin onto PLLA disks for the culturing of biotinylated hepatic cells. However, rigid PLLA scaffolds are a very different material type compared with soft hydrogels and their range of application. [22] Leppiniemi et al. (2017) have shown that avidin-functionalized nanocellulose combined with alginate is suitable for 3D printing. Cell culture Visibly there are more elongated cells in NaGG-avd+bRGD than in the non- PLOS ONE | https://doi.org/10.1371/journal.pone.0221931 August 30, 2019 12 / 22 Avidin-functionalized gellan gum hydrogels for cell culture applications functionalized NaGG, which is more evident with the stained actin (additional cytoskeleton images in S7 Appendix). The hBMSC culture test was analyzed with only staining and micro- scope images, while no quantitative analysis was carried out. Discussion The presented results support the concept of an avidin-modified hydrogel material for 2D and 3D cell culture applications. We have shown that the functionalized gellan gum can bind bioti- nylated compounds, thus equipping the hydrogel with bioactive factors and enabling cell attachment. The gellan gum-based material shows little to no degradation and has been found to be feasible for long-term cell culture. Furthermore, it has suitable mechanical properties and convenient preparation. PLOS ONE | https://doi.org/10.1371/journal.pone.0221931 August 30, 2019 Structure analysis In our project, the concentration of avidin coupled to gellan gum has been determined through fluorescence titration with a biotinylated dye. From the fluorescence titration we can conclude a concentration of 0.075 μM (L)/0.375 μM (H) avidin in 1 mg/mL NaGG solution. In dry formulation (before dissolving and adding biotinylated species) 0.004 (L)/ 0.021 (H) mg/mg avidin:NaGG ratio. The functionalization yield is 21% (L) and 54% (H), respectively, l l d f h l f CNCA d i h f i li i i In our project, the concentration of avidin coupled to gellan gum has been determined through fluorescence titration with a biotinylated dye. From the fluorescence titration we can conclude a concentration of 0.075 μM (L)/0.375 μM (H) avidin in 1 mg/mL NaGG solution. μ μ g In dry formulation (before dissolving and adding biotinylated species) 0.004 (L)/ 0.021 (H) mg/mg avidin:NaGG ratio. The functionalization yield is 21% (L) and 54% (H), respectively, as calculated from the total amount of CNCA used in the functionalization reaction. Ferris et al. (2015) directly coupled RGD peptide to GG via carbodiimide coupling, report a functionalization yield of roughly 20% of carboxyl group concentration.[23] This peptide sequence (0.65 kDa) is significantly smaller than the protein avidin (57 kDa) and steric effects preventing higher coupling efficiency with carbodiimide strategy are plausible for avidin. As reported by Ferris et al. (2015), the purification of GG and the removal of divalent counterions increases activation yield with EDC and NHS, and thus convinced us to employ the same strat- egy.[23] The functionalization yield is further crucial for the number and density of available cell cues in the hydrogel matrix. It has been shown that cells are sensitive to the presentation, spacing, and clustering of ligands in 2D cell culture systems and the interplay of substrate stiffness and availability of adhesion factors is well understood. In 2D, an RGD ligand spacing of less than 60 nm exhib- ited significantly improved adhesion and was found to be optimal.4 However, the reality for 3D matrices is less explored and likely more complex. Functionalization strategies In their study, the protein was covalently bound to the cellulose fibrils and confirmed through electrophoresis. Their range of applications included biomedical devices, wearable sensors, and drug-releasing materials for wound healing, and therefore no cytotoxicity assay or cell culture experiments were presented.[19] Nanocellulose forms weak hydrogels without macroscopic hierarchy, and dimensionality was achieved by the addition of alginate and an additional freeze-drying step. On the other hand, gellan gum can PLOS ONE | https://doi.org/10.1371/journal.pone.0221931 August 30, 2019 13 / 22 Avidin-functionalized gellan gum hydrogels for cell culture applications form true hydrogels applicable to long-term disease modeling.[41] Further, Wylie et al. (2011) created an advanced system using two different proteins, namely avidin and barnase, for the orthogonal functionalization of agarose hydrogels for 3D patterning. Indeed, the use of a pro- tein-binding phenomenon enables the flexible modification of a 3D cell culture material.[21] Their approach is more complex and encompasses a more time-consuming set-up, whereas our system is applicable to larger scale use and requires no laser equipment. Hobzova et al. (2011) showed the covalent grafting of avidin to planar 2-hydroxyethyl methacrylate (pHEMA) surfaces, and they also compared non-specific surface adsorption and electrostatic interaction as a means of avidin immobilization. Their preliminary cell culture results rudi- mentarily show the attachment of keratinocytes on surface-modified avidin-pHEMA, but no 3D system or in-depth cell response analysis.[20] In contrast to the strategies mentioned above, our system aims to provide 3D soft tissue mimic for true cell encapsulation. Recently, Silva et al. (2018) published their development of metalloproteinase-1 degradable GG hydrogels, which were equipped with divinyl-sulfone groups to enable modular attach- ment of cell-adhesive peptides, namely, T1 and C16, both derived from ECM. According to the authors, elongation of the cells is achieved when coupled with a peptide sequence from the Cyr61/CCN1 protein at a concentration of 800 μM in the gel.[42] PLOS ONE | https://doi.org/10.1371/journal.pone.0221931 August 30, 2019 Crosslinking and physical properties Mechanical testing revealed that functionalization does not significantly alter the ability of the material to form gels. Forming true gels of NaGG-avd with the relevant mechanical properties for tissue engineering has proven to be uncomplicated and similar to other GG-based hydro- gels.[25,43–45] The stiffness and mechanical behavior of the bulk hydrogel should mimic nat- ural tissue, as it has been shown numerous times in the literature to influence, e.g., the differentiation of anchorage-dependent stem cells.[3,8] To prove their adequate mechanical properties, the loss modulus G” and storage modulus G’ determined through rheological assessment is often reported for hydrogels. The drawbacks of rheology include problematic sample preparation, complicated data interpretation, and GG is possibly too brittle a material to be analyzed through rheology.[24] Thus, in this study we employed compression testing of the hydrogel, and thereby benefitted from easy sample preparation and straight-forward data interpretation of the fracture point. The determination of the compression modulus is, how- ever, challenging and prone to errors due to the softness of the material and instrument limita- tions as well as the unclear elastic region or the nonlinear elasticity of the hydrogel material. [46,47] Regardless of the above-mentioned limitations, compression testing offers an easy and fast method of comparison between different hydrogels and hydrogel compositions, especially when comparing different compositions using the exact same test parameters, or within the same study. Because the goal of this study is to develop a non-specialized 3D hydrogel platform for cell culture, our system needs to have the ability to adapt not only to a wide range of biochemical cues, e.g. growth factors and peptides, but also a wide range of biophysical cues and mechani- cal stiffness requirements. Gellan gum based hydrogels have already been shown to provide a flexible platform and an adequate stiffness range which can be adjusted by polymer concentra- tion and crosslinker type and concentration. Koivisto et al. (2017) demonstrate a linear corre- lation between crosslinker concentration and final stiffness of gellan gum hydrogels using the bioamines spermine and spermidine.[24] It must be noted that the functionalization reaction uses carboxyl groups of glucuronic acid, which are also needed to form crystalline junction zones as crosslinking sites and to form the hydrogel. However, NaGG-avd forms true gels with very similar compression behavior to non-functionalized NaGG. This clearly indicates that a sufficient number of carboxyl groups is still available for crosslinking even after the avidin-functionalization. Structure analysis One of the core issues for this study was the nature of the binding of avidin to the polymer chain because a covalent attachment of avidin is substantial for the modular approach.4,5,10 In the literature, it has been shown that unattached compounds can also enhance cell response.25 However, anchoring the attachment cue to the network is required to generate the mechano- transduction effect, and the ECM receptors of the cell “pull” on the cue rather than the net- work. If the avidin were merely entrapped via unspecific binding and electrostatic interactions, the biotinylated cell cues would not appropriately convey the matrix stiffness to the cells. Further, the diffusion of the attachment cue out of the matrix system would be antici- pated. Our analysis of the functionalized material with electrophoresis showed sufficiently that avidin is covalently tethered to the polymer network. interactions, the biotinylated cell cues would not appropriately convey the matrix stiffness to the cells. Further, the diffusion of the attachment cue out of the matrix system would be antici- pated. Our analysis of the functionalized material with electrophoresis showed sufficiently that avidin is covalently tethered to the polymer network. PLOS ONE | https://doi.org/10.1371/journal.pone.0221931 August 30, 2019 14 / 22 Avidin-functionalized gellan gum hydrogels for cell culture applications Crosslinking and physical properties The addition of DMEM to the gels increases their fracture strength, which can be explained by the formation of tighter crystalline junction zones in the presence of higher cation concentration. Ions present in the swelling medium increase the crosslink density, cause densification, and thus increase the frac- ture strength of the hydrogels. On a more practical note, the formed NaGG-avd hydrogels can be manipulated and moved with ease. The gelation time of the hydrogels has been determined to be between 30 seconds and one minute. This indicates a possibility to use the hydrogel as an injectable scaffold, which has not yet been further explored. Cell response The functionalized material was tested as cell culture support, while using biotinylated RGD and biotinylated fibronectin for bioactive modification. All the required components are non- toxic and biocompatible. The crosslinking method is gentle and does not disturb cells because there are no side products of the crosslinking reaction. Further, the applied gel preparation achieves a true 3D structure, where the cells are homogeneously distributed throughout the hydrogel. This homogenous cell distribution is achieved by mixing the hydrogel sol with the PLOS ONE | https://doi.org/10.1371/journal.pone.0221931 August 30, 2019 15 / 22 Avidin-functionalized gellan gum hydrogels for cell culture applications cell suspension using a magnetic stirrer at 300 rpm, before casting the mixture to the wells of the well plate. Supported by the high viability results, this treatment does not harm the cells. Indeed, the short-term test with human WI-38 fibroblasts demonstrated high cell viability throughout different compositions. GG has previously been shown to be suitable for soft tissue culture, but it is rather bioinert. [23–26,48] Here, we have shown that after functionalization, GG retains its good mechanical properties, such as soft tissue mimicking, while adding biofunctionalization. A direct comparison of the cell culture results is challenging due to the ubiquity of cell lines and analysis methods used in the literature. Regarding the fibroblasts, we can point out similar viability of the same WI-38 cells studied by Pacelli et al. (2016) who report 74% viability with GG-based hydrogel after 4 days.[48] However, their cells were seeded in a 2D layer for 24 h more than in our study, and viability was asserted through optical density with a so-called neu- tral red assay. For 3D cultures, however, dye-based assays require optimization because the hydrogel affects both light propagation and the diffusion of the dye during incubation. In the GG-based hydrogel study by da Silva et al. (2018) mentioned earlier[42], image analysis was used to determine the cell viability of human umbilical vein endothelial cells (HUVECs) in thiol-functionalized gellan gum. The authors found a 65% viability of HUVECs after 3 days encapsulated in the hydrogel. Overall, our observation of WI-38 fibroblast viability was between 85% and 90% in different formulations of NaGG-avd, which exceeds or at least matches the values found in the literature for GG-based hydrogels. Therefore, the excellent biocompatibility of NaGG-avd can be affirmed. Cell response There appears to be no overall visible difference in WI-38 cell viability between 2D and 3D conditions, with live and dead cells being present in either culture method with substantial standard deviation. 2D culture promotes the growth of cell aggregates and the cells possibly migrate to form larger clusters, possibly due to the inert properties of the hydrogel surface. When encapsulated in 3D, the cells seemingly cannot migrate, and are therefore limited to grow within the hydrogel in the area of initial original deposition. There is no visible difference in 3D cell distribution between the different biotinylated compounds added to NaGG-avd. Overall, no statistical significance in cell spreading of could be found between all tested gellan- gum compositions (GG and NaGG-avd samples). From Fig 6B a trend is visible favoring the avidin-functionalized samples, however the large standard deviation prevents any decisive statement. A higher degree of cell adhesion and spreading would have been anticipated from the presence of ECM derived factors. However, this effect could be explained by the presence of serum proteins in the culture medium that compete with the cells to reach the provided attachment sites. This is largely due to the Vroman effect[49] that describes the mobility- dependent adsorption of proteins onto hydrophilic surfaces. Thus, serum components, such as albumin, fibrinogen, and fibronectin, attach faster to the biomaterial than the cells and favor- ing them may prevent the cell receptors from recognizing any available cell adhesive cues cova- lently bound to the biomaterial. In order to avoid the Vroman effect, some studies have successfully used serum starving conditions to show enhanced cell attachment and survival in biomaterials that contain cell adhesive cues.[50,51] The same tendency of cell behavior was visible in hBMSC 3D culture in our functionalized gellan gum. NaGG-avd had been equipped with biotinylated RGD to stimulate cell adhesion because RGD is known to be a crucial and prevalent peptide sequence for cell integrin recogni- tion of ECM. RGD has been used frequently for BMSC studies and positive effects have been reported in the literature[11,12,52], and thus was chosen here as a model cell adhesive site. We can correlate our findings to a study by Anjum et al. PLOS ONE | https://doi.org/10.1371/journal.pone.0221931 August 30, 2019 Avidin-functionalized gellan gum hydrogels for cell culture applications Table 1. Component concentration of hydrogel composition. Component Mass of component Weight percent Concentration Avidin (L)/(H) 0.02/ 0.11 mg 0.002/0.011 wt% 0.375/ 1.875 μM NaGG 5 mg 0.50% 10 μM bRGD/bFn 0.3 x10-3/0.03 mg 0.03x10-3/0.003 wt% 0.27/ 0.08 μM H2O 995 mg 99.5% https://doi org/10 1371/journal pone 0221931 t001 Table 1. Component concentration of hydrogel composition. in viability to our findings and interestingly also show a similar obfuscation with time. Simi- larly, Blache et al. (2016) used RGD-functionalized PEG-based hydrogel and co-cultured HUVEC+hMSC. The authors reported good cell viability and definite cell elongation with a three-times higher cell concentration compared with our study.[11] Another comparable study has been published by Tsaryk et al. (2014) using ionically crosslinked methacrylated GG (iGG-MA) with human mesenchymal stem cells (hMSC) and nasal chondrocytes. [54] They achieved good viability and adequate differentiation towards chondrogenic and osteogenic lin- eages. No attachment factors had been added for this encapsulation study, and the differentia- tion is steered solely through medium composition. After 14 days encapsulated in iGG-MA, the hMSC showed a round shape, while the hydrogel opacity increased, and the confocal microscope image clarity deteriorated similar to our study. In summary, different approaches to design hydrogels for the encapsulation of human mesenchymal stem cells have been pre- sented in the literature. Our proof-of-concept with avidin-functionalized NaGG provides an easy modification of hydrogel material for cell culture applications. In the laboratory, the hydrogel is rather simple to handle and requires the combination of only three components. The persisting problem in the presented results is the small amount of avidin coupled to the gellan gum network. This in turn leads to a rather low number of bioactive cues available for the cells. As a brief compari- son, Broguiere et al. (2018) report that a fibrin hydrogel at 3 mg/mL provides a sufficient con- centration of RGD binding sites at 75 μM, whereas our results show a concentration of 0.3 μM bRGD in the final hydrogel (Table 1).[55] Further research is therefore needed to increase functionalization degree and overcome the problem of the low number of bioactive cues in the hydrogel. A range of other functionalization reaction is conceivable, such as bio-orthogonal click reactions.[56] However, an interference of high functionalization degree with crosslink- ing capability must be considered. Cell response (2016) who cultured hBMSC over 14 days in their chondroitin sulfate-poly(ethylene glycol) (PEG) hydrogel and found 80% to 95% via- bility between different compositions.[53] The LIVE/DEAD1 stained images appear similar PLOS ONE | https://doi.org/10.1371/journal.pone.0221931 August 30, 2019 16 / 22 PLOS ONE | https://doi.org/10.1371/journal.pone.0221931 August 30, 2019 Conclusions Gellan gum was successfully functionalized with the avidin protein (charge neutralized chime- ric avidin). This functionalized hydrogel provides a general, modular platform capable of teth- ering biotinylated compounds and aiding the attachment and proliferation of cells in 3D. Gellan gum was successfully functionalized with the avidin protein (charge neutralized chime- ric avidin). This functionalized hydrogel provides a general, modular platform capable of teth- ering biotinylated compounds and aiding the attachment and proliferation of cells in 3D. Contrasting the common strategy of direct attachment, our modular hydrogel is intended to be off-the-shelf ready for any cell culture application, while still being consistent in physical properties. The current proposed avidin functionalization system is not targeted at a specific cell type or application, but rather intended to form the base material for further studies. Fur- ther modification by the addition of a cell type specific biotinylated compound is necessary to enhance the positive cell response. The presented data using two different cell types support that the avidin-modified gellan gum is a useful tool applicable for cell culture. The hydrogel is biocompatible, sterilizable, and retains adequate mechanical properties and stability over sev- eral weeks. This long-term stability is essential for disease or tissue models involving cell types with a slow metabolism and development, such as bone and cartilage derived cells. Another prospect could be the functionalization of the poly- mer chain end-groups to avoid blocking the carboxyl groups of gellan gum, as proposed by Bondalapati et al. (2014).[57] While considering an alternative functionalization reaction, the carbodiimide strategy will always have the advantage of being well-known and heavily used in the literature[14], and thus we can rely on its efficacy. As an example, the end-group modifica- tion strategy would have to be tested and refined for gellan gum and avidin, and additionally the cytotoxic substance aniline is used as a catalyst, which may prove problematic. With the proposed avidin-biotin modification system, the modularity hinges solely on the availability of the biotinylated compounds. There is a multitude of these compounds already commercially available, but they can also be synthesized by biotinylating the desired com- pound, if such a compound is not yet available. As proof-of-concept, these results underline the viability of stromal cells in a gellan gum-based 3D hydrogel cell culture system. Our approach is suitable for disease modeling applications due to its capacity to image cells inside the hydrogel and the ability to control the 3D biochemical environment through avidin-biotin modularity. Future work will focus on the effect of biotinylated species and quantitative assess- ment of stromal cells encapsulated in our modular hydrogel. 17 / 22 PLOS ONE | https://doi.org/10.1371/journal.pone.0221931 August 30, 2019 Avidin-functionalized gellan gum hydrogels for cell culture applications Supporting information S1 Appendix. ICP-OES data. Elemental composition of counterions in commercial GelzanTM (GG) and purified product (NaGG) has been determined with ICP-OES. The ion concentra- tion of the purified product matches values found in literature (24,28). (PDF) S2 Appendix. SDS PAGE. Full uncropped image of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) blot. (PDF) S2 Appendix. SDS PAGE. Full uncropped image of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) blot. (PDF) S3 Appendix. Compression data of functionalized and non-functionalized samples. Data are the same as presented in Fig 4B and are presented as means ± SD. The graphs (stress vs. strain) show all compression curves, from which the fracture points are averaged. The photo- graph shows cylindrical hydrogel samples, cell-free but incubated in DMEM. (PDF) S4 Appendix. Swelling degree of NaGG-avd(L) hydrogel samples. Hydrogels were prepared in Eppendorf tubes with either SPD or CaCl2 as crosslinker and the initial mass of each sample was taken. Swelling media, either PBS or DMEM, were added on top after the gels had set and the samples were incubated at 37˚C up to 3 weeks. Swelling degree was calculated through monitoring change in mass according to Eq (3). Data are shown as means ± SD (n = 3). (PDF) S5 Appendix. Cell viability from particle counting. Averaged values of particle counting algorithm results for all LIVE/DEAD1 images. The bar graph shows average values of image- by-image comparison for ‘total area’ value. (PDF) S6 Appendix. Flow cytometry surface markers analysis results of hBMSCs. n = 1. The flow cytometry analysis confirmed the mesenchymal origin of the hBMSCs. (PDF) S7 Appendix. Cytoskeleton images of hBMSC after 21 days. Blue channel = cell nuclei (DAPI) and red channel = actin filaments (TRITC-phalloidin). (A) Cells in NaGG and (B) NaGG-avd+bRGD. (PDF) 18 / 22 PLOS ONE | https://doi.org/10.1371/journal.pone.0221931 August 30, 2019 Avidin-functionalized gellan gum hydrogels for cell culture applications S8 Appendix. Washing test and immunofluorescence straining. Description of washing test for hydrogel samples formed with NaGG and NaGG-avd using biotinylated fibronection (bFn). Immuncytochemistry staining for fibronectin shows 4-fold retention of fibronectin in avidin-functionalized gel. (PDF) S9 Appendix. Microscope images of WI-38 cell culture experiment for counting analysis. Exemplary images of 3D samples between day 0 (light microscope) and day 3 (LIVE/DEAD1 fluorescence images) for comparison. (PDF) S9 Appendix. Microscope images of WI-38 cell culture experiment for counting analysis. Supporting information Exemplary images of 3D samples between day 0 (light microscope) and day 3 (LIVE/DEAD1 fluorescence images) for comparison. (PDF) Acknowledgments We wish to thank Tampere CellTech Laboratories and Tampere Imaging Facility (TIF) for their services, as well as the group of Assoc. Prof. Jonathan Massera for assistance with the ICP analysis. References References 1. Tirrell DA, Langer R. Designing materials for biology and medicine. Nature. 2004; 428: 487–492. https:/ doi.org/10.1038/nature02388 PMID: 15057821 2. Levental I, Georges PC, Janmey PA. 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McKee C, Chaudhry GR. Advances and challenges in stem cell culture. Author Contributions Conceptualization: Christine Gering, Janne T. Koivisto, Jenny Parraga, Jenni Leppiniemi, Kaisa Vuornos, Vesa P. Hyto¨nen, Susanna Miettinen, Minna Kelloma¨ki. Formal analysis: Christine Gering, Janne T. Koivisto, Kaisa Vuornos. Investigation: Christine Gering. Formal analysis: Christine Gering, Janne T. Koivisto, Kaisa Vuornos. Investigation: Christine Gering. Resources: Vesa P. Hyto¨nen, Susanna Miettinen, Minna Kelloma¨ki. Resources: Vesa P. Hyto¨nen, Susanna Miettinen, Minna Kelloma¨ki. Supervision: Vesa P. Hyto¨nen, Susanna Miettinen, Minna Kelloma¨ki. Supervision: Vesa P. Hyto¨nen, Susanna Miettinen, Minna Kelloma¨ki. Writing – original draft: Christine Gering, Janne T. Koivisto, Jenny Parraga. Writing – review & editing: Christine Gering, Janne T. Koivisto, Jenny Parraga, Jenni Leppi- niemi, Kaisa Vuornos, Vesa P. Hyto¨nen, Susanna Miettinen, Minna Kelloma¨ki. 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RESEARCH ARTICLE OPEN ACCESS To evaluate the potential value of using a serological assay to quantitate the hepatitis C virus core antigen (HCV-Ag) when monitoring patients with chronic hepatitis C being treated with direct-acting antivirals (DAAs). Citation: Loggi E, Galli S, Vitale G, Di Donato R, Vukotic R, Grandini E, et al. (2017) Monitoring the treatment of hepatitis C with directly acting antivirals by serological and molecular methods. PLoS ONE 12(11): e0187755. https://doi.org/ 10.1371/journal.pone.0187755 Results Copyright: © 2017 Loggi 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. A sustained viral response (SVR) was achieved in 82 patients (91%), whereas 11 relapsed (R) and 1 showed a virological breakthrough while receiving treatment. HCV-RNA and HCV-Ag showed good concordance (kappa = 0.62) and correlation. No significant differ- ences between SVR and R was observed in either assay at 2 and 4 weeks after the start of treatment. At 8 weeks, HCV-Ag showed higher accuracy than HCV-RNA (AUC: 0.74 vs. 0.55) and there was a significantly greater decrease from baseline in SVR than in R (4.01 vs. 3.36 log10; p<0.05). Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: The laboratory reagents (serological determinations) have been provided by Abbott Diagnostics, free of charge. The Abbott Diagnostics partially supported the research providing the research materials, following an agreement for an Investigator Initiated Study (IIS), but did not play a role in the study design, data collection, decision to publish, or preparation of the manuscript. No Methods Ninety-six patients treated with DAAs, either alone (91) or in combination with PEG inter- feron (5), were tested for HCV-RNA and for HCV-Ag at baseline and at weeks 2, 4, 8 and 12 during treatment and 12 weeks after completion. The concordance and correlation between the viral parameters as well as the respective kinetics during and after treatment were evaluated. Editor: Ming-Lung Yu, Kaohsiung Medical University Chung Ho Memorial Hospital, TAIWAN Received: July 13, 2017 Accepted: October 25, 2017 Published: November 10, 2017 Received: July 13, 2017 Accepted: October 25, 2017 Published: November 10, 2017 Monitoring the treatment of hepatitis C with directly acting antivirals by serological and molecular methods Elisabetta Loggi1¤, Silvia Galli2, Giovanni Vitale1, Roberto Di Donato1, Ranka Vukotic1, Elena Grandini1, Marzia Margotti1, Valeria Guarneri1, Giuliano Furlini2, Claudio Galli3, Maria Carla Re2, Pietro Andreone1* 1 Dipartimento di Scienze Mediche e Chirurgiche, Centro di Ricerca per lo Studio delle Epatiti, Università degli Studi di Bologna, Bologna, Italy, 2 Unità di Microbiologia, Policlinico S.Orsola-Malpighi, Bologna, Italy, 3 Medical Scientific Liaison, Abbott Diagnostics, Roma, Italy ¤ Current address: UOC Patologia Clinica, ASUR Area Vasta 4, Fermo (FM), Italy * pietro andreone@unibo it ¤ Current address: UOC Patologia Clinica, ASUR Area Vasta 4, Fermo (FM), Italy * [email protected] ¤ Current address: UOC Patologia Clinica, ASUR Area Vasta 4, Fermo (FM), Italy * [email protected] Introduction Authors’ salaries have been provided. The Co- Author, Dr. Claudio Galli is currently employed by Abbott Diagnostics as the Associate Director, Medical Scientific Liaison Europe contributed to data analysis, as stated in Author roles in the Author Contributions section. The treatment of chronic hepatitis C virus infection (CHC) has been revolutionized by the intro- duction of direct-acting antivirals (DAAs), which are agents that can interfere with different steps of the replicative cycle of a virus [1, 2]. This molecular approach replaces the standard treatment based on a combination of pegylated interferon α (peg-IFNα) and ribavirin (RBV), which acts on several non-specific pathways to boost the antiviral immune response [3]. DAAs lead to viral erad- ication in more than 90% of patients [2]. In addition to being significantly more effective than interferon-based therapy for curing the infection, these treatments have important additional ben- efits, including a tolerability profile that makes them suitable for previously excluded patients [4], simplified management due to the shorter treatment duration and an oral route of administration. Unfortunately, the high costs of these therapies currently limit the access to these drugs, thereby requiring strict patient selection and blocking the drugs’ widespread use [5]. Competing interests: Dr. Claudio Galli is currently employed by Abbott Diagnostics as the Associate Director, Medical Scientific Liaison Europe. The other Authors have no conflicting interests to declare. The Commercial affiliation does not alter our adherence to PLOS ONE policies on sharing data and materials. The providing of laboratory reagents does not alter our adherence to PLOS ONE policies on sharing data and materials. Measuring HCV-RNA by using sensitive molecular techniques has been the gold standard for treatment monitoring in the era of IFN-based treatment. Baseline viremia and an early drop in HCV-RNA levels are the strongest elements for predicting the treatment outcome, and viremia measurement during treatment is crucial for establishing the treatment duration and assessing the response-guided therapy [6]. The introduction of DAA regimens has changed virological monitoring because the base- line HCV-RNA levels no longer seem to be response-predictors and because detectable resid- ual HCV-RNA at the end of DAA therapies can occur frequently but does not have an association with subsequent viral relapse [7]. Consistent with this assumption, no futility/stop- ping rules have been established to date. To simplify treatment monitoring, hepatitis C core antigen (HCV-Ag) is emerging as a new tool for diagnosis and treatment monitoring in CHC. HCV-RNA and HCVAg in DAAs treatment Conclusions Monitoring during treatment with DAAs by using either HCV-RNA or HCV-Ag has only a lim- ited predictive value for SVR. Since those assays are equivalent for identifying a virological relapse, HCV-Ag may be preferred from an economical and organizational perspective. 1 / 13 PLOS ONE | https://doi.org/10.1371/journal.pone.0187755 November 10, 2017 Introduction HCV-Ag is a highly conserved and antigenic protein that is released into the plasma [8, 9] and can be easily quantified due to the availability of an automated platform. HCV-Ag quanti- fication is an indirect measure of viral replication [10, 11], and it has been proven to be useful for treatment monitoring of IFN-based therapy [12, 13] and has even recently been used to monitor DAA-based therapy [14, 15]. In this study, we aimed to assess the accuracy of HCV-Ag for monitoring therapy efficacy compared to RT-PCR in a population of multi-genotype CHC patients who were undergoing treatment with different DAA regimens in a real-life clinical setting. PLOS ONE | https://doi.org/10.1371/journal.pone.0187755 November 10, 2017 HCV-RNA testing and HCV-Ag determinations The HCV-RNA levels were measured with the COBAS AmpliPrep/COBAS TaqMan HCV Quantitative Test, version 2.0 (Roche; lower limit of quantitation = 15 IU/mL) at baseline as well as after 2, 4, 8, 12, 16 and 24 weeks for patients treated until the 24th week (W2, W4, W8, W12, W16, W24) during treatment. Follow-up measurements were collected at 4 and 12 weeks post- treatment. HCV-RNA was positive if the level was >15 IU/mL, whereas detectable but not mea- surable HCV-RNA levels were reported as <15 IU/mL and scored as a gray zone. Only samples with an undetectable level of HCV-RNA were considered negative. The HCV genotype and sub- type were determined using the Siemens VERSANT HCV Genotype INNO-LiPA 2.0 Assay. HCV-Ag was retrospectively measured in serum samples obtained at the same time points as the HCV-RNA using an automated chemiluminescent HCV-Ag assay (Abbott Diagnostics, Wiesbaden, Germany) that was performed on the Abbott ARCHITEC T i2000SR platform according to the manufacturer’s instructions. The cut-off value for HCV-Ag detection was 3.0 fmol/L. Levels below 3.0 fmol/L were considered non-reactive, levels between 3 and 10 fmol/L represented the “gray” zone, and levels above 10 fmol/L were positive for HCV-Ag. The dynamic range of the test was 3–20,000 fmol/L, and an automated dilution extended this range to 180,000 fmol/L. Study assessments SVR was defined as HCV-RNA that was undetectable 12 weeks after completing treatment (SVR12). Post-treatment relapse (R) was defined as confirmed HCV-RNA 15 IU/ml during a follow-up in patients with undetectable HCV-RNA at the end of treatment. Viral breakthrough was defined as a 1 log10 IU/ml increase from the nadir of HCV-RNA or when HCV-RNA 15 IU/ml after HCV-RNA was undetectable during the treatment. Results Patients and response to treatment HCV-RNA and HCVAg in DAAs treatment The DAA regimen, treatment duration and addition of weight-based ribavirin (RBV) were determined by the treating physician according to current guidelines. The study was approved by the Ethical Committee of Azienda Ospedaliera di Bologna, (Bologna, Italy), and the patients provided written informed consent. Statistical analysis Quantitative variables were expressed as the median and range, and categorical variables were expressed as a number count and proportions. Chi-square or Fisher’s exact test and the Mann- Whitney test were used to compare categorical and continuous variables when appropriate. Correlations were determined with a non-parametric Spearman correlation and kappa statis- tics. The accuracy of HCV-RNA and HCV-Ag for assessing SVR was evaluated by receiver- operating characteristics (ROC) curves. A p value of <0.05 was considered statistically significant. All analyses were performed using SPSS for Windows (Statistical Package for the Social Sciences, version 21.0, Armonk, New York, NY, USA), and GraphPad Prism, version 5. Patients Among all the consecutive all-genotype CHC patients who received a DAA-based treatment in between June 2013 and December 2015 at the ITEC Outpatient Clinics of Azienda Ospeda- liero-Universitaria di Bologna (Bologna, Italy), 96 CHC patients were selected for the analysis based on the availability of longitudinal serum samples. The inclusion criteria were adult age (18 years), CHC infection confirmed by serum HCV-RNA using an RT-PCR-based method and, in particular, fulfilling the criteria to be eligible for treatment with DAA according to the indications established by the Italian government (Agenzia Italiana del Farmaco-AIFA). The patient population consisted of the following 3 groups: 1. Patients with advanced disease with METAVIR stage F3 (advanced fibrosis) or F4 (cirrhosis); 2. Patients with HCV recurrence after liver transplantation; 3. Patients with extrahepatic manifestations, such as cryoglobulinemia and B-cell lymphopro- liferative disease. 2 / 13 PLOS ONE | https://doi.org/10.1371/journal.pone.0187755 November 10, 2017 HCV-RNA and HCVAg in DAAs treatment and 24 weeks for 31 patients. Most patients (63%) were cirrhotic, and twelve had received a liver transplantation with HCV recurrence. Overall, SVR12 was achieved in 82 of the 96 patients (85% intention to treat analysis), 11 patients experienced a relapse (11%) and only one showed a breakthrough after 6 weeks from the start of treatment. The remaining 2 patients died due to causes unrelated to liver disease before the completion of the follow-up period, but they were HCV-RNA negative until the last available time point. Relapses were diagnosed in 8 patients after 4 weeks from treatment com- pletion and in 3 patients after 12 weeks. The occurrence of viral relapse was unrelated to the previous treatment (10.5% among treatment-naïve patients, 12.5% among treated patients). HBV: hepatitis B virus; 3D: Ombitasvir-Paritaprevir-Ritonavir and Dasabuvir; SOF: Sofosbuvir; SMV: p p simeprevir; LDV: Ledipasvir; DCV: Daclatasvir; pegIFNα: Pegylated Interferon alpha; RBV: Ribavirin. HBV: hepatitis B virus; 3D: Ombitasvir-Paritaprevir-Ritonavir and Dasabuvir; SOF: Sofosbuvir; SMV: simeprevir; LDV: Ledipasvir; DCV: Daclatasvir; pegIFNα: Pegylated Interferon alpha; RBV: Ribavirin. PLOS ONE | https://doi.org/10.1371/journal.pone.0187755 November 10, 2017 Patients and response to treatment The demographics and clinical and virological features of the enrolled patients are listed in Table 1. The treatment regimens were quite heterogeneous, although most patients were treated with an IFN-free course (91, or 95%), but 5 patients (5%) were treated with a combina- tion of Peg-IFN, Ribavirin and DAA. The duration of treatment was 12 weeks for 65 patients 3 / 13 PLOS ONE | https://doi.org/10.1371/journal.pone.0187755 November 10, 2017 https://doi.org/10.1371/journal.pone.0187755.t001 Baseline HCV-RNA and HCV-Ag values and correlation All patients were positive for both parameters at the start of treatment. The baseline values of HCV-RNA ranged from 6,14x103 to 533x106 IU/mL (Table 1). The distribution of levels showed that most patients (54/96; 56.3%) displayed levels higher than 1x106 IU/mL, whereas 39 of the 96 patients (40.6%) showed values ranging from 1x105 to 1x106, and the remaining 3 had values lower than 105 IU/mL. The baseline values for HCV-Ag ranged from 10.79 to >20,000 fmol/L (Table 1). The levels distribution showed that most patients (71/96, 74%) dis- played levels higher than 1,000 fmol/L, whereas 20 of the 96 patients (20.8%) showed values ranging from 100 to 1,000 and only 5 (5.2%) had values lower than 100 fmol/L. Interestingly, the patients with low HCV-RNA or low HCV-Ag levels at baseline were not the same patients. Table 1. Demographics, clinical and virological features and type of treatment of enrolled patients. Parameter(s) Data Age, years: Median (range) 60.5 (31–86) Male/Female: N (%) 61/35 (64/36) Naives/Experienced: N (%) 38/58 (60/40) Baseline HCV-RNA IU/mL: median (range) 1.2x106 (6.14x103-533x106) Baseline HCV-Ag fmol/L: median (range) 3,376 (10.79->20,000) HCV Genotype: N (%) 1a 11 (11) 1b 60 (62.5) 1 (subtype not available) 1 (1) 2 6 (6) 3 14 (14.5) 4 4 (4) Treatment duration 12/24 weeks: N (%) 65/31 (68/32) Anti-HBV core positivity: N (%) 35 (36) Cirrhosis: N (%) 61 (63%) Previous liver transplantation: N (%) 12 (12.5) Treatment regimen: N (%) 3D ± RBV 16 (17) SOF ± RBV 23 (24) SOF + SMV ± RBV 32 (33) SOF + LDV ± RBV 6 (6) SOF + DCV 14 (15) SOF + pegIFNα + RBV 5 (5) Demographics, clinical and virological features and type of treatment of enrolled patients. 4 / 13 PLOS ONE | https://doi.org/10.1371/journal.pone.0187755 November 10, 2017 PLOS ONE | https://doi.org/10.1371/journal.pone.0187755 November 10, 2017 HCV-RNA and HCVAg in DAAs treatment Fig 1. Frequency of negative results for HCV-RNA (blue columns) and HCV-Ag (red columns) during on-treatment monitoring and follow-up of 96 patients treated with DAAs for chronic HCV-infection. https://doi.org/10.1371/journal.pone.0187755.g001 Fig 1. Frequency of negative results for HCV-RNA (blue columns) and HCV-Ag (red columns) during on-treatment monitoring and follow-up of 96 patients treated with DAAs for chronic HCV-infection. Baseline HCV-RNA and HCV-Ag values and correlation https://doi org/10 1371/journal pone 0187755 g001 Neither baseline HCV-RNA nor HCV-Ag levels were different between SVR and non-SVR patients (median HCV-RNA: 1.18x106 vs 2.2 x106 IU/mL p = 0.219; median HCV-Ag: 2,974 vs 4,170 fmol/L, p = 0.358, respectively). The correlation between HCV-RNA and HCV-Ag at baseline was good (Spearman r = 0.767, 95% confidence interval 0.66–0.84, p = 0.000). e overall agreement (kappa statistic) was good (0.62; 95% confidence limits 0.56–0.68). GZ = gray zone results. HCV-RNA and HCV-Ag: patterns, concordance and accuracy As expected, the decrease in HCV-RNA during treatment was very rapid since the rate of neg- ativity for HCV-RNA went from 16% after 2 weeks to 55% after 4 weeks and 99% after 8 weeks of treatment (Fig 1). The kinetics of HCV-Ag were different because negativity was already seen at 63% after 2 weeks but showed a slower progression during treatment (74% after 4 weeks, 83% after 8 weeks) (Fig 1). At the end of treatment, all patients except the breakthrough case were negative for both HCV-RNA and HCV-Ag. At the time of virological relapse, all 11 patients were positive for both HCV-RNA (range: 5,244 to 1.74x107 IU/mL) and HCV-Ag (range: from 10.13 to >20,000 fmol/L). Over the complete course of treatment, including baseline and follow-up, a total of 690 samples were assayed for both HCV-RNA and HCV-Ag. Of those, the qualitative concordance between the measures was good (92%; kappa statistics 0.62) (Table 2). Discordant results were Table 2. Qualitative comparison between HCV-RNA and HCV-Ag on 690 samples from 96 patients treated with DAAs. Qualitative virological parameters HCV-RNA positive HCV-RNA GZ HCV-RNA negative Total HCV-Ag positive 139 5 13 157 HCV-Ag GZ 12 6 25 43 HCV-Ag negative 39 37 414 490 Total 190 48 452 690 The overall agreement (kappa statistic) was good (0.62; 95% confidence limits 0.56–0.68). GZ = gray zone results. https://doi.org/10.1371/journal.pone.0187755.t002 PLOS ONE | https://doi.org/10.1371/journal.pone.0187755 November 10, 2017 5 / 13 CV-RNA and HCV-Ag on 690 samples from 96 patients treated with DAAs. ualitative comparison between HCV-RNA and HCV-Ag on 690 samples from 96 patients treated with DAAs. Table 2. Qualitative comparison between HCV-RNA and HCV-Ag on 690 samples from 96 patients treate 5 / 13 HCV-RNA and HCVAg in DAAs treatment observed on both sides, with 39 samples being positive for HCV-RNA and with no detectable HCV-Ag and 13 samples showing opposite outcomes. The latter samples were obtained during treatment and at 4 and 8 weeks after the start of therapy. The median level of HCV-RNA in the 39 samples negative for HCV-Ag was 55 IU/mL with only one specimen (2.6%) yielding a value >1,000 IU/mL. Conversely, the median level of HCV-Ag in the 13 samples negative for HCV-RNA was 15 fmol/L, with only one sample (7.7%) yielding a value >100 fmol/L. HCV-RNA and HCV-Ag: patterns, concordance and accuracy Those discrepancies did not account for the different behavior of HCV-RNA and HCV-Ag over the monitoring course because the levels varied with a consistent pattern in SVR and in R (Fig 2). The only difference was a slower decrease in HCV-Ag, which explains how samples were posi- tive for HCV-Ag and had undetectable HCV-RNA. The accuracy of HCV-RNA and HCV-Ag towards SVR was assessed at W2, W4 and W8 during treatment. HCV-RNA levels did not show a strong predictive value for SVR because the area under the ROC curve (AUC) was 0.65 at W2, 0.70 at W4 and 0.55 at W8 (Fig 3A). Indeed, the latter figure is a purely mathematical estimate because all patients with SVR or R did not show detectable viral RNA at that time. HCV-Ag was similarly a poor predictor of SVR at W2 and W4, with AUCs of 0.63 and 0.62, respectively (Fig 3B). However, at W8, the AUC for HCV-Ag was the highest (0.74); this occurred at the same time point when the AUC for HCV-RNA was the lowest, as observed previously. When the kinetics of HCV-RNA and HCV-Ag in the 82 patients infected by HCV genotype 1 (84.5%) were compared to the 14 infected by other genotypes (Table 1), no significant differences were observed. Finally, as a further measure of the efficacy of antiviral treatment, the decrease (expressed in log10 values) in HCV-RNA and HCV-Ag at different time points compared to baseline was calculated (Fig 4). Again, the differences between SVR and R were not significant for HCV- RNA, whereas for HCV-Ag, a significant difference (<0.05) was observed at W8, with a median decrease of 4.23 log10 in SVR compared to 3.36 in R (p<0.05). However, when consid- ering the patients treated for 12 weeks separately from those treated for 24 weeks, this differ- ence was no longer significant. PLOS ONE | https://doi.org/10.1371/journal.pone.0187755 November 10, 2017 Discussion The outcome of any pharmacological therapy for liver disease due to HCV is still based on test- ing for HCV-RNA with sensitive techniques because the absence of circulating viral RNA defines both patient responsiveness to drugs and the sustained viral response after treatment. The purpose of HCV-RNA testing may differ according to the type of therapy. Although mon- itoring patients after 2 and/or 4 weeks of treatment is still recommended for regimens such as interferon in interferon-free DAA schedules, testing after 2/4 weeks is optional and is used to verify adherence [16]. Indeed, negativity for HCV-RNA is obtained early by almost all subjects due to the direct effects of DAAs on viral replication. This conclusion was confirmed in the present study, in which 84% of the patients showed undetectable RNA after 4 weeks and 100% of the patients had the same result after 8 weeks, regardless of the treatment outcome. However, the same guidelines [16][17] suggest that although doing so provides less strength (A2 level), monitoring may be simplified to increase access to care by skipping assessments dur- ing treatment and determining HCV-RNA only at baseline and 12 or 24 weeks after the end of therapy (SVR12 or SVR24, respectively) (A2). In contrast, the most recent guidelines from the American Association for the Study of Liver Disease (AASLD) still recommend quantitative HCV viral load testing after 4 weeks of therapy [17], and the Asian Pacific Association for the Study of the Liver (APASL) guidelines that were issued last year indicate generically that moni- toring the HCV loads during treatment is important for response-guided therapy to determine the futility, treatment protocol, and duration [18]. Therefore, a consensus on monitoring for PLOS ONE | https://doi.org/10.1371/journal.pone.0187755 November 10, 2017 6 / 13 HCV-RNA and HCVAg in DAAs treatment https://doi.org/10.1371/journal.pone.0187755 November 10, 2017 7 / 13 PLOS ONE | https://doi.org/10.1371/journal.pone.0187755 November 10, 2017 PLOS ONE | https://doi.org/10.1371/journal.pone.0187755 November 10, 2017 7 / 13 HCV-RNA and HCVAg in DAAs treatment Fig 2. Monitoring profiles for HCV-RNA and HCV-Ag of a representative patient with sustained viral response after 24 weeks of treatment with Sofosbuvir + Ribavirin (2a) and of a patient with relapse after 24 weeks of treatment with Sofosbuvir + Simeprevir + Ribavirin (2b). Discussion The horizontal axis reports the dates of blood draws (month, day) from June 2015 to April 2016 after the start of treatment https://doi.org/10.1371/journal.pone.0187755.g002 https://doi.org/10.1371/journal.pone.0187755.g002 DAA treatment has yet to be reached, and the positive predictive value for SVR of HCV-RNA testing still needs to be ascertained. For this purpose, Maasoumy et al [7] studied the HCV-RNA kinetics from 298 patients with HCV genotypes 1 to 5a who were treated with different Sofosbu- vir-based regimens, and they reported that lower levels were attained by SVR patients compared to R in both genotype 1 and genotype 3 infections. For the latter group, the authors indicated that HCV-RNA levels <45 IU/mL or <60 IU/mL (according to the method employed for HCV-RNA quantitation) after 2 weeks of treatment were predictive of treatment outcome and yielded 100% sensitivity. Conversely, the specificity was not outstanding because levels below the proposed thresholds were also attained in 29% and 33% or R, respectively. We were not able to confirm these results, at least for patients with HCV genotype 1 infection (73.5% of our cases), because no significant differences in HCV-RNA levels or decreases could be observed between SVR and R at week 2 or 4 during treatment. This result is relevant because the percent- age rates of SVR may currently be lower than the rates reported in the literature, which most likely occurred because of advanced disease and a suboptimal regimen (according to the infor- mation available at the time of therapy starting) for some patients (Table 1). In our experience, we observed an 11% rate of R that was unrelated to previous treatment failure and/or other clin- ical data, and a similar rate (14%) was recently reported by Aghemo et al. [14]. A new approach for DAA monitoring has also been included in the EASL 2017 guidelines. Although the standard methodology is still real-time PCR with high sensitivity (15 IU/ml), measuring HCV-Ag has been recommended (A1 level) as an alternative to PCR “when HCV- RNA is not available or not affordable” [16]. HCV-Ag is also recommended in the same guide- lines as a way to identify people with ongoing HCV infection (i.e., viral replication) among those individuals who are found to be positive for anti-HCV antibodies. https://doi.org/10.1371/journal.pone.0187755.g002 PLOS ONE | https://doi.org/10.1371/journal.pone.0187755 November 10, 2017 Discussion Consistently, the same approach has also been envisioned by the World Health Organization in a very recent document designed to update the diagnostic criteria for both hepatitis B virus and HCV infec- tions [19]. The timing for HCV-Ag testing in treatment monitoring will be the same as HCV-RNA, i.e., at baseline, optional between weeks 2 and 4, at the end of treatment and at the end of post- treatment follow-up [16]. The data reported here are more complete because we could assess both markers of HCV replication at multiple time points during treatment. Overall, the HCV-Ag trends mimicked those of HCV-RNA with different kinetics. The antigen levels decrease more rapidly in the early stages (63% of negativity vs. 16% for RNA) due to the lower analytical sensitivity compared to the real-time PCR for HCV-RNA [10, 11]. The antigen is cleared later, which possibly occurred because the circulating HCV core protein is available not only from virions but also from antigen-antibody complexes [8, 20] that have a longer half-life. When HCV-Ag has been related to treatment outcomes, its overall accuracy was the same as HCV-RNA. Serological testing for HCV-Ag in patients treated for chronic HCV infection has already been described by several papers, either on patients treated with IFN-based regimens [11–13] or with DAAs [14, 15, 21]. The results of the latter treatments reported high consistency between HCV-RNA and HCV-Ag results both during treatment and at the end of the follow- up period. Our experience complements those studies with more details since we enrolled patients treated with different regimens and infected by different HCV genotypes, and the behavior of the two markers in SVR and in R was assessed separately. 8 / 13 PLOS ONE | https://doi.org/10.1371/journal.pone.0187755 November 10, 2017 HCV-RNA and HCVAg in DAAs treatment PLOS ONE | https://doi.org/10.1371/journal.pone.0187755 November 10, 2017 9 / 13 HCV-RNA and HCVAg in DAAs treatment Fig 3. Receiver-operating characteristics (ROC) curves for HCV-RNA (3a) and HCV-Ag (3b) after 2, 4 and 8 weeks after starting treatment with DAAs. The horizontal axis reports the dates of blood draws (month, day) from June 2015 to April 2016 after the start of treatment. HCV-RNA and HCVAg in DAAs treatment diagnosis of acute hepatitis C. Additionally, for treatment monitoring, HCV-Ag allows savings because it is less expensive than RNA [14, 23], does not need specialized technicians and, most of all, has a much faster turnaround time that allows a result to be available in 1 hour compared to up to 7 hours for HCV-RNA [12, 14]. Even though the economic savings may be small com- pared to the treatment cost, they may be help decrease the overall cost in economically devel- oped countries and will definitely improve access to care for HCV worldwide with the aim of decreasing the prevalence and eventually the social and economic burden of the disease. A possible limitation of our study is represented by the variety of DAA-based treatment reg- imens for our patients. Our choice was driven by the objective to report real-life experiences from a reference Hepatology Center, where patients are most frequently referred from other institutions and present with different clinical histories. The rate of virological failure that we observed depends on the heterogeneity of the study population (post-OLT, high percentage of advanced disease) and relies on the fact that a substantial number of patients were treated with a regimen that is no longer considered optimal according to current guidelines. This subset, albeit small, gave us the chance to observe perfect synchronism between viral and serological rebound, which confirms the results from previous studies [14, 21]. The barely significant dif- ference in which there was an HCV-Ag decrease between SR and RR at W8 should not, in our opinion, lead clinicians to check HCV-Ag at this time point because this time point will not represent a stopping rule and will not lead to a change in treatment. However, it is suggested that RNA decay/clearance is linked to the halt in replicative activity due to DAAs action (phar- macological effect), whereas HCV-Ag clearance is linked to the elimination of infected cells that still contain virion components, so a negative HCV-Ag can reflect the complete clearance of infected cells. Considering that viral relapse was diagnosed equally by HCV-RNA and by HCV-Ag, the latter may represent an alternative clinical value in settings where HCV-RNA is still the pre- ferred or routine method for HCV monitoring. Supporting information S1 Table. Study dataset. (XLSX) In conclusion, our study provides further evidence of the clinical equivalency between HCV-RNA and HCV-Ag testing for assessing the response to DAA treatment for chronic HCV infection. A decision on what assay(s) to use in treatment monitoring will rely on the specific setting for the country/region where the therapy is dispensed, including reimburse- ment policies and continuous interaction and updates between the clinical and microbiology specialists of any given institution. https://doi.org/10.1371/journal.pone.0187755.g003 https://doi.org/10.1371/journal.pone.0187755.g003 Summarizing this evidence, HCV-RNA and HCV-Ag present similar kinetics in DAA treatment, either during treatment or during follow-up. As expected, HCV-Ag is less sensitive and is often negative in cases with very low levels of HCV-RNA. However, the current sched- ules do not include stopping rules because all patients are expected to complete the treatment course. Similarly, compared to the Peg-IFN-based treatment, a value for the baseline levels of HCV-RNA is no longer needed in order to tailor therapy, except in select cases [16], so the lower sensitivity of HCV-Ag does not seem to limit the performance of the treatment monitor- ing. Furthermore, low levels of HCV RNA during treatment or at the end of treatment are not indicative of non-adherence and do not predict a relapse [21, 22], and these are some of the reasons for moving towards a simplified monitoring strategy. Furthermore, the HCV-Ag assay has a very high specificity ranging from 99.8% in the original study [9] to 100% in subsequent observations [10, 19]. However, the potential advantages of using a serological test instead of a virological one is almost clear [9–12, 23]. Cresswell et al [23] demonstrated absolute clinical equivalence and substantial savings for utilizing HCV-Ag in place of HCV-RNA for the Fig 4. Decrease (logarithmic mean values) from baseline levels of HCV-RNA and HCV-Ag during treatment; SVR = sustained viral response; RR = response with relapse; W2, W4, W8 = weeks 2, 4 and 8 after the start of treatment; * = p<0.05. W = weeks after the start of treatment. https://doi.org/10.1371/journal.pone.0187755.g004 Fig 4. Decrease (logarithmic mean values) from baseline levels of HCV-RNA and HCV-Ag during treatment; SVR = sustained viral response; RR = response with relapse; W2, W4, W8 = weeks 2, 4 and 8 after the start of treatment; * = p<0.05. W = weeks after the start of treatment. https://doi.org/10.1371/journal.pone.0187755.g004 Fig 4. Decrease (logarithmic mean values) from baseline levels of HCV-RNA and HCV-Ag during treatment; SVR = sustained viral response; RR = response with relapse; W2, W4, W8 = weeks 2, 4 and 8 after the start of treatment; * = p<0.05. W = weeks after the start of treatment. https://doi.org/10.1371/journal.pone.0187755.g004 PLOS ONE | https://doi.org/10.1371/journal.pone.0187755 November 10, 2017 10 / 13 References 1. Kohli A, Shaffer A, Sherman A, Kottilil S. Treatment of hepatitis C: a systematic review. Jama. 2014; 312(6):631–40. Epub 2014/08/15. https://doi.org/10.1001/jama.2014.7085 PMID: 25117132. 2. European Association for Study of L. EASL Recommendations on Treatment of Hepatitis C 2015. J Hepatol. 2015; 63(1):199–236. Epub 2015/04/26. https://doi.org/10.1016/j.jhep.2015.03.025 PMID: 25911336. 3. Chung RT, Gale M Jr., Polyak SJ, Lemon SM, Liang TJ, Hoofnagle JH. Mechanisms of action of inter- feron and ribavirin in chronic hepatitis C: Summary of a workshop. Hepatology. 2008; 47(1):306–20. Epub 2007/12/29. https://doi.org/10.1002/hep.22070 PMID: 18161743; PubMed Central PMCID: PMC2799164. 4. Lens S, Marino Z, Forns X. Efficacy of new direct acting antivirals in transplant recipients and patients with advanced disease. Digestive and liver disease: official journal of the Italian Society of Gastroenter- ology and the Italian Association for the Study of the Liver. 2014; 46 Suppl 5:S197–205. Epub 2014/12/ 03. https://doi.org/10.1016/j.dld.2014.10.002 PMID: 25458782. 5. Craxi A, Perno CF, Vigano M, Ceccherini-Silberstein F, Petta S, AdHoc Working P. From current status to optimization of HCV treatment: Recommendations from an expert panel. Digestive and liver disease: official journal of the Italian Society of Gastroenterology and the Italian Association for the Study of the Liver. 2016. Epub 2016/07/09. https://doi.org/10.1016/j.dld.2016.06.004 PMID: 27388261. 6. Sarrazin C, Dierynck I, Cloherty G, Ghys A, Janssen K, Luo D, et al. An OPTIMIZE study retrospective analysis for management of telaprevir-treated hepatitis C virus (HCV)-infected patients by use of the Abbott RealTime HCV RNA assay. Journal of clinical microbiology. 2015; 53(4):1264–9. Epub 2015/02/ 06. https://doi.org/10.1128/JCM.03030-14 PMID: 25653396; PubMed Central PMCID: PMC4365219. 7. Maasoumy B, Vermehren J, Welker MW, Bremer B, Perner D, Honer Zu Siederdissen C, et al. Clinical value of on-treatment HCV RNA levels during different sofosbuvir-based antiviral regimens. J Hepatol. 2016. Epub 2016/04/18. https://doi.org/10.1016/j.jhep.2016.04.006 PMID: 27085252. 8. Reed KE, Rice CM. Overview of hepatitis C virus genome structure, polyprotein processing, and protein properties. Current topics in microbiology and immunology. 2000; 242:55–84. Epub 1999/12/11. PMID: 10592656. 9. Bouvier-Alias M, Patel K, Dahari H, Beaucourt S, Larderie P, Blatt L, et al. Clinical utility of total HCV core antigen quantification: a new indirect marker of HCV replication. Hepatology. 2002; 36(1):211–8. Epub 2002/06/27. https://doi.org/10.1053/jhep.2002.34130 PMID: 12085367. 10. Medici MC, Furlini G, Rodella A, Fuertes A, Monachetti A, Calderaro A, et al. Hepatitis C virus core anti- gen: analytical performances, correlation with viremia and potential applications of a quantitative, auto- mated immunoassay. Writing – review & editing: Pietro Andreone. Writing – review & editing: Pietro Andreone. Author Contributions Conceptualization: Pietro Andreone. Data curation: Elisabetta Loggi, Giovanni Vitale. Formal analysis: Elisabetta Loggi, Giuliano Furlini, Claudio Galli. Formal analysis: Elisabetta Loggi, Giuliano Furlini, Claudio Galli. Investigation: Silvia Galli, Giovanni Vitale, Roberto Di Donato, Ranka Vukotic, Elena Grand- ini, Marzia Margotti, Valeria Guarneri. Methodology: Claudio Galli. Project administration: Elisabetta Loggi, Giuliano Furlini, Maria Carla Re. Project administration: Elisabetta Loggi, Giuliano Furlini, Maria Carla Re. 11 / 13 PLOS ONE | https://doi.org/10.1371/journal.pone.0187755 November 10, 2017 HCV-RNA and HCVAg in DAAs treatment Resources: Giuliano Furlini, Claudio Galli, Maria Carla Re. Supervision: Giuliano Furlini, Maria Carla Re, Pietro Andreone. Validation: Pietro Andreone. Writing – original draft: Elisabetta Loggi. Writing – review & editing: Pietro Andreone. Resources: Giuliano Furlini, Claudio Galli, Maria Carla Re. Resources: Giuliano Furlini, Claudio Galli, Maria Carla Re. Supervision: Giuliano Furlini, Maria Carla Re, Pietro Andreone. Validation: Pietro Andreone. Validation: Pietro Andreone. Writing – original draft: Elisabetta Loggi. References Omata M, Kanda T, Wei L, Yu ML, Chuang WL, Ibrahim A, et al. APASL consensus statements and rec- ommendations for hepatitis C prevention, epidemiology, and laboratory testing. Hepatology interna- tional. 2016; 10(5):681–701. Epub 2016/05/28. https://doi.org/10.1007/s12072-016-9736-3 PMID: 27229718; PubMed Central PMCID: PMC5003900 the ethical standards of the responsible committee on human experimentation (institutional and national) and with the Declaration of Helsinki 1975, as revised in 2008 (5). Informed consent was obtained from all patients included in the study. This article does not contain any studies with animal subjects. Conflict of interest Alaaeldin Ibrahim, Cosmas Rinaldi Adithya Lesmana, Mamun Al-Mahtab, George K. K. Lau, Barjesh C. Sharma, Jose Sollano, Manoj Kumar, Ankur Jindal, A. Kadir Dokmeci, Geofferey W. McCaughan, Darrell H. G. Crawford, Jafri Wasim, and Shiv Kumar Sarin declare that they have no conflict of interest. Masao Omata received fees for being a speaker, consultant, and advisory board member for Bayer Co., Boehringer Ingelheim, Bristol-Myers Squibb, Otsuka, Astellas, Gilead Sciences, Chugai, Mitsubishi Tanabe, Kyorin, Merck Sharp & Dohme, Dainippon Sumitomo, Vertex Pharmaceuticals, Takeda, and Zeria. Tatsuo Kanda received lecture fees from Chugai Pharmaceutical, MSD, Tanabe-Mitsubishi, Daiichi-Sankyo, Bristol- Myers Squibb, Gilead Sciences, and AbbVie and a research grant from Chugai and MSD. Lai Wei has research grants from BMS and Roche and received consulting fees from Abbott, AbbVie, BMS, Gilead, and Novartis. Ming-Lung Yu is a consultant and advisory board member and has grant support from AbbVie, BMS, Gilead, Roche, MSD, and Abbott. Wang-Long Chuang received speaker fees from Gil- ead, BMS, MSD, Roche, and Novartis and is a member of the following advisory boards: Gilead, Abb- Vie, and Roche. Saeed S. Hamid has conference travel support from Gilead. Jia-Horng Kao has served as a consultant for AbbVie, Bristol-Myers Squibb, Gilead Sciences, and Roche and has also served on speakers’ bureaus for Roche, Bristol-Myers Squibb, Gilead Sciences, and Novartis. Osamu Yokosuka has research grants from Chugai Pharmaceutical, Bayer, MSD, Daiichi-Sankyo, Tanabe-Mitsubishi, and Bristol-Myers Squibb and received speaking fees from Merck Sharp and Dohme, Kowa Souku, Sysmex, Chugai Pharmaceutical Co., GlaxoSmithKline, Bristol-Myers Squibb, Ajinomoto-Seiyaku, Bayer, Abbott, Given Imaging, Mitsubishi Tanabe Pharm, Taiho Yakuhin, Dainippon Sumitomo Pharm, and Igaku-Seibutsugaku Institute. 19. Organization WH. Guidelines on hepatitis B and C testing. Geneva: World Health Organization. 2017. 19. 20. Morota K, Fujinami R, Kinukawa H, Machida T, Ohno K, Saegusa H, et al. References Journal of clinical virology: the official publication of the Pan American Society for Clinical Virology. 2011; 51(4):264–9. Epub 2011/05/31. https://doi.org/10.1016/j.jcv.2011.05.003 PMID: 21621454. 11. Chevaliez S, Soulier A, Poiteau L, Bouvier-Alias M, Pawlotsky JM. Clinical utility of hepatitis C virus core antigen quantification in patients with chronic hepatitis C. Journal of clinical virology: the official publication of the Pan American Society for Clinical Virology. 2014; 61(1):145–8. Epub 2014/06/29. https://doi.org/10.1016/j.jcv.2014.05.014 PMID: 24973282. 12. Loggi E, Cursaro C, Scuteri A, Grandini E, Panno AM, Galli S, et al. Patterns of HCV-RNA and HCV core antigen in the early monitoring of standard treatment for chronic hepatitis C. Journal of clinical virol- ogy: the official publication of the Pan American Society for Clinical Virology. 2013; 56(3):207–11. Epub 2012/12/19. https://doi.org/10.1016/j.jcv.2012.11.012 PMID: 23245628. 13. Kamal SM, Kassim S, El Gohary E, Fouad A, Nabegh L, Hafez T, et al. The accuracy and cost-effective- ness of hepatitis C core antigen assay in the monitoring of anti-viral therapy in patients with chronic hep- atitis C genotype 4. Alimentary pharmacology & therapeutics. 2015; 42(3):307–18. Epub 2015/05/29. https://doi.org/10.1111/apt.13261 PMID: 26018116. 14. Aghemo A, Degasperi E, De Nicola S, Bono P, Orlandi A, D’Ambrosio R, et al. Quantification of Core Antigen Monitors Efficacy of Direct-acting Antiviral Agents in Patients With Chronic Hepatitis C Virus PLOS ONE | https://doi.org/10.1371/journal.pone.0187755 November 10, 2017 12 / 13 HCV-RNA and HCVAg in DAAs treatment Infection. Clinical gastroenterology and hepatology: the official clinical practice journal of the American Gastroenterological Association. 2016. Epub 2016/04/06. https://doi.org/10.1016/j.cgh.2016.03.035 PMID: 27046484. Infection. Clinical gastroenterology and hepatology: the official clinical practice journal of the American Gastroenterological Association. 2016. Epub 2016/04/06. https://doi.org/10.1016/j.cgh.2016.03.035 PMID: 27046484. Infection. Clinical gastroenterology and hepatology: the official clinical practice journal of the American Gastroenterological Association. 2016. Epub 2016/04/06. https://doi.org/10.1016/j.cgh.2016.03.035 PMID: 27046484. 15. 15. Chevaliez S, Feld J, Cheng K, Wedemeyer H, Sarrazin C, Maasoumy B, et al. Clinical utility of HCV core antigen detection and quantification in the diagnosis and management of patients with chronic hep- atitis C receiving an all-oral, interferon-free regimen. Antiviral therapy. 2016. Epub 2016/04/27. https:// doi.org/10.3851/IMP3042 PMID: 27115431. 16. European Association for the Study of the Liver. Electronic address eee. EASL Recommendations on Treatment of Hepatitis C 2016. J Hepatol. 2016. Epub 2016/09/27. https://doi.org/10.1016/j.jhep.2016. 09.001 PMID: 27667367 17. America TAAftSoLDatIDSo. Recommendations for Testing, Managing, and Treating Hepatitis C. Last Updated: April 12, 2017. www.hcvguidelinesorg. 2017. 18. PLOS ONE | https://doi.org/10.1371/journal.pone.0187755 November 10, 2017 References A new sensitive and auto- mated chemiluminescent microparticle immunoassay for quantitative determination of hepatitis C virus core antigen. Journal of virological methods. 2009; 157(1):8–14. Epub 2009/01/13. https://doi.org/10. 1016/j.jviromet.2008.12.009 PMID: 19135481. 21. Rockstroh JK, Feld JJ, Chevaliez S, Cheng K, Wedemeyer H, Sarrazin C, et al. HCV core antigen as an alternate test to HCV RNA for assessment of virologic responses to all-oral, interferon-free treatment in HCV genotype 1 infected patients. Journal of virological methods. 2017; 245:14–8. Epub 2017/04/01. https://doi.org/10.1016/j.jviromet.2017.03.002 PMID: 28359920. 22. Sarrazin C, Wedemeyer H, Cloherty G, Cohen DE, Chevaliez S, Herman C, et al. Importance of very early HCV RNA kinetics for prediction of treatment outcome of highly effective all oral direct acting anti- viral combination therapy. Journal of virological methods. 2015; 214:29–32. Epub 2014/12/23. https:// doi.org/10.1016/j.jviromet.2014.11.027 PMID: 25528998. 23. Cresswell FV, Fisher M, Hughes DJ, Shaw SG, Homer G, Hassan-Ibrahim MO. Hepatitis C core antigen testing: a reliable, quick, and potentially cost-effective alternative to hepatitis C polymerase chain reac- tion in diagnosing acute hepatitis C virus infection. Clinical infectious diseases: an official publication of the Infectious Diseases Society of America. 2015; 60(2):263–6. Epub 2014/10/11. https://doi.org/10. 1093/cid/ciu782 PMID: 25301216. 13 / 13 PLOS ONE | https://doi.org/10.1371/journal.pone.0187755 November 10, 2017

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English

Influence of Vitamin D-Related Gene Polymorphisms (CYP27B and VDR) on the Response to Interferon/Ribavirin Therapy in Chronic Hepatitis C

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Abstract Background and Aims: Vitamin D exerts immunomodulatory effects on the host response against infection with hepatitis C virus (HCV). This study was performed to assess the putative influence of polymorphisms in vitamin D-related genes on the response to antiviral therapy in patients with chronic hepatitis C (CHC). Methods: Single nucleotide polymorphisms (SNPs) in CYP27B-1260 gene promoter (rs10877012AC) and in vitamin D receptor (VDR) gene rs2228570TC, rs1544410CT, rs7975232AC and rs731236AT were analyzed in a cohort of 238 Caucasian CHC patients treated with pegylated interferon (Peg-IFN) plus ribavirin (RBV). Multivariate analyses were performed to exclude confounding effects of well-known baseline predictors of response to therapy (HCV genotype and load, IL28B genotype, age, and GGT and serum cholesterol). Results: Three SNPs at the VDR gene (rs1544410, rs7975232 and rs731236) were in strong linkage disequilibrium, with the CCA haplotype predicting therapeutic failure [Odds ratio 2.743; (95% C.I. 1.313–5.731), p = 0.007]. The carrier state of the VDR rs2228570 T allele was inversely related to the probability of therapeutic failure [Odds ratio 0.438; 95 C.I. (0.204–0.882), p = 0.021]. No relation existed between CYP27B-1260 rs10877012 polymorphism and response to therapy. The area under the operating curve (AUROC) based on the model including all variables significantly related to the response to therapy was 0.846 (95% confidence interval = 0.793–0.899). Conclusion: VDR gene polymorphisms are independently related to the response to Peg-IFN+RBV therapy in chronic hepatitis C and could be used as complementary biomarkers of response when included in a prediction algorithm in association with demographic, virologic, biochemical and genetic traits. Citation: Garcı´a-Martı´n E, Agu´ndez JAG, Maestro ML, Sua´rez A, Vidaurreta M, et al. (2013) Influence of Vitamin D- elated Gene Polymorphisms (CYP27B and VDR) on the Response to Interferon/Ribavirin Therapy in Chronic Hepatitis C. PLoS ONE 8(9): e74764. doi:10.1371/journal.pone.0074764 R Editor: Matias A. Avila, University of Navarra School of Medicine and Center for Applied Medical Research (CIMA), Spain Received July 4, 2013; Accepted August 5, 2013; Published September 20, 2013 Copyright:  2013 Garcı´a-Martı´n 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: This work was supported by PS09/00943, PS09/00469, PI12/00241, PI12/00324, RETICS RIRAAF RD12/0013/0002 from Fondo de Investigacio´n Sanitaria, Instituto de Salud Carlos III, Spain, and GR10068 from Junta de Extremadura, Spain. Elena Garcı´a-Martı´n1, Jose´ A. G. Agu´ ndez2, Marı´a L. Maestro3, Avelina Sua´rez4, Marta Vidaurreta3, Carmen Martı´nez2, Cristina Ferna´ndez-Pe´rez5, Luis Ortega6, Jose´ M. Ladero7* Elena Garcı´a-Martı´n1, Jose´ A. G. Agu´ ndez2, Marı´a L. Maestro3, Avelina Sua´rez4, Marta Vidaurreta3, Carmen Martı´nez2, Cristina Ferna´ndez-Pe´rez5, Luis Ortega6, Jose´ M. Ladero7* 1 Department of Biochemistry and Molecular Biology, University of Extremadura, Ca´ceres, Spain, 2 Department of Pharmacology, University of Extremadura, Ca´ceres, Spain, 3 Genomics Unit, Clinical Laboratory Department, Hospital Clı´nico San Carlos, Instituto de Investigacio´n Sanitaria del Hospital Clı´nico San Carlos (IdISSC), Madrid, Spain, 4 Service of Clinical Microbiology, Hospital Clı´nico San Carlos, Instituto de Investigacio´n Sanitaria del Hospital Clı´nico San Carlos (IdISSC), Madrid, Spain, 5 Clinical Research and Methodology Unit. Hospital Clı´nico San Carlos, Medical School, Universidad Complutense, Instituto de Investigacio´n Sanitaria del Hospital Clı´nico San Carlos (IdISSC), Madrid, Spain, 6 Service of Pathology, Hospital Clı´nico San Carlos, Instituto de Investigacio´n Sanitaria del Hospital Clı´nico San Carlos (IdISSC), Madrid, Spain, 7 Service of Gastroenterology, Hospital Clı´nico San Carlos, Department of Medicine, Medical School, Universidad Complutense, Instituto de Investigacio´n Sanitaria del Hospital Clı´nico San Carlos (IdISSC), Madrid, Spain Abstract Financed in part with FEDER funds from the European Union. 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: [email protected] Citation: Garcı´a-Martı´n E, Agu´ndez JAG, Maestro ML, Sua´rez A, Vidaurreta M, et al. (2013) Influence of Vitamin D- elated Gene Polym on the Response to Interferon/Ribavirin Therapy in Chronic Hepatitis C. PLoS ONE 8(9): e74764. doi:10.1371/journal.pone.0074764 R Patients Analyses of Vitamin D-related genes. Genotyping was carried out by the use of TaqMan probes designed to detect the following SNPs: CYP2B7B1, rs10877012 (custom TaqMan SNP assay; Applied Biosystems, Madrid, Spain); VDR, rs2228570, rs731236, rs1544410 and rs7975232 (TaqMan SNP assays C__12060045_20, C__2404008_10, C___8716062_10 and C__28977635_10, respectively; Applied Biosystems, Madrid, Spain). The SNPs were selected on the basis of allele frequencies and functional of clinical implications [12–14]. The only nonsynonymous SNP confirmed for the VDR gene was included in the study. No other nonsynonymous SNPs have been described to occur in Caucasian individuals with minor allele frequencies over 0.001 (see the website http://www.ncbi.nlm.nih.gov/ projects/SNP/snpref.cgi?showRare= on&chooseRs = coding&go= Go&locusId = 7421). Analyses of Vitamin D-related genes. Genotyping was carried out by the use of TaqMan probes designed to detect the following SNPs: CYP2B7B1, rs10877012 (custom TaqMan SNP assay; Applied Biosystems, Madrid, Spain); VDR, rs2228570, rs731236, rs1544410 and rs7975232 (TaqMan SNP assays C__12060045_20, C__2404008_10, C___8716062_10 and C__28977635_10, respectively; Applied Biosystems, Madrid, Spain). The SNPs were selected on the basis of allele frequencies and functional of clinical implications [12–14]. The only nonsynonymous SNP confirmed for the VDR gene was included in the study. No other nonsynonymous SNPs have been described to occur in Caucasian individuals with minor allele frequencies over 0.001 (see the website http://www.ncbi.nlm.nih.gov/ projects/SNP/snpref.cgi?showRare= on&chooseRs = coding&go= Go&locusId = 7421). The population study included all patients with CHC who were scheduled to receive combined double therapy with pegylated interferon and ribavirin in accordance with current guidelines [19], and who attended since June 2003 to the outpatient clinic of the Gastroenterology Department (Liver Unit), San Carlos University Hospital, Madrid, Spain, Only patients who had completed a full course of therapy or who had stopped therapy due to therapeutic failure, defined with widely accepted criteria [19], were included in the study. Patients who had had to stop therapy due to drug intolerance, and those co-infected with HIV or HBV or with superimposed hepatocellular carcinoma were not included. The study was designed in accordance with the guidelines of the Declaration of Helsinki and approved by the ethics committee of the San Carlos University Hospital, Madrid, Spain. Written informed consent was obtained for all participants. The final study group was composed of 238 patients of Caucasian descent (221 Spaniards). Baseline and clinical characteristics are summarized in Table 1. Vitamin D Gene Polymorphism in Chronic Hepatitis C Calcitriol - 1,25(OH)2 Vitamin D - is a component of the hormonal system that maintains calcium and phosphorus homeo- stasis. In addition, calcitriol shows immunomodulatory effects as it reduces the levels of proinflammatory cytokines and promotes innate immune response. Calcitriol is synthesized in many tissues and may act as a ubiquitous immune regulating factor [4–6]. AmpliPrep, and the viral load was detected using Real-Time polymerase chain reaction (PCR) with Cobas TaqMan (Roche Diagnostics, Madrid, Spain) which has a detection range of between 15 IU/mL and 26108 IU/mL [20]. Viral load was classified as low (,400.000 IU/ml) or high ($400.000 IU/ml), according to Wittho¨ft et al. [21]. Vitamin D deficiency is very common in patients with CHC [7– 9]. This deficiency may reduce the rate of success of interferon plus ribavirin combined antiviral therapy [10–11], although two recent reports [12,13] have challenged the existence of this relation. The biological effects of calcitriol depend on its rate of synthesis, which is catalyzed by several enzymes involved in the 1– 25 double hydroxylation of calciferol. Cytochrome P-450 CYP27B1 generates 1–25 (OH)2 vitamin D, mainly in the kidney. Active Vitamin D must interact with its specific transmembrane receptor (VDR) to exert its physiological functions [6]. Both CYP27B1 and VDR genes have polymorphisms which possibly influence the efficacy of antiviral therapy, as it has recently been suggested by two independent groups [14–16]. In a previous study we did not identify any immediate effect of isolated vitamin D therapy on HCV viral load, although we did confirm that vitamin D deficiency is very common in Spanish CHC patients [17], but at a similar level to that found in the general Spanish population [18]. In the present study we aim to evaluate polymorphisms in Vitamin D-related genes as baseline biomarkers of response to interferon-ribavirin therapy in patients with CHC. The Hepatitis C viral genome is highly variable and is classified into 6 genotypes groups, or clades, based on phylogenetic analyses of the genomic sequence. HCV genotypes were determined by a reverse hybridization assay (VERSANT HCV Genotype 2.0 Assay (LiPA); Siemens, Tarrytown, NY USA). Biotinilated DNA PCR product generated by RT-PCR amplification of the 59UTR and core region of the HCV RNA is hybridized to immobilized oligonucleotide probes. The probes, which are bound to a nitrocellulose strip by a poli(dT) tail, are specific for the 59UTR and core region of different genotypes. Laboratory Methods Routine hematological and biochemical tests were performed with the standard methods at our laboratory. HCV analysis. Quantitative analysis of HCV-RNA was performed with the Cobas Amplicor HCV Monitor version 2.0 (Roche Molecular Diagnostic, Madrid, Spain). The detection range was 600 IU/mL to 8.56105 IU/mL. Starting from July 2005, viral RNA was extracted automatically using Cobas Patients Blood samples for biochemical, virologic and genetic studies were obtained during the month previous to the onset of therapy. Histological data were collected when a liver biopsy performed within the previous six months was available. In the remaining cases the results of transient elastography were included, when available. For every SNP analysed, twenty samples with heterozygous genotypes and up to twenty samples with homozygous genotypes (homozygous non-mutated and homozygous mutated when available), were determined as blind duplicates by direct sequenc- ing of the amplified fragments. In all cases the genotypes fully corresponded to those obtained with TaqMan probes. As a prerequisite, only individuals with full genotyping were scheduled to be included in the study. All possible haplotypes combining the four VDR SNPs analyzed were constructed and their frequencies were analyzed by using PHASE as described elsewhere [24]. Putative departures of Hardy-Weinberg Equilibrium and SNP linkages were calculated by using the software Haploview 4.1. In addition, the rs12979860 IL28B gene polymorphism was deter- mined in 236 patients as described previously [25]. The final study group was composed of 238 patients of Caucasian descent (221 Spaniards). Baseline and clinical characteristics are summarized in Table 1. Blood samples for biochemical, virologic and genetic studies were obtained during the month previous to the onset of therapy. Histological data were collected when a liver biopsy performed within the previous six months was available. In the remaining cases the results of transient elastography were included, when available. Subset analyses were performed in patients infected with viral genotype 1. Vitamin D Gene Polymorphism in Chronic Hepatitis C The VERSANT HCV Genotype 2.0 Assay (LiPA) is a line probe assay, for in vitro diagnostic use, which identifies Hepatitis C Virus genotypes 1 to 6 and subtypes a and b of the genotype 1 in human serum or EDTA plasma samples. Chen et al. [22] communicate that the accuracy of 1a and 1b subtyping ranges from 80% to 95% by 59UTR analysis, depending on the specific isolated tested, so the VERSANT HCV Genotype 2.0 Assay (LiPA) uses sequence motifs from the core region in addition to the 59UTR to improve the accuracy of the identification of 1a and 1b. Liver biopsy specimens were scored using the METAVIR system [23]. All biopsies were evaluated and scored by the same pathologist. Liver biopsy specimens were scored using the METAVIR system [23]. All biopsies were evaluated and scored by the same pathologist. Introduction biomarkers at baseline [3] it is possible to make an approximate prediction of the probability of achieving sustained virologic response (SVR, defined as non-detectable HCV RNA in serum 6 months after the end of therapy). The adjunction of new direct anti-HCV drugs is changing the therapeutic approach in CHC, but at present a still significant number of CHC patients are treated with the classic two-drug approach. To refine the predictive capacity biomarkers of response to pegylated interferon plus ribavirin additional baseline predictors of clinical response would be welcome. The success rate of current therapy for chronic hepatitis C (CHC) that combines pegylated interferon plus ribavirin is related to a variety of predictive factors that can be used as response biomarkers. Some of these depend on the hepatitis C virus (HCV), such as viral genotype, changes in critical regions of the viral genome and viral load. Other are host-related, either genetic (IL28B gene polymorphisms, gender and race) or acquired (insulin resistance, obesity, liver steatosis, iron overload and liver fibrosis stage) [1,2]. Hence, by combining the most reliable of these September 2013 | Volume 8 | Issue 9 | e74764 1 PLOS ONE | www.plosone.org Vitamin D Gene Polymorphism in Chronic Hepatitis C Statistical Analysis A database was created to include clinical and demographical data, and baseline biochemical, virologic, histological and genomic results. September 2013 | Volume 8 | Issue 9 | e74764 PLOS ONE | www.plosone.org 2 Vitamin D Gene Polymorphism in Chronic Hepatitis C Table 1. Baseline characteristics of the 238 patients with chronic hepatitis C. Variable(1) All patients Sustained virological response Failure of therapy Statistics (Univariate) Gender Male, n(%) 148 (62.2) 57 (62.0) 91 (62.3) Odss ratio (95% C.I.) = 0.984 (0.575–1.685) Female, n(%) 90 (37.8) 35 (38.0) 55 (37.7) Age (years) Mean (range) 49.9 (20–77) 46.6 (20–74) 52.0 (24–77) 1.04 (1.01–1.07). p,0.001 HCV genotype, n (%) 1 208 (87.4) 73 135 Chi2 = 13.92 (p = 0.003) 1b 157 (66.0) 53 104 1non b 51 (21.4) 20 31 2 2 (0.8) 2 0 3 14 (5.9) 11 3 4 13 (5.5) 6 7 n.a.2 1 (0.4) 0 1 Genotypes 1–4/genotypes 2–3 221/16 79/13 142/3 Odss ratio (95% C.I.) = 7.789 (2.144–28.16) HCV RNA ,400.000 IU/ml, n (%) 46 (19.2) 27 19 Odss ratio (95% C.I.) = 2.798 (1.447–5.410) $400.000 IU/ml (%) 190 (79.8) 64 126 n.a.2 2 (0.8) 1 1 Fibrosis stage (METAVIR), n (%)3 0 20 (8.4) 10 10 Chi2 = 8.46 (p = 0.076) 1 61 (25.6) 24 37 2 35 (14.7) 13 22 3 53 (22.3) 17 36 4 33 (13.9 5 28 n.a.2 36 (15.1) 23 13 Necroinflammatory grade (METAVIR), n (%) 1 19 (8.0) 10 9 Chi2 = 2.89 (p = 0.236) 2 67 (28.2) 21 46 3 77 (32.4) 28 49 n.a.2 75 (31.5) 33 42 Steatosis, n (%) No 103 (43.4) 37 66 Odds ratio (95% C.I.) = 1.189 (0.618–2.289) Yes 60 (25.1) 24 36 n.a.2 75 (31.5) 31 44 ALT, U/L 104 (75) 108 (69) 100 (79) 0.999 (0.995–1.002). p = 0.134 GGT, U/L 89 (95) 62 (63) 146 (105) 1.008 (1.003–1.013). p,0.001 Cholesterol, mg/dL 176 (35) 185 (36) 171 (32) 0.998 (0.980–0.996). p = 0.006 IL28B rs12979860 genotype n (%) CC 79 (33.2) 54 25 Chi2 = 43.56 (p,0.001) CT 124 (52.1) 31 93 TT 34 (14.3) 7 27 n.a.2 1 (0.4) 0 1 T allele non carriers/T allele carriers 79/158 54/38 25/120 Odds ratio (95% C.I.) = 6.82 (3.75–12.41) 1Continuous variables are given as mean (SD) except age. 2n.a.: Data not available. 3The stage of fibrosis was established by liver biopsy in 163 cases and by transient elastography in 39 cases. ta not available. ge of fibrosis was established by liver biopsy in 163 cases and by transient elastography in 39 cases. 371/j l 0074764 t001 Vitamin D Gene Polymorphism in Chronic Hepatitis C the allelic combination rs1544410C - rs7975232C - rs731236A, defined as the risk haplotype in agreement with previously published evidence [14]. The remaining SNP at the VDR gene (rs 2228570 TC) was not in linkage disequilibrium with the others and was included in the analysis as an independent variable. Statistical analysis was performed using SPSS 17.0 (SPSS Inc, Chicago, Il, USA). Therapy response was divided into two categories: responder patients obtaining SVR and non-responder patients. This latter category including patients with primary failure, partial viral response and relapse after end-of-therapy viral response. Statistical associations were calculated by univariate analysis comparing the two groups defined according to therapy response for age, sex, viral genotype (classified as difficult- (1 and 4) vs. easy-to-treat (2 and 3)), viral load (low vs. high), serum ALT, GGT, and cholesterol and the studied polymorphisms at CYP27B, VDR and IL28B genes. Continuous variables, expressed as mean (SD), were compared with the Student’ t test or the Mann-Whitney U test, each when adequate, depending on their Gaussian distribution. A p value ,0.05 was considered significant. Categorical variables were compared with the x2 or the Fisher exact tests, each when appropriate, and the effect of differences was established by calculating the odds ratio with the 95% confidence interval. In the univariate analysis the following variables were signifi- cantly linked with therapeutic failure: older age, IL28B rs12979870T allele carrier state, higher viral load, viral genotype 1 or 4, higher GGT levels and lower serum cholesterol levels. These variables were included in the multivariate analysis along with the CYP27B and VDR SNPs and the risk haplotype. In the multivariate analysis, all variables previously identified in the univariate analysis were confirmed as predictors of therapy failure at a significance level equal to p,0.05, with the exception of advanced age, as was, in addition, the carrier state of the of the risk CCA haplotype (combined rs1544410C - rs7975232C - rs731236A alleles) (Odds ratio = 2.743 - 95% confidence inter- val = 1.313–5.731- p = 0.007). On the contrary, the carrier state of the minor T allele at VDR rs2228570 was significantly related to the probability of obtaining sustained viral response to therapy Odds ratio = 0.438 295% confidence interval = 0.204–0.882- p = 0.021 (Table 2). Vitamin D Gene Polymorphism in Chronic Hepatitis C The variables different from a p value ,0.05 in the univariate analysis were included in a multivariate analysis based on a logistic regression model by exact methods (maximum likelihood tests) to identify which were independently related to the result of therapy. CYP27B and VDR gene polymorphisms were forced into the multivariate analysis except when strong linkage disequilibrium was detected between VDR SNPs (see below). The probability of therapeutic failure (P) was estimated with the formula: P~ 1 1ze{({1:65z0:83|az1:01|bz2:17|cz1:84|dz1:46|7z0:01|f {0:02|g) Statistical Analysis d i 10 1371/j l 0074764 t001 September 2013 | Volume 8 | Issue 9 | e74764 September 2013 | Volume 8 | Issue 9 | e74764 PLOS ONE | www.plosone.org 3 Vitamin D Gene Polymorphism in Chronic Hepatitis C Vitamin D Gene Polymorphism in Chronic Hepatitis C Discussion This study reveals that a common nonsynonymous SNP in the VDR gene (rs2228570 T/C), which is studied here for the first time in chronic hepatitis C patients, is a predictor of the clinical outcome of combined interferon plus ribavirin therapy. This polymorphism causes a threonine-metionine change in the Vitamin D receptor and, in the multivariate analysis that included the most potent predictors of viral response to combined therapy at baseline, those patients carrying the minor T allele in homo- or heterozygosis obtained SVR at a higher rate than patients with the rs2228570 CC genotype (p = 0.015). Most of these patients were infected with difficult-to-treat viral genotypes (87.4% genotype 1 and 5.5% genotype 4). When the analysis was restricted to the The retrospective design of this study precluded us to determine serum vitamin D and hence to analyze the possible influence of the studied polymorphisms on serum concentrations of the vitamin. Available data on the CYP27B1-1260 promoter polymorphism (rs10877012) are contradictory, as Lange et al. suggested that this polymorphism could influence serum concentrations of 1–25(OH)2 vitamin D [15] whereas Kitanaka et al. [26] did not confirm such a relationship. However, these authors reported that the BsmI, ApaI, TaqI haplotype at the VDR gene (that we have designed as CCA haplotype) is found 5.61 times more frequently among Vitamin-D deficient Japanese children than in the control group. None of the Vitamin D-related polymorphisms included in our study was found to be related with serum vitamin D levels in a recent GWAS analysis [27]. Figure 1. Receiver-operating curve provided by the model constructed to establish the predictive value for therapeutic failure. Area under the receiver-operating curve = 0.846 (95% confi- dence interval = 0.793–0.899). doi:10.1371/journal.pone.0074764.g001 Lange et al. in a complementary study based on a greater series of CHC patients (701 cases), detected an association between CYP27B1-1260 promoter polymorphism (rs10877012) and the rate of response to antiviral therapy (p = 0.06) which reached significance (p = 0.02) when the analysis was limited to patients with the IL28B genotype associated with poor response [16]. In the present study we have not confirmed such association although the sample size of our study group was only a third of that included by Lange et al. Results September 2013 | Volume 8 | Issue 9 | e74764 PLOS ONE | www.plosone.org 4 Vitamin D Gene Polymorphism in Chronic Hepatitis C where the substitution values were as follow: a : VDR rs10877012T: non carrier=1; carrier=0; b : VDR rs1544410BsmIC/ rs7975232ApaIC/rs731236TaqIA haplotype: non carrier = 0; carrier = 1; c : IL28Brs1297860T: non carrier = 0; carrier = 1; d : Viral genotype: 1 or 4 = 1; 2 or 3 = 0; e : Viral loal: ,400.000 IU/mL = 0; $400.000 IU/mL = 1; f : GGT (U/L); g : Serum cholesterol (mg/dL). where the substitution values were as follow: a : VDR rs10877012T: non carrier=1; carrier=0; b : VDR rs1544410BsmIC/ rs7975232ApaIC/rs731236TaqIA haplotype: non carrier = 0; carrier = 1; c : IL28Brs1297860T: non carrier = 0; carrier = 1; d : Viral genotype: 1 or 4 = 1; 2 or 3 = 0; e : Viral loal: ,400.000 IU/mL = 0; $400.000 IU/mL = 1; f : GGT (U/L); g : Serum cholesterol (mg/dL). subset of genotype 1 infected patients, these differences remained significant. In addition, we also analyzed three highly linked SNPs in the VDR gene previously studied by Baur et al. in 155 Swiss patients with chronic hepatitis C [12]. Only 62.9% of their patients were infected with genotype 1. These authors reported a significant association (p = 0.028) between failure of the combined therapy and the haplotype bAt. In our study we found that these three SNPs are in strong linkage disequilibrium (95%), leading us to categorize our patients as non carriers/carriers of the rs1544410C - rs7975232C - rs731236A allelic combination. Eighty three patients carrying this allelic assortment had a highly significant lower rate of SVR in multivariate analysis (Pc = 0.009). The receiving operating curve (ROC) was plotted in accordance with the same model to establish specificity and sensitivity values (Figure 1). The best cut-off value for this model was 0.620 that provided a sensitivity of 78.3% and a specificity of 79.2% for predicting therapeutic failure. In the analysis restricted to the 208 patients infected with viral genotype 1 the differences detected in the whole series remained significant in the multivariate analysis although at a lower level, both for the carrier state of the VDR rs2228570 T allele (Odds ratio for SVR = 0.469 (0.223–0.990). p = 0.042 and for the VDR CCA haplotype (Odds ratio for therapy failure = 2.179 (1.000– 4.762). Results p = 0.044). Vitamin D 1a-hydroxylase, the CYP27B1 gene product, catalyzes the synthesis of 1–25 (OH)2 Vitamin D, the active form of vitamin D that binds to the vitamin D receptor. Its immediate precursor, 25(OH) Vitamin D, is considered to be an adequate marker of vitamin D state [6], but not necessarily of vitamin D physiologic activity because a genetic-induced reduction of 1– 25(OH) vitamin D synthesis may result in a functional deficit of vitamin D, as occurs in vitamin D-dependent rickets type I. However, in a recent study, the CYP27B1-1260 promoter polymorphism (rs10877012) also included in our study is not related with vitamin deficiency in children [26]. Results Three of the four SNPs at the VDR gene (rs1544410, rs7975232, and rs731236) were in strong linkage disequilibrium and hence patients were categorized as non carriers/carriers of Table 2. Logistic regression analysis of demographic, biochemical and virological variables at baseline and of CYP27B1, VDR and IL28B gene polymorphisms in relation with the lack of response (failure to antiviral therapy) in 238 patients with chronic hepatitis C. Variable (1) Result of therapy Statistical analysis Sustained virological response Primary failure Univariate(2) Multivariate(3) Age 46.6 (10.4) 52.5 (9.4) 1.04 (1.01–1.07). 1.031 (0.995–1.068). p = 0.133 CYP27B_rs10877012 T allele carrier (no/yes) 47/45 57/39 0.654 (0.386–1.111) 0.650 (0.327–1.290). p = 0.218 VDR_rs2228570 T allele carrier (no/yes) 33/60 38/58 0.640 (0.375–1.096) 0.438 (0.204–0.882). p = 0.021 Carrier of rs1544410 BsmI C/rs7975232 ApaI C/rs731236 TaqI A VDR allelic combination (no/yes) 66/27 64/32 1.498 (0.856–2.620) 2.743 (1.313–5.731). p = 0.007 IL28B rs12979860 T allele carrier (no/yes) 54/39 9/84 6.82 (3.75–12.41) 8.724 (4.031–18.88). p,0.001 Viral load (low/high/unknown) 28/64/1 9/87/0 2.632 (1.348–5.137) 4.286 (1.392–9.507). p,0.001 Viral genotype 1/non-1 74/20 91/5 0.129 (0.036–0.464 6.268 (1.365–28.79). p = 0.018 GGT 61 (61) 125 (118) 1.008 (1.003–1.013) 1.006 (1.001–1.011). p = 0.025 Cholesterol 185 (37) 167 (32) 0.998 (0.980–0.996) 0.982 (0.971–0.992). p = 0.001 (1)Continuous variables are given as mean (SD). (2)Odds ratio (95% confidence interval). (3)Odds ratio (95% confidence interval) and p value in all significant variables in the univariate analysis. CYP27B1and VDR gene polymorphisms were forced into the analysis. doi:10.1371/journal.pone.0074764.t002 Table 2. Logistic regression analysis of demographic, biochemical and virological variables at baseline and of CYP27B1, VDR and IL28B gene polymorphisms in relation with the lack of response (failure to antiviral therapy) in 238 patients with chronic Table 2. Logistic regression analysis of demographic, biochemical and virological variables at baseline and of CYP27B1, VDR and IL28B gene polymorphisms in relation with the lack of response (failure to antiviral therapy) in 238 patients with chronic Table 2. Logistic regression analysis of demographic, biochemical and virological variables at baseline and of CYP27B1, VDR and IL28B gene polymorphisms in relation with the lack of response (failure to antiviral therapy) in 238 patients with chronic hepatitis C. References Arteh J, Narra S, Nair S (2010) Prevalence of vitamin D deficiency in chronic liver disease. Dig Dis Sci 55: 513–520. [PMID: 19960254]. [ ] 20. Heid CA, Stevens J, Livak KJ, Williams PM. (1996) Real time quantitative PCR. Genome Res 6: 989–994. [PMID: 8908518]. 8. Fisher L, Fisher A (2007). Vitamin D and parathyroid hormone in outpatients with noncholestatic liver disease. Clin Gastroenterol Hepatol 5: 513–520. [PMID: 17222588]. 21. Wittho¨ft TH, Mo¨ller B, Wiedmann KH, Mauss S, Link R, et al. (2007) Safety, tolerability and efficacy of peginterferon alpha-2a and ribavirin in chronic hepatitis C in clinical practice: The German Open Safety Trial. J Viral Hepat 14: 788–796. [PMID: 17927615]. 9. Miroliaee A, Nasiri-Toosi M, Khalilzadeh O, Esteghamati A, Abdollahi A, et al. (2010) Disturbances of parathyroid hormone-vitamin D axis in non-cholestatic chronic liver disease: a cross-sectional study. Hepatol Int 4: 634–640. [PMID: 21063488]. 22. Chen Z, Weck KE (2002) Hepatitis C virus genotyping interrogation of the 59untranslated region cannot accurately distinguish genotypes 1a and 1b. J Clin Microbiol 40: 3127–3134. [PMID: 12202542]. 10. Petta S, Camma` C, Scazzone C, Tripodo C, Di Marco V, et al. (2010) Low vitamin D serum level is related to severe fibrosis and low responsiveness to interferon-based therapy in genotype 1 chronic hepatitis C. Hepatology 51: 1158–1167. [PMID: 20162613]. 23. The French METAVIR Cooperative Study Group (1994). Intraobserver and interobserver variations in liver biopsy interpretation in patients with chronic hepatitis C. Hepatology 20: 15–20. [PMID: 80200885]. 24. Agu´ndez JA, Golka K, Martı´nez C, Selinski S, Blaszkewicz M, et al. (2008) Inravelin ambiguous NAT2 genotyping data. Clin Chem 54: 1390–1394. [PMID: 18864443]. 11. Bitetto D, Fattovich G, Fabris C, Ceriani A, Falleti E, et al. (2011) Complementary role of vitamin D deficiency and the interleukin-28B rs12979860 C/T polymorphism in predicting antiviral response in chronic hepatitis C. Hepatology 53: 1118–1126. [PMID: 21480318]. hepatitis C. Hepatology 53: 1118–1126. [PMID: 21480318]. 25. Ladero JM, Martin EG, Ferna´ndez C, Carballo M, Devesa MJ, et al. (2012) Predicting response to therapy in chronic hepatitis C: an approach combining interleukin-28B gene polymorphisms and clinical data. J Gastroenterol Hepatol 27: 279–285. [PMID: 21722179]. 12. Kitson MT, Dore GJ, George J, Button P, McCaughan GW, et al. (2013) Vitamin D status does not predict sustained viral response or fibrosis stage in chronic hepatitis C genotype 1 infection. J Hepatol 58: 467–472. [PMID: 23183524]. 26. Acknowledgments We are grateful to Prof. James McCue for assistance in language editing, and to Ms. Gara Esguevillas and Ms. Isabel Salas for their technical assistance. References pegylated-interferon/ribavirin-based therapy in chronic hepatitis C patients. Antiviral Ther 17: 541–547. [PMID: 22300961]. 1. Maekawa S, Sakamoto M, Miura M, Kadokura M, Sueki R, et al. (2012) Comprehensive analysis for viral elements and interleukin-28B polymorphisms in response to pegylated interferon plus ribavirin therapy in hepatitis C virus 1B infection. Hepatology 56: 1611–1621. [PMID: 22577043]. pegylated-interferon/ribavirin-based therapy in chronic hepatitis C patients. Antiviral Ther 17: 541–547. [PMID: 22300961]. 15. Lange CM, Bojunga J, Ramos-Lopez E, von Wagner M, Hassler A, et al. (2011) Vitamin D deficiency and CYP27B1–1260 promoter polymorphism are associated with chronic hepatitis C and poor response to interferon-alfa based therapy. J Hepatol 54: 887–893. [PMID: 21145801]. 2. Romero-Go´mez M, Del Mar Viloria M, Andrade RJ, Salmero´n J, Diago M, et al. (2005) Insulin resistance impairs sustained response rate to peginterferon plus ribavirin in chronic hepatitis C patients. Gastroenterology 128: 636–641. [PMID: 19845037]. therapy. J Hepatol 54: 887–893. [PMID: 21145801]. 16. Lange CM, Bibert S, Kutalik Z, Burgisser P, Cerny A, et al. (2012) A genetic validation study reveals a role of vitamin D metabolism in the response to interferon-alfa-based therapy of chronic hepatitis C. PloS ONE 7: e40159. [PMID: 22808108]. 3. Cuenca F, Ferna´ndez C, Devesa MJ, Lo´pez-Alonso G, Mayol J, et al. (2010) Predictive baseline criteria of primary therapeutic failure in chronic hepatitis C genotype 1. Rev Esp Enferm Dig 102: 234–238. [PMID: 20486745]. 17. Ladero JM, Torrejo´n M, Sa´nchez-Pobre P, Sua´rez A, Cuenca F, et al. (2013) Vitamin D deficiency and vitamin D therapy in chronic hepatitis C. Ann Hepatol 12: 199–204. [PMID: 23396730]. g yp p g 4. Bikle D (2009). Nonclassic actions of vitamin D. J Clin Endocrinol Metab 94: 26–34. [PMID: 18854395]. 5. Gutierrez JA, Parikh N, Branch AD (2011) Classical and emerging roles of vitamin D in hepatitis C virus infection. Sem Liver Dis 31: 387–398.[PMID: 22189978]. 18. Gonza´lez-Molero I, Morcillo S, Valde´s S, Pe´rez-Valero V, Botas P, et al. (2011) Vitamin D deficiency in Spain: a population-based cohort study. Eur J Clin Nutr 65: 321–328. [PMID: 21179052]. ] 6. Rosen CJ, Adams JS, Bikle DD, Black DM, Demay MB, et al. (2012) The nonskeletal effects of vitamin D: an Endocrine Society Scientific Statement. Endocrinol Rev 33: 456–492. [PMID: 22596255]. 19. European Association for the Study of the Liver (2011). EASL Clinical Practice Guidelines: Management of hepatitis C virus infection. J Hepatol 56: 245–264. [PMID: 21371579]. 7. Discussion A sample size of 370 cases (a ,0.05; power .80%) would have been necessary to exclude unit from the 95% confidence interval of the observed odds ratio (1,539), which is similar to that found by Lange et al [16]. Advanced liver fibrosis negatively influences the probability of response to antiviral combined therapy in chronic hepatitis C [27]. In the present study a liver biopsy was available in 163 (68.5%) patients, with data provided by transient elastography in other 39 patients (see table 1). Data on the stage of fibrosis were lacking in 36 patients and this kept us to include this variable in the multivariate analysis aimed to detect the possible influence of the studied vitamin D-related polymorphisms on the results of antiviral therapy. However, we found that the relative frequencies of the studied polymorphisms were quite similar in the group of patients classified according to their fibrosis stage as null-low (F0– F1) vs. moderate-advanced (F2–F4). (Data not shown). Figure 1. Receiver-operating curve provided by the model constructed to establish the predictive value for therapeutic failure. Area under the receiver-operating curve = 0.846 (95% confi- dence interval = 0.793–0.899). doi:10.1371/journal.pone.0074764.g001 September 2013 | Volume 8 | Issue 9 | e74764 September 2013 | Volume 8 | Issue 9 | e74764 PLOS ONE | www.plosone.org 5 Vitamin D Gene Polymorphism in Chronic Hepatitis C In this study, data on insulin resistance, that is a known predicting factor of therapeutic failure, are lacking. Prospective studies taking into consideration all these factors are warranted. the refinement in the prediction of clinical response based on ancillary baseline data. the refinement in the prediction of clinical response based on ancillary baseline data. We conclude that two polymorphic sites in the VDR gene that influence the response rate to interferon-ribavirin therapy in chronic hepatitis C exist. The association of these polymorphisms with the response rate is independent from other well known predictors of response at baseline that were included in the multivariate analysis as potentially confounding factors (IL28B gene polymorphism, viral genotype and load, age, and serum GGT and cholesterol levels). In addition to the mechanistic implications of alteration in VDR with regard to clinical response to interferon-ribavirin therapy in chronic hepatitis C, which deserves further studies, the use of these new criteria may add to Author Contributions Conceived and designed the experiments: JML JAGA. Performed the experiments: EGM MLM AS MV CM LO. Analyzed the data: CFP JML. Conceived and designed the experiments: JML JAGA. Performed the experiments: EGM MLM AS MV CM LO. Analyzed the data: CFP JML. Contributed reagents/materials/analysis tools: LO. Wrote the paper: JML JAGA. Conceived and designed the experiments: JML JAGA. Performed the experiments: EGM MLM AS MV CM LO. Analyzed the data: CFP JML. Contributed reagents/materials/analysis tools: LO. Wrote the paper: JML JAGA. p y J Contributed reagents/materials/analysis tools: LO. Wrote the paper: JML JAGA. Contributed reagents/materials/analysis tools: LO. Wrote the paper: JML JAGA. References Kitanaka S, Isojima T, Takaki M, Numakura C, Hayasaka K, et al. (2012) Association of vitamin-D related gene polymorphisms with manifestations of vitamin D deficiency in children. Endocr J 59: 1007–1014. [PMID: 22785457]. ] 13. Bitetto D, Bortolotti N, Falleti E, Vescovo S, Fabris C, et al. (2013) Vitamin A deficiency is associated with HCV chronic infection and with unresponsiveness to interferon based antiviral therapy. Hepatology 2013; 57: 925–933. [PMID: 23213086]. 27. Wang TJ, Zhang F, Richards JB, Kestenbaum B, van Meurs JB, et al. (2010) Common genetic determinants of vitamin D insufficiency: a genome-wide association study. Lancet 376: 180–188. [PMID: 20541252]. 14. Baur K, Mertens JC, Schmitt J, Iwata R, Stieger B, et al. (2012) The vitamin D receptor gene bAt (CCA) haplotype impairs the response to September 2013 | Volume 8 | Issue 9 | e74764 PLOS ONE | www.plosone.org 6

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de

Zum Nachweis von Cadmium und Nickel

Analytical and bioanalytical chemistry/Analytical & bioanalytical chemistry

public-domain

186 Bericht: Chemische Analyse a.norganischer Stoffe. Bteifolie gewiekelt, in die Schmelze eingetragen, unmittelbar darauf ftigt man etwa 5 g Cadmium hinzu. Durcti leichtes Drehen des Scherbens wird innerhalb dreier Minuten ein vollstitndiges Legieren erzielt, Der Inhalt des Scherbens wird nun in eine Form ausgegossen, der Regulus ~om Cyankalium befreit, m i t heissem Wasser gewaschen, und in starker Sall?eters'~ure gel6st. Man kocht die nitrosen DSmpfe weg, verdtinn~ die LSsung auf 100 c c m und titriert in Gegenwart yon 5 c c m gesattigter Eisenalaunl6sung mit Sulfocyankalium. B r z e z i n e r. Cam N a c h w e i s yon Cadmium a n d N i c k e l verwendet A. A g r e st in i '1 konz. LSsungen yon Alkalijodiden, besonders Jodkalium. 'Versetzt man eine ammoniakatische CadmiumlSsung mit 2 0 - - 3 0 ~ JodkaliumlSsung I so erh~lt man einen weissen, kristallinischen Niederschlag yon der Zusammensetzung Cd(~H~)~J 2. Kupferl6sungen geben keinen entspreehenden iNiederschlag. Ebenso erh~ilt man bei Kobalt- und ~Niekelsa]zen in stark ammoniakalischer LSsung analog zusammengesetzte Doppeljodide. Will man Nickel neben Kobali naehweisen, so muss man das Kobalt mit Ammoniak und Wasserstqffsuperoxyd in die Kobaltamminverbindung Oberfiihren oder auf andere Weise abscheiden, z. B. mit Katiumnitrit, und fSllt hie,'auf das Nickel als ~iolettblauen, kristallinischen Niederschlag, dem ~lie Zusammensetzung Ni(NH~),)~ zukommt. Die Reaktion erreicht bei weitem nicht die Empfind]ichkeit der mit Dimethylglyoximl Aueh das yon R. S a 1v a d o r i ~) zum Nachweis ~on Cadmium empfohlene Perchlorat gibt in ammoniakalischer LSsung mit Ni und Co eharakteristische ~Niederschl~ge. Mit Kobaltamminen jedoch nicht. Brzeziner. Zink. D e r C h e m i k e r - F a c h a u s s c h u s s tier Gesellsch~ft Deutseher Metallh~ttenund Bergleute hat t'or die Z i n k bestimmungin E r z e n und R S s t b l e n d e n nach S c h a f f n e r folgende yon F. B u 11 n h e i m e r t ~) vorgeschlagene Arbeitsweise angenommen m~d sie als N o r m a l - S c h a f f n e r - M e t h o d e fiirSchiedsanaiysen, anerkannt: Prinzip. 1, Das Zinkerz wird derartig mit S~uren auf'geschlossen. d~ss ein zinkfi'eier R~lckstand bleibt. 2. Es wird eine ammoniakalische Zinkl~sung hergestel]t, welehe frei is[ yon Bestandteilen, die be[ der Titration mit Schwefetnatrium st6re~. 3. E i s e n wird nur einmal gef~.llt. ,~. Der durch, die einmalige Eisenf~llung bedingte Zinkverlust wird durch Zusatz yon Eisen zum Titer Uusgeglichen. 5. Titer und :Probe werden gleiehzeitig nebeneinander titriert. Bereitung der ErzlSsung. 1 , 2 5 g des fein gepulverten und bei 100 o getrockneten E r z e s werden zuerst mit KSnigswasser aufgeschlossen und dann mit 5 c c m Sehwefels~ure ( 1 : 1 u eingedampft, his keine Schwefels~ured~mpfe mehr entweichen. Den Trockenr~ckstand 1) Gazz. ehim. itaL 48, 30 (1918); d. Chem. Zentrb]. 90, II, 6~4 (1919). -~) Vergl. diese Ztschrft. 58, 415 (1919). - - ~) Metall und Erz 17, 45~ (1920). 

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English

Tuberculosis in the Aged and the Diagnostic Value of Increased Whisper in the Interscapular Space

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TUBERCULOSIS IN THE AGED AND THE DIAGNOSTIC VALUE OF INCREASED WHISPER IN THE INTERSCAPULAR SPACE BY HENRY FARNUM STOLL, M.D., HARTFORD, CONN. Assistant Physician to the Hartford Hospital, Hartford, Conn. Pulmonary tuberculosis in the el- derly, because of its chronicity and periods of arrest when symptoms are more or less in abey- ance, is diagnosed less frequently perhaps than at any other time of life. This is in part due to the fact that chronic bronchitis and em- physema are so common in the elderly, and also because the clinical picture differs considerably from that which we are accustomed to see in early life. In elderly people cough is the chief complaint. The other symptoms are frequently slight or lacking altogether. In looking over my private cases for the past two years, I find that I have examined sixteen patients of 60 or over, who consulted me because of cough. This was due to the following causes : inhalation ol dust (tracheitis) 1; chronic bronchitis (with or without emphysema) 5 ; nephritis and myo- carditis, 1 ; possible tuberculosis, 2 ; tuberculosis, 7. The recital of several case histories will best illustrate certain features of the disease that characterize the latter decades. g p g Thus we find that though the total deaths from tuberculosis between 44 and 64 years of age are only one-half what they are between 24 and 44, yet when estimated on the basis of 1,000 living at those two decades, the percentage is practically the same. And it is twice as high for those of 65 as for those between 15 and 20. p There was very slight retraction of the right apex and dullness to the right of the fourth and fifth dorsal vertebra, with small moist rales at the right apex on coughing both in front and behind, and a few over the right lower lobe and at the left apex posteriorly. The whispered voice was heard over the vertebral column as low as the 8th dorsal and also to the right and left of the vertebral column from the fourth to the sixth dorsal vertebra. Two weeks later the right apex showed the same signs and a wet leather rub had developed over the right lower lobe. No tubercle bacilli were found in either of two specimens consisting chiefly of blood. In both these cases there was an exposure to tuberculosis over 40 years before.. Jour, of Physiology, April 1, 1911. , , ; , g , , p 0 Cannon: Boston Med. and Surg. Jour., 1912, clxvi, 563; also with D. de la Paz: "Emotional Stimulation of Adrenal Secre- tion." Amer. Jour, of Physiology, April 1, 1911. g p 0 Cannon: Boston Med. and Surg. Jour., 1912, clxvi, 563; also with D. de la Paz: "Emotional Stimulation of Adrenal Secre- tion." Amer. Jour, of Physiology, April 1, 1911. y gy, p , 7 Cannon: "The Influence of Emotional States on the Functions of the Alimentary Canal." Amer. Jour. Med. Sciences, April, 1909. y gy, p , 7 Cannon: "The Influence of Emotional States on the Functions of the Alimentary Canal." Amer. Jour. Med. Sciences, April, 1909. y p , s Jones: "A Modern Conception of the Psychoneuroses." Inter- state Med. Jour., 1910, xvii, no. 8. y p , s Jones: "A Modern Conception of the Psychoneuroses." Inter- state Med. Jour., 1910, xvii, no. 8. TUBERCULOSIS IN THE AGED AND THE DIAGNOSTIC VALUE OF INCREASED WHISPER IN THE INTERSCAPULAR SPACE BY HENRY FARNUM STOLL, M.D., HARTFORD, CONN. Assistant Physician to the Hartford Hospital, Hartford, Conn. Case 2. Amelia H. Aged 64. Father died at 65 of tuberculosis, 41 years ago. Well and strong as a girl; thirty-two years ago while pregnant de- veloped a cough. This continued after confinement, and her strength was slowly regained. At that time she had several hemorrhages. Following the next confinement, three years later, the patient was very much run down with a cough, night sweats and hemorrhages, and it was six months before she re- gained her health. Some of these hemorrhages came on while sleeping, others after a hard attack of coughing. Her cough is frequently so severe as to cause gagging. She has had a number of attacks of left sided pleurisy. Her health was good for a con- siderable period of time prior to three years ago, when she developed a severe illness diagnosed "typho-pneumonia." She was ill at this time for three months with a severe cough and hemorrhages. At the suggestion of a lady friend she slept out of doors for a number of months with great benefit, and this past year has been unusually well until two weeks ago, when she began to cough and expector- ate bright blood and have a slight afternoon tem- perature. So great is the death toll from tuberculosis during the early years of life that we are apt to think of it only as a disease of young adults. And this is but natural since nearly one-third of those who die are between 15 and 40 years of age. g These figures, however, do not give us any idea of the relative importance of tuberculosis as a cause of death at any one period. That can only be determined when estimations are based on the number living at that particular age. Th h h h living particular age. Thus we find that though the total deaths from tuberculosis between 44 and 64 years of age are only one-half what they are between 24 and 44, yet when estimated on the basis of 1,000 living at those two decades, the percentage is practically the same. And it is twice as high for those of 65 as for those between 15 and 20. This is a fact of the greatest importance, the full significance of which is not generally ap- preciated. The Boston Medical and Surgical Journal as published by The New England Journal of Medicine. Downloaded from nejm.org at SAN DIEGO (UCSD) on July 9, 2016. For personal use only. No other uses without permission. From the NEJM Archive. Copyright © 2010 Massachusetts Medical Society. and the specialists, who are the modern handmaids of practical medicine; knowing the history of his trusting friends and taking an interest in their wholeness and wholesomeness,—the chum of the old peop.e, the intimate of confiding girlhood, and the uncle and oracie of the kids." Jacobi: "Significance of the General Practitioner." Boston Med. and Sdrg. Jour., 1912, clxvi, 439. and the specialists, who are the modern handmaids of practical medicine; knowing the history of his trusting friends and taking an interest in their wholeness and wholesomeness,—the chum of the old peop.e, the intimate of confiding girlhood, and the uncle and oracie of the kids." Jacobi: "Significance of the General Practitioner." Boston Med. and Sdrg. Jour., 1912, clxvi, 439. Case 1. Mary S. Aged 60. A brother died of tuberculosis 40 years ago. The patient had good health up to the time of her marriage at the age of 20. The second year after her marriage she had a hard cough, which the neighbors diagnosed as con- sumption. She has worked hard much of her life, though frail, and each winter for the past four or five years has had a cough which has been diagnosed a number of times by her physician as grippe. The past winter the "grippe" was more severe and per- sistent and for the past few months the patient has been somewhat hoarse and has lost weight and strength. Examinations showed an old fibroid con- dition at the right apex with infiltration throughout the whole of the left lung and a cavity below the apex. Patient also had tuberculosis of the larynx and many tubercle bacillus in the sputum. , , , -Putnam: Proc. Mass. Med. Society, 1899; Boston Med. and Suro. Jour., 1899, cxli, 53. 3 Dr. P. C. Knapp has recently presented the arguments for this general viewpoint. "The Rehabilitation of Neurasthenia." Boston Med. and Surg. Jour., 1910, clxii, 269. , , , 4 A good resume will be found in Janet: Major Symptoms of Hysteria. The Macmillan Co., 1909. y , ECushing: "The Hypophysis Cerebri." Jour. Amer. Med. Asso., 1909, liii, 249; also, Proc. Amer. Neurolog. Asso., 1911, p. 202. y ECushing: "The Hypophysis Cerebri." Jour. Amer. Med. Asso., 1909, liii, 249; also, Proc. Amer. Neurolog. Asso., 1911, p. 202. 0 Cannon: Boston Med. and Surg. Jour., 1912, clxvi, 563; also with D. de la Paz: "Emotional Stimulation of Adrenal Secre- tion." Amer. TUBERCULOSIS IN THE AGED AND THE DIAGNOSTIC VALUE OF INCREASED WHISPER IN THE INTERSCAPULAR SPACE BY HENRY FARNUM STOLL, M.D., HARTFORD, CONN. Assistant Physician to the Hartford Hospital, Hartford, Conn. This sign, first called attention to by D 'Espine,1 as being indicative of enlarged bron- chial glands, has not received the attention it deserves. It is elicited as follows : The stetho- scope is placed over the vertebral spines, and the patient requested to whisper "three-thirty- three." In children the trachéal quality nor- mally ceases at the seventh cervicle. Occasion- ally in an apparently normal adult the trachéal quality, though of diminished intensity, persists over the upper three or four dorsal spines. The sign is said to be positive when the final " e " of the last word, "three," persists for an appre- ciable time after phonation ceases. It is notoriously hard many times to detect a tuberculosis lesion of the lungs when emphy- sema or asthma co-exists. The x-ray is of great value here but unfortunately is available to only a few. Asthma is sometimes due to enlarged bronchial glands, and we should consider the possibility of their presence in those patients who have a great deal of dyspnoea without pulmonary, cardiac, or renal lesion. p The diganostic value of this sign I have con- sidered elsewhere.2 In both children and adults, but especially in the latter, its signifi- cance is considerably increased when the whis- pered bronchophony is also heard at one or both sides of the vertebrae and its import is greater when heard down to, or below, the level of the fifth dorsal vertebral. In the adult when it is only heard over the upper three or four thoracic spines it is of questionable significance. I have had numerous radio- graphic confirmations of its value, and just Case 5. An old gentleman, over 60 years of age, had a severe cough, and was extremely emaciated, so much so that examination of the lungs was diffi- cult; no definite signs of disease could be detected. No attempt was made, unfortunately, to a elicit a whispered vertebral bronchophony. Within a short time after first coming under observation, he had a chill and fever every other day, very typical of ma- laria, and that diagnosis in fact had been made by another physician. No plasmodia were detected in the blood and his leucocytes were 13,000. He sub- sequently devloped an effusion of the left pleural cavity and a 1000 cc. of straw colored was with- drawn; sp. gr. 1020; coagulum slight; many small mononuclear cells, no polynuclears seen. TUBERCULOSIS IN THE AGED AND THE DIAGNOSTIC VALUE OF INCREASED WHISPER IN THE INTERSCAPULAR SPACE BY HENRY FARNUM STOLL, M.D., HARTFORD, CONN. Assistant Physician to the Hartford Hospital, Hartford, Conn. su e gs Our conception of the duration of tuberculo- sis would be materially changed did we devote more time to obtaining our histories. The mere fact that a cough has been present for a number of years is too often taken as sufficient proof of its non-tubercular character. Case 3. Margaret IN. Aged 61. Was delicate to her 45th year, every spring running down with "malaria." The past two winters she has had a se- vere cough which caused gagging. Two months ago she developed a pleurisy of the left lung which has continued, and for about six weeks has had a cough of increasing severity, often causing gagging. She has lost weight, had night sweats, complains of weakness, loss of appetite, increasing dyspnoea, and more recently, severe pain over the lower part of the sternum. The patient was extremely ill and suffered much from dyspnoea. The chief signs were those of a resolving pneumonia over the left lower lobe. Slightly prolonged expiration over right apex pos- teriorly was also noted. In addition, the whispered voice was present over the upper four dorsal ver- tebra. The patient died in 48 hours. At the post- mortem an extensive pericarditis was found, the whole of the pericardium being adherent to the heart. A caseous area was present in the left lower lobe. A few tubercles were present on the viceral pleura and a partly caseous gland about one 1% inches in diameter was found at the junction of the right bronchus and trachea. Case 4. Annie H. Aged 65. Consulted me be- cause of chronic cough and attacks of asthma. She was a typical asthmatic who wheezed markedly while her history was being taken. The physical signs were those of emphysema and chronic bronchitis, except that the rales were somewhat more numer- ous and moist at the right apex, where a slight amount of dullness was detected. There was marked increase of whispered voice in the interscapular re- gion and especially over the vertebral column. Nu- merous tubercle bacilli were found in the sputum. Apart from a questionable infection dating back to childhood, the chief interest in this case centers in the enlarged bronchial gland, whose presence was suspected because of the whis- pered voice heard over the upper dorsal verte- bra. The Boston Medical and Surgical Journal as published by The New England Journal of Medicine. Downloaded from nejm.org at SAN DIEGO (UCSD) on July 9, 2016. For personal use only. No other uses without permission. From the NEJM Archive. Copyright © 2010 Massachusetts Medical Society. TUBERCULOSIS IN THE AGED AND THE DIAGNOSTIC VALUE OF INCREASED WHISPER IN THE INTERSCAPULAR SPACE BY HENRY FARNUM STOLL, M.D., HARTFORD, CONN. Assistant Physician to the Hartford Hospital, Hartford, Conn. It is note- worthy that in one case the disease first mani- fested itself 32 years ago, and in the other the first symptoms appeared 38 years before. Case 2 is living and in excellent condition today, thanks to a lay friend who suggested the out-of- door life. Case 1 is dead because her physician either failed to make the diagnosis, or lacked the moral courage to tell the truth. And there is no more despicable example of moral cowardice than the physician who shrinks from inflicting the momentary pain the truth would cause, pre- ferring rather to let his patient drift along to an untimely death or chronic invalidism with its sufferings and lost illusions. Our conception of the duration of tuberculo- sis would be materially changed did we devote more time to obtaining our histories. The mere fact that a cough has been present for a number of years is too often taken as sufficient proof of its non-tubercular character. an untimely death or chronic invalidism with its sufferings and lost illusions. O recently a case came to autopsy whose only constant sign for several weeks had been in- creased whispered voice in the interscapular space. The trachéal and bronchial glands were found to be very much enlarged. Several pa- tients with chronic bronchitis and emphysema have been studied and in no instance was there an increase of the whispered voice. Paravertebral dullness from bronchial glands, often demonstrated in children, is more diffi- cult to elicitate in adults because of muscu- lar development but it can frequently be de- tected in those of spare frame if very light per- cussion be employed. The demonstration of en- larged bronchial glands does not prove their tuberculous nature, though such is usually the case, nor can one affirm with absolute positive- ness, even when they are tuberculous, that they are causing the symptoms complained of as good health is compatible with bronchial adenitis if the disease is in an inactive state. Unfortu- nately we are forced many times in medicine to deal with probabilities rather than certainties, and so in an adult with the history of ' ' running down" associated with a persistent cough, the presence of marked whispered bronchophony over the mid thoracic vertebral, and to one or both sides thereof, greatly increases the proba- bility of tuberculosis if syphilis and a neoplasm can be excluded. 'D'Espine: Bull. Acad. de Med. de Paris, 1907, vol. lvii, p. 167. 2 Stoll, H. F. : Amer. Jour. Med. Science, 1911, vol. cxli. p. 83 ; also, Amer. Jour. Diseases of Children (not yet published). president's address. gland enlargement. When an individual has enough resistance to fight a drawn battle with the tubercle bacillus in his early life, the truce may be prolonged many years with only an occasional skirmish to indicate that a complete victory has not been won ; but never was a skirmish frought with more grave peril to non-combatants, for it is in these flare-ups when cough recurs that tubercle bacilli are scattered broadcast, often with dire results. Case two contracted the disease from her father, aged 65, and she probably infected her daughter who died with tuberculosis five years ago. Case five has a son who has had hemorrhages, and his daughter-in-law with whom he lived when I saw him. had an active pulmonary lesion. The husband of case one had moist rales above his right clavicle, though no symptoms, and a grown son and daughter not examined, are very delicate in appearance. Not infrequently it is very difficult to convince the patient that he has tuberculosis, and must exercise great care in the disposal of his sputum. For this reason it is hazardous for children to be in intimate association with them. l i b l i Dr. J. George Adami, Montreal: As a step in advance I would suggest that our Association might take the initiative in a plan to have the Govern- ment provide a building in Washington in which various national and international societies might hold their meetings and in which their archives might be safely kept. The Association might also provide a medal which could be bestowed as occa- sion arises upon those who in the opinion of our body have made some advance of the first order in medicine. Also, for the same object, an annual lectureship could be founded as a means of honor- ing those who make notable advances in our sci- ence. It is my sad duty to recite the loss from our ranks of John H. Musser, who has died since our last meeting. A keen student, enthusiastic cli- nician, a cheery friend, beloved alike by his patients and professional brethren, we mourn his untimely death. TUBERCULOSIS IN THE AGED AND THE DIAGNOSTIC VALUE OF INCREASED WHISPER IN THE INTERSCAPULAR SPACE BY HENRY FARNUM STOLL, M.D., HARTFORD, CONN. Assistant Physician to the Hartford Hospital, Hartford, Conn. The high 'D'Espine: Bull. Acad. de Med. de Paris, 1907, vol. lvii, p. 167. 2 Stoll, H. F. : Amer. Jour. Med. Science, 1911, vol. cxli. p. 83 ; also, Amer. Jour. Diseases of Children (not yet published). gravity and the type of the cells made the diagnosis of tuberculous pleuritis probable. A The detection of bronchial gland enlargement in adults speaks for tuberculosis rather than chronic bronchitis or emphysema. p p At the autopsy the apices were adherent, and were moderately infiltrated. The left parietal pleura was studded with tubercles, the bronchial glands much enlarged, and a large mass of glands was situated near the pylorus. Reports of Societies In several of these cases mention is made of the fact that the cough was severe enough to cause gagging. In a series of 169 cases I found that cough followed by gagging occurs twice as often with tuberculosis as with all other diseases combined—pertuesis excepted. It seems to be especially common in patients presenting signs of bronchial gland enlargement. A clinical study of the effects of sleep and rest ON BLOOD PRESSURE. Drs. Harlow Brooks and John Carroll, New York: Numerous physiological researches on both man and the lower animals have shown that there is a marked fall of blood pressure during sleep. In our experiments we have found that the night pres- sure, taken in groups of night sleepers, was almost invariably lower than the day pressure in the same individual, but that, conversely, in night workers and day sleepers the finding was reversed. The pressure was found to vary from 6 2-3 mm. to 44.8 mm. lower during sleeping than during waking hours, being slightest in those whose usual pressure was lowest. The maximum fall took place about two hours after sleep began. Attempts to secure even a temporarily lower twenty-four hour pressure by prolonging or deepening the sleep were appar- ently without avail; nor was the degree or persist- ence of the drop increased by artificial means, as, by large dosage of bromide or chloral. It may be said that attempts at lowering blood pressure are perhaps as harmful as they are futile. In conclusion: 1. Pulmonary tuberculosis is not an uncommon disease in persons past 60 years of age. Laënnaconee encountered it in a person aged 99. g II. It is more prevalent than the mortality suggests as an acute pneumonia is often the ul- timate cause of death. III. In persons in the latter decade of life the disease is chiefly characterized by its ex- treme chronicity and by periods of relatively good health. g IV. Manv people contract tuberculosis each year from the intimate association with some elderly member of the household who has had a "stomach cough" or "catarrh" for "as long as he can remember." V. The examination of the sputum of elderly people for tubercle bacilli is a much neglected procedure. ASSOCIATION OF AMERICAN PHYSICIANS Twenty-seventh Annual Meeting, Held at At- lantic City, N. J., May 14-15, 1912. First Day: Tuesday, May H. The Boston Medical and Surgical Journal as published by The New England Journal of Medicine. Downloaded from nejm.org at SAN DIEGO (UCSD) on July 9, 2016. For personal use only. No other uses without permission. From the NEJM Archive. Copyright © 2010 Massachusetts Medical Society. DISCUSSION. Dr. Theodore C. Janeway, New York: Fall in systolic blood pressure is not at all synonymous with fall in the mean blood pressure. The exces- sive fall in the high pressure cases obtained during sleep I think about corresponds to the fall in mean pressure and is largely due to the diminished size of the pulse wave at the periphery during the sleep. While Dr. Brooks does not feel that he has seen p VI. The recognition of the pulmonary le- sion is often difficult as the typical physical signs are frequently marked by some other con- dition, notably asthma or emphysema. y p y VII. In these cases the key to the diagnosis will often be found in the interscapular space.

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Autologous Fat Grafting Reduces Pain in Irradiated Breast: A Review of Our Experience

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Clinical Study Autologous Fat Grafting Reduces Pain in Irradiated Breast: A Review of Our Experience Fabio Caviggioli,1 Luca Maione,2 Francesco Klinger,1 Andrea Lisa,2 and Marco Klinger2 1Reconstructive and Aesthetic Plastic Surgery School, University of Milan, MultiMedica Holding S.p.A., Plastic Surgery Unit, Via Milanese 300, Sesto San Giovanni, 20099 Milan, Italy 2Reconstructive and Aesthetic Plastic Surgery School, Department of Medical Biotechnology and Translational Medicine (BIOMETRA), University of Milan, Plastic Surgery Unit, Humanitas Research Hospital, Via Alessandro Manzoni 56, Rozzano, 20089 Milan, Italy Fabio Caviggioli,1 Luca Maione,2 Francesco Klinger,1 Andrea Lisa,2 and Marco Klinger2 1Reconstructive and Aesthetic Plastic Surgery School, University of Milan, MultiMedica Holding S.p.A., Plastic Surgery Unit, Via Milanese 300, Sesto San Giovanni, 20099 Milan, Italy 2Reconstructive and Aesthetic Plastic Surgery School, Department of Medical Biotechnology and Translational Medicine (BIOMETRA), University of Milan, Plastic Surgery Unit, Humanitas Research Hospital, Via Alessandro Manzoni 56, Rozzano, 20089 Milan, Italy Fabio Caviggioli,1 Luca Maione,2 Francesco Klinger,1 Andrea Lisa,2 and Marco Klinger2 1Reconstructive and Aesthetic Plastic Surgery School, University of Milan, MultiMedica Holding S.p.A., Plastic Surgery Unit, Via Milanese 300, Sesto San Giovanni, 20099 Milan, Italy 2Reconstructive and Aesthetic Plastic Surgery School, Department of Medical Biotechnology and Translational Medicine (BIOMETRA), University of Milan, Plastic Surgery Unit, Humanitas Research Hospital, Via Alessandro Manzoni 56, Rozzano, 20089 Milan, Italy Correspondence should be addressed to Fabio Caviggioli; [email protected] Received 29 June 2015; Revised 7 September 2015; Accepted 29 September 2015 Received 29 June 2015; Revised 7 September 2015; Accepted 29 September 2015 Academic Editor: Coralie Sengen`es Copyright © 2016 Fabio Caviggioli 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. Introduction. Pain syndromes affect women after conservative and radical breast oncological procedures. Radiation therapy influences their development. We report autologous fat grafting therapeutical role in treating chronic pain in irradiated patients. Materials and Methods. From February 2006 to November 2014, we collect a total of 209 patients who meet the definition of “Postmastectomy Pain Syndrome” (PMPS) and had undergone mastectomy with axillary dissection (113 patients) or quadrantectomy (96 patients). Both procedures were followed by radiotherapy. We performed fat grafting following Coleman’s procedure. Mean amount of adipose tissue injected was 52 cc (±8.9 cc) per breast. Seventy-eight in 209 patients were not treated surgically and were considered as control group. Data were gathered through preoperative and postoperative VAS questionnaires; analgesic drug intake was recorded. Results. Clinical Study Autologous Fat Grafting Reduces Pain in Irradiated Breast: A Review of Our Experience The follow-up was at 12 months (range 11.7–13.5 months). In 120 treated patients we detected pain decrease (mean ± SD point reduction, 3.19 ± 2.86). Forty-eight in 59 patients stopped their analgesic drug therapy. Controls reported a mean ± SD decrease of pain of 1.14 ± 2.72. Results showed that pain decreased significantly in patients treated (𝑝< 0.005, Wilcoxon rank-sum test). Conclusion. Our 8-year experience confirms fat grafting effectiveness in decreasing neuropathic pain. Hindawi Publishing Corporation Stem Cells International Volume 2016, Article ID 2527349, 5 pages http://dx.doi.org/10.1155/2016/2527349 2. Patients and Methods From February 2006 to November 2014, a total of 209 patients who had undergone mastectomy with axillary dissection (113 patients) or quadrantectomy (96 patients) came to our Institution. This effect has been observed since two weeks from the procedure. Both procedures were followed by radiotherapy for onco- logical reasons. In the treated group a total of 48 patients stopped their analgesic drug therapy. We selected patients who presented severe scar retraction, radiodystrophy, and chronic pain meeting the definition of “Postmastectomy Pain Syndrome.” In this group mean VAS before fat grafting was 7.7 (±2.7) and 3.4 (±2.4) after autologous fat grafting; thus the mean pain decrease was 4.23 (SD ± 2.14). All patients enlisted had a normal follow-up with no com- plications, such as dehiscence, infection, or scar anomalies. In the group of patients that continued analgesic therapy mean VAS before treatment was 7.9 (±1.9), while after treat- ment mean value was 4.2 (±2.3) with a mean pain decrease of 1.15 (SD ± 2.79). In our study population, we performed autologous fat grafting following Coleman’s procedure. After routine preop- erative examination and clinical assessment, patients under- went liposuction of the subumbilical area under sedation and local anesthesia. The adipose tissue was harvested from abdominal subcutaneous fat which is an easy accessible and abundant reservoir.ht Control group at first outpatient visit showed a mean VAS of 6.9 (±2.2), while at follow-up visit they showed a mean VAS of 5.8 (±1.9) reporting a mean pain decrease of 1.14 (SD ± 2.72).f Statistical analysis of the difference between VAS values was made before and after treatment in case patients and at first outpatient visit and at follow-up for controls. In both groups the follow-up was at 12 months (range 11.7–13.5 months). The fat graft was injected using 18-gauge angiographic needle with a snap-on wing (Cordis, a Johnson & Johnson Company, N. V, Roden, Netherlands) at the dermohypo- dermal junction in the painful and radiodystrophic scar areas. The mean amount of adipose tissue injected was 52 cc (±8.9 cc) per breast. In particular, in patients submitted to mastectomy, mean amount of adipose tissue was 55 cc (±9.3 cc) per breast, while in patients submitted to quadran- tectomy mean value was 39 cc per breast (±5.4 cc). 1. Introduction Beyond all these multifactorial risk factors, radiotherapy strongly influences the development of PMPS as already noted by Tasmuth et al. [7]. Chronic pain affects from 25% to 60% of women submitted to breast surgery, both mastectomy and conservative procedures for oncological reasons, and represents an important clinical problem involving intra- and postoperative factors [1].h Medications commonly used to treat nociceptive pain, such as opioids, have proved to be ineffective for neuropathic pain and a chronic analgesic therapy is burdened with complications and high rate of drop-off. This condition, also named Postmastectomy Pain Syn- drome (PMPS), is situated in the anterior side of the thorax, in the axilla, and/or in the upper half of the arm and lasts more than 3 months after mastectomy or quadrantectomy surgical procedures [2]. In our experience, autologous fat graft obtained following Coleman’s technique has proven to be an efficient and safe procedure in treating the clinical field of breast reconstructive surgery, providing an important tool for correction of breast size and shape. Several risk factors have been advocated to explain the development of PMPS [3] such as axillary lymph node dissection [4], chemotherapy agents [5], and postsurgical complications such as infection, seroma, or hematoma. In 2006, our group started treating patients submitted to mastectomy with axillary dissection and radiotherapy [8] demonstrating the capability of lipostructure to relieve chronic pain. We subsequently extended its indications to Moreover, BMI, age, and physiological status could also be a predictive factor in postsurgical pain [6]. 2 Stem Cells International patients submitted to quadrantectomy followed by radiother- apy [9].i 2. Patients and Methods Wilcoxon rank-sum test was employed with a level of significance fixed at 𝑝< 0.005.i ii Results showed that pain decreased significantly in patients with Postmastectomy Pain Syndrome treated with autologous fat tissue grafting (𝑝< 0.005), while in control patients no statistical difference was found (𝑝> 0.05). Seventy-eight in 209 patients with Postmastectomy Pain Syndrome (41 submitted to mastectomy and 37 to quadran- tectomy) not treated surgically were considered as a control group for statistical analysis. f In addition to that we compared VAS values in case patients before treatment with control patients at first out- patient visit. Eventually we compared again VAS values in case patients after treatment with control patients at follow- up visit. Mann and Whitney test was employed with a level of significance fixed at 𝑝< 0.005. While in the first comparison no statistically significant difference was noted (𝑝> 0.05), in the latter a statistical significant difference was observed (𝑝< 0.005). By performing this test selection bias was excluded. Results are listed in Table 1. Patients were assigned to control group due to their refusal to be submitted to surgical procedure, although they present comparable VAS scores.i During the first visit, a trained research assistant explained the purpose and methods of this study to each eligible patient; patients who were willing to participate signed an informed agreement. Data were gathered through preoperative and postopera- tive questionnaires. Before undergoing the surgical procedure, patients were required to score their spontaneous pain using a visual analogue scale ranging from 0 to 10; analgesic drug intake was also recorded.i 3. Results We collected a total of 209 patients; 113 patients were submitted to mastectomy with axillary dissection, while 96 patients were submitted to quadrantectomy. In both groups we observed a significant decrease in pain after procedure and a reduction of intake of analgesic drug therapy.h In our population we lost a total of nineteen patients in the follow-up (11 were in case group and 9 were submitted to mastectomy and 2 were submitted to quadrantectomy, while 8 were controls, 6 who underwent previous mastectomy and 2 who underwent quadrantectomy). The present review aims to report our experience in treating chronic pain in irradiated breasts collected after an 8-year experience confirming autologous fat grafting role in improving pain control in patients affected by PMPS. In 120 treated patients we observed a mean VAS before fat grafting of 7.2 (SD ± 2.1) and a value of 3.3 (SD ± 3.1) after treatment so that we detected a mean pain decrease of 3.19 (SD ± 2.86). 4. Discussion Persistent pain as a consequence of surgical treatment has been established for several common surgical procedures and represents a clinical problem of great magnitude.i During follow-up patients were required to fill out the same questionnaire and possible modification in analgesic intake was also accurately recorded. Postmastectomy Pain Syndrome was firstly reported in the 1970s by Wood [10]. Subsequently the International 3 Stem Cells International Stem Cells International Table 1: Study population description. We differentiate between patients who report PMPS after mastectomy and patients who report PMPS after quadrantectomy. Analgesic drug intake was recorded for both groups. We report mean VAS values before and after treatment or at follow-up for controls; mean and median VAS values decrease together with ranges. Analysis of pain before and after treatment in both case and control patients was performed by means of Wilcoxon test; 𝑝value significance was fixed at < 0.005. We obtain 𝑝< 0.005 in cases and 𝑝> 0.05 in controls. Moreover, by means of Mann and Whitney, we compare VAS values in case patients before treatment with controls (𝑝> 0.05) and in case patients after treatment with controls test (𝑝< 0.005). Table 1: Study population description. We differentiate between patients who report PMPS after mastectomy and patients who report PMPS after quadrantectomy. Analgesic drug intake was recorded for both groups. We report mean VAS values before and after treatment or at follow-up for controls; mean and median VAS values decrease together with ranges. Analysis of pain before and after treatment in both case and control patients was performed by means of Wilcoxon test; 𝑝value significance was fixed at < 0.005. We obtain 𝑝< 0.005 in cases and 𝑝> 0.05 in controls. Moreover, by means of Mann and Whitney, we compare VAS values in case patients before treatment with controls (𝑝> 0.05) and in case patients after treatment with controls test (𝑝< 0.005). 4. Discussion PMPS after mastectomy Mean VAS before treatment PMPS after quadrantectomy Mean VAS after treatment or at follow-up Mean VAS decrease Median VAS decrease Range VAS decrease Treated 63 (120 total treated patients) 7.2 (±2.1) 57 (120 total treated patients) 3.3 (±3.1) 3.19 (±2.86) 2.63 −2.1–9.6 Control 35 (70 total control patients) 6.9 (±2.2) 35 (70 total control patients) 5.8 (±1.9) 1.14 (±2.72) 1.09 −4.2–6.3 Stop pharmacologic therapy 28 (34 total patients who assumed therapy) 7.7 (±2.7) 20 (25 total patients who assumed therapy) 3.4 (±2.4) 4.23 (±2.14) 4.86 −1.6–9.3 Continue pharmacologic therapy 6 (34 total patients who assumed therapy) 7.9 (±1.9) 5 (25 total patients who assumed therapy) 4.2 (±2.3) 1.15 (±2.79) 1.0 2.2–2.5 Association for the Study of Pain defined it as a chronic pain condition with characteristics resembling neuropathic pain. reaction can induce peripheral and central sensitization with a failed nociception system leading to pain augmentation. In addition to that, radiotherapy stimulates local fibrosis which could result in a strong adherence to the deeper muscular layer, sustaining a painful syndrome. In patients submitted to quadrantectomy or mastectomy, it presents common features and for this reason it is con- sidered as a unique pathological condition characterized by a dull, burning, and aching sensation exacerbated by movement of the shoulder girdle [11].f Actually, the mainstay of treatment for PMPS as any form of neuropathic pain is pharmacological, including the use of antidepressants [15], antiepileptics, and topical anesthetics, such as lidocaine patches and opioids. Since a pharmacologic therapy lasting for a long period is poorly tolerated, non- pharmacological treatments have been proposed including psychological approaches, physical therapy, interventional therapy, spinal cord stimulation, and surgical procedures. Nevertheless, neuropathic pain remains difficult to treat, and a combination of therapies may be more effective than monotherapy.it Women affected by PMPS may present signs and symp- toms such as neck pain, shoulder pain, reduced mobility, and bad body image, demonstrating a multidimensional character that affects psychological and physical aspects of the patient’s life [12].h Though the exact pathogenesis of PMPS remains unclear, many etiological theories have been postulated. During the surgical procedure, dissection of the inter- costobrachial nerve or damage of axillary nerve pathways could favor development of chronic pain after mastectomy or quadrantectomy. For this reason, chronic pain has a greater rate of occurrence in patients who underwent axillary lymph node dissection [4]. Stem Cells International to notice that mesenchymal stem cells could also down- regulate radiation therapy induced inflammatory response as they inhibit production and release of proinflammatory cytokines, which are the main mediators of radio-induced tissue damage. [5] K. G. Andersen, M. B. Jensen, H. Kehlet, R. G¨artner, L. Eckhoff, and N. Kroman, “Persistent pain, sensory disturbances and functional impairment after adjuvant chemotherapy for breast cancer: cyclophosphamide, epirubicin and fluorouracil compared with docetaxel + epirubicin and cyclophosphamide,” Acta Oncologica, vol. 51, no. 8, pp. 1036–1044, 2012. g In the present review we report our 8-year experience in treating Postmastectomy Pain Syndrome with autologous fat grafting in patients submitted to mastectomy with axillary dissection and quadrantectomy and radiotherapy.f [6] K. L. Schreiber, M. O. Martel, H. Shnol et al., “Persistent pain in postmastectomy patients: comparison of psychophysical, medical, surgical, and psychosocial characteristics between patients with and without pain,” Pain, vol. 154, no. 5, pp. 660– 668, 2013. We considered patients affected as a unique study popu- lation as they all meet the definition of Postmastectomy Pain Syndrome.h [7] T. Tasmuth, C. Blomqvist, and E. Kalso, “Chronic post- treatment symptoms in patients with breast cancer operated in different surgical units,” European Journal of Surgical Oncology, vol. 25, no. 1, pp. 38–43, 1999. y The main limitation of this study is the fact that we did not consider the placebo effect to give further strength to our protocol. [8] F. Caviggioli, L. Maione, D. Forcellini, F. Klinger, and M. Klinger, “Autologous fat graft in postmastectomy pain syn- drome,” Plastic and Reconstructive Surgery, vol. 128, no. 2, pp. 349–352, 2011. In addition to that our research’s lack of data regarding patients’ age, BMI, and estrogen status as increasing evidence seems to correlate these variables to clinical outcome [24]. [9] L. Maione, V. Vinci, F. Caviggioli et al., “Autologous fat graft in postmastectomy pain syndrome following breast conservative surgery and radiotherapy,” Aesthetic Plastic Surgery, vol. 38, no. 3, pp. 528–532, 2014. Comparing data with a wide study population of 120 patients, to our knowledge the largest study population presented in a scientific report about this condition, we confirm autologous fat grafting’s promising therapeutic role in the treatment of Postmastectomy Pain Syndrome. [10] K. M. Wood, “Intercostobrachial nerve entrapment syndrome,” Southern Medical Journal, vol. 71, no. 6, pp. 662–663, 1978. [11] K. Kwekkeboom, “Postmastectomy pain syndromes,” Cancer Nursing, vol. 19, no. 1, pp. 37–43, 1996. 5. Conclusions [12] I. Cantarero-Villanueva, C. Fern´andez-Lao, C. Fern´andez-De- Las-Pe˜nas, L. D´ıaz-Rodr´ıguez, E. Sanchez-Cantalejo, and M. Arroyo-Morales, “Associations among musculoskeletal impair- ments, depression, body image and fatigue in breast cancer survivors within the first year after treatment,” European Journal of Cancer Care, vol. 20, no. 5, pp. 632–639, 2011. Fat graft has already proven its effectiveness in terms of pain decrease in the treatment of different forms of neuropathic pain as a result of its effect of scar entrapment release and anatomical remodeling.fi Because of its safety, efficacy, and optimal tolerability, we support its adoption as a standard procedure in each Breast Unit in patients who report chronic pain after breast surgery especially if previously submitted to radiotherapy. [13] C. Blunt and A. Schmiedel, “Some cases of severe post- mastectomy pain syndrome may be caused by an axillary haematoma,” Pain, vol. 108, no. 3, pp. 294–296, 2004. [14] J. B. Hamner and M. D. Fleming, “Lymphedema therapy reduces the volume of edema and pain in patients with breast cancer,” Annals of Surgical Oncology, vol. 14, no. 6, pp. 1904– 1908, 2007. Conflict of Interests The authors declare that there is no conflict of interests regarding the publication of this paper. [15] H. J. McQuay, M. Tram`er, B. A. Nye, D. Carroll, P. J. Wiffen, and R. A. Moore, “A systematic review of antidepressants in neuropathic pain,” Pain, vol. 68, no. 2-3, pp. 217–227, 1996. [16] G. Rigotti, A. Marchi, M. Gali`e et al., “Clinical treatment of radiotherapy tissue damage by lipoaspirate transplant: a healing process mediated by adipose-derived adult stem cells,” Plastic and Reconstructive Surgery, vol. 119, no. 5, pp. 1409–1424, 2007. 4. Discussion Our group firstly proposed autologous fat graft as an effective complementary approach to relieve PMPS. Mov- ing from the experience of Rigotti et al. [16] who treated radiotherapy-induced tissue damage, several studies focused on the capability of fat graft to induce scar tissue architectural remodelling and regeneration with neoangiogenesis. Chemotherapy agents, such as taxanes, platinum agents, and vinca alkaloids, are neurotoxic, while endocrine therapy may induce musculoskeletal pain and arthralgia [5]. In our previous researches, histological proofs [17–19] have demonstrated how fat grafting could be responsible for scar remodelling inducing release of fibrotic tissue with nerve liberation and loose connective regeneration, leading to increased scar softness.t Another possible explanation seems to be related to the scarring process which leads to fibrosis, strong adherence to the deeper muscular layer, and possible nervous entrapment which would represent a continuous trigger of nerve excita- tion. For this reason, postsurgical complications [13, 14] such as infection, seroma, and hematoma may be potential sources of the development of persistent pain altering physiological scarring. t We have also hypothesized that autologous fat graft could induce molecular changes in the microenvironment of posttraumatic scar [20], which is hostile to regeneration of the nervous system because of intrinsic inhibitory factors expressed by extracellular matrix, as shown by experimental model of spinal cord injuries [21], determining pain control as described in patients affected by Arnold’s Neuralgia [22, 23].t g Radiation therapy represents a major risk factor for devel- oping of PMPS since it leads to an inflammatory reaction accompanied by increased production of proinflammatory cytokines, such as IL-1, IL-6, TNF-alfa, and TGF-beta, and chemokines such as IL-8 and eotaxin. This inflammatory f It is possible to hypothesize that fat grafting in PMPS induces analgesia by inhibition of inflammation. It is relevant 4 Stem Cells International References [1] K. G. Andersen and H. Kehlet, “Persistent pain after breast cancer treatment: a critical review of risk factors and strategies for prevention,” The Journal of Pain, vol. 12, no. 7, pp. 725–746, 2011. [17] M. Klinger, M. Marazzi, D. Vigo, and M. Torre, “Fat injection for cases of severe burn outcomes: a new perspective of scar remodeling and reduction,” Aesthetic Plastic Surgery, vol. 32, no. 3, pp. 465–469, 2008. [2] International Association for the Study of Pain: Task Force on Taxonomy, Classification of Chronic Pain: Descriptions of Chronic Pain Syndromes and Definition of Pain Terms, IASP Press, Seattle, Wash, USA, 2nd edition, 1994. [18] M. Klinger, A. Lisa, F. Klinger et al., “Regenerative approach to scars, ulcers and related problems with fat grafting,” Clinics in Plastic Surgery, vol. 42, no. 3, pp. 345–352, 2015. [3] K. G. Andersen and H. Kehlet, “Persistent pain after breast cancer treatment: a critical review of risk factors and strategies for prevention,” Journal of Pain, vol. 12, no. 7, pp. 725–746, 2011. [19] M. Klinger, F. Caviggioli, F. Klinger, A. V. Pagliari, F. Villani, and V. Bandi, “Scar remodeling following burn injuries,” in Fat Injection: From Filling to Regeneration, S. R. Coleman and R. F. Mazzola, Eds., Quality Medical Publishing, St. Louis, Mo, USA, 2009.t [4] M. A. Steegers, B. Wolters, A. W. Evers, L. Strobbe, and O. H. Wilder-Smith, “Effect of axillary lymph node dissection on prevalence and intensity of chronic and phantom pain after breastcancer surgery,” Journal of Pain, vol. 9, no. 9, pp. 813–822, 2008. [20] L. Maione, A. Memeo, L. Pedretti et al., “Autologous fat graft as treatment of post short stature surgical correction scars,” Injury, vol. 45, no. 6, pp. S126–S132, 2014. Stem Cells International 5 [21] J. W. Rowland, G. W. J. Hawryluk, B. Kwon, and M. G. Fehlings, “Current status of acute spinal cord injury pathophysiology and emerging therapies: promise on the horizon,” Neurosurgical Focus, vol. 25, no. 5, article E2, 2008. [22] M. Klinger, F. Villani, F. Klinger, P. Gaetani, R. Rodriguez Y Baena, and D. Levi, “Anatomical variations of the occipital nerves: implications for the treatment of chronic headaches,” Plastic and Reconstructive Surgery, vol. 124, no. 5, pp. 1727–1728, 2009. [23] A. Lisa, L. Maione, V. Vinci, F. Caviggioli, and M. E. Klinger, “A systematic review of peripheral nerve interventional treatments for chronic headaches,” Annals of Plastic Surgery, vol. 74, no. 4, article 515, 2015. References [24] P. J. Geissler, K. Davis, J. Roostaeian, J. Unger, J. Huang, and R. J. Rohrich, “Improving fat transfer viability: the role of aging, body mass index, and harvest site,” Plastic and Reconstructive Surgery, vol. 134, no. 2, pp. 227–232, 2014. References Submit your manuscripts at http://www.hindawi.com Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Anatomy Research International Peptides International Journal of Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Hindawi Publishing Corporation http://www.hindawi.com  International Journal of Volume 2014 Zoology Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Molecular Biology International Genomics International Journal of Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 The Scientific World Journal Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Bioinformatics Advances in Marine Biology Journal of Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Signal Transduction Journal of Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 BioMed Research International Evolutionary Biology International Journal of Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Biochemistry Research International Archaea Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Genetics Research International Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Advances in Virology Hindawi Publishing Corporation http://www.hindawi.com Nucleic Acids Journal of Volume 2014 Stem Cells International Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Enzyme Research Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 International Journal of Microbiology Submit your manuscripts at http://www.hindawi.com Peptides International Journal of Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Hindawi Publishing Corporation http://www.hindawi.com  International Journal of Volume 2014 Zoology Genomics International Journal of Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Th S i tifi Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 BioMed Research International Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Advances in Virology Hindawi Publishing Corporation http://www.hindawi.com Nucleic Acids Journal of Volume 2014 Stem Cells International Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Genomics International Journal of Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 The Scientific World Journal Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Submit your manuscripts at http://www.hindawi.com The Scientific World Journal Hindawi Publishing Corporation Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Anatomy Research International Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Molecular Biology International Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Bioinformatics Advances in Marine Biology Journal of Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Evolutionary Biology International Journal of Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Biochemistry Research International Archaea Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Genetics Research International Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Enzyme Research Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 International Journal of Microbiology

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Hot Flashes, CTCAE

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Qeios · Definition, February 2, 2020 Open Peer Review on Qeios Hot Flashes, CTCAE National Cancer Institute National Cancer Institute Qeios ID: 6GH7XB · https://doi.org/10.32388/6GH7XB Source National Cancer Institute. Hot Flashes, CTCAE. NCI Thesaurus. Code C143549. A disorder characterized by an uncomfortable and temporary sensation of intense body warmth, flushing, sometimes accompanied by sweating upon cooling. Qeios ID: 6GH7XB · https://doi.org/10.32388/6GH7XB 1/1

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Online Japanese-Language Information on Lifestyle Factors Associated With Reduced Fertility: Content Analysis (Preprint)

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Abstract Background: Approximately one-third of Japanese couples currently worry or previously worried about infertility. To develop strategies for the primary prevention of infertility as a population approach, it is important for the general population to be knowledgeable about fertility and infertility. The internet may contribute to the dissemination of information regarding infertility and fertility. However, few studies have examined online information about fertility. Objective: This study aimed to quantitatively examine online Japanese-language information about lifestyle factors associated with reduced fertility. Methods: We conducted online searches, using the 10 search terms with the highest numbers of searches that people hoping to conceive are likely to input in two major search engines in Japan (Google Japan and Yahoo! Japan). From the 2200 retrieved websites, 1181 duplicates and 500 websites unrelated to our objective were excluded, resulting in a final dataset of 519 websites. Coding guidelines were developed for the following lifestyle factors associated with reduced fertility: sexually transmitted diseases, psychological stress, cigarette smoking, alcohol use, nutrition and diet, physical activity and exercise, underweight, overweight and obesity, and environmental pollutants. Results: In terms of the website author’s professional expertise, 69.6% of the coding instances for the selected lifestyle factors were mentioned by hospitals, clinics, or the media, whereas only 1.7% were mentioned by laypersons. Psychological stress (20.1%) and sexually transmitted diseases (18.8%) were the most frequently mentioned lifestyle factors associated with reduced fertility. In contrast, cigarette smoking, alcohol use, nutrition and diet, physical activity and exercise, underweight, overweight and obesity, and environmental pollutants were mentioned relatively infrequently. The association between reduced fertility and sexually transmitted diseases was mentioned significantly more frequently by hospitals and clinics than by the media (P<.001). The association between reduced fertility and nutrition and diet was mentioned significantly more frequently by the media than by hospitals and clinics (P=.008). With regard to the sex of the target audience for the information, female-specific references to psychological stress, sexually transmitted diseases, nutrition and diet, underweight, physical activity and exercise, and overweight and obesity were significantly more frequent than were male-specific references to these lifestyle factors (psychological stress: P=.002, sexually transmitted diseases: P<.001, nutrition and diet: P<.001, underweight: P<.001, physical activity and exercise: P<.001, overweight and obesity: P<.001). Online Japanese-Language Information on Lifestyle Factors Associated With Reduced Fertility: Content Analysis Rie Yokota1, MPH; Tsuyoshi Okuhara2, PhD; Haruka Ueno3, PhD; Hiroko Okada2, PhD; Emi Furukawa1, MD; Takahiro Kiuchi2, MD, PhD 1Department of Health Communication, Graduate School of Medicine, The University of Tokyo, Bunkyo-ku, Tokyo, Japan 2Department of Health Communication, School of Public Health, The University of Tokyo, Bunkyo-ku, Tokyo, Japan 3Department of Health and Dietetics, Faculty of Health and Medical Science, Teikyo Heisei Unievrsity, Toshima-ku, Tokyo, Japan Corresponding Author: Rie Yokota, MPH Department of Health Communication Graduate School of Medicine The University of Tokyo 7-3-1 Hongo Bunkyo-ku, Tokyo, 113-8655 Japan Phone: 81 358006549 Email: [email protected] Original Paper Online Japanese-Language Information on Lifestyle Factors Associated With Reduced Fertility: Content Analysis Rie Yokota1, MPH; Tsuyoshi Okuhara2, PhD; Haruka Ueno3, PhD; Hiroko Okada2, PhD; Emi Furukawa1, MD; Takahiro Kiuchi2, MD, PhD 1Department of Health Communication, Graduate School of Medicine, The University of Tokyo, Bunkyo-ku, Tokyo, Japan 2Department of Health Communication, School of Public Health, The University of Tokyo, Bunkyo-ku, Tokyo, Japan 3Department of Health and Dietetics, Faculty of Health and Medical Science, Teikyo Heisei Unievrsity, Toshima-ku, Tokyo, Japan Corresponding Author: Yokota et al JOURNAL OF MEDICAL INTERNET RESEARCH JOURNAL OF MEDICAL INTERNET RESEARCH Yokota et al Original Paper https://www.jmir.org/2020/8/e19777/ Background At the 1994 United Nations International Conference on Population and Development, reproductive health was defined as “a state of complete physical, mental and social well-being and not merely the absence of disease or infirmity, in all matters relating to the reproductive system and to its functions and processes” [1]. Infertility is increasingly acknowledged as a global public health issue by the World Health Organization [2], and reproductive health implies that “people are able to have the capability to reproduce and the freedom to decide if, when and how often to do so” [1]. To aid decision making concerning fertility and assist the reproductive-aged population in optimizing their fertility and reproductive health, knowledge of the lifestyle factors associated with reduced fertility is crucial [3,4]. Infertility is defined as the failure to achieve conception following at least 12 months of unprotected sexual intercourse [5]. Infertility affects as many as 186 million people worldwide [6], and about 10% to 15% of couples experience infertility [7]. In 2015, as many as one in three Japanese couples (approximately 35.0%) reported currently or previously worrying about infertility, and more than one-sixth (approximately 18.2%) reported currently or previously undergoing screening for infertility or trying to achieve pregnancy through assisted reproduction technologies [8]. Currently, the internet is a preferred and common source of health information [33]. About 72% of internet users access health-related information via the web [32]. Health information is conveyed to targeted audiences to influence their attitudes or behaviors [34], and the presentation of information and framing used in media portrayals affect the general public’s understanding of lifestyle factors associated with reduced fertility [35]. Thus, online information about fertility influences the general population’s knowledge about conception. Numerous factors may contribute to reduced fertility in men and women. In addition to genetic background and reproductive history, environmental factors and current lifestyle habits have been proposed as causes of male and female infertility [9]. According to large systematic reviews and meta-analyses, the lifestyle factors associated with reduced fertility are (1) sexually transmitted diseases, (2) psychological stress, (3) cigarette smoking, (4) alcohol use, (5) nutrition and diet, (6) physical activity and exercise, (7) underweight, (8) overweight and obesity, and (9) environmental pollutants [10-27]. (J Med Internet Res 2020;22(8):e19777) doi: 10.2196/19777 content analysis; online information; lifestyle factor; fertility; infertility; reproductive health overweight and obesity among the reproductive-aged population in Japan. Background Given the situation of one-third of Japanese couples currently or previously worrying about infertility, for those who are trying to conceive or hope to have a child in the future, knowledge about the lifestyle factors associated with reduced fertility may help to prevent infertility. Introduction In terms of sex differences, several studies have shown that, compared with women, men are less knowledgeable regarding the associations between lifestyle factors and reduced fertility [3,29]. Although most men (88.5%) regarded themselves as knowledgeable on this topic, only half of the men (53.1%) participating in a population-based survey were able to identify the lifestyle factors associated with reduced fertility [4]. A recent survey in Japan found that only 46.4% of reproductive-aged men and 56.7% of reproductive-aged women were knowledgeable about male infertility factors, which constitute approximately 50% of infertility cases [30]. Moreover, according to the same survey, 38.0% of men reported that they did not intend to undergo a semen examination at a medical institution because they thought they had no infertility problems [30]. Because infertility is presumed to be a women’s issue [31], the reproductive-aged population has relatively low knowledge about issues related to male fertility [32]. https://www.jmir.org/2020/8/e19777/ J Med Internet Res 2020 | vol. 22 | iss. 8 | e19777 | p. 2 (page number not for citation purposes) Abstract Conclusions: Of the lifestyle factors known to be related to reduced fertility, cigarette smoking, alcohol use, and male-specific lifestyle factors are mentioned relatively infrequently in online information sources in Japan, and these factors should be discussed more in information published on websites. https://www.jmir.org/2020/8/e19777/ J Med Internet Res 2020 | vol. 22 | iss. 8 | e19777 | p. 1 (page number not for citation purposes) J Med Internet Res 2020 | vol. 22 | iss. 8 | e19777 | p. 1 (page number not for citation purposes) XSL•FO RenderX JOURNAL OF MEDICAL INTERNET RESEARCH Yokota et al Prior Work Previous studies regarding information on infertility and fertility in the media have examined (1) newspaper reports about assisted reproductive technology [35]; (2) information on clinic websites [36]; (3) the readability, suitability, and quality of online information [32,37]; (4) online videos made by laypersons and informational infertility-related educational videos [38]; and (5) online emotional support and social media on infertility-seeking to reduce isolation [39,40]. As mentioned above, although people who are trying to conceive tend to have relatively low levels of knowledge about lifestyle factors associated with reduced fertility, especially male infertility, to our knowledge, few studies have investigated the content of online information on the lifestyle factors associated with reduced fertility. A recent study found that about 60% to 70% of the reproductive-aged population in Japan responded incorrectly to a question about the association between cigarette smoking and reduced fertility [28]. Similarly, when asked about the association between female overweight and reduced fertility, approximately 80% to 90% of the reproductive-aged population in Japan answered incorrectly [28]. These findings indicate a lack of knowledge about the associations between reduced fertility and lifestyle factors such as smoking cigarettes and People who are diagnosed with infertility and those receiving infertility treatment may receive accurate information on fertility from health care professionals. However, no studies have examined what kinds of online content are accessible to the general population, although online information is especially important for people who do not visit medical institutions but are trying to conceive or hoping to have a child in the future. Thus, it is crucial to examine online information on the lifestyle factors associated with reduced fertility. J Med Internet Res 2020 | vol. 22 | iss. 8 | e19777 | p. 2 (page number not for citation purposes) XSL•FO RenderX JOURNAL OF MEDICAL INTERNET RESEARCH Yokota et al search terms from the previous literature was difficult. The main search terms thought to be used by people who are trying to conceive were derived in the following manner. First, we listed terms such as “ninshin” (pregnancy), “ninkatsu” (trying to conceive), and “funin” (infertility) to capture the most common and basic keywords that those hoping to get pregnant would be likely to input into search engines. We identified the number of searches for each of these keywords using a keyword search calculation tool [44]. Prior Work Although both Google (Keyword Planner) and Yahoo (keyword advice tool) have tools to check the number of monthly searches, we were not able to use these services because we would need to register as a corporation to do so. Therefore, we used the keyword search calculation tool produced by Devo Inc [44]. This keyword search tool provides the total number of searches in one month for the search term, as well as the total number of searches for the top 50 webpages, per Google Japan and Yahoo! Japan [44]. After carefully reading websites found through Google Japan and Yahoo! Japan, we listed additional keywords such as “bebimachi” (waiting for a baby) that people who are trying to conceive would be likely to input. Then, we described the number of searches for each keyword using the abovementioned keyword search calculation tool. The top 6 keywords in terms of the number of searches were “funin” (infertility), “ninkatsu” (trying to conceive), “ninshindekinai” (I cannot get pregnant), “akachanhosii” (hoping to have a baby), “shizenninshin” (natural conception), and “bebimachi” (waiting for a baby). Goal of the Study The study aimed to (1) quantitatively examine the information on lifestyle factors associated with reduced fertility accessible to people hoping to have a child who seek information regarding fertility on the internet and (2) identify the characteristics of this information in terms of lifestyle factors, the webpage author’s professional expertise, and the sex of the target audience (ie, information for men, women, or both). Search Engine We used a Japanese-language search string input into the two most popular search engines in Japan, Google Japan [41] and Yahoo! Japan [42]. Google Japan and Yahoo! Japan accounted for roughly 75% and 19% of all internet searches, respectively, at the end of October 2019 [43]. JOURNAL OF MEDICAL INTERNET RESEARCH Yokota et al JOURNAL OF MEDICAL INTERNET RESEARCH pregnant), “shizenninshin” (natural conception), and “huninsho genin” (infertility cause). As a next step, we entered these 6 keywords into Google Japan and Yahoo! Japan and listed the related keywords shown by the search engines (eg, “ninkatsu sapurimento” [trying to conceive AND supplement]). Related keywords are words that are frequently searched in combination with the main keywords [45]. Additionally, we described the number of searches for related keywords such as “funin genin” (infertility cause) by entering the top 6 keywords into the keyword search calculation tool [44]. The objective of this study was not to examine the information available to people who have already been diagnosed with infertility or those who are receiving infertility treatment. Rather, the objective was to examine the information accessible to those who are trying to conceive or hoping to have a child in the future. Therefore, we excluded the following from the listed search phases keywords concerning specific commercial products or services, financial support for infertility, and specific risk factors for infertility such as endometriosis or azoospermia. Of the remaining keywords, the top 10 were listed using the keyword search calculation tool. Using Google Trends, we then confirmed that the number of searches for the top 10 search terms did not represent a surge, compared with the history of searches over the last 10 years [46]. J Med Internet Res 2020 | vol. 22 | iss. 8 | e19777 | p. 4 (page number not for citation purposes) Search Terms A flow diagram depicting the search terms generation procedure is presented in Figure 1. The search terms were determined using the following procedure. Few studies have used content analysis to examine online information about the lifestyle factors associated with reduced fertility. Although English speakers frequently refer to “fertility,” Japanese speakers do not commonly use the word “ninyousei” (fertility). Thus, deriving Figure 1. Flow diagram of the search terms generation procedure. e 1. Flow diagram of the search terms generation procedure. https://www.jmir.org/2020/8/e19777/ XSL•FO RenderX Material Collection The unit of analysis in this study was the webpages suggested by the search engines. Because websites differ considerably in size, coding entire websites could introduce biases based on size [47,48]. Moreover, it has been found that visitors rarely look though all the pages of a website [47,48]. Additionally, a previous study indicated that those who used search engines stayed on each website for a mean of only 1 minute and 9 seconds (median 37 seconds) [49]. Therefore, for each webpage suggested by the search engines, this study analyzed the major part of the website that visitors were likely to read. The first 100 results retrieved using each search engine were collected by the first author. Each time we input search keywords or changed search engines, the search history and cookies were cleared. We conducted online searches from October 21 to November 3, 2019. The flow diagram of the webpage selection is presented in Figure 2. Of the total 2200 webpages, 1181 duplicates were excluded. Additionally, 500 webpages not directly related to the objective of this study were excluded. Ultimately, 519 webpages were included in the analysis. The URL and ranking of each result for the 2200 webpages were saved in Excel (Microsoft Corporation). The 519 webpages to be analyzed were stored as PDFs and, if that was impossible, they were copied and pasted into Word (Microsoft Corporation). At the end of this process, the final search was performed using the following keywords: “ninkatsu” (trying to conceive), “ninshinshitai” (hoping to get pregnant), “funin” (infertility), “bebimachi” (waiting for a baby), “funin genin” (infertility cause), “kodomohosii” (hoping to have a child), “akachanhosii” (hoping to have a baby), “ninshindekinai” (I cannot get https://www.jmir.org/2020/8/e19777/ XSL•FO RenderX Yokota et al JOURNAL OF MEDICAL INTERNET RESEARCH e 2. Flow diagram of the webpage selection. ing Guidelines and Procedures e are no current clinical practice guidelines in Japan ding lifestyle factors associated with reduced fertility. We ed a list of lifestyle factors associated with reduced fertility viewing information from the Japan Society of Obstetrics Gynecology [50], Ministry of Health, Labour and Welfare h ib 5 i i h ili i 5 research [10-27]. This list was then reviewed obstetrician-gynecologists. https://www.jmir.org/2020/8/e19777/ J Med Internet Res 2020 | vol. 22 | iss. 8 | e19777 | p. 5 (page number not for citation purposes) Material Collection Through discussion, we consensus to include the following 9 lifestyle factors a with reduced fertility: (1) sexually transmitted dise psychological stress, (3) cigarette smoking, (4) alcoho nutrition and diet, (6) physical activity and exer underweight, (8) overweight and obesity, and (9) envir Yo RNAL OF MEDICAL INTERNET RESEARCH Figure 2. Flow diagram of the webpage selection. Figure 2. Flow diagram of the webpage selection. research [10-27]. This list was then reviewed by two obstetrician-gynecologists. Through discussion, we reached consensus to include the following 9 lifestyle factors associated with reduced fertility: (1) sexually transmitted diseases, (2) psychological stress, (3) cigarette smoking, (4) alcohol use, (5) nutrition and diet, (6) physical activity and exercise, (7) underweight, (8) overweight and obesity, and (9) environmental pollutants. These lifestyle factors and the major references supporting their inclusion are summarized in Table 1. Coding Guidelines and Procedures There are no current clinical practice guidelines in Japan regarding lifestyle factors associated with reduced fertility. We created a list of lifestyle factors associated with reduced fertility by reviewing information from the Japan Society of Obstetrics and Gynecology [50], Ministry of Health, Labour and Welfare [51], Cochrane Library [5], British Fertility Society [52], American Society for Reproductive Medicine [53], and previous J Med Internet Res 2020 | vol. 22 | iss. 8 | e19777 | p. 5 (page number not for citation purposes) https://www.jmir.org/2020/8/e19777/ XSL•FO RenderX XSL•FO RenderX Yokota et al JOURNAL OF MEDICAL INTERNET RESEARCH company, recruiting company, children’s goods retail business, cooking school, counseling organization, architectural firm, stationery company, or hospital search service company. Academic and medical societies indicated that the content appeared on the website of a medical society such as an association of physicians or medical institutes. Pharmaceutical and medical device firms indicated that the content appeared on the website of a pharmaceutical company, medical device firm, or pharmacy. Alternative medicine practitioners indicated that the content appeared on the website of a practitioner of alternative medicine such as osteopathy, herbal medicine Kampo, acupuncture, moxibustion, or yoga or on the website of a health food company. Laypersons meant that the content was written by persons who were currently trying or had previously tried to conceive, including patients. Each webpage was categorized according to the author’s professional expertise: hospitals and clinics, media organizations, public administration institutions, business and service entities, academic and medical societies, pharmaceutical and medical device firms, alternative medicine practitioners, or laypersons. J Med Internet Res 2020 | vol. 22 | iss. 8 | e19777 | p. 6 (page number not for citation purposes) J Med Internet Res 2020 | vol. 22 | iss. 8 | e19777 | p. 7 (page number not for citation purposes) Sexually transmitted diseases Sexually transmitted diseases Cochrane Library Japan Society of Obstetrics and Gynecology American Society for Reproductive Medicine Ministry of Health, Labour, and Welfare Psychological stress Japan Society of Obstetrics and Gynecology American Society for Reproductive Medicine Ministry of Health, Labour, and Welfare Cigarette smoking Cochrane Library Japan Society of Obstetrics and Gynecology British Fertility Society American Society for Reproductive Medicine Alcohol use Cochrane Library British Fertility Society American Society for Reproductive Medicine Nutrition and diet Cochrane Library Physical activity and exercise Cochrane Library Underweight Cochrane Library Japan Society of Obstetrics and Gynecology Ministry of Health, Labour, and Welfare Overweight and obesity Cochrane Library Japan Society of Obstetrics and Gynecology British Fertility Society American Society for Reproductive Medicine Ministry of Health, Labour, and Welfare Environmental pollutants Cochrane Library American Society for Reproductive Medicine Ministry of Health, Labour, and Welfare We created coding rules for the selected lifes coding guidelines are summarized in Tab included expressions directly related to the Table 1. Lifestyle factors and major references supporting their inclusion. Lifestyle factor and reference lly transmitted diseases x ane Library x Society of Obstetrics and Gynecology x x can Society for Reproductive Medicine x x ry of Health, Labour, and Welfare ological stress x Society of Obstetrics and Gynecology x can Society for Reproductive Medicine x ry of Health, Labour, and Welfare ette smoking x x ane Library x Society of Obstetrics and Gynecology x Fertility Society x x can Society for Reproductive Medicine ol use x x ane Library x Fertility Society x can Society for Reproductive Medicine ion and diet x x ane Library al activity and exercise x x ane Library weight x x ane Library x Society of Obstetrics and Gynecology x ry of Health, Labour, and Welfare weight and obesity x x ane Library x Society of Obstetrics and Gynecology x Fertility Society x can Society for Reproductive Medicine x x ry of Health, Labour, and Welfare onmental pollutants x x ane Library x can Society for Reproductive Medicine x x ry of Health, Labour, and Welfare ated coding rules for the selected lifestyle factors. These guidelines are summarized in Table 2. Our coding ed expressions directly related to the selected lifestyle factors associated with reduced fertility. However, expressions exclusively concerning the influence of these factors on fetuses or babies were excluded. Material Collection Hospitals and clinics indicated that the content appeared on the website of a hospital or clinic, including blogs written by clinicians. Media organizations indicated that the content appeared on the website of a mass media organization such as a newspaper, magazine, or news site. Public administration institutions indicated that the content appeared on the website of a public organization such as the government, a municipality, public health care center, or specialized public consultation support center. Business and service entities indicated that the content appeared on the website of an enterprise such as a marriage support service company, bridal https://www.jmir.org/2020/8/e19777/ XSL•FO RenderX Table 1. Lifestyle factors and major references supporting their inclusion. Relevant sex Lifestyle factor and reference Unknown Female Male Sexually transmitted diseases x Cochrane Library x Japan Society of Obstetrics and Gynecology x x American Society for Reproductive Medicine x x Ministry of Health, Labour, and Welfare Psychological stress x Japan Society of Obstetrics and Gynecology x American Society for Reproductive Medicine x Ministry of Health, Labour, and Welfare Cigarette smoking x x Cochrane Library x Japan Society of Obstetrics and Gynecology x British Fertility Society x x American Society for Reproductive Medicine Alcohol use x x Cochrane Library x British Fertility Society x American Society for Reproductive Medicine Nutrition and diet x x Cochrane Library Physical activity and exercise x x Cochrane Library Underweight x x Cochrane Library x Japan Society of Obstetrics and Gynecology x Ministry of Health, Labour, and Welfare Overweight and obesity x x Cochrane Library x Japan Society of Obstetrics and Gynecology x British Fertility Society x American Society for Reproductive Medicine x x Ministry of Health, Labour, and Welfare Environmental pollutants x x Cochrane Library x American Society for Reproductive Medicine x x Ministry of Health Labour and Welfare Yokota et a JOURNAL OF MEDICAL INTERNET RESEARCH https://www.jmir.org/2020/8/e19777/ J Med Internet Res 2020 | vol. 22 | iss. 8 | e19777 | p. 7 JOURNAL OF MEDICAL INTERNET RESEARCH JOURNAL OF MEDICAL INTERNET RESEARCH Environmental pollutants Additionally, expressions concerning alcohol drinking are included. Content directly related to nutrition and diet is included. Additionally, expressions concerning nutrient factors are included. Content only related to nutrient factors for the purpose of marketing (eg, information about supplement only) is excluded. Content directly related to physical activity and exercise is included. Additionally, expressions concerning obesity prevention and exercise in regular life are included. Content directly related to underweight is included. Additionally, expressions concerning precipitous weight loss, dieting, and underweight as indicated by body mass index are included. Content directly related to underweight is included. Additionally, expressions concerning precipitous weight loss, dieting, and underweight as indicated by body mass index are included. Content directly related to overweight and obesity is included. Additionally, expressions concerning precipitous weight gain, and overweight and obesity as indicated by body mass index are included. Content directly related to overweight and obesity is included. Additionally, expressions concerning precipitous weight gain, and overweight and obesity as indicated by body mass index are included. Content directly related to environmental pollutants is included. Additionally, expressions concerning environ- mental hormones are included. Environmental pollutants lifestyle factors associated with reduced fertility by categorizing the information into two groups: (1) information for men plus information for both men and women and (2) information for women plus information for both men and women. Lifestyle factors that could not be classified in this way were treated as missing in the above tests. Differences in the author’s professional expertise, lifestyle factors, and sex of the target audience were assessed using count data analyzed by using the chi-square test and Fisher exact test. Statistical significance was set at P<.05 for all comparisons. Analyses were performed using R for Windows version 3.5.1 (R Foundation for Statistical Computing). We analyzed the textual data on the websites retrieved using the search terms described above. First, we read the text carefully. Second, all data were coded on the selected lifestyle factors, author’s professional expertise, and sex of the target audience (ie, information for men, women, or both). Finally, data from all webpages were assembled and pooled in Microsoft Excel. When information on lifestyle factors was provided, the code of 1 was assigned. When no information on lifestyle factors was provided, we assigned the code of 0. The URL and title of each webpage were saved as a reference during the data analysis. Interrater Reliability Approximately 20% of the final dataset (100/519, 19.3%) was evaluated by two independent, blinded raters (RY and EF) to examine interrater reliability. Using the coding guidelines created by the first author of this study (RY), EF was instructed on applying the coding system in a training session that lasted about 1 hour. In a pilot test phase, the two raters applied the coding system to 10 webpages randomly selected from the full sample. No problems were identified during this phase. After a formal reliability assessment phase was completed, the first author (RY) calculated the interrater reliability index. Environmental pollutants Because multiple types of lifestyle factors could be listed on a single website, instead of calculating the number of webpages mentioning a particular lifestyle factor, we calculated the number of mentions (codes) for each selected lifestyle factor associated with reduced fertility. Ethical Considerations This study was granted an exemption from the requirement of ethics approval by the ethical review committee at the Graduate School of Medicine, University of Tokyo, because we aimed to analyze online information, meaning that this study was not medical research involving human subjects, and because the authors had no conflicts of interest related to this study. Distributions of Author’s Professional Expertise The assignment of all codes ranged from 0 to 9 (mean 1.017) per page. The assignment of codes ranged from 0 to 8 (mean 1.058) for hospitals and clinics, from 0 to 8 (mean 0.974) for media organizations, from 0 to 9 (mean 1.147) for public administration institutions, from 0 to 6 (mean 1.029) for business and service entities, from 0 to 6 (mean 1.389) for academic and medical societies, from 0 to 7 (mean 2.105) for pharmaceutical and medical device firms, from 0 to 3 (mean 0.367) for alternative medicine practitioners, and from 0 to 3 (mean 0.391) for laypersons. Of the webpages retrieved, 0 codes were assigned to 60.7% (315/519). The number of webpages for which 0 codes https://www.jmir.org/2020/8/e19777/ J Med Internet Res 2020 | vol. 22 | iss. 8 | e19777 | p. 8 (page number not for citation purposes) Environmental pollutants Cochrane Library American Society for Reproductive Medicine Ministry of Health, Labour, and Welfare factors associated with reduced fertility. However, expressions exclusively concerning the influence of these factors on fetuses or babies were excluded. We created coding rules for the selected lifestyle factors. These coding guidelines are summarized in Table 2. Our coding included expressions directly related to the selected lifestyle https://www.jmir.org/2020/8/e19777/ XSL•FO RenderX JOURNAL OF MEDICAL INTERNET RESEARCH JOURNAL OF MEDICAL INTERNET RESEARCH Yokota et al Table 2. Coding guidelines. Description Lifestyle factor Content directly related to sexually transmitted diseases, including Chlamydia trachomatis and gonorrhea, is in- cluded. Additionally, content related to sexually transmitted disease screening or examination for causes of infer- tility is included. Sexually transmitted diseases Content related to psychological stress in daily life or occupational life is included. Additionally, content related to psychological stress caused by infertility is included. Psychological stress Content directly related to cigarette smoking is included. Additionally, expressions concerning quitting smoking are included. Cigarette smoking Content directly related to alcohol is included. Additionally, expressions concerning alcohol drinking are included. Alcohol use Content directly related to nutrition and diet is included. Additionally, expressions concerning nutrient factors are included. Content only related to nutrient factors for the purpose of marketing (eg, information about supplement only) is excluded. Nutrition and diet Content directly related to physical activity and exercise is included. Additionally, expressions concerning obesity prevention and exercise in regular life are included. Physical activity and exercise Content directly related to underweight is included. Additionally, expressions concerning precipitous weight loss, dieting, and underweight as indicated by body mass index are included. Underweight Content directly related to overweight and obesity is included. Additionally, expressions concerning precipitous weight gain, and overweight and obesity as indicated by body mass index are included. Overweight and obesity Content directly related to environmental pollutants is included. Additionally, expressions concerning environ- mental hormones are included. Environmental pollutants Table 2. Coding guidelines. Content directly related to sexually transmitted diseases, including Chlamydia trachomatis and gonorrhea, is in- cluded. Additionally, content related to sexually transmitted disease screening or examination for causes of infer- tility is included. Content related to psychological stress in daily life or occupational life is included. Additionally, content related to psychological stress caused by infertility is included. Content directly related to cigarette smoking is included. Additionally, expressions concerning quitting smoking are included. Content directly related to alcohol is included. Distribution of Lifestyle Factors were assigned was 53.4% (110/206) for hospitals and clinics, 66.2% (102/154) for media organizations, 68% (23/34) for public administration institutions, 60% (21/35) for business and service entities, 50% (9/18) for academic and medical societies, 53% (10/19) for pharmaceutical and medical device firms, 77% (23/30) for alternative medicine practitioners, and 74% (17/23) for laypersons. Of the coding instances concerning lifestyle factors associated with reduced fertility, 20.1% (106/528) related to psychological stress and 18.8% (99/528) related to sexually transmitted diseases. Together, codes on these two lifestyle factors accounted for 38.9% (205/528) of all lifestyle factor-coding instances. Websites referring to the associations between reduced fertility and nutrition and diet, cigarette smoking, underweight, physical activity and exercise, overweight and obesity, alcohol use, and environmental pollutants were relatively rare. The distributions of the webpage author’s professional expertise are summarized in Multimedia Appendix 1. Of the webpages retrieved, 39.7% (206/519) were produced by hospitals and clinics, 29.7% (154/519) by media organizations, 6.6% (34/519) by public administration institutions, 6.7% (35/519) by business and service entities, 5.8% (30/519) by alternative medicine practitioners, 4.4% (23/519) by laypersons, 3.7% (19/519) by pharmaceutical or medical device firms, and 3.5% (18/519) by academic and medical societies. Of the codes assigned for the examined lifestyle factors associated with reduced fertility, 41.2% (218/528) were published by hospitals and clinics, 28.4% (150/528) by media organizations, 7.6% (40/528) by pharmaceutical and medical device firms, 7.4% (39/528) by public administration institutions, 6.8% (36/528) by business and service entities, 4.7% (25/528) by academic and medical societies, 2.1% (11/528) by alternative medicine practitioners, and 1.7% (9/528) by laypersons. Distribution of Lifestyle Factors by Author’s Professional Expertise Figure 3 illustrates the distribution of codes for the examined lifestyle factors associated with reduced fertility by author’s professional expertise. Of the coding instances referring to the association between reduced fertility and sexually transmitted diseases, 67% (66/99) were published by hospital or clinics and 14% (14/99) were published by media organizations. We assessed differences in the frequency of each lifestyle factor code between the two most common types of author’s professional expertise (ie, hospitals/clinics and media organizations) using the chi-square test and Fisher exact test (Table 3). The results of these tests showed that the association between sexually transmitted diseases and reduced fertility was mentioned more frequently by hospitals and clinics than by media organizations (P<.001). In contrast, of the coding instances referring to the association between nutrition and diet and reduced fertility, 26% (18/68) were published by hospitals and clinics and 40% (27/68) were published by media organizations; the association between nutrition and diet and reduced fertility was significantly more frequently mentioned by the media than by hospitals and clinics (P=.008). The other lifestyle factors associated with reduced fertility did not show statistically significant differences between hospitals/clinics and media organizations. Statistical Analysis To assess interrater reliability, the Gwet agreement coefficient (AC1) statistic, which is less affected by prevalence compared with the Cohen kappa [54], was used to assess interrater agreement of the coding. We also conducted a test to assess differences in frequency between the two most common types of professional expertise. In addition, we compared differences between male-specific and female-specific information on XSL•FO RenderX Yokota et al JOURNAL OF MEDICAL INTERNET RESEARCH Interrater Reliability A review of the coding by the two independent, blinded raters on the randomly selected subsample of 100 webpages revealed that the interrater agreement was excellent. The two raters agreed on the assignment of codes in 98.00% (3528/3600) of coding instances. The Gwet AC1 statistic for the assignment of codes ranged from 0.969 to 1.000 (mean AC1 0.985) for the author’s professional expertise, from 0.957 to 0.992 (mean AC1 0.979) for lifestyle factors, and from 0.950 to 0.986 (mean AC1 0.978) for the sex of the target audience. For the data analysis in this study, we used the coding of the first author. Figure 3. Distribution of codes for lifestyle factors associated with reduced fertility by the author’s professional expertise. J Med Internet Res 2020 | vol. 22 | iss. 8 | e19777 | p. 9 https://www.jmir.org/2020/8/e19777/ (page number not for citation purposes) •FO derX Figure 3. Distribution of codes for lifestyle factors associated with reduced fertility by the author’s professional expertise. Figure 3. Distribution of codes for lifestyle factors associated with reduced fertility by the author’s professional expertise. J Med Internet Res 2020 | vol. 22 | iss. 8 | e19777 | p. 9 (page number not for citation purposes) https://www.jmir.org/2020/8/e19777/ https://www.jmir.org/2020/8/e19777/ JOURNAL OF MEDICAL INTERNET RESEARCH Yokota et al Table 3. Lifestyle factors and their associations with webpage author’s professional expertise for hospitals or clinics and media organizations. Interrater Reliability P valueb df a Chi-square Author’s professional expertise, n (%) Lifestyle factor Media organizations (n=150) Hospitals and clinics (n=218) <.001c 1 21.7 Sexually transmitted diseases 14 (9.3) 66 (30.3) Number of webpages with codes 136 (90.7) 152 (69.7) Number of webpages without codes .64c 1 0.2 Psychological stress 32 (21.3) 41 (18.8) Number of webpages with codes 118 (78.7) 177 (81.2) Number of webpages without codes .31c 1 1 Cigarette smoking 22 (14.7) 23 (10.6) Number of webpages with codes 128 (85.3) 195 (89.4) Number of webpages without codes .35c 1 0.9 Alcohol use 12 (8.0) 11 (5.0) Number of webpages with codes 138 (92.0) 207 (95.0) Number of webpages without codes .008c 1 7 Nutrition and diet 27 (18.0) 18 (8.3) Number of webpages with codes 123 (82.0) 200 (91.7) Number of webpages without codes .23c 1 1.4 Physical activity and exercise 14 (9.3) 12 (5.5) Number of webpages with codes 136 (90.7) 206 (94.5) Number of webpages without codes .96c 1 0 Underweight 12 (8.0) 19 (8.7) Number of webpages with codes 138 (92.0) 199 (91.3) Number of webpages without codes >.99c 1 0 Overweight and obesity 13 (8.7) 18 (8.3) Number of webpages with codes 137 (91.3) 200 (91.7) Number of webpages without codes .42d — — Environmental pollutants 4 (2.7) 10 (4.6) Number of webpages with codes 146 (97.3) 208 (95.4) Number of webpages without codes aDegrees of freedom. bP values compare media organizations with hospitals and clinics. cChi-square test. with webpage author’s professional expertise for hospitals or clinics and media organizations. Number of webpages without codes aDegrees of freedom. bP values compare media organizations with hospitals and clinics. cChi-square test. dFisher exact test. dFisher exact test. Appendix 2). Across all examined types of lifestyle factors, female-specific information was observed more frequently than was male-specific information. Information referring to psychological stress, sexually transmitted diseases, nutrition and diet, underweight, physical activity and exercise, and overweight and obesity was significantly more frequently directed toward women than toward men (Table 4; psychological stress: P=.002, sexually transmitted diseases: P<.001, nutrition https://www.jmir.org/2020/8/e19777/ J Med Internet Res 2020 | vol. 22 | iss. 8 | e19777 | p. 10 (page number not for citation purposes) J Med Internet Res 2020 | vol. 22 | iss. 8 | e19777 | p. 11 (page number not for citation purposes) Distribution of Lifestyle Factors by the Sex of the Target Audience Figure 4 illustrates the distribution of codes for lifestyle factors associated with reduced fertility by the sex of the target audience. Of the total lifestyle factor-coding instances, 12.9% (68/528) were about men’s lifestyle factors, 60.2% (318/528) were about women’s lifestyle factors, and 17.4% (92/528) were about both women’s and men’s lifestyle factors (see Multimedia J Med Internet Res 2020 | vol. 22 | iss. 8 | e19777 | p. 10 (page number not for citation purposes) J Med Internet Res 2020 | vol. 22 | iss. 8 | e19777 | p. 10 (page number not for citation purposes) XSL•FO RenderX JOURNAL OF MEDICAL INTERNET RESEARCH Yokota et al Yokota et al Yokota et al Lifestyle factors and their associations with the sex of the target audience. aWe categorized the information into two groups: (1) information for men plus information for both men and women and (2) information for women plus information for both men and women. Lifestyle factors that could not be classified in this way were treated as missing. b cP values compare the information for men with the information for women. dChi-square test. eFisher exact test. was disseminated by hospitals, clinics, and the media. Furthermore, the findings show that the frequencies of descriptions provided by different entities varied by the particular lifestyle factor examined. was disseminated by hospitals, clinics, and the media. Furthermore, the findings show that the frequencies of descriptions provided by different entities varied by the particular lifestyle factor examined. https://www.jmir.org/2020/8/e19777/ J Med Internet Res 2020 | vol. 22 | iss. 8 | e19777 | p. 12 (page number not for citation purposes) Yokota et al and diet: P<.001, underweight: P<.001, physical activity and exercise: P<.001, overweight and obesity: P<.001). Figure 4. Distribution of codes for lifestyle factors associated with reduced fertility by the sex of the target audience. and diet: P<.001, underweight: P<.001, physical activity and exercise: P<.001, overweight and obesi Figure 4. Distribution of codes for lifestyle factors associated with reduced fertility by the sex of the target audience. https://www.jmir.org/2020/8/e19777/ https://www.jmir.org/2020/8/e19777/ XSL•FO RenderX JOURNAL OF MEDICAL INTERNET RESEARCH Yokota et al Table 4. Lifestyle factors and their associations with the sex of the target audience. P valuec df b Chi-square Sex of target audience Lifestyle factor Femalea, n (%) Malea, n (%) <.001d 1 121.5 Sexually transmitted diseases (n=108) 95 (88.0) 13 (12.0) Number of webpages with codes 13 (12.0) 95 (88.0) Number of webpages without codes .002d 1 9.8 Psychological stress (n=108) 66 (61.1) 42 (38.9) Number of webpages with codes 42 (38.9) 66 (61.1) Number of webpages without codes .45d 1 0.6 Cigarette smoking (n=86) 46 (53.5) 40 (46.5) Number of webpages with codes 40 (46.5) 46 (53.5) Number of webpages without codes .82d 1 0.1 Alcohol use (n=38) 20 (52.6) 18 (47.4) Number of webpages with codes 18 (47.4) 20 (52.6) Number of webpages without codes <.001d 1 45.1 Nutrition and diet (n=71) 56 (78.9) 15 (21.1) Number of webpages with codes 15 (21.1) 56 (78.9) Number of webpages without codes <.001d 1 15.7 Physical activity and exercise (n=46) 33 (71.7) 13 (28.3) Number of webpages with codes 13 (28.3) 33 (71.7) Number of webpages without codes <.001e — — Underweight (n=53) 50 (94.3) 3 (5.7) Number of webpages with codes 3 (5.7) 50 (94.3) Number of webpages without codes <.001d 1 28.8 Overweight and obesity (n=47) 37 (78.7) 10 (21.3) Number of webpages with codes 10 (21.3) 37 (78.7) Number of webpages without codes >.99e — — Environmental pollutants (n=13) 7 (53.8) 6 (46.2) Number of webpages with codes 6 (46.2) 7 (53.8) Number of webpages without codes aWe categorized the information into two groups: (1) information for men plus information for both men and women and (2) information for women plus information for both men and women. Lifestyle factors that could not be classified in this way were treated as missing. bDegrees of freedom. cP values compare the information for men with the information for women. dChi-square test. Table 4. Distribution of Author’s Professional Expertise In terms of the professional expertise of the author publishing online information on lifestyle factors associated with reduced fertility, following the categorization of professional expertise described in the Methods section, our study demonstrated that, of the codes assigned for the examined lifestyle factors associated with reduced fertility, 41.2% (218/528) were published by hospitals and clinics, 28.4% (150/528) by media organizations, and 1.7% (9/528) by laypersons. This may be because of the strategy of search engine optimization used by hospitals and clinics to make their websites appear higher in user lists of search results generated by the search engines. This may also be because lifestyle factors associated with reduced fertility are discussed in the context of infertility examinations or treatments. The large number of references to lifestyle factors associated with reduced fertility by media organizations may be associated with the mass media coverage of the public debate [55] after the Japanese government’s proposal for the need for fertility education in 2013 [28]. Conversely, the low number of references to lifestyle factors associated with reduced fertility by laypersons may be associated with perceived stigma around infertility [56]: Laypersons may avoid discussing these factors because of concerns about stigma related to infertility or not being able to get pregnant. In contexts where the desire for children is generally regarded as the social norm, higher levels of stigma consciousness may be associated with reductions in disclosure of one’s own infertility [56]. Those who disclose their infertility may also have more negative social experiences associated with their infertility in such contexts [56] and consequently avoid further disclosing their infertility in the future. Therefore, the stigma around infertility may explain why laypersons were found to rarely mention lifestyle factors associated with reduced fertility in this study. Distribution of Lifestyle Factors Psychological stress and sexually transmitted diseases were the two most frequently mentioned lifestyle factors associated with reduced fertility. In contrast, the associations between reduced fertility and cigarette smoking, alcohol use, physical activity and exercise, underweight, and overweight and obesity were less frequently discussed. Our findings are consistent with those of a previous study showing that people lack a general understanding of fertility, including the associations between reduced fertility and cigarette smoking and overweight and obesity [28]. In 2017, the National Health and Nutrition Survey demonstrated that (1) 40% to 50% of the reproductive-aged population in Japan drank alcohol [57], (2) more than one-quarter of reproductive-aged men were obese [57], (3) 10% to 20% of reproductive-aged women were underweight [57], and (4) approximately 20% of the population smoked cigarettes [57]. Despite this situation, the associations between reduced fertility and alcohol consumption of both men and women, male overweight and obesity, female underweight, and cigarette smoking of both men and women were mentioned relatively infrequently on the websites analyzed in this study. These lifestyle factors should be discussed more frequently. We also believe that public initiatives for the reproductive-aged population and a fertility-related information strategy should be established in Japan. Several exemplary websites with Principal Findings This study quantitatively examined online information on the lifestyle factors associated with reduced fertility. The main findings of the study confirm that a large proportion of the information on lifestyle factors associated with reduced fertility J Med Internet Res 2020 | vol. 22 | iss. 8 | e19777 | p. 12 (page number not for citation purposes) XSL•FO RenderX JOURNAL OF MEDICAL INTERNET RESEARCH Yokota et al evidence-based information exist that could serve as a model for these changes [58,59]. Distribution of Lifestyle Factors by Author’s Professional Expertise Our research has revealed that associations between reduced fertility and both psychological stress and sexually transmitted diseases were more frequently discussed by hospitals and clinics than by the other types of webpage author. This may be because lifestyle factors associated with reduced fertility are discussed in the context of infertility distress regarding infertility examinations or treatments [32,60]. We also found that the association between reduced fertility and nutrition and diet was more frequently discussed by the media than by hospitals and clinics. This may be because lifestyle factors associated with reduced fertility are presented less as health-oriented information, with media outlets orienting health messages toward entertainment. Providing treatment is the main role of hospitals and clinics, and these entities may use their websites as a tool for patient acquisition. However, for individuals seeking fertility information on the internet because they are trying to conceive, the content of hospital and clinic websites may be a major source of information on fertility. This idea is supported by the finding that hospital and clinic websites rank higher in lists of search results when users use web search engines. Hence, various kinds of information provided by hospitals and clinics may be beneficial for people who are trying to conceive. In previous work, scholars have recommended that, despite the pressures of a competitive environment, hospitals and clinics offering fertility treatment should present educational information in an ethically balanced manner [61]. The same scholars have also recommended that medical experts lead the way for best practices among doctors by creating guidelines regarding the provision of online information [61]. We additionally recommend the ethically balanced presentation of information on fertility in the media. Media websites should incorporate benchmarks for collaboration between experts and the media. J Med Internet Res 2020 | vol. 22 | iss. 8 | e19777 | p. 13 (page number not for citation purposes) https://www.jmir.org/2020/8/e19777/ Distribution of Lifestyle Factors by the Sex of the Target Audience We found that there were fewer mentions of lifestyle factors associated with reduced fertility for men than for women. This may be because (1) men are less likely to seek information on infertility [32], (2) male and female infertility is highly stigmatized, and (3) men have a relatively low level of knowledge regarding male infertility [62]. Regarding stigma around male infertility, reproductive health is often regarded as a women’s issue [31]. This may be because fertility treatment has mainly focused on women’s bodies, although male factors also contribute to infertility [31]. Likewise, previous work indicates that infertility tends to be regarded as a women’s issue in Japan [63]. Message senders may be influenced by this social norm, and they may therefore convey messages regarding infertility in a way that is consistent with this topic being a women’s issue. Conversely, male infertility may be related to stigma stemming from ideas about masculinity that many men consider to be a social norm [60,64]. Attempts to hide stigmatized conditions often lead to delays in https://www.jmir.org/2020/8/e19777/ XSL•FO RenderX XSL•FO RenderX JOURNAL OF MEDICAL INTERNET RESEARCH Yokota et al Yokota et al should be explored as a potential factor associated with reduced fertility in future research. Fifth, when identifying lifestyle factors associated with reduced fertility, we checked for critical threshold levels for the contribution of particular factors, but study designs, outcomes, and sample sizes varied across the examined studies. Because there are presently no guidelines concerning lifestyle factors associated with reduced fertility in any country, we could not identify unified critical thresholds for judging the impact of each lifestyle factor on reduced fertility. Sixth, we did not evaluate the accuracy of the information presented on the webpages. It is possible that some websites provided accurate information and others provided incorrect information. The accuracy of information provided on this topic should be explored in future research. Seventh, our study was conducted via online search at the end of October 2019. Although we confirmed that the number of searches for the top 10 most frequent main search terms did not represent a surge, we did not explore how the number and frequency of search results concerning the examined lifestyle factors associated with reduced fertility changed over the time. Finally, our analysis was restricted to Japanese-language online information, which may limit its generalizability to other contexts. Infertility and Stigma As mentioned above, infertility may harm the self-esteem of people who are trying to conceive because of its latently stigmatizing nature [56]. People with a stigmatized condition tend to use the internet to seek health information more often compared with people with nonstigmatized conditions [65]. Those who experience infertility may feel a sense of isolation and be less likely to seek social support because of reduced self-esteem [56]. Advantages of the internet include user anonymity, optional disclosure, and the lack of geographical barriers [66]. These advantages could mitigate the threat of social stigma and distress around disclosure, providing much needed information on fertility. It is also important to address online discussion boards in terms of informational, emotional, and appraisal support [66]. Moreover, because the internet may be a good public education tool for people with stigmatized conditions [65], we recommend the use of online media for fertility-related education [67]. Distribution of Lifestyle Factors by the Sex of the Target Audience However, considering context is crucial when examining media messages, which is an advantage of focusing the analysis on Japanese-language websites [35]. Although there are limitations to the study, to our knowledge, this study is the first to examine online information on the lifestyle factors associated with reduced fertility using quantitative content analysis. information-seeking behavior [65]. Therefore, it is important that published discussions on the lifestyle factors associated with reduced fertility include information for both men and women. Conclusions In terms of the webpage author’s professional expertise, authors from hospitals, clinics, and the media relatively frequently mentioned lifestyle factors associated with reduced fertility, whereas laypersons mentioned them relatively rarely. Regarding the specific lifestyle factors associated with reduced fertility, psychological stress and sexually transmitted diseases were more frequently discussed compared with the other factors. The association between reduced fertility and sexually transmitted diseases was more frequently discussed by hospitals and clinics than by the media. Conversely, the association between reduced fertility and nutrition and diet was significantly more frequently mentioned by the media than by hospitals and clinics. With regard to the sex of the target audience, male lifestyle factors were less frequently discussed than were female lifestyle factors. The authors of fertility-related websites should more frequently mention information on lifestyle factors associated with reduced fertility overall, moving beyond only psychological stress and sexually transmitted diseases to also discuss nutrition and diet, cigarette smoking, underweight, physical activity and exercise, overweight and obesity, alcohol use, and environmental pollutants. These authors should also make an effort to provide specific information on men’s lifestyle factors. Limitations This study has several limitations. First, we limited our examination to online information, which does not capture all circulating public messages. Television broadcasts and print newspapers and magazines may also be widely used sources of information about lifestyle factors associated with reduced fertility. Second, although a substantial number of websites (n=519) were retrieved for this analysis, availability, accessibility, and time limitations made it unfeasible to analyze all relevant sites. Third, despite the fact that we selected search terms based on related words indicated by the selected search engines, these search terms may have reflected our own biases. For example, it is possible that search terms were relatively easy for women to use. However, because men were less likely to seek information regarding infertility [32], the webpages for women should include information for men. Fourth, it is possible that the selected lifestyle factors associated with reduced fertility may also have reflected our biases; however, this study examined several publications from academic societies, Cochrane Library, and previous studies to determine which lifestyle factors to investigate. For example, age was not treated as a lifestyle factor associated with reduced fertility in this study because Cochrane Library did not directly consider age when defining lifestyle factors that may both influence fertility and affect the chances of a healthy, live birth [5]. Additionally, a recent study found that most of the Japanese reproductive-aged population (60% to 70%) were knowledgeable about the association between age and reduced fertility [28]. Therefore, this study did not include age in the analysis. However, age https://www.jmir.org/2020/8/e19777/ J Med Internet Res 2020 | vol. 22 | iss. 8 | e19777 | p. 14 (page number not for citation purposes) JOURNAL OF MEDICAL INTERNET RESEARCH the final manuscript for publication. The authors would like to thank Ritsuko Shirabe and Saeko Higuchi for their assistance in identifying lifestyle factors associated with reduced fertility. We also thank Jennifer Barrett, PhD, from Edanz Group for editing a draft of this manuscript. 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[doi: 10.3109/14647273.2014.942390] [Medline: 25116275] J Med Internet Res 2020 | vol. 22 | iss. 8 | e19777 | p. 17 (page number not for citation purposes) https://www.jmir.org/2020/8/e19777/ XSL•FO RenderX XSL•FO RenderX JOURNAL OF MEDICAL INTERNET RESEARCH J Med Internet Res 2020 | vol. 22 | iss. 8 | e19777 | p. 18 (page number not for citation purposes) 10.1016/j.socscimed.2005.03.025] [Medline: 16029778] 66. Richard J, Badillo-Amberg I, Zelkowitz P. “So much of this story could be me”: men’s use of support in online infertility discussion boards. Am J Mens Health 2017 Dec;11(3):663-673 [FREE Full text] [doi: 10.1177/1557988316671460] [Medline: 27702886] Abbreviations AC1: Gwet agreement coefficient 1 ©Rie Yokota, Tsuyoshi Okuhara, Haruka Ueno, Hiroko Okada, Emi Furukawa, Takahiro Kiuchi. Originally published in the Journal of Medical Internet Research (https://www.jmir.org), 25.08.2020. 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A Geographical Analysis for the war Demography and its effect on Population Structure of Anbar Governorate (1997 – 2007 )

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جملةذجامعةذاألنبارذللعلومذاإلندانوة ذ ذ (العددذ4ذ )ذ (كانونذاألول)ذ3102ذذذذذذذذذذ اجمللدذالثاني ذ حتلولذجغرايفذلدميوغراػوةذاحلربذوأثرهاذعلىذالرتكوبذالدكانيذلدكانذحماػظةذ (االنبارذللمدةذ0991ذ- 3111 ) أ.م..ذ د حدنيذعليذعبدذالراوي جامعةذاألنبارذ–ذ كلوةذالرتبوةذللعلومذاإلندانوة ذذذذذذذذذذذذذذذذذذذذذ جملةذجامعةذاألنبارذللعلومذاإلندانوة ذ ذ (العددذ4ذ )ذ (كانونذاألول)ذ3102ذذذذذذذذذذ اجمللدذالثاني ذ حتلولذجغرايفذلدميوغراػوةذاحلربذوأثرهاذعلىذالرتكوبذالدكانيذلدكانذحماػظةذ (االنبارذللمدةذ0991ذ- 3111 ) أ.م..ذ د حدنيذعليذعبدذالراوي جامعةذاألنبارذ–ذ كلوةذالرتبوةذللعلومذاإلندانوة ذذذذذذذذذذذذذذذذذذذذذ جملةذجامعةذاألنبارذللعلومذاإلندانوة ذ ذ (العددذ4ذ )ذ (كانونذاألول)ذ3102ذذذذذذذذذذ اجمللدذالثاني ذ حتلولذجغرايفذلدميوغراػوةذاحلربذوأثرهاذعلىذالرتكوبذالدكانيذلدكانذحماػظةذ (االنبارذللمدةذ0991ذ- 3111 ) أ.م..ذ د حدنيذعليذعبدذالراوي جامعةذاألنبارذ–ذ كلوةذالرتبوةذللعلومذاإلندانوة ذذذذذذذذذذذذذذذذذذذذذ (العددذ4ذ )ذ (كانونذاألول)ذ3102ذ اجمللدذالثاني ذ ادلدتخلص جاء البحث تحت عنكاف (تحميؿ جغرافي لديمكغرافية الحرب كأثرىا عمى التركيب السكاني لسكاف محافظة االنبار لممدة7991 - 7001 ) , إذ تحتؿ المقكمات البشرية مكانة ميمة في بناء قكة الدكلة كالمنطػػػػػػػػػػ قة الجغرافية المراد بحثيا , لذلؾ أخذت أقالـ المفكريف كالمختصيف في دراسة المككنات السكانية كبػػػػحثيا, كتقكيـ نتائجيا كتحميميا كصكالن إلى تشخيص أشكاليا كعالجيا , إذ إف حػػجـ السكاف يتزايد كيتفاقـ باستمرار, كاستغالليـ العشكائي لمبيئة كمكاردىا متسارع في خطكاتو أيضا, إذا ما تعرضت الى حرب التي بدكرىا ينعكس أثرىا عمى التركيب السكاني مكضكع البحث. لذا بدأ الجغرافيكف يكلكف أىمية كبيرة في دراسة الخصائص السكانية كا ظيار تباينػػػػػيا اإلقميمي, أك بيف الكحدات اإلدارية لمدكلة , ك منيا محافظة االنبار مكضكع البحث , اذ تساعد دراسة التركيب السكاني عمى تفسير بعض المشاكؿ الت نمكية داخؿ المنطقة المراد بحثيا , كما تفسر التبايف بيف المناطؽ الحضرية كالريفية , فضال عف معرفة ما يممػػػػكو المجتمع السكاني المعني بالبحث مف مكارد بشرية كتصنيفيا حسب األنشطة االقتػػػػ صادية المختمفة , لما لو مف عال قة مباشرة بتكزيع السكاف كنمكىـ , فت كزيع السكاف بحسػػػػػػػب فئات األعمار كالنكع يحدد تطكرىـ , كيرتبط ذلؾ بالقكة اإلنتاجية لمسكاف كمػػػػػػقدار فعالياتيـ االقػػػػ تصادية التي تؤثر بدكرىا عمى حجـ األسرة, لذا أصبح االىتماـ بتطكر حجـ السكاف كنمكىـ كتركيبيـ العمرم كالنكعي كالنشاط االقتصادم كاالجتماع ي عمى مستكل محافظة االنبار, كاثر ديمكغرافية الحرب عمى .ذلؾ التركيب السكاني في منطقة البحث A Geographical Analysis for the war Demography and its effect on Population Structure of Anbar Governorate (1997 – 2007 ) Abstract The research entitled (A Geographical Analysis for the war Demography and its effect on Population Structure of Anbar Governorate (1997 – 2007 ), as human characteristics represent a prominent significance in building the power of the country and the Geographic area understudy. Thus, scientists and specialists concerned with the study of population have began expling, evalutuaing and analy in order to diagnose their forms and find solutions for their problems as the size of population increases and becomes complicated by time, also their random use for the environment and its natural resources is also continually increasing, When ever it faces a war which within its role reflect its effect into population structure the research subject. Geographers, therefore, devotes much of their attention studying their population characteristics and showing their regional diversity, also between the administrative units of the country, among which is Anbar Governorate, topic of the research understudy as the study of population structure helps in interpreting some development problems of the area of research, also it shows the variation between rural and urban areas. In addition, it shows the human resources of the society in the area understudy, and to classify their various economic activities since they have a direct relation with distribution and growth of society. Distribution of population according to age type determines their development and this is related to population productive power and their economic activities that, in turn, determines size of the family. thus, the concern is devoted to the development of population size, their growth, age and type structure and economic activity on the level of Anbar, topic of the study, And war demography effect on that population structure in research area. د ادلدتخلص ادلدتخلص جاء البحث تحت عنكاف (تحميؿ جغرافي لديمكغرافية الحرب كأثرىا عمى التركيب السكاني لسكاف محافظة االنبار لممدة7991 - 7001 ) , إذ تحتؿ المقكمات البشرية مكانة ميمة في بناء قكة الدكلة كالمنطػػػػػػػػػػ قة الجغرافية المراد بحثيا , لذلؾ أخذت أقالـ المفكريف كالمختصيف في دراسة المككنات السكانية كبػػػػحثيا, كتقكيـ نتائجيا كتحميميا كصكالن إلى تشخيص أشكاليا كعالجيا , إذ إف حػػجـ السكاف يتزايد كيتفاقـ باستمرار, كاستغالليـ العشكائي لمبيئة كمكاردىا متسارع في خطكاتو أيضا, إذا ما تعرضت الى حرب التي بدكرىا ينعكس أثرىا عمى التركيب السكاني مكضكع البحث. لذا بدأ الجغرافيكف يكلكف أىمية كبيرة في دراسة الخصائص السكانية كا ظيار تباينػػػػػيا اإلقميمي, أك بيف الكحدات اإلدارية لمدكلة , ك منيا محافظة االنبار مكضكع البحث , اذ تساعد دراسة التركيب السكاني عمى تفسير بعض المشاكؿ الت نمكية داخؿ المنطقة المراد بحثيا , كما تفسر التبايف بيف المناطؽ الحضرية كالريفية , فضال عف معرفة ما يممػػػػكو المجتمع السكاني المعني بالبحث مف مكارد بشرية كتصنيفيا حسب األنشطة االقتػػػػ صادية المختمفة , لما لو مف عال قة مباشرة بتكزيع السكاف كنمكىـ , فت كزيع السكاف بحسػػػػػػػب فئات األعمار كالنكع يحدد تطكرىـ , كيرتبط ذلؾ بالقكة اإلنتاجية لمسكاف كمػػػػػػقدار فعالياتيـ االقػػػػ تصادية التي تؤثر بدكرىا عمى حجـ األسرة, لذا أصبح االىتماـ بتطكر حجـ السكاف كنمكىـ كتركيبيـ العمرم كالنكعي كالنشاط االقتصادم كاالجتماع ي عمى مستكل محافظة االنبار, كاثر ديمكغرافية الحرب عمى .ذلؾ التركيب السكاني في منطقة البحث ( 88 ( أ.م.د.ذحدنيذعليذعبدذ الراوي ذ ذ حتلولذجغرايفذلدميوغراػوةذاحلربذوأثرهاذ علىذالرتكوبذالدكاني ذ ذ A Geographical Analysis for the war Demography and its effect on Population Structure of Anbar Governorate (1997 – 2007 ) Abstract أ.م.د.ذحدنيذعليذعبدذ الراوي :ادلقــــــــــدمةذ د بدأت الدراسات السكانية تحتؿ مكانان بارزان في األدبيات الجغرافية كاالجتماعي ة ,كاالقتصادية كمنذ عاـ7997 , أصبحت دراسة عمـ جغرافية السكاف مف المناىج األساسية ) 89 ) جملةذجامعةذاألنبارذللعلومذاإلندانوة ذ ذ (العددذ4ذ )ذ (كانونذاألول)ذ3102ذذذذذذذذذذذ اجمللدذالثاني ذ في أقساـ الجغرافي ة , كبذلؾ أخذت أقالـ المفكريف كالمختصيف تتنافس مف اجؿ تغطية مككنات ىذه الدراسات السكانية كتقكيـ نتائجيا كصكالن إلى تشخيص إشكاليا كعالجيا , لككف ىذه الدراسات ارتبطت بكاقع العاـ الحاضر كما سيؤكؿ إليو في المستقبؿ , إذ إف حجـ السكاف يتزايد كبتفاقـ مستمر. كاستغالليـ العشكائي لم بيئة كمكاردىا متسارع في خطكاتو أيضا, األمر الذم خمؼ مشكالت سكانية كبيئية معقدة , لـ تكف مكجكدة قبؿ اآلف, منيا اختالؿ التكازف البيئي بعنصرية الطبيعي كالبشرم , فالبيئة الطبيعية أخذت مقاكمتيا تضعؼ أماـ تحديات قدرات اإلنساف المتزايدة , كىذا ما جاء بدكر الدراسات ا لسكانية التي أخذت عمى عاتقيا التأثير الكبير في التخطيط لمتنمية االقتصاد ية كاالجتماعية في كثير مف الدكؿ. لقد بدأ الجغرافيكف يكلكف أىمية كبيرة في دراسة الخصائص السكانية كا ظيار تباينيا اإلقميمي بيف األقاليـ, اذ تساعد دراسة التركيب السكاني عمى تفسير بعض المشاك ؿ التنمكية د اخؿ اإلقميـ, كما تفسر التبايف بيف المناطؽ الحضرية كالمناطؽ الريفية مف جية, كالمجمكعات العرقية في الدكلة الكاحدة مف جية أخرل, فضال عف دراسة العكامؿ المؤثرة في ىذا التبايف كمدل ارتباطيا بالظركؼ الديمكغرافية األخرل, ىذا باإلضافة إلى معرفة ما يممكو المجتمع المعني بالدراسة مف مػػػػكارد بشرية كتصػػنيفي ا حسب النشاط االقتصادم المختمفة , لما لو مف عال قة مباشرة بتكزيع السكاف كنمكىـ , يكاد يككف مف أىـ العكامؿ الم ؤثرة في المتغيرات الديمكغرافية , فتكزيع السكاف حسب فئات األعمار كالنكع يحدد تطكرىـ, فيك يؤثر مف ج ية عمى تككيف الكالدات كالكفيات , كمف ثـّ عمى اتجاه الخصكبة كمقدار الزيادة الطبيعية لمسكاف كاليجرة مف جية أخرل, كذلؾ الرتباطيـ بالقكة اإلنتاجية لمسكاف كمقدار فعالياتيـ االقتصادية التي تؤثر بدكرىا عمى حجـ األسرة, لذا ال أصبحت تيتـ بتطكر حجـ السكاف كنمكىـ , كال تركيب النكعي كالعمرم كالنشاط االقتصادم كاالجتماعي عمى م ستكل محافظة االنبار مكضكع البحث. مشكلةذالبحث:ذ لمظركؼ التي مر بيا القطر العراقي كمنطقة البحث : منذ عاـ7980 اكحتى عاـ جملةذجامعةذاألنبارذللعلومذاإلندانوة ذ ذ (العددذ4ذ )ذ (كانونذاألول)ذ3102ذذذذذذذذذ اجمللدذالثاني ذ في أقساـ الجغرافي ة , كبذلؾ أخذت أقالـ المفكريف كالمختصيف تتنافس مف اجؿ تغطية مككنات ىذه الدراسات السكانية كتقكيـ نتائجيا كصكالن إلى تشخيص إشكاليا كعالجيا , لككف ىذه الدراسات ارتبطت بكاقع العاـ الحاضر كما سيؤكؿ إليو في المستقبؿ , إذ إف حجـ السكاف يتزايد كبتفاقـ مستمر. :ادلقــــــــــدمةذ كاستغالليـ العشكائي لم بيئة كمكاردىا متسارع في خطكاتو أيضا, األمر الذم خمؼ مشكالت سكانية كبيئية معقدة , لـ تكف مكجكدة قبؿ اآلف, منيا اختالؿ التكازف البيئي بعنصرية الطبيعي كالبشرم , فالبيئة الطبيعية أخذت مقاكمتيا تضعؼ أماـ تحديات قدرات اإلنساف المتزايدة , كىذا ما جاء بدكر الدراسات ا لسكانية التي أخذت عمى عاتقيا التأثير الكبير في التخطيط لمتنمية االقتصاد ية كاالجتماعية في كثير مف الدكؿ. جملةذجامعةذاألنبارذللعلومذاإلندانوة ا ي يا لقد بدأ الجغرافيكف يكلكف أىمية كبيرة في دراسة الخصائص السكانية كا ظيار تباينيا اإلقميمي بيف األقاليـ, اذ تساعد دراسة التركيب السكاني عمى تفسير بعض المشاك ؿ التنمكية د اخؿ اإلقميـ, كما تفسر التبايف بيف المناطؽ الحضرية كالمناطؽ الريفية مف جية, كالمجمكعات العرقية في الدكلة الكاحدة مف جية أخرل, فضال عف دراسة العكامؿ المؤثرة في ىذا التبايف كمدل ارتباطيا بالظركؼ الديمكغرافية األخرل, ىذا باإلضافة إلى معرفة ما يممكو المجتمع المعني بالدراسة مف مػػػػكارد بشرية كتصػػنيفي ا حسب النشاط االقتصادم المختمفة , لما لو مف عال قة مباشرة بتكزيع السكاف كنمكىـ , يكاد يككف مف أىـ العكامؿ الم ؤثرة في المتغيرات الديمكغرافية , فتكزيع السكاف حسب فئات األعمار كالنكع يحدد تطكرىـ, فيك يؤثر مف ج ية عمى تككيف الكالدات كالكفيات , كمف ثـّ عمى اتجاه الخصكبة كمقدار الزيادة الطبيعية لمسكاف كاليجرة مف جية أخرل, كذلؾ الرتباطيـ بالقكة اإلنتاجية لمسكاف كمقدار فعالياتيـ االقتصادية التي تؤثر بدكرىا عمى حجـ األسرة, لذا ال أصبحت تيتـ بتطكر حجـ السكاف كنمكىـ , كال تركيب النكعي كالعمرم كالنشاط االقتصادم كاالجتماعي عمى م ستكل محافظة االنبار مكضكع البحث. مشكلةذالبحث:ذ مشكلةذالبحث:ذ لمظركؼ التي مر بيا القطر العراقي كمنطقة البحث : منذ عاـ7980 كحتى عاـ 7001, أثرىا الكبير عمى ال تركيب السكاني في محافظة االنبار , كمما أفرزتو تمؾ الظركؼ مف حرب دامت ثماف سنكات كحصار منذ عاـ7997 كحرب عاـ7002 , لذا تعرض ىذا ( 90 ( أ.م.د.ذحدنيذعليذعبدذ الراوي :ػرضوةذالبحث أثرت العكامؿ الجغرافية المختمفة عمى الخصائص الديم كغرافية كال سيما الحرب كاثرىا عمى التركيب السكاني لسكاف م حافظة االنبار , مما خمؽ تبايننان زمانيان, كمف ثـّ انعكس ىذا .عمى المحافظة اقتصاديان كاجتماعيان كحيكيان تأتي أىمية مكضكع ديمكغرافية الحرب كاثرىا عمى التركيب السكاني, لما لمتغيرات التي تحصؿ في تركيب السكاف خالؿ مدة معينة مف تأثيرات عمى تركيب سكاف محافظة االنبار, يمكف أف تقكد إلى مجمكعة مف المتغيرات في عدد مف المجاالت االقتصادية كاالجتماعية كالحيكية , كما يمكف مف خاللو المقارنة بيف البناء السكاني الكاحد في مراحؿ زمنية مختمفة كتحديد اتجاىات التغيرات العددية لكؿ منيا , مع استخداـ بعض المؤشرات .اإلحصائية البسيطة االزمة هدفذالدراسة:ذ 7 - التعرؼ عمى أنَّكاع خصائص الترك يب السكاني لسكاف محافظة االنبار , انطالقا مف معرفة تطكر حجـ السكاف كنمكىـ كتكزيعيـ البيئي كتركيبيـ النكعي كالنشاط االقتصادم كالميني.كالتعميمي كالتككيف االثني 7 - التعرؼ عمى أنَّكاع خصائص الترك يب السكاني لسكاف محافظة االنبار , انطالقا مف معرفة تطكر حجـ السكاف كنمكىـ كتكزيعيـ البيئي كتركيبيـ النكعي كالنشاط االقتصادم كالميني.كالتعميمي كالتككيف االثني 7 - البحث عف عكامؿ تبايف خصائص التركيب السكاني, فيما بيف التبايف الزماني كالكشؼ عف العالقات المكانية التي تفسر ىذا التبايف كمعرفة التغير الذم طرأ عمى ( التركيب السكاني لمنطقة البحث لممدة7991 – 7001 ) , كتشخيص عكامؿ التغير بسبب ال حرب ك اثارىا المختمفة. 7 - البحث عف عكامؿ تبايف خصائص التركيب السكاني, فيما بيف التبايف الزماني كالكشؼ عف العالقات المكانية التي تفسر ىذا التبايف كمعرفة التغير الذم طرأ عمى ( التركيب السكاني لمنطقة البحث لممدة7991 – 7001 ) , كتشخيص عكامؿ التغير بسبب ال حرب ك اثارىا المختمفة. ر قمة الدراسات التي تناكلت مكضكع ديمكغرافية الحرب كاثرىا عمى التركيب السكاني بشكؿ خاص كمفصؿ كمتخصصة لدراسة تركيب سكاف محافظة االنبار , كالتعرؼ عمى التغيرات التي أصابت خصائص التركيب السكاني سمبان اك ايجابان التي ىي نتيجة لتداعيات الحرب العراقية اإليرانية كالحصار االقتصادم كأحداث عاـ7002 .كما بعدىا ) 97 ) جملةذجامعةذاألنبارذللعلومذاإلندانوة ذ ذ (العددذ4ذ )ذ (كانونذاألول)ذ3102ذذذذذذذذذ اجمللدذالثاني ذ منهجوةذالبحث:ذ اعتمد الباحث المنيج الجغرافي في التحميؿ الكصفي كالكمي البسيط , كربط التبايف في خصائص التركيب السكاني بالعكامؿ الجغرافية التي تحدده, كتعد البيانات اإلحصائية الخاصة بمحافظة االنبار لمتعداد العاـ لسكاف محافظة االنبار(عاـ7991 كتقديرات عاـ7001 ) , كالدراسات الجغرافية مصدر أساسي ليذا التحميؿ , كقد كانت مص ادر البيانات الجغرافية السكانية , ىي إحصاءات التعدادا ت السكانية أعاله كالرسائؿ كاألطاريح الجامعية كالبحكث العممية المتاحة. جملةذجامعةذاألنبارذللعلومذاإلندانوة ذ ذ (العددذ4ذ )ذ (كانونذاألول)ذ3102ذذذذذذ اجمللدذالثاني ذ منهجوةذالبحث:ذ جملةذجامعةذاألنبارذللعلومذاإلندانوة ذ ذ (العددذ4ذ )ذ (كانونذاألول)ذ3102ذذذذذذ اجمللدذالثاني ذ منهجوة البحث: (العددذ4ذ )ذ (كانونذاألول)ذ3102ذذ اجمللدذالثاني ذ اعتمد الباحث المنيج الجغرافي في التحميؿ الكصفي كالكمي البسيط , كربط التبايف في خصائص التركيب السكاني بالعكامؿ الجغرافية التي تحدده, كتعد البيانات اإلحصائية الخاصة بمحافظة االنبار لمتعداد العاـ لسكاف محافظة االنبار(عاـ7991 كتقديرات عاـ7001 ) , كالدراسات الجغرافية مصدر أساسي ليذا التحميؿ , كقد كانت مص ادر البيانات الجغرافية السكانية , ىي إحصاءات التعدادا ت السكانية أعاله كالرسائؿ كاألطاريح الجامعية كالبحكث العممية المتاحة. حدودذا:لبحث مكانيا تمثمت بالحيز الجغرافي الذم تشغمو محافظة االنبار كبحدكدىا اإلدارية , ( خارطة7 ), كزمانيا فقد أخذت المدة الممتدة ما ( بيف7991 – 7001 ). م تعد دراسة حجـ السكاف كنمكىـ مف القضايا الكطنية الميمة التي ليا انعكاسات عمى مختمؼ األصعدة االقتػػصادية كاالجتماعية كالسػػػياسية , فالزياد ة السكانية تككف ليا تبعات سمبية , إذا لـ يتـ استثمار مكارد اقتصادية إضافية تسد حا جة السكاف كمتطمباتيـ المستقبمية , أما التب عات االيجابية لزيادة حجـ السكاف , فتتمثؿ في بناء قكة اقتصادية التي تخدـ الدكلة كت ككف مسؤكلة عف تأ ميف احتياجاتيا. كعمى ىذا األساس ركزت العديد مف البحكث في دراسة العالقة بيف نمك السكاف كالنمك االقتصادم األمثؿ لسد احتياجات السكاف المستقبمية , كما إف مف غير الممكف التنبؤ باتجاىات النمك االقتصادم مف دكف دراسة التكقعات المستقبمية لنمك السكاف كتطكر حجميـ بكصف يـ القكة االقتصادية كاالجتماعية. ( 97 ( ذ ي ى رت ب ( اذ بمغ عدد سكاف محافظة االنبار7072121 ) نسمة( , مشكميف نسبة1 . 4 % كاف ال راؽ(البالغ77048744ة عا ) ن7991يف بمغ عددى , ف ( اذ بمغ عدد سكاف محافظة االنبار7072121 ) نسمة( , مشكميف نسبة1 . 4 % ) مف اجمالي سكاف العراؽ( البالغ77048744 ) نسمة عاـ7991 , في حيف بمغ عددىـ عاـ ( اذ بمغ عدد سكاف محافظة االنبار7072121 ) نسمة( , مشكميف نسبة1 . 4 % ) مف اجمالي سكاف العراؽ( البالغ77048744 ) نسمة عاـ7991 , في حيف بمغ عددىـ عاـ ) 92 ) جملةذجامعةذاألنبارذللعلومذاإلندانوة ذ ذ (العددذ4ذ )ذ (كانونذاألول)ذ3102ذذذذذذذذذذ اجمللدذالثاني ذ 7001 ( 7489989) نسمة( , مشكميف نسبة9 ) مف اجمالي سكاف العراؽ% ( 79187087 ) نس مة حسب تقديرات سكاف العراؽ7001 . أما فيما يخص تطكر حجـ السكاف في محافظة االنبار , حسب كحداتيا اإلدارية ( )عمى مستكل القضاء , يتبايف حػػجـ السكاف مف كحػػدة إدارية إلى أخرل, كذلؾ لجممة مف ,األسباب منيا طبيعة السطح , إذ إف معظـ السطح في منطقة البحث ت تخممو اليضػػ بة الغربية )(الصحراكية , كىي ذات طبيعة صخرية في األغمب مع كجكد شريط ضيؽ مف السيؿ الفيضي مع اتصالو ب السيؿ الرسكبي ضمف حدكد المحافظة( , خارطة7 ,) فضال عف العكامؿ االقتصادية. جملةذجامعةذاألنبارذللعلومذاإلندانوة ذ ذ (العددذ4ذ )ذ (كانونذاألول)ذ3102ذذذذذذذذذ اجمللدذالثاني ذ 7001 ( 7489989) نسمة( , مشكميف نسبة9 ) مف اجمالي سكاف العراؽ% ( 79187087 ) نس مة حسب تقديرات سكاف العراؽ7001 . جملةذجامعةذاألنبارذللعلومذاإلندانوة ذ ذ (العددذ4ذ )ذ (كانونذاألول)ذ3102ذذذذذذذذذذ اجمللدذالثاني ذ 7001 ( 7489989) نسمة( , مشكميف نسبة9 ) مف اجمالي سكاف العراؽ% ( 79187087 ) نس مة حسب تقديرات سكاف العراؽ7001 . حدودذا:لبحث (العددذ4ذ )ذ (كانونذاألول)ذ3102ذذ اجمللدذالثاني ذ أما فيما يخص تطكر حجـ السكاف في محافظة االنبار , حسب كحداتيا اإلدارية ( )عمى مستكل القضاء , يتبايف حػػجـ السكاف مف كحػػدة إدارية إلى أخرل, كذلؾ لجممة مف ,األسباب منيا طبيعة السطح , إذ إف معظـ السطح في منطقة البحث ت تخممو اليضػػ بة الغربية )(الصحراكية , كىي ذات طبيعة صخرية في األغمب مع كجكد شريط ضيؽ مف السيؿ الفيضي مع اتصالو ب السيؿ الرسكبي ضمف حدكد المحافظة( , خارطة7 ,) فضال عف العكامؿ االقتصادية. ( 94 ( أ.م.د.ذحدنيذعليذعبدذ الراوي ذ ذ حتلولذجغرايفذلدميوغراػوةذاحلربذوأثرهاذ علىذالرتكوبذالدكاني ذ ذ ( إذ نالحظ مف الجدكؿ7 ( ) الشكؿ7 ), إف أعمى حػػجـ السكاف يتركز في قضاء الرمادم (مركز )المحافظة إذ ( بمغ حجـ السكاف217797 )نسمة( , كبنسبة21,4 )% , مف مجمكع سكاف المحافظة لعاـ7991, كذلؾ لتركز الخدمات اإلدارية كاالجتماعية كاالقتصادية , المتمثمة بكجكد المعامؿ كالسكؽ المركزم كشبكة النقؿ كالمؤسسا ت الحككمية. كيأتي قضاء الفمكجة بالمرت( بة الثانية بحػجـ سػػكانو البالغ212819 نسمة ), بنػسبة ( 29,9 )%, مف إجمالي سكاف المحافظة , كذلؾ بسبب مكقعيا الجغرافي الذم يتكسط بيف الػ عاصمة بغداد مف جية الشرؽ, كمركز المحافظة مف جية الغرب , فضال عف تكفر فرص العمؿ لكجكد العديد مف المنشآت كالمعامؿ الصناعية, بين ما يأتي قضاء القائـ بالمرتبة الثالثة ( بحجـ سكانو البػػػالغ99081 )نسمة( , بنػػسبة9,2 )%, مف مجمكع سكاف المحافظة , ثـ ( قضاء ىيت بالمرتبة الرابعة بحجـ سكانو البالغ88897 )نسمة( , كبنسبة8,1 % ) , مف إجمالي سكاف محافظة االنبار, ثـ يأتي قضاء حػػديثة بالػػػمرتبة الخ امػس ة بحجـ سكانو البالغ ( 94804 )نسمة( , كبنسبة9,2 ), مف مجمكع سكاف المحافظة% , كبالمرتبة األخيرة يأتي كؿ مف قضاء (الرطبة7,7 , عنو% 7,9 , راكه% 7,2 )% , مف مجمكع سػػكاف المػحافظة ( جدكؿ7 ). أ.م.د.ذحدنيذعليذعبدذ الراوي أما في تقديرات السكاف لعاـ7001 , فقد ظمت االقضية بنفس المراتب مع ارتف اع في حجـ السكاف , كبنسب ال ت ختمؼ كثيرا عف عاـ7991 , بسبب الزيادة الطبيعية لمسكاف كاليجرة الكافدة بسبب ظركؼ الحرب عاـ7002 . أما بالنسبة لمعدؿ النمك السكاني فكاف في قضائي الرمادم كالفمكجة, قد سػػجؿ المػعدؿ نفسو كالػػ( بالغ2,8), لكؿ منيما%, إال إف اختالؼ حجـ السكاف بينيما , يأتي نتيجة الحركة المكانية لمسكاف , بسبب عكامؿ الجذب المكاني كلتركز خدمة التعميـ الجامعي في مرك)ز قضاء الرمادم (مركز المحافظة , أما باقي االقض ية فقد سجمت معدؿ نمك بمغ ( 2,1 )% ( , باستثناء قضاء حديثة إذ بمغ معدؿ نمكىـ الػػسكاني2,1 ( ) جدكؿ% 7 ). حدودذا:لبحث ( 91 ( أ.م.د.ذحدنيذعليذعبدذ الراوي ذ ذ حتلولذجغرايفذلدميوغراػوةذاحلربذوأثرهاذ علىذالرتكوبذالدكاني ذ ذ ( شكؿ7 ) تطكر حجـ السكاف كنمكىـ في محافظة االنبار بحسب كحداتو اإلدارية لممدة ( 7991 – 7001 ) المصدر: ( باالعتماد عمى جدكؿ7 ) ( جدكؿ7 ) إسقاطات السكاف)في العراؽ كمحافظة االنبار (نسمة ( لممدة7001 – 7071 ) 0 100000 200000 300000 400000 500000 600000 الفلوجة.ق الرمادي.ق هيت.ق حديثة.ق عنه.ق راوه.ق القائم.ق الرطبة.ق الوحدة االدارية نسمة ( عدد السكان 1997)نسمة( عدد السكان2007)نسمة( عدد السكان أ.م.د.ذحدنيذعليذعبدذ الراوي ذ ذ حتلولذجغرايفذلدميوغراػوةذاحلربذوأثرهاذ علىذالرتكوبذالدكاني ذ ذ ( شكؿ7 ) تطكر حجـ السكاف كنمكىـ في محافظة االنبار بحسب كحداتو اإلدارية لممدة ( 7991 – 7001 ) ذ علىذالرتكوبذالدكاني ذ ذ ( شكؿ7 ) تطكر حجـ السكاف كنمكىـ في محافظة االنبار بحسب كحداتو اإلدارية لممدة ( 7991 – 7001 ) المصدر: ( باالعتماد عمى جدكؿ7 ) ( جدكؿ7 ) إسقاطات السكاف)في العراؽ كمحافظة االنبار (نسمة ( لممدة7001 – 7071 ) المصدر: باالعتماد عمى استخداـ :المعادلة التالية PN = PO ( 1 + r \ 100 ) n : حيث إف PN = التعداد الالح ؽ . PO = التعداد السابؽ . r = معدؿ النمك . n = عدد السنكات بيف التعداديف. إ أ 0 100000 200000 300000 400000 500000 600000 الفلوجة.ق الرمادي.ق هيت.ق حديثة.ق عنه.ق راوه.ق القائم.ق الرطبة.ق الوحدة االدارية نسمة ( عدد السكان 1997)نسمة( عدد السكان2007)نسمة( عدد السكان السنة عدد سكاف العراؽ (نسمة) عدد سكاف محافظة )االنبار (نسمة السنة عدد سكاف العراؽ (نسمة) عدد سكاف محافظة االنبار )(نسمة 7001 79187087 7489989 7072 29447994 7841922 7008 20917942 7940911 7074 21909777 7971201 7009 27489179 7991987 7079 21100218 7981709 7070 27424470 7191701 7071 21100218 7010121 7077 22401447 7174878 7071 29890720 7721982 7077 24409119 7187999 ( شكؿ7) تطكر حجـ السكاف كنمكىـ في محافظة االنبار بحسب كحداتو اإلد ( 7991 – 7001 ) المصدر: ( باالعتماد عمى جدكؿ7 ) 0 100000 200000 300000 400000 500000 600000 الفلوجة.ق الرمادي.ق هيت.ق حديثة.ق عنه.ق راوه.ق القائم.ق الرطبة.ق الوحدة االدارية نسمة ( عدد السكان 1997)نسمة( عدد السكان2007)نسمة( عدد السكان المصدر: ( باالعتماد عمى جدكؿ7 ) المصدر: ( باالعتماد عمى جدكؿ7 ) ( جدكؿ7 ) إسقاطات السكاف)في العراؽ كمحافظة االنبار (نسمة ( لممدة7001 – 7071 ) المصدر: باالعتماد عمى استخداـ :المعادلة التالية PN = PO ( 1 + r \ 100 ) n : حيث إف PN = التعداد الالح ؽ . PO = التعداد السابؽ . r = معدؿ النمك . n = عدد السنكات بيف التعداديف. حدودذا:لبحث ك عمى أساس حساب معدؿ النمك السكاني يمكف معرفة حجـ السكاف في المستقبؿ مف خالؿ اإلسقاطات السكانية , إذ ( يبيف الجدكؿ7 ) , تطػػكر حػػجـ السكاف لمحافظة االنبار لممدة ( 7001 – 7071 ) , إذ يتبيف باف عدد سػػػػػكاف المػػػحافظة لعاـ7071 سػػكؼ يػػػػػػبمغ ( 7,721,982نسمة) , ( كبنسبة9,2 )% , مف مجمكع سكاف العراؽ الذم سكؼ يبمغ ) 99 ) ذ ي لمج ( 29890720 نسمة), لممدة المذككرة , إف ىذه الزيادة السكانية ستشكؿ عبئان ديمكغرافيان في ظؿ الظركؼ كالمعطيات االقتصا دية التي تتسـ بالرككد االقتصادم , كسيؤثر ىذا النمك السكاني عمى جممة مف اإلجراءات المعنية لتحقيؽ مستكيات أعمى في نك عية حياة السكاف كمستكل رفاىيتيـ , كتؤدم بدكرىا السػػمبي عمى التعميـ كالصحة كالمياه كالغذاء كالنقؿ كالبيئة , ما لـ يتـ كضع خطط إستراتيجية في المستقبؿ الستيعاب النمك السكاني , فضالن عف إجراء التعداد السكاني في مكعدة المحدد بعد مركر عشرة سنكات بشكؿ دكرم , كما كاف معمكؿ بو ( سابقا خالؿ تعدادم7981 – 7991 ). ( جدكؿ7 ) تطكر حجـ السكاف كمعدؿ نمكىـ في محافظة االنبار بحسب كحداتو اإلدارية ( لممدة7991 – 7001 ) ( جدكؿ7 ) تطكر حجـ السكاف كمعدؿ نمكىـ في محافظة االنبار بحسب كحداتو اإلدارية ( لممدة7991 – 7001 ) ال ا ال ا ة اإل ا لإل ال ك ا ال ة ال ط ط ال كا ا الكحدات اإلدارية 7991 7001 معدؿ النمك عدد السكاف )(نسمة نسبتيـ )%( عدد السكاف )(نسمة نسبتيـ )%( .ؽ الفمكجة 212819 29,9 979998 29,1 2,8 .ؽ الرمادم 217797 21,4 940419 21,4 2,8 .ؽ ىيت 88897 8,1 779002 8,1 2,1 .ؽ حديثة 94804 9,2 18191 9,7 2,1 .ؽ عنو 79747 7,9 77811 7,9 2,1 .ؽ راكه 77981 7,2 78199 7,2 2,1 .ؽ القائـ 99081 9,2 721911 9,2 2,1 .ؽ الرطبة 70848 7,7 20019 7,0 2,1 المجمكع 7072121 700 7489989 700 2,1 المصدر: جميكرم العراؽ, ىيئة التخطيط, الجياز المركزم لإلحصاء, مديرية اإلحصاء السكاني , نتائج التعداد العاـ لمسكاف, لمحافظة االنبار , 7998 . ج ميكرم العراؽ, ,كزارة التخطيط كالتعاكف اإلنمائي الجياز المركزم لإلحصاء, مديرية اإلحصاء السكاني , تقديرات سكاف العراؽ , لعاـ7001 . اب ر ف ـ ج ميكرم العراؽ, ,كزارة التخطيط كالتعاكف اإلنمائي الجياز المركزم لإلحصاء, مديرية اإلحصاء السكاني , تقديرات سكاف العراؽ , لعاـ7001 . حدودذا:لبحث المصدر: محمد فتحي أبك عيانو, مدخؿ إلى التحميؿ اإلحصائي في الجغرافية البشرية, دار المعرفة الجامعية ,لمطبع, اإلسكندرية7981 , ص721 . السنة عدد سكاف العراؽ (نسمة) عدد سكاف محافظة )االنبار (نسمة السنة عدد سكاف العراؽ (نسمة) عدد سكاف محافظة االنبار )(نسمة 7001 79187087 7489989 7072 29447994 7841922 7008 20917942 7940911 7074 21909777 7971201 7009 27489179 7991987 7079 21100218 7981709 7070 27424470 7191701 7071 21100218 7010121 7077 22401447 7174878 7071 29890720 7721982 7077 24409119 7187999 ( جدكؿ7 ) إسقاطات السكاف)في العراؽ كمحافظة االنبار (نسمة ( لممدة7001 – 7071 ) المصدر: باالعتماد عمى استخداـ :المعادلة التالية PN PO ( 1 \ 100 ) السنة عدد سكاف العراؽ (نسمة) عدد سكاف محافظة )االنبار (نسمة السنة عدد سكاف العراؽ (نسمة) عدد سكاف محافظة االنبار )(نسمة 7001 79187087 7489989 7072 29447994 7841922 7008 20917942 7940911 7074 21909777 7971201 7009 27489179 7991987 7079 21100218 7981709 7070 27424470 7191701 7071 21100218 7010121 7077 22401447 7174878 7071 29890720 7721982 7077 24409119 7187999 PN = PO ( 1 + r \ 100 ) n إف ي PN = التعداد الالح ؽ . PO = التعداد السابؽ . r = معدؿ النمك . n = عدد السنكات بيف التعداديف. المصدر: محمد فتحي أبك عيانو, مدخؿ إلى التحميؿ اإلحصائي في الجغرافية البشرية, دار المعرفة الجامعية ,لمطبع, اإلسكندرية7981 , ص721 . ) 91 ) جملةذجامعةذاألنبارذللعلومذاإلندانوة ذ ذ (العددذ4ذ )ذ (كانونذاألول)ذ3102ذذذذذذذذذذ اجمللدذالثاني ذ اخلصائصذالدميوغراػوةذللرتكوبذالدكانيذلدكانذحماػظةذ:االنبار إف تركيب السكاف لو مدلكؿ كاسع كتأثير مباشر عمى النشاط االقتصادم كاالجتماعي, باعتباره عامال ديمكغرافيا متغيرا( 7 ) . إذ يمكف مف خاللو تحديد تأثيرات السكاف عمى مسرح الجغرافية االقتصادية , كىك يعني بدراسة خصائص السكاف التي يتككف منيا المجتمع( 7 ) , كغالبا ما تككف طبيعة البيانات التي يمكف الحصكؿ عمييا مف التعدادا ت السكانية تتمثؿ بالسكاف مف حيث الجنس كالعمر كالتركيب االقتصادم كالتككيف االجتماعي كالثقافي كالديف كالمغة كالقكمية( 2 ). جملةذجامعةذاألنبارذللعلومذاإلندانوة ذ ذ (العددذ4ذ )ذ (كانونذاألول)ذ3102ذذذذذذذذذ اجمللدذالثاني ذ اخلصائصذالدميوغراػوةذللرتكوبذالدكانيذلدكانذحماػظةذ:االنبار إف تركيب السكاف لو مدلكؿ كاسع كتأثير مباشر عمى النشاط االقتصادم كاالجتماعي, باعتباره عامال ديمكغرافيا متغيرا( 7 ) . إذ يمكف مف خاللو تحديد تأثيرات السكاف عمى مسرح الجغرافية االقتصادية , كىك يعني بدراسة خصائص السكاف التي يتككف منيا المجتمع( 7 ) , كغالبا ما تككف طبيعة البيانات التي يمكف الحصكؿ عمييا مف التعدادا ت السكانية تتمثؿ بالسكاف مف حيث الجنس كالعمر كالتركيب االقتصادم كالتككيف االجتماعي كالثقافي كالديف كالمغة كالقكمية( 2 ). (العددذ4ذ )ذ (كانونذاألول)ذ3102ذذ اجمللدذالثاني ذ (العددذ4ذ )ذ (كانونذاألول)ذ3102ذذ اجمللدذالثاني ذ (العددذ4ذ )ذ (كانونذاألول)ذ3102ذذ اجمللدذالثاني ذ جملةذجامعةذاألنبارذللعلومذاإلندانوة كؿ ىذه الخصائص تكسب المجتمع ميزات خاصة ينفرد بيا سكاف المحافظة قيد البحث, إذ ينب غي دراستيا مف اجؿ الكصكؿ إلى البيان ات كالفكارؽ المكجكدة بيف إقميـ كآخر اك محافظة كاخرل ضمف الدكلة الكاحدة, مما تعػػػكسو تمؾ الفكارؽ مف أثار سػػمبية كايجابية تؤثر عمى قكة الدكلة اقتصاديان, كلمكقكؼ عمى تحميؿ تركيب السكاف لمحافظة االنبار كدكره في دعـ المحافظة اقتصاديا , ينبغي دراسة التركيب العمرم كالنكعي كاالقتصادم كالميني كالتعميمي :كالتككيف االثني عمى النحك األتي أوال : التركيب العمري لمسكان: يعد التركيب العمرم لمسكاف مف أىـ الخصائص الديمكغرافية التي تشير في داللتيا إلى قكة السكاف اإلنتاجية كمقدار حيكيتي ـ , كينعكس عمى حجـ الناتج المحمي كالدخؿ القكمي( 4 ) . كىك كثيؽ الصمة بالظركؼ ا الجتماعية كاالقتصادية كالسياسية , الف دراسة عمر السكاف يعتبر األساس في رسـ الدكلة لخططيا التنمكية عمى أسس عممية مدركسة لتحديد المالمح الميم ة لميرـ السكاني , كما إذا كاف المجتمع فتيان, ك ىػػـ الذيف في الغالب ما تككف ( أعمارىـ بيف79 – 49 سنة) ( 9 ). الذيف تقع عمييـ إعالة األطفاؿ ككبار السف , كعمى ىذا األساس ينقسـ المجتمع السكاني لمحافظة االنبار إلى ثالث فئات عمرية , جدك( ؿ2 ) كشكؿ ( 7), كىي: 1 ( : فئة صغار السن0 - 14 سنة:) كىـ ا لفئة العمرية الذيف يمثمكف قاعدة اليرـ السكاني كىـ الذيف تنحصر أعمارىـ بيف اليكـ األكؿ ( لكالدتيـ كصكال إلى سف74 ) سنة , كىذه الفئة غالبا ما تككف غير منتجة كتتأ ثر ( بعاممي الكالدات كالكفيات1 ) ( . كقد بمغت نسبتيا48,9), مف مجمكع سكاف المحافظة% , ( 98 ( أ.م.د.ذحدنيذعليذعبدذ الراوي ( كاف نصػػيب الذككر74,1 )% ( , كاإلناث72,9 ) ك% ( حسب تعداد عاـ7991 ) , في حيف بمغ نسبة مجمكع اإلناث كالذككر ( 44,7 ) مف مجمكع سكاف المحافظة, إذ يشكؿ الذككر% ( نسبة77,1 ( ), كاإلناث% 77,9 )%, كذلؾ بحػػػسب تق( ديرات الػػػػػسكاف لعاـ7001 ) , كمف خالؿ ذلؾ سجؿ الفارؽ بالنقصاف ل( نسبتييما لممدة بيف7991 - 7001 ) ( , بحدكد4,4 )% , كتعكد األسباب في تراجع نسبة فئة صغار السف إلى الظركؼ االقتصادية الصعبة التي مر بيا العراؽ خالؿ حقبة التسعينات , فضال عف الحرب التي شنتيا القكات األمريكية كمف حالفيا عمى العراؽ مما أدل إلى تناقص فئة الذكك ر فضال عف عامؿ حركة ىجرة السكاف. (العددذ4ذ )ذ (كانونذاألول)ذ3102ذذ اجمللدذالثاني ذ 2 : ف ئة متوسطي السن (البالغين( ) 15 - 64 سنة:) تشمؿ ىذه الفئة العمرية السكاف العامم يف في مختمؼ النشاطات االقتصادية , كيطمؽ عمييـ المصطم ح ( القكة العاممةman power lobourtion ) ( 1 ) . أك التي تعرؼ بالنشيطيف اقتصاديان, فيـ يمثمكف الفئة العاممة كالمنتجة في الكقت نفسو , كتقع عمييـ أعباء إعالة فئة صغ( ار السف0 - 74 )سنة( , كفئة كبار السف19 )سنة فأكثر , فضال عف أ ف ىذه الفئة , تعد العنصر األساسي في تحقيؽ التقدـ كالتطكر اإلنساني في نمك ال مجتمع مف خالؿ الزيادة الطبيعية( , إذ بمغت نسبتيـ48,4), مف إجمالي سكاف المحافظة, تشكؿ الذ% ككر ( 74,7 )%, كاإلنا ( ث74,2 )% , كذلؾ بحسب التعداد العاـ لمسكاف عاـ7991 , في حيف ( بمغت النسبة92,7 )%, مف إجمالي سكاف المحافظة ( حسب تقديرات السكاف لعاـ7001 ) , ( كاف مف نصيب الذككر منيا71,4 ( ) كاإلناث% 71,1 ) كمف مجمكع سكاف محافظة% .االنبار 3 : ( فئة كبار السن65 )سنة فأكثر: تمثؿ ىذه الفئة العمرية قمة اليرـ السكاني , إذ تككف نسبتػػػيـ قميمة في اغمب األحياف , كيعد دليالن عمى عدـ االىتماـ بيـ مف قبؿ المؤسسات االجتماعية , إذ تفتقر منطقة البحث إلى دكر رعاية المسنيف بسبب األكضاع االجتماعية , كتمتاز ىذه ال فئة بتفكؽ إعداد اإل ناث ( عمى الذككر, إذ بمغ مجمكعيـ20791 ( نسمة), مشكميف7,9 )% ( , منيا7,2 ) لمذككر% ك( 7,1 ) %, لإلناث م ف مجمكع سكاف المحافظة لعاـ7991 , في حيف بمغ مجمكعيـ ( 40491 )نسمة( , بنسبة7,1 ( ) منيا% 7,7 () لمذككر ك% 7,9 ) % , لإلناث بحسب تقديرات السكاف لعاـ( 7001 ) , فعمى الرغـ مف قمة أعدادىـ , إال أف ليـ تأثيران مباشران مف خالؿ ) 99 ) جملةذجامعةذاألنبارذللعلومذاإلندانوة ذ ذ (العددذ4ذ )ذ (كانونذاألول)ذ3102ذذذذذذذذ اجمللدذالثاني ذ اإلدالء بأصكاتيـ االنتخابية التي برزت بشكؿ كاضح بعد إحداث حرب عاـ7002 . ( جدكؿ2 ( ) تكزيع سكاف محافظة االنبار بحسب الفئات العمرية لممدة7991 - 7001 ) جملةذجامعةذاألنبارذللعلومذاإلندانوة ذ ذ (العددذ4ذ )ذ (كانونذاألول)ذ3102ذذذذذذذ اجمللدذالثاني ذ اإلدالء بأصكاتيـ االنتخابية التي برزت بشكؿ كاضح بعد إحداث حرب عاـ7002 . ( جدكؿ2 ( ) تكزيع سكاف محافظة االنبار بحسب الفئات العمرية لممدة7991 - 7001 ) (العددذ4ذ )ذ (كانونذاألول)ذ3102ذذ اجمللدذالثاني ذ اإلدالء بأصكاتيـ االنتخابية التي برزت بشكؿ كاضح بعد إحداث حرب عاـ7002 . ( جدكؿ2 ( ) تكزيع سكاف محافظة االنبار بحسب الفئات العمرية لممدة7991 - 7001 ) :المصدر7 - جميكرية العراؽ, ىيئة التخطيط, الجياز المركزم لإلحصاء السكاني , نتائج التعداد العاـ لمسكاف7991, لمػحاف ظة االنبار, ص19 . (العددذ4ذ )ذ (كانونذاألول)ذ3102ذذ اجمللدذالثاني ذ 7 -جميكرية العراؽ ,كزارة التخطيط كالتعاكف اإلنمائي , الجياز المركزم لإلحصاء كتكنكلكجي ا المعمكمات , تقديرات سكاف العراؽ لسنة7001 ( جدكؿ77 ), ص72 . الفئبث الؼوشيت 7991 7001 ركْس )%( إًبد )%( ركْس )%( إًبد )%( 0 – 4 99159 9,4 94997 9,7 757715 8,8 774124 8,4 9 – 9 89481 8,5 87027 8,0 770929 1,9 709779 1,0 70 – 74 10891 1,0 21298 2,2 95557 2,5 90097 2,7 الوجوْع 797019 74,2 744217 75,9 559929 77,2 570080 77,9 79 – 79 20242 2,0 98751 9,2 87789 9,9 19494 9,5 70 – 74 41588 4,2 48111 4,9 29242 4 ّ 1 21979 4,9 79 – 79 59107 4,0 58872 5,1 98999 4,0 91415 4.0 50 – 54 78991 5,0 79888 7,9 48521 5,5 48509 5,7 59 - 59 72018 7,2 70725 7,9 59012 7,2 59948 7,1 40 – 44 79792 7,0 79709 7,8 79844 7,0 57859 7,7 49 – 49 77478 7,7 77425 7,7 75421 7,2 79989 7,1 90 - 94 70999 7,0 9917 0,9 71818 7,7 79870 7,5 99 - 99 1147 0,1 2925 0,2 75121 0,9 79599 7,0 20 – 24 4847 0,4 9479 0,9 70579 0,2 77547 0,1 الوجوْع 741717 74,7 749278 74,5 595744 72,4 592259 72,1 29 – 29 9702 0,4 9497 0,9 1575 0,4 8710 0,9 10 – 14 5015 0,5 4790 0,4 4281 0,5 9995 0,5 19 – 19 7700 0,7 7159 0,5 7897 0,7 5270 0,7 80 فأكثش 5501 0,5 4755 0,4 5209 0,7 4971 0,5 الوجوْع الفئت 75982 7,5 72270 7,2 78491 7,7 77000 7,9 الوجوْع 977851 90,7 970899 49,9 141710 90,5 158179 49,1 هجوْع عكبى الوحبفظت 7075152 7489989 7991 :المصدر7 - جميكرية العراؽ, ىيئة التخطيط, الجياز المركزم لإلحصاء السكاني , نتائج التعداد العاـ لمسكاف7991, لمػحاف ظة االنبار, ص19 . 7 -جميكرية العراؽ ,كزارة التخطيط كالتعاكف اإلنمائي , الجياز المركزم لإلحصاء كتكنكلكجي ا المعمكمات , تقديرات سكاف العراؽ لسنة7001 ( جدكؿ77 ), ص72 . (العددذ4ذ )ذ (كانونذاألول)ذ3102ذذ اجمللدذالثاني ذ ج- إف ارتفاع شكؿ اليرـ السكاني تدر يجيان, يدؿ عمى أفَّ معدؿ المكاليد أعمى مف معدؿ .الكفيات عمى الرغـ مف الظركؼ التي نمر بيا العراؽ عامة كالمحافظة بشكؿ خاص د- ( ظير في شكؿ اليرـ السكاني تخصرا كاضحا في الفئة العمرية29 – 29 سنة ), مما يدؿ ذلؾ عمى كجكد حركة ىجرة سكانية مغادرة مف محافظة االنبار , نتي جة لمظركؼ الصعبة االقتصادية كاالجتماعية السيما ظركؼ الحرب التي مر بيا العراؽ عامة كالمحافظة بشكؿ خاص منيا حرب عاـ7980 - 7988 كما تبعيا مف حصار ) 707 ) ) 707 ) جملةذجامعةذاألنبارذللعلومذاإلندانوة ذ ذ (العددذ4ذ )ذ (كانونذاألول)ذ3102ذذذذذذذذذ اجمللدذالثاني ذ اقتصادم االمر الذم ادل الى ىجرة عدد مف الشباب القادريف عمى العمؿ الى بعض دكؿ الخميج كما تبعيا اخيرا مف احداث عاـ7002 . ق- أما بالنسبة لميرـ السكاني في الشكؿ الثاني فيدؿ عمى انتظاـ ارتفاعو بالتدرج عمى عدـ تأثر ن مك السكاف بحركة اليجرة المغادرة , كا نما بحركة ال يجرة الكافدة إلى محافظة االنبار , نتيجة لالستقرار األمني الن سبي, فضال إلى العامؿ االجتماعي. نسبة اإلعالة: جملةذجامعةذاألنبارذللعلومذاإلندانوة ذ ذ (العددذ4ذ )ذ (كانونذاألول)ذ3102ذذذذذذذذذ اجمللدذالثاني ذ اقتصادم االمر الذم ادل الى ىجرة عدد مف الشباب القادريف عمى العمؿ الى بعض دكؿ الخميج كما تبعيا اخيرا مف احداث عاـ7002 . ق- أما بالنسبة لميرـ السكاني في الشكؿ الثاني فيدؿ عمى انتظاـ ارتفاعو بالتدرج عمى عدـ تأثر ن مك السكاف بحركة اليجرة المغادرة , كا نما بحركة ال يجرة الكافدة إلى محافظة االنبار , نتيجة لالستقرار األمني الن سبي, فضال إلى العامؿ االجتماعي. نسبة اإلعالة: (العددذ4ذ )ذ (كانونذاألول)ذ3102ذ اجمللدذالثاني ذ (العددذ4ذ )ذ (كانونذاألول)ذ3102ذذ اجمللدذالثاني ذ ( 700 ( أ.م.د.ذحدنيذعليذعبدذ الراوي ذ ذ حتلولذجغرايفذلدميوغراػوةذاحلربذوأثرها علىذالرتكوبذالدكاني ذ ذ ( شكؿ7 ( ) يكضح تكزيع سكاف محافظة االنبار بحسب الفئات العمرية لممدة7991 – 7001 ) ( المصدر: باالعتماد عمى جدكؿ2 ) 0 20000 40000 60000 80000 100000 120000 140000 0 – 4 5 – 9 10 – 14 15 – 19 20 – 24 25 – 29 30 – 34 35- 39 40 – 44 45 – 49 50 -54 55 -59 60 – 64 65 – 69 70 – 74 75 – 79 80 فأكثر الفئات العمرية )نسمة ( عدد السكان 1997 ذكور1997 اناث2007 ذكور2007 اناث أ.م.د.ذحدنيذعليذعبدذ الراوي ذ ذ حتلولذجغرايفذلدميوغراػوةذاحلربذوأثرهاذ علىذالرتكوبذالدكاني ذ ذ ( شكؿ7 ( ) يكضح تكزيع سكاف محافظة االنبار بحسب الفئات العمرية لممدة7991 – 7001 ) ( كعند النظر إلى الشكؿ2 ) ( , 4 ) , الذم يمثؿ اليرـ السكاني لسكاف محاف ظة االنبار ( لممدة7991 - 7001 ), نالحظ ما يأتي : َ ( كعند النظر إلى الشكؿ2 ) ( , 4 ) , الذم يمثؿ اليرـ السكاني لسكاف محاف ظة االنبار ( لممدة7991 - 7001 ), نالحظ ما يأتي : أ- في عاـ7991 نالحظ أ ف قاعدة اليرـ السكاني عريضة َّ, مما يدؿ عمى أف المجتمع فتي شاب. أ- في عاـ7991 نالحظ أ ف قاعدة اليرـ السكاني عريضة َّ, مما يدؿ عمى أف المجتمع فتي شاب. أ- في عاـ7991 نالحظ أ ف قاعدة اليرـ السكاني عريضة َّ, مما يدؿ عمى أف المجتمع فتي شاب. ب- يدؿ عرض قاعدة اليرـ السكاني , عمى أفَّ نسبة إعالة ( فئة األطفاؿ0 - 74), مرتفعة كثيرا. ب- يدؿ عرض قاعدة اليرـ السكاني , عمى أفَّ نسبة إعالة ( فئة األطفاؿ0 - 74 ), مرتفعة كثيرا. ب- يدؿ عرض قاعدة اليرـ السكاني , عمى أفَّ نسبة إعالة ( فئة األطفاؿ0 - 74 ), مرتفعة كثيرا. نسبة اإلعالة: تثمؿ نسبة السكاف غير القادريف عمى العمؿ ( دكف سف79 سنة كأكثر مف19 سنة ,) ( عمى السكاف الذيف ىـ في سف العمؿ79 – 14 )سنة , مضركبا في مئة . كعمى افتراض تساكم الظركؼ االقتصادي ة كاالجتماعية في أم مجتمع سكاني , فاف ارتفاع نسبة السكاف المنتجيف فيو , يعد أفضؿ اقتصاديان مف الم ,جتمع الذم تقؿ نسبتيـ فيو كلقياس نسبة اإلعالة العمرية, ليا إبعاد اقتصادية ميمة , إذ تكشؼ لنا إمكانية تحمؿ السكاف المنتجيف أعباء إعالة السكاف الباقيف( مف الفئتيف الصغار دكف السف79 ) ( , ككبار السف أكثر مف19 )سنة , الذيف ىـ خارج قكة العػمؿ لتككف مؤشرا في دراسة تحميؿ قكة السكاف اقتصاديا. ( كلقياس نسبة اإلعالة لمحافظة االنبار, تـ احتسابيا اعتمادا عمى جدكؿ2 ) كتـ استخراج نسبة اإلعالة كفؽ الصيغ ة اآلتية : ك ؽ لإ آرج 491190 + 20791 آ 491190 + 20791 نسبة اإلعالة لعاـ7991 = ػػػػػػػػػػػػػػػػػػ ػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػ ػػػػػػػػػػػػػػػػ× 700 = 701 نسمة 491190 199149 + 40491 نسبة اإلعالة لعاـ7001 = ػػػػػػػػػػػػػ ػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػ ػػػػػػػػػػػػػػػػػػػ ػػػػػػػػػػػػػػػػػػػػػػػػ× 700 = 88 نسمة 819819 نسبة اإلعالة لعاـ7991 = ػ نسبة اإلعالة لعاـ7991 = ػ× 700 = 701 نسمة ػ× 700 = 701 نسمة 491190 199149 + 40491 نسبة اإلعالة لعاـ7001 = ػ 819819 كمف خالؿ تطبيؽ المعادلة الخاصة ب نسبة اإلعالة , يتبيف باف كؿ مئة شخص مف ال( ذيف في سف العمؿ يعيمكف701 نسمة), مف الذيف ىـ خارج سف العمؿ, كذلؾ حسب تعداد 7991, في حيف حسب تقديرات السكاف لعاـ7001 ( , بمغت نسبة اإلعالة نحك88 )نسمة. ( 707 ( أ.م.د.ذحدنيذعليذعبدذ الراوي ذ ذ حتلولذجغرايفذلدميوغراػوةذاحلربذوأثرهاذ علىذالرتكوبذالدكاني ذ ذا أ.م.د.ذحدنيذعليذعبدذ الراوي ثانيا: الترك يب النوعي لمسكان محافظة االنبار: ( ينقسـ السكاف بطبيعة الحاؿ إلى قسميف ذككرmale ) ( , كا ناثfemal ) , كيعبر عف ( نسبة النكعsix ratio ), بعدد الذككر لكؿ مئة مف اإلناث( 8 ) . إذ تعمؿ الظكاىر الديمكغرافية عمى خمؽ فركؽ بيف معدؿ الذككر كاإلناث, كتشير طبيعة نسبة النكع التي تؤثر بشكؿ مباشر عمى األنشطة االقتصادية في أم مجتمع سكاني( 9 ) . ففي الظركؼ االعتيادية يتفكؽ عدد الذ( ككر عمى اإلناث بمعدؿ بمغ4 - 9 % ), غير إف كفيات اإلناث اقؿ منيا في الذككر, أما في الظركؼ غير االعتيادي ة كالم تمثمة بالحرب كحركة اليجرة , فاف الييكؿ النكعي لمسكاف يختؿ ليصبح لصالح اإلناث, كذلؾ أل ف في حالة الحرب كحركة اليجرة , غالبا ما يشمالف ( الذككر دكف اإلناث70 ). نسبة اإلعالة: مما يؤدم في قمة في نػػسبة النػػكع في عػػمكـ سكػػاف محافظة ,االنبار ( كمف معطيات جدكؿ4 ( ) كشكؿ9 ), يتضح اف نسبة النكع تتبايف حػػسب كحػػػػداتيا ( اإلدارية)االقضية , إذ بمغت( نسبة النكع في المحافظة700,2 )%, كك اف ىناؾ تبايف عمى مستكل االقضية , إذ بمغ اعمى ن( سبة لمنكع في قضاء الرطبة702 )% , حسب تعداد7991 بسبب الظركؼ التي يعيشيا مف استتبا ب .األمف كاالستقرار ) 702 ) ب ي ي ي ي ف ب رر أل ف كال ) 702 ) جملةذجامعةذاألنبارذللعلومذاإلندانوة ذ ذ (العددذ4ذ )ذ (كانونذاألول)ذ3102ذذذذذذذذذذذذذ اجمللدذالثاني ذ (العددذ4ذ )ذ (كانونذاألول)ذ3102ذذ اجمللدذالثاني ذ (العددذ4ذ )ذ (كانونذاألول)ذ3102ذذ اجمللدذالثاني ذ جملةذجامعةذاألنبارذللعلومذاإلندانوة ( 704 ( أ.م.د.ذحدنيذعليذعبدذ الراوي ذ ذ حتلولذجغرايفذلدميوغراػوةذاحلربذوأثرهاذ علىذالرتكوبذالدكاني ذ ذ ( شكؿ9 ) يكضح نسبة النكع لسكاف محافظة االنبار بحسب ك( حداتيا اإلدارية لممدة7991 – 7001 ) ( المصدر: باالعتماد عمى جدكؿ4 ) كاقؿ ن( سبة لمنكع في قضاء الفمكجة99 )%, السبب ي عكد الى عامؿ حػػركة ىجرة السكاف, غالبان ما يفضؿ الذككر اليجرة اكثر مف االناث اما إلكماؿ الدراسة اك لتحسيف ظركؼ المعيشة كىي خاصية مف خصائص السكاف المياجريف , كما أف انخفاض نسبة النكع لو أثار سمبية مف الناحية االقتصادية خاصة كاف األيدم العاممة في مختمؼ القطاعات االقتصادية تعت مد عمى العمالة الذكرية بشكؿ خاص. نسبة اإلعالة: إذ إف بعض السكاف ال يعطكف معمكمات صحيحة عف عدد الذككر خكفا مف انخراط أبنائيـ في الخدمة العسكرية اإللزامية التي كانت في مدة ما قبؿ االحتالؿ األمريكي لمعراؽ معمكالن بيا كخاصة خالؿ التعداد العاـ لمسكاف عاـ7991 . كمما تقدـ يتضح باف زيادة النكع في المجتمع السكاني يعد عامال أساسيا في دعـ قكة الدكلة اقتصاديا كعسكريا. :ثالثا: التركيب االقتصادي (العددذ4ذ )ذ (كانونذاألول)ذ3102ذ اجمللدذالثاني ذ ت - ( األخطاء البيانية لمتعداد العاـ لمسكاف77 ). إذ إف بعض السكاف ال يعطكف معمكمات صحيحة عف عدد الذككر خكفا مف انخراط أبنائيـ في الخدمة العسكرية اإللزامية التي كانت في مدة ما قبؿ االحتالؿ األمريكي لمعراؽ معمكالن بيا كخاصة خالؿ التعداد العاـ لمسكاف عاـ7991 . كمما تقدـ يتضح باف زيادة النكع في المجتمع السكاني يعد عامال أساسيا في دعـ قكة الدكلة اقتصاديا كعسكريا. الدكلة اقتصاديا كعسكريا. نسبة اإلعالة: أ( ما في تقديرات السكاف لعاـ7001 ), نجد بقي قضاء الرطبة بالمرتبة األكلى مف حيث ( نسبة النكع البالغة702 )%, كذلؾ قضاء الفمكجة الذم ظؿ بالمرتبة األخيرة, إذ بمغت 97% 98% 99% 100% 101% 102% 103% 104% الفلوجه .ق الرمادي .ق هيت .ق حديثة .ق عنه .ق راوه .ق القائم.ق الرطبة .ق الوحدة االدارية % نسبة النوع 1997 نسبة النوع2007 نسبة النوع أ.م.د.ذحدنيذعليذعبدذ الراوي ذ ذ حتلولذجغرايفذلدميوغراػوةذاحلربذوأثرهاذ علىذالرتكوبذالدكاني ذ ذ ( شكؿ9 ) يكضح نسبة النكع لسكاف محافظة االنبار بحسب ك( حداتيا اإلدارية لممدة7991 – 7001 ) ( المصدر: باالعتماد عمى جدكؿ4 ) 97% 98% 99% 100% 101% 102% 103% 104% الفلوجه .ق الرمادي .ق هيت .ق حديثة .ق عنه .ق راوه .ق القائم.ق الرطبة .ق الوحدة االدارية % نسبة النوع 1997 نسبة النوع2007 نسبة النوع ( شكؿ9 ) يكضح نسبة النكع لسكاف محافظة االنبار بحسب ك( حداتيا اإلدارية لممدة 7001 ) كاقؿ ن( سبة لمنكع في قضاء الفمكجة99 )%, السبب ي عكد الى عامؿ حػػركة ىجرة السكاف, غالبان ما يفضؿ الذككر اليجرة اكثر مف االناث اما إلكماؿ الدراسة اك لتحسيف ظركؼ المعيشة كىي خاصية مف خصائص السكاف المياجريف , كما أف انخفاض نسبة النكع لو أثار سمبية مف الناحية االقتصادية خاصة كاف األيدم العاممة في مختمؼ القطاعات االقتصادية تعت مد عمى العمالة الذكرية بشكؿ خاص. أ( ما في تقديرات السكاف لعاـ7001 ), نجد بقي قضاء الرطبة بالمرتبة األكلى مف حيث ( نسبة النكع البالغة702 )%, كذلؾ قضاء الفمكجة الذم ظؿ بالمرتبة األخيرة, إذ بمغت ( ,نسبة النكع فيو99 )%, كتتأثر عادة نسبة النكع بالمجتمعات السكانية , سكاء أكانت زيادة أـ نقصاف بمجمكعة مف العكامؿ منيا ما يأتي: أ - حركة اليجرة الكافدة كالمغادرة لكال الذككر ك اإلناث , إذ تعمؿ حركة اليجرة الكافدة عمى زيادة النكع كبالعكس تؤدم حركة اليجرة المغادرة إلى قمتيا. ب - تبايف معدؿ الكفيات لكال النكعيف مف الجنس الذككر كاإلناث , كبحسب الظر كؼ التي يمر بيا المجتمع السكاني , فعمى سبيؿ المثاؿ تؤد م الحركب إلى زيادة كفيات الذككر دكف اإلناث , فضالن عف الحكادث المركرية ككذلؾ األمراض كاألكبئة كغيرىا كخاصة ما تعرض لو قضاء الفمكجة مف آثار معركتيف مف قبؿ القكات األمريكية المحتمة بعد عاـ7002 . ) 709 ) ) 709 ) جملةذجامعةذاألنبارذللعلومذاإلندانوة ذ ذ (العددذ4ذ )ذ (كانونذاألول)ذ3102ذذذذذذذذذ اجمللدذالثاني ذ ت - ( األخطاء البيانية لمتعداد العاـ لمسكاف77 ). :ثالثا: التركيب االقتصادي إف دراسة التركيب االقتصادم لمسكاف , ييدؼ إلى معرفة السكاف النشطيف اقتصاديا كغير النشطيف اقتصاديا كالقكل العاممة , الذم يعد أساسان لكضع خطط المستقبؿ سكا ء في مشركعات التنمية االقتصادية , أـ في مجاؿ( الخدمة االجتماعية77 ). كما يعد السكاف النشطيف اقتصادي ا القكل العاممة ألم مجتمع سكاني , كيندرج في إطار القكل البشرية لمسكاف الذيف تجاكزا عمرا محددا , بحيث يمكف أف تستغؿ قكتيـ البدنية كالفعمية في العمؿ كاإلنتاج ( كتتراكح أعمارىـ بيف79 – 19 )سنة , مف الجد( كؿ9 ), نالحظ أف عدد السكاف الن شطيف اقتصاديا في محافظة االنبار( , بمغ729708 )نسمة( , كىـ يشكمكف نسبة72,2 ) مف% إجمالي س كاف المحافظة البالغ7072121نسمة , إذ إف إجمالي الذككر بالنسبة لمسك اف ( الحضر كالريؼ بمغ770799 نسمة( ), كبنسبة9 . 77 )%, مف إجمالي عدد س كاف المحافظة لعاـ7991, في ( حيف بمغ عدد اإلناث78909 )نسمة , كبنسبة ( 7,8 ) مف% إجمالي سكاف المحافظة. كما تتبايف أيضا نسبة السكاف النشطي ف اقتصاديان فيما بيف الحضر كالريؼ , فمف ( الجدكؿ9 ) ( كالشكؿ1 )أ ك ب , نالحظ ارتفاع نسبة السكاف النشطيف اقتصاديان التي بمغت ( 1 . 90) بالنسب%ة لمسكاف الحضر( , بينما في الريؼ بمغت2 . 49 )%, مف إجم الي عدد السكاف النشطيف اقتصاديا , أما بالنسبة لمسكاف غير النشطي ف( , فقد بمغ مجمكعيـ970724 )نسمة( , مشكميف نسبة1 . :ثالثا: التركيب االقتصادي 90 ) مف إجمالي%عدد سكاف محافظة االنبار , كبناءن عمى ما تقدـ يمكف أف نستخرج معدؿ اإلعالة االقتص ا دية لمسكاف كفؽ المعادلة اآلتية: ( 701 ( أ.م.د.ذحدنيذعليذعبدذ الراوي ذ ذ حتلولذجغرايفذلدميوغراػوةذاحلربذوأثرهاذ علىذالرتكوبذالدكاني ذ ذ )مجمكع السكف غير النشطيف اقتصاديا (سبع سنكات فأكثر معدؿ اإلعالة االقتصادية =ػػػػػػػػػػػػػػػػ ػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػ ػػػػػػػػػػػػػػػػػػػػػػػػػػػ ػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػ× 700 )مجمكع السكف النشطيف اقتصاديا (سبع سنكات فأكثر 970724 = ػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػ× 700 = 771 نسمة 729708 729708 ( جدكؿ9) ت كزيع السكاف( في سف1سنكات فاكثر ) بحسب الفئات العمرية كالجنس كالنشطيف اقتصاديا كالبيئة في محافظة االنبار لعاـ7991 ( جدكؿ9) ت كزيع السكاف( في سف1سنكات فاكثر ) بحسب الفئات العمرية كالجنس كالنشطيف اقتصاديا كالبيئة في محافظة االنبار لعاـ7991 البيئت الفئبث الؼوشيت (عٌت)/الجٌظ الغكبى الٌشطيي الخصبديب )(ًغوت ) هي%( هجوْع الٌشطيي ًغبخِن هي عكبى الوحبفظت الغكبى غيش الٌشطيي الخصبديب ) هي%( هجوْع غيش الٌشطيي ًغبخِن هي عكبى الوحبفظت حضش 1 - 74 9991 7,5 0,9 772875 77,4 77,4 79 – 24 777872 71,7 70,9 791997 50,7 79,4 29 فأكثش 7102 7,7 0,5 77109 7,4 7,7 غيش هبيي 97 0,05 0,07 745 0.07 0,07 الوجوْع 777770 90,1 - 781792 99,7 78,0 ركْس 777751 42,8 70,9 94799 78,7 9,7 إًبد 9085 5,8 0,9 795727 51,7 78,8 الوجوْع 777770 90,1 77,8 781792 99,7 78,0 سيف 1 - 74 75999 9,8 7,4 91054 78,2 9,4 79 – 24 99957 47,2 9,1 779881 74,7 77,5 29 فأكثش 4744 7,8 0,4 9904 4,7 0,9 غيش هبيي 775 0.04 0,07 795 0,05 0,07 الوجوْع 771888 49,5 - 718792 99,7 77,1 ركْس 708027 49,7 70,9 29079 77,4 2,5 إًبد 9872 4,7 0,7 721999 57,5 72,4 الوجوْع 771888 49,5 77,9 757918 44,8 77,1 الوجوْع الكلي للوحبفظت 759708 700 % 75,5 970754 700 90,8 المصدر: جميكرم,العراؽ ىيئة التخطيط , الجياز المركزم لإلحصاء , مديرية اإلحصاء السكاني, نتائج ,التعداد العاـ لمسكاف 7998 ,, لمحافظة االنبار7998 ( , جدكؿ77 ), ص777 . ( جدكؿ9) ت كزيع السكاف( في سف1سنكات فاكثر ) بحسب الفئات العمرية كالجنس كالنشطيف اقتصاديا كالبيئة في محافظة االنبار لعاـ7991 المصدر: جميكرم,العراؽ ىيئة التخطيط , الجياز المركزم لإلحصاء , مديرية اإلحصاء السكاني, نتائج ,التعداد العاـ لمسكاف 7998 ,, لمحافظة االنبار7998 ( , جدكؿ77 ), ص777 . :ثالثا: التركيب االقتصادي ) 701 ) جملةذجامعةذاألنبارذللعلومذاإلندانوة ذ ذ (العددذ4ذ )ذ (كانونذاألول)ذ3102ذذذذذذذذ اجمللدذالثاني ذ ( شكؿ1 ( أ) يكضح تكزيع السكاف في سف1 سنكات فأكثر) حسب الفئات العمرية النشطيف اقتصاديا لسكاف حضر محافظة االنبار لعاـ7991 ( المصدر: باالعتماد عمى جدكؿ9 ) ( شكؿ1 ب) يكضح تكز( يع السكاف في سف1 سنكات فاكثر ) حسب الفئات العمرية النشطيف اقتصاديا لسكاف ريؼ محافظة االنبار لعاـ7991 ( المصدر : باالعتماد عمى جدكؿ9 ) بمعنى أف كؿ مئة مف السكاف ال( نشطيف اقتصاديا يعيمكف771 )شخصا , مف السكاف غير النشطيف اقتصاديان كيعد ىذا المعدؿ كبيران نسبيا كلو لثار سمبية عم ى قكة العمؿ داخؿ 1997 السكان الحضر النشطين اقتصاديا لعام 0 20000 40000 60000 80000 100000 120000 140000 160000 180000 5 – 14 15 – 64 65 فأكثر الفئات العمرية عدد السكان النشطين اقتصاديا غير النشطين اقتصاديا 1997 السكان الريف النشطين اقتصاديا لعام 0 20000 40000 60000 80000 100000 120000 140000 5 – 14 15 – 64 65 فأكثر الفئات العمرية عدد السكان النشطين اقتصاديا غير النشطين اقتصاديا ملةذجامعةذاألنبارذللعلومذاإلندانوة ذ ذ (العددذ4ذ )ذ (كانونذاألول)ذ3102ذذذذذ اجمللدذالثاني ذ ( شكؿ1 ( أ) يكضح تكزيع السكاف في سف1 سنكات فأكثر) حسب الفئات العمرية النشطيف اقتصاديا لسكاف حضر محافظة االنبار لعاـ7991 ( المصدر: باالعتماد عمى جدكؿ9 ) 1997 السكان الحضر النشطين اقتصاديا لعام 0 20000 40000 60000 80000 100000 120000 140000 160000 180000 5 – 14 15 – 64 65 فأكثر الفئات العمرية عدد السكان النشطين اقتصاديا غير النشطين اقتصاديا (العددذ4ذ )ذ (كانونذاألول)ذ3102ذذ اجمللدذالثاني ذ ( 708 ( ( شكؿ1 ب) يكضح تكز( يع السكاف في سف1 سنكات فاكثر ) حسب الفئات العمرية النشطيف اقتصاديا لسكاف ريؼ محافظة االنبار لعاـ7991 ( المصدر : باالعتماد عمى جدكؿ9 ) بمعنى أف كؿ مئة مف السكاف ال( نشطيف اقتصاديا يعيمكف771 )شخصا , مف السكاف غير النشطيف اقتصاديان كيعد ىذا المعدؿ كبيران نسبيا كلو لثار سمبية عم ى قكة العمؿ داخؿ محافظة االنبار , كبما إف المرأة تمثؿ نصؼ المجتمع السكا ني , فاف انخراطيا في إطار العمؿ , 1997 السكان الريف النشطين اقتصاديا لعام 0 20000 40000 60000 80000 100000 120000 140000 5 – 14 15 – 64 65 فأكثر الفئات العمرية عدد السكان النشطين اقتصاديا غير النشطين اقتصاديا ( شكؿ1 ب) يكضح تكز( يع السكاف في سف1 سنكات فاكثر ) حسب الفئات العمرية النشطيف اقتصاديا لسكاف ريؼ محافظة االنبار لعاـ7991 ( المصدر : باالعتماد عمى جدكؿ9 ) 1997 السكان الريف النشطين اقتصاديا لعام 0 20000 40000 60000 80000 100000 120000 140000 5 – 14 15 – 64 65 فأكثر الفئات العمرية عدد السكان النشطين اقتصاديا غير النشطين اقتصاديا ( 708 ( بمعنى أف كؿ مئة مف السكاف ال( نشطيف اقتصاديا يعيمكف771 )شخصا , مف السكاف غير النشطيف اقتصاديان كيعد ىذا المعدؿ كبيران نسبيا كلو لثار سمبية عم ى قكة العمؿ داخؿ محافظة االنبار , كبما إف المرأة تمثؿ نصؼ المجتمع السكا ني , فاف انخراطيا في إطار العمؿ , ( 708 ( أ.م.د.ذحدنيذعليذعبدذ الراوي ذ ذ حتلولذجغرايفذلدميوغراػوةذاحلربذوأثرهاذ علىذالرتكوبذالدكاني ذ ذ الذم يعد مساىمة فعالة في دا خؿ المجتمع الس كاني لمنطقة البحث , كىي نسبة مساىمة المرأة ضعيفة جدا بسبب الكاقع العشائرم المعركؼ ضمف محافظة ا النبار بشكؿ خاص , ففي حالة استغالؿ األيدم العاممة األنثكية في األعماؿ اإلدارية كالكظائؼ الخدمية سيؤدم إلى تحسف الكضع االقتصادم بشكؿ عاـ( 74 ) . :ثالثا: التركيب االقتصادي التركيب المهني لسكان م حافظة االنبار: أ.م.د.ذحدنيذعليذعبدذ الراوي ذ ذ حتلولذجغرايفذلدميوغراػوةذاحلربذوأثرهاذ علىذالرتكوبذالدكاني ذ ذ الذم يعد مساىمة فعالة في دا خؿ المجتمع الس كاني لمنطقة البحث , كىي نسبة مساىمة المرأة ضعيفة جدا بسبب الكاقع العشائرم المعركؼ ضمف محافظة ا النبار بشكؿ خاص , ففي حالة استغالؿ األيدم العاممة األنثكية في األعماؿ اإلدارية كالكظائؼ الخدمية سيؤدم إلى تحسف الكضع االقتصادم بشكؿ عاـ( 74 ) . التركيب المهني لسكان م حافظة االنبار: أ.م.د.ذحدنيذعليذعبدذ الراوي التركيب المهني لسكان م حافظة االنبار: يعد مكضكع تكزيع السكاف عبر المسرح الجغرافي سكاء عمى مستكل الدكلة أك أقاليميا, أك حسب الدكلة ككحداتيا اإلدارية كمنيا محافظة االنبار, عمى درجة كبيرة مف األىمية لمكقكؼ عمى معالـ االنتشار السكاني كمدل التجانس كالتبايف في تكزيع السكاف ىذا مف جية كمف جية أخرل ضر ,كرة معرفة القكل العاممة حسب الدكلة ككحداتيا اإلدارية لتكطيف مختمؼ المشاريع اإلنمائية كالخدمية كاستصالح المجاؿ الذم يشكؿ احد جكانب الضعؼ في انتشار السكاف بعدِّ السكاف احد العناصر األساسية الداخمة في استراتجيات التنمية االقتصاد ية, كتعرؼ القكل العاممة البشري ة , عمى أنَّيا جزء مف السكاف يمكف استثماره في النشاط االقتصادم( 79 ). أما النشاط االقتصادم فيك المجاؿ الذم يمارس فيو ال فرد عممة , كذلؾ تزاكلو المنشآت حيث يعمؿ الفرد( 71 ) . كمما الشؾ فيو إف القكل العاممة تمثؿ احد العناصر الميمة في تقدير الكزف االقتصادم لمدكلة كنال( حظ مف معطيات الجدكؿ1 ), إف النشاط االقتصادم ين قسـ عادة إلى ثالثة قطاعات رئيسة , إذ يتكزع جزء مف سكاف المحاف ظة عمى تمؾ القطاعات بنسب مختمفة. ( فمف خالؿ الجدكؿ1 ( ) كالشكؿ1 ), نالحظ أف عد د العامميف في قطاع الزراعة الذم يمثؿ العامميف بالزراعة كالصيد كىي الحرفة األساسية ا لتي يزاكليا سكاف محافظة ( االنبار, اذ بمغ عددىـ17729 ( نسمة), كشكمكا نسبة71 )% , مف مجمكع القكل العاممة في ( محافظة االنبار, البالغة729708 )نسمة( , كبنسبة7,2 ) مف مجمكع القكل العاممة في% العراؽ كالبالغ عددى( ـ4,817094 نسمة) حسب تعداد7991, كفي تمؾ ال مدة شجعت الحككمة العراقية آنذاؾ السكاف عمى تكثيؼ الجيكد الستغالؿ األراضي الزراعية مف خالؿ تقديـ المنح كالقركض المالي ة كتكفير األسمدة كالبذكر كغبرىا. التركيب المهني لسكان م حافظة االنبار: أما القطاع الصناعي الذم يشمؿ العامميف في التعديف كالمحاجر كالصناعات التحكيمي ة كالغاز كالكيرباء كخدمات الماء , فضال عف التشيي د كالبناء , إذ بمغ مجمكعيـ ) 709 ) جملةذجامعةذاألنبارذللعلومذاإلندانوة ذ ذ (العددذ4ذ )ذ (كانونذاألول)ذ3102ذذذذذذذذذ اجمللدذالثاني ذ ( 74121 )نسمة , كشكمك ا ( نسبة7,7) مف مجمكع الق%كل العاممة في منطقة الدراسة , في ( حيف بمغت نسبتيـ0,9) مف إجمالي ا% لقكل العاممة في العراؽ عاـ7991 , كيعكد قمة العامميف في الصناعة إلى سياسة الدكلة آنذاؾ, رغـ الدعـ المادم ليذا القطاع , إال إف إعداد ال ككادر المؤىمة لمعمؿ في الصناعة, تعد قمػػيمة نسبيان , إذ إف اغمب العامميف في ىذا القطاع ىـ مف غير المؤىميف عمميا , لذا فاف أجرة الػػػعامؿ تككف قمػػيمة مقا رنة بباقي القطػػاعات االقتصادية , فضال عف قمة استخداـ تكنكلكجيا المعمكمات في إدارة المصانع التي تعيؽ عممية التطكر الصناعي , ففي المدة نفسيا أىممت الحككمة الجانب الصناعي بسبب تكقؼ استيراد المكاد األكلية , عدا الصناعات التي تككف مكادىا األكلية محمية, حيث انصب اىتماـ الحككمة .عمى الجانب الزراعي لسد حاجة السكاف مف الغذاء اأا (العددذ4ذ )ذ (كانونذاألول)ذ3102ذذذ اجمللدذالثاني ذ اأا 7991 المطبع الٌشبط االلخصبدي ػذد الؼبهليي )(ًغوت ًْٓغبخِن هي الم الؼبهلت )%( في الوحبفظت ًْٓغبخِن هي الم الؼبهلت )%( في الؼشاق الضساػت الضساػت ّالصيذ 27759 72 7,5 الوجوْع الوجوْع 27759 72 % 7,5 الصٌبػي الخؼذيي ّالوحبجش 7187 7,7 0,09 الصٌبػت الخحْيليت 8770 5,4 0,7 الكِشببء ّالغبص ّالوبء 7091 0,4 0,07 الخشيي ذ ّالبٌبء 77949 9,7 0,7 الوجوْع 74251 70,7 % 0,9 الخجبسي حجبسة الجولت ّالوفشد ّالوطبػن ّالفٌبدق 55444 75,9 0,1 الٌمل ّالوْاصالث ّالخخضيي 71504 1,7 0,5 الخحْيل ّالخأهيي ّالؼمبساث 142 0,5 0,07 خذهبث الوجخوغ 20890 79,9 7,7 الوجوْع 777584 41 % 5,7 غيش هبيي 459 0,5 0,07 الؼبطليي الزيي لن يغبك لِن الؼول 59475 72,9 0,8 الوجوْع الكلي 759708 700 % 9,9 المصدر: جميكرم العراؽ, ىيئة التخطيط, الجياز المركزم لإلحصاء , مديرية اإلحصاء السكاني , نتائج التعداد العاـ لمسكاف, لمحافظة االنبار , 7991, لمحافظة االنبار , 7998 ,جدكؿ , ص729 المصدر: جميكرم العراؽ, ىيئة التخطيط, الجياز المركزم لإلحصاء , مديرية اإلحصاء السكاني , نتائج التعداد العاـ لمسكاف, لمحافظة االنبار , 7991, لمحافظة االنبار , 7998 ,جدكؿ , ص729 ( 770 ( أ.م.د.ذحدنيذعليذعبدذ الراوي ذ ذ حتلولذجغرايفذلدميوغراػوةذاحلربذوأثرهاذ علىذالرتكوبذالدكاني ذ ذ ( شكؿ1 ( ) تكزيع القكل العاممة لسكاف محافظة االنبار نسمة) عمى االنشطة االقتصادية لعاـ7991 ( المصدر: باالعتماد عمى جدكؿ1 ) 0 10000 20000 30000 40000 50000 60000 70000 الزراعة والصيد التعدين والمحاجر الصناعة التحويلية الكهرباء والغاز والماء التشييد والبناء تجارة الجملة والمفرد والمطاعم والفنادق النقل والمواصالت والتخزين التحويل والتأمين والعقارات خدمات المجتمع غير مبين العاطلين الذين لم يسبق لهم العمل النشاط االقتصادي عدد السكان أ.م.د.ذحدنيذعليذعبدذ الراوي ذ ذ حتلولذجغرايفذلدميوغراػوةذاحلربذوأثرهاذ علىذالرتكوبذالدكاني ذ ذ ( شكؿ1 ( ) تكزيع القكل العاممة لسكاف محافظة االنبار نسمة) عمى االنشطة االقتصادية لعاـ7991 ( شكؿ1 ( ) تكزيع القكل العاممة لسكاف محافظة االنبار نسمة) عمى االنشطة االقتص لعاـ7991 ( المصدر: باالعتماد عمى جدكؿ1 ) 0 10000 20000 30000 40000 50000 60000 70000 الزراعة والصيد التعدين والمحاجر الصناعة التحويلية الكهرباء والغاز والماء التشييد والبناء تجارة الجملة والمفرد والمطاعم والفنادق النقل والمواصالت والتخزين التحويل والتأمين والعقارات خدمات المجتمع غير مبين العاطلين الذين لم يسبق لهم العمل النشاط االقتصادي عدد السكان أما القطاع اآلخر المتمثؿ بالقطاع التجارم الذم يشمؿ تجارة ا لجممة كالمفرد كالمطاعـ كالفنادؽ , كالنقؿ كالمكاصالت كا لتخزيف كخدمات التمكيؿ كالعقارات, فضال عف خدمات المجتمع األخرل , اذ بمغ( مجمكع العامميف777284 ) نسمة ك( شكمكا41 % ) مف مجمكع القكل العاممة في محافظة االنبار( , في حيف شكمكا2,7) مف% إجمالي القكل العاممة في العراؽ , كيعكد السبب في ارتفاع نسبة العامميف في ىذا النشاط االقتصادم إلى مشاركة القطاع الخاص كالمختمط الستثمار الخدمات في المح افظة إذ إف اغمب سكاف المحافظة يزاكلكف أعماؿ التجارة كذلؾ الف محافظة االنبار تعد مف المحافظات الحدكدية التي تشترؾ بالحدكد مع ثالث دكؿ عربية مف جية الغرب فضال عف أف المردكد االقتصادم لمقطاع التجارم أعمى نس بيا مف باقي القطاعات االقتصادية. التركيب المهني لسكان م حافظة االنبار: ) 777 ) جملةذجامعةذاألنبارذللعلومذاإلندانوة ذ ذ (العددذ4ذ )ذ (كانونذاألول)ذ3102ذذذذذذذذذذذذذ اجمللدذالثاني ذ كما يمكف تكزيع القكل العاممة البشرية في محافظة االنبار بحسب كحداتيا اإلدا رية (االقضية ) ليكشؼ لنا مدل مساىمة أم مف الكحدات اإلدارية في ضمنيا القكل العاممة أكثر مف غيرىا مف الكحدات اإلدارية األخرل كبياف أسباب ذلؾ , فمف خالؿ مالحظ( ة الجدكؿ1 ) ( كالشكؿ8 ,) يتضح أف قضاء الفمكجة استحكذ عمى المرتبة األكلى مف حيث عدد العامميف فيو ,كبمختمؼ القطا عات الزراعية كالصناعية كالخدمات( , إذ بمغ مجمكعيـ14719 ) نسمة ( كبنسبة27 ) مف أجمالي%القكل العاممة في محافظة االنبار , بينما جاء قضاء الرمادم ( بالمرتبة الثانية اذ بمغ عدد العامميف10244 ( ) نسمة كشكمكا نسبة79,4) مف% مجمكع عدد العامميف في المحافظة , ثـ يأتي قضاء القائـ بالمرتبة الثالثة إذ بمغ عدد العامميف فيو ( 78177) نسمة كبنسبة ( 1.8) مف إجمالي القكل العاممة% , يميو قضاء ىيت بالمرتبة ( الرابعة بعدد العامميف البالغ71271 ( ) نسمة ليشكمكا نسبة1.8) مف إ% جمالي القكل العاممة في المحافظة , ثـ جاء قضاء حديثة ب( المرتبة الخامسة بعدد العامميف البالغ9007 )نسمة , ( كبنسبة4) كثـ جاء كؿ مف أقضية الرط%بة كعنو ك راكه بالمراتب األخيرة , إذ بمغت بنسبة ( العامميف7,1ك7,4ك7,2 )% , عمى التكالي, كذلؾ حسب تعداد عاـ7991 , إذ يتزايد األعباء االقتصادية كاالجتماعية عمى الفئات العمرية التي تندرج تحت تصنيؼ فئة الشباب , إذ تككف الطاقة الميدرة مف عنصر العمؿ أكثر كفاية قدرة عمى العمؿ , في حيف أف البطا لة ليذه الفئة )العمرية (الكسيطة , يخمؽ حالة العنؼ كالجريمة بحكـ الخبرة الحياتية المحددة كاإلحباط الذم جملةذجامعةذاألنبارذللعلومذاإلندانوة ذ ذ (العددذ4ذ )ذ (كانونذاألول)ذ3102ذذذذذذذ اجمللدذالثاني ذ (العددذ4ذ )ذ (كانونذاألول)ذ3102ذ اجمللدذالثاني ذ جملةذجامعةذاألنبارذللعلومذاإلندانوة كما يمكف تكزيع القكل العاممة البشرية في محافظة االنبار بحسب كحداتيا اإلدا رية (االقضية ) ليكشؼ لنا مدل مساىمة أم مف الكحدات اإلدارية في ضمنيا القكل العاممة أكثر مف غيرىا مف الكحدات اإلدارية األخرل كبياف أسباب ذلؾ , فمف خالؿ مالحظ( ة الجدكؿ1 ) ( كالشكؿ8 ,) يتضح أف قضاء الفمكجة استحكذ عمى المرتبة األكلى مف حيث عدد العامميف فيو ,كبمختمؼ القطا عات الزراعية كالصناعية كالخدمات( , إذ بمغ مجمكعيـ14719 ) نسمة ( كبنسبة27 ) مف أجمالي%القكل العاممة في محافظة االنبار , بينما جاء قضاء الرمادم ( بالمرتبة الثانية اذ بمغ عدد العامميف10244 ( ) نسمة كشكمكا نسبة79,4) مف% مجمكع عدد العامميف في المحافظة , ثـ يأتي قضاء القائـ بالمرتبة الثالثة إذ بمغ عدد العامميف فيو ( 78177) نسمة كبنسبة ( 1.8) مف إجمالي القكل العاممة% , يميو قضاء ىيت بالمرتبة ( الرابعة بعدد العامميف البالغ71271 ( ) نسمة ليشكمكا نسبة1.8) مف إ% جمالي القكل العاممة في المحافظة , ثـ جاء قضاء حديثة ب( المرتبة الخامسة بعدد العامميف البالغ9007 )نسمة , ( كبنسبة4) كثـ جاء كؿ مف أقضية الرط%بة كعنو ك راكه بالمراتب األخيرة , إذ بمغت بنسبة ( العامميف7,1ك7,4ك7,2 )% , عمى التكالي, كذلؾ حسب تعداد عاـ7991 , إذ يتزايد األعباء االقتصادية كاالجتماعية عمى الفئات العمرية التي تندرج تحت تصنيؼ فئة الشباب , إذ تككف الطاقة الميدرة مف عنصر العمؿ أكثر كفاية قدرة عمى العمؿ , في حيف أف البطا لة ليذه الفئة )العمرية (الكسيطة , يخمؽ حالة العنؼ كالجريمة بحكـ الخبرة الحياتية المحددة كاإلحباط الذم تكلده حالة التعطؿ التي تصدـ كؿ طمكحات التحق ؽ لمشباب( 79 ). التركيب المهني لسكان م حافظة االنبار: أما القطاع اآلخر المتمثؿ بالقطاع التجارم الذم يشمؿ تجارة ا لجممة كالمفرد كالمطاعـ كالفنادؽ , كالنقؿ كالمكاصالت كا لتخزيف كخدمات التمكيؿ كالعقارات, فضال عف خدمات المجتمع األخرل , اذ بمغ( مجمكع العامميف777284 ) نسمة ك( شكمكا41 % ) مف مجمكع القكل العاممة في محافظة االنبار( , في حيف شكمكا2,7) مف% إجمالي القكل العاممة في العراؽ , كيعكد السبب في ارتفاع نسبة العامميف في ىذا النشاط االقتصادم إلى مشاركة القطاع الخاص كالمختمط الستثمار الخدمات في المح افظة إذ إف اغمب سكاف المحافظة يزاكلكف أعماؿ التجارة كذلؾ الف محافظة االنبار تعد مف المحافظات الحدكدية التي تشترؾ بالحدكد مع ثالث دكؿ عربية مف جية الغرب فضال عف أف المردكد االقتصادم لمقطاع التجارم أعمى نس بيا مف باقي القطاعات االقتصادية. بناء ن عمى ما تقدـ نجد أف طابع السياسة االقتصادية يغمب عميو نمط االستيالؾ أكثر مف نمط اإلنتاج كاالعتماد عمى االستيراد لسد االحتياجات المحمية كىك تكريس إلحدل السمات السمبية في السياسة االقتصادية( 71 ) التي احتؿ فييا القطاع العاـ األىمية األساس في عممية التنمية االقتصادية المنش كدة عمى كفؽ االستراتيجيات التي تبنتيا الحككمة العراقية آنذاؾ التي أثبتت انخفاض الكفاءة اإلنتاجية لقطاعات األنشطة االقتصادية( 78 ) بسبب ظركؼ الحرب كالحصار االقتصادم كآ خرىا حرب عاـ7002 . بناء ن عمى ما تقدـ نجد أف طابع السياسة االقتصادية يغمب عميو نمط االستيالؾ أكثر مف نمط اإلنتاج كاالعتماد عمى االستيراد لسد االحتياجات المحمية كىك تكريس إلحدل السمات السمبية في السياسة االقتصادية( 71 ) التي احتؿ فييا القطاع العاـ األىمية األساس في عممية التنمية االقتصادية المنش كدة عمى كفؽ االستراتيجيات التي تبنتيا الحككمة العراقية آنذاؾ التي أثبتت انخفاض الكفاءة اإلنتاجية لقطاعات األنشطة االقتصادية( 78 ) بسبب ظركؼ الحرب كالحصار االقتصادم كآ خرىا حرب عاـ7002 . التركيب المهني لسكان م حافظة االنبار: ( شكؿ8 ) تكزيع عدد العامميف باألنشطة االقتصادية في محافظة االنبار بحسب الكحدات اإلدارية (االقضية ) لعاـ7991 ( المصدر: باالعتماد عمى جدكؿ1 ) 0 5000 10000 15000 20000 25000 30000 35000 40000 45000 50000 الفلوجه .ق الرمادي .ق هيت .ق حديثة .ق عنه .ق راوه .ق القائم.ق الرطبة .ق الوحدة االدارية عدد العاملين العاملين في الزراعة العاملين في الصناعة العاملين في الخدمات الْحذاث اإلداسيت ػذد الؼبهليي في لطبع الضساػت )(ًغوت ػذد الؼبهليي في لطبع الصٌبػت )(ًغوت ػذد الؼبهليي في لطبع الخذهبث )(ًغوت هجوْع الؼبهليي )%( ق. الفلْجت 72858 1287 59149 14729 57 ق. الشهبدي 71917 9759 45209 10544 79,4 ق. ُيج 4924 7592 9592 72572 2,8 ق. حذيثت 7417 7779 2507 9007 4,0 ٌَق. ػ 7724 794 7191 5799 7,4 ٍّق. سا 7191 797 7745 5750 7,5 .ق المبئن 1794 5527 8092 78177 1,8 ق. الشطبت 7947 592 7740 4751 7,1 غيش هبيي --- --- --- 459 0,7 ػبطلْى لن يغبك لِن الؼول --- --- --- 59475 72,4 هجوْع الوحبفظت 27759 74251 777584 759708 700 % ذ ( جدكؿ1 ) يكضح تكزيع عدد العامميف باألنشطة االقتصادية في محافظة االنبار بحسب الكحدات اإلدارية (االقضية) لعاـ7991 ) 772 ) ( جدكؿ1 ) يكضح تكزيع عدد العامميف باألنشطة االقتصادية في محافظة االنبار بحسب الكحدات اإلدارية (االقضية) لعاـ7991 ,المصدر: جميكرية العراؽ, ىيئة التخطيط, الجياز المركزم لإلحصاء السكاني ,مديرية اإلحصاء المركزم نتائج التعداد العاـ لمسكاف7991 ,لمػحافظة االنبار7998 ( جدكؿ24 ) ص729 . (*) تـ اعتبار ق ضاء لتكحيد البيانات حسب االقضية. ( شكؿ8 ) تكزيع عدد العامميف باألنشطة االقتصادية في محافظة االنبار بحسب الكحدات اإلدارية (االقضية ) لعاـ7991 ( المصدر: باالعتماد عمى جدكؿ1 ) 0 5000 10000 15000 20000 25000 30000 35000 40000 45000 50000 الفلوجه .ق الرمادي .ق هيت .ق حديثة .ق عنه .ق راوه .ق القائم.ق الرطبة .ق الوحدة االدارية عدد العاملين العاملين في الزراعة العاملين في الصناعة العاملين في الخدمات الْحذاث اإلداسيت ػذد الؼبهليي في لطبع الضساػت )(ًغوت ػذد الؼبهليي في لطبع الصٌبػت )(ًغوت ػذد الؼبهليي في لطبع الخذهبث )(ًغوت هجوْع الؼبهليي )%( ق. الفلْجت 72858 1287 59149 14729 57 ق. الشهبدي 71917 9759 45209 10544 79,4 ق. ُيج 4924 7592 9592 72572 2,8 ق. حذيثت 7417 7779 2507 9007 4,0 ٌَق. ػ 7724 794 7191 5799 7,4 ٍّق. سا 7191 797 7745 5750 7,5 .ق المبئن 1794 5527 8092 78177 1,8 ق. التركيب المهني لسكان م حافظة االنبار: ( إذ يبمغ عدد العاطميف عف العمؿ29472 ( نسمة), بنسبة71,4 ), مف إجمالي% القكل العاممة في محافظة االنبار , ىـ مؤىمكف كقادركف عمى العمؿ , كلـ يجدكا فرصة العمؿ في المحافظة, مما يعني أف ىؤالء العاطميف يشكمكف عبء كعالة عمى أسرتيـ, كمف ثـّ شيكع االضطراب ا ت األسرية كالصراعات عمى الميراث , كالتزايد في مستكل العنؼ كالجريمة كىـ في ( سف الشباب, بينما تبمغ نسبة عمالة األطفاؿ مف الذيف تتراكح أعمارىـ1 - 74 )سنة , ( كمشكميف نسبة7,2 )% , إذ يجرم تشغيميـ عمى كفؽ شركط مجحفة كبأجكر زىيدة جدا , فازدادت بي نيـ حاالت الجنكح كمما رسة التسكؿ , بما يتنافى كاتفاقية منظمة العمؿ الدكلية رقـ ( 28 ) لعاـ7912 ( , كاتفاقية منظمة العمؿ الدكلية رقـ787 ), لعاـ7998 , لمقضاء عمى عمالة األطفاؿ, كىذه الظاىرة تنتشر في مركز محافظة االنبار (مركز قضاء الرمادم) بشكؿ ( 777 ( ذ ير ذ ( جدكؿ1 ) يكضح تكزيع عدد العامميف باألنشطة االقتصادية في محافظة االنبار بحسب الكحدات اإلدارية (االقضية) لعاـ7991 ,المصدر: جميكرية العراؽ, ىيئة التخطيط, الجياز المركزم لإلحصاء السكاني ,مديرية اإلحصاء المركزم نتائج التعداد العاـ لمسكاف7991 ,لمػحافظة االنبار7998 ( جدكؿ24 ) ص729 . (*) تـ اعتبار ق ضاء لتكحيد البيانات حسب االقضية. الْحذاث اإلداسيت ػذد الؼبهليي في لطبع الضساػت )(ًغوت ػذد الؼبهليي في لطبع الصٌبػت )(ًغوت ػذد الؼبهليي في لطبع الخذهبث )(ًغوت هجوْع الؼبهليي )%( ق. الفلْجت 72858 1287 59149 14729 57 ق. الشهبدي 71917 9759 45209 10544 79,4 ق. ُيج 4924 7592 9592 72572 2,8 ق. حذيثت 7417 7779 2507 9007 4,0 ٌَق. ػ 7724 794 7191 5799 7,4 ٍّق. سا 7191 797 7745 5750 7,5 .ق المبئن 1794 5527 8092 78177 1,8 ق. الشطبت 7947 592 7740 4751 7,1 غيش هبيي --- --- --- 459 0,7 ػبطلْى لن يغبك لِن الؼول --- --- --- 59475 72,4 هجوْع الوحبفظت 27759 74251 777584 759708 700 % ذ ) 772 ) ( جدكؿ1 ) يكضح تكزيع عدد العامميف باألنشطة االقتصادية في محافظة االنبار بحسب الكحدات اإلدارية (االقضية) لعاـ7991 ,المصدر: جميكرية العراؽ, ىيئة التخطيط, الجياز المركزم لإلحصاء السكاني ,مديرية اإلحصاء المركزم نتائج التعداد العاـ لمسكاف7991 ,لمػحافظة االنبار7998 ( جدكؿ24 ) ص729 . (*) تـ اعتبار ق ضاء لتكحيد البيانات حسب االقضية. (العددذ4ذ )ذ (كانونذاألول)ذ3102 اجمللدذالثاني ذ (العددذ4ذ )ذ (كانونذاألول)ذ3102ذ اجمللدذالثاني ذ جملةذجامعةذاألنبارذللعلومذاإلندانوة ذ ذ (العددذ4ذ )ذ (كانونذاألول)ذ3102ذذذذذذذذذ اجمللدذالثاني ذ ممحكظ كالعراؽ بشكؿ عاـ, إذ تؤدم إلى تكلد الخكؼ كاالنطكاء العاـ, كنمك مشاعر النقص عند األطفاؿ, كعميو أسيـ االحتالؿ األمريكي بعد عاـ7002 كتسمط العقمية العنصرية في ,مضاعفة ىذه الظاىرة الغير حضارية في حيف نجد اف البعد الجغرافي كالديمكغرافي لقضاء الفمكجة قد أسيما بشكؿ فاعؿ في ارتفاع نسبة عدد العامميف في القطاعات االقتصادية المختمفة, إذ إف العكامؿ الجغرافية الطبيعية لقضاء الفمكجة المتمثمة بشكؿ خاص بالمكقع )الجغرافي القريب إداريا مف مكقع العاصمة (بغداد), (السكؽ االستيالكي األكبر مف جية الشرؽ, كمركز المحافظة مكضكع البحث (السكؽ االستيالكي ,األصغر ), مف جية الغرب قد خمؽ عامال مؤثرا لجذب األنشطة االقتصادية إليو, فضال عف عكامؿ التربة كالمكرد المائي كطرؽ النقؿ, إذ يقع القضاء عند مفترؽ الطرؽ الداخمية كالدكلية, ككؿ تمؾ العكامؿ أسيمت بشكؿ فاعؿ مف حيث التركز السكاني المتمثؿ بالكثافة السكانية العال,ية, كاألنشطة االقتصادية ,(زراعة, صناعة تجارة), كميا تفاعمت فيما بينيا لتكجد منطقة التركز السكاني عمى الرغـ مف ما تعرض لو القضاء مف معركتيف مدمرتيف بعد عاـ7002 . جملةذجامعةذاألنبارذللعلومذاإلندانوة ذ اجمللدذالثاني ذ ممحكظ كالعراؽ بشكؿ عاـ, إذ تؤدم إلى تكلد الخكؼ كاالنطكاء العاـ, كنمك مشاعر النقص عند األطفاؿ, كعميو أسيـ االحتالؿ األمريكي بعد عاـ7002 كتسمط العقمية العنصرية في ,مضاعفة ىذه الظاىرة الغير حضارية في حيف نجد اف البعد الجغرافي كالديمكغرافي لقضاء الفمكجة قد أسيما بشكؿ فاعؿ في ارتفاع نسبة عدد العامميف في القطاعات االقتصادية المختمفة, إذ إف العكامؿ الجغرافية الطبيعية لقضاء الفمكجة المتمثمة بشكؿ خاص بالمكقع )الجغرافي القريب إداريا مف مكقع العاصمة (بغداد), (السكؽ االستيالكي األكبر مف جية الشرؽ, كمركز المحافظة مكضكع البحث (السكؽ االستيالكي ,األصغر ), مف جية الغرب قد خمؽ عامال مؤثرا لجذب األنشطة االقتصادية إليو, فضال عف عكامؿ التربة كالمكرد المائي كطرؽ النقؿ, إذ يقع القضاء عند مفترؽ الطرؽ الداخمية كالدكلية, ككؿ تمؾ العكامؿ أسيمت بشكؿ فاعؿ مف حيث التركز السكاني المتمثؿ بالكثافة السكانية العال,ية, كاألنشطة االقتصادية ,(زراعة, صناعة تجارة), كميا تفاعمت فيما بينيا لتكجد منطقة التركز السكاني عمى الرغـ مف ما تعرض لو القضاء مف معركتيف مدمرتيف بعد عاـ7002 . أما فيما يخص تكزيع السكاف حسب المينة, اذ تعبر المينة عف طبيعة العمؿ الذم يؤديو الفرد في مجاالت األنشطة االقتصادية المختمفة, فعمى حد قكؿ االقتصادم كينز (بأف الطمب عمى ((المينة)) ىك طمب مف اجؿ إنتاج السمع كالخدمات التي يتـ بيعيا( 70 ) . لذا يصنؼ السكاف الداخميف في قكة العمؿ عمى أساس المينة أك نكع الحرفة التي يمارسيا, يمكف تقسيـ العامميف حسب الميف كفقا ل:مجمكعات الميف التالية 7 - االختصاصيكف كالفنيك ف كمف يرتبط بيـ. التركيب المهني لسكان م حافظة االنبار: الشطبت 7947 592 7740 4751 7,1 غيش هبيي --- --- --- 459 0,7 ػبطلْى لن يغبك لِن الؼول --- --- --- 59475 72,4 هجوْع الوحبفظت 27759 74251 777584 759708 700 % ) 772 ) ( شكؿ8) تكزيع عدد العامميف باألنشطة االقتصادية في محافظة االنبار بحسب الكحدات اإلدارية (االقضية ) لعاـ7991 ( المصدر: باالعتماد عمى جدكؿ1 ) 0 5000 10000 15000 20000 25000 30000 35000 40000 45000 50000 الفلوجه .ق الرمادي .ق هيت .ق حديثة .ق عنه .ق راوه .ق القائم.ق الرطبة .ق الوحدة االدارية عدد العاملين العاملين في الزراعة العاملين في الصناعة العاملين في الخدمات ) 772 ) ( شكؿ8) تكزيع عدد العامميف باألنشطة االقتصادية في محافظة االنبار بحسب الكحدات اإلدارية (االقضية ) لعاـ7991 ( المصدر: باالعتماد عمى جدكؿ1 ) 0 5000 10000 15000 20000 25000 30000 35000 40000 45000 50000 الفلوجه .ق الرمادي .ق هيت .ق حديثة .ق عنه .ق راوه .ق القائم.ق الرطبة .ق الوحدة االدارية عدد العاملين العاملين في الزراعة العاملين في الصناعة العاملين في الخدمات ( المصدر: باالعتماد عمى جدكؿ1 ) ) 772 ) (العددذ4ذ )ذ (كانونذاألول)ذ3102ذذ اجمللدذالثاني ذ (العددذ4ذ )ذ (كانونذاألول)ذ3102 اجمللدذالثاني ذ 7 - .التشريعيكف كالرؤساء كاإلداريكف كالمدراء 2 - .المكظفكف كالتنفيذيكف كالكتبة كمف يرتبط بيـ 4 - العاممكف في البيع. 9 - .العاممكف في الخدمات 1 - .العاممكف في الزراعة كالغابات كتربية الحيكاف كالقنص كالصيد 1 - العاممكف في.اإلنتاج كمف يرتبط بيـ كمشغمك معدات النقؿ كالشغيمة (ف خالؿ تمؾ التق ي ات يظير الجد ؿ8ال زعة عم الييكؿ ال ين ), نتائ أما فيما يخص تكزيع السكاف حسب المينة, اذ تعبر المينة عف طبيعة العمؿ الذم يؤديو الفرد في مجاالت األنشطة االقتصادية المختمفة, فعمى حد قكؿ االقتصادم كينز (بأف الطمب عمى ((المينة)) ىك طمب مف اجؿ إنتاج السمع كالخدمات التي يتـ بيعيا( 70 ) . لذا يصنؼ السكاف الداخميف في قكة العمؿ عمى أساس المينة أك نكع الحرفة التي يمارسيا, يمكف تقسيـ العامميف حسب الميف كفقا ل:مجمكعات الميف التالية ا 7 - االختصاصيكف كالفنيك ف كمف يرتبط بيـ. إ 7 - .التشريعيكف كالرؤساء كاإلداريكف كالمدراء 2 - .المكظفكف كالتنفيذيكف كالكتبة كمف يرتبط بيـ 4 - العاممكف في البيع. 9 - .العاممكف في الخدمات 9 - .العاممكف في الخدمات 1 - .العاممكف في الزراعة كالغابات كتربية الحيكاف كالقنص كالصيد 1 - العاممكف في.اإلنتاج كمف يرتبط بيـ كمشغمك معدات النقؿ كالشغيمة ( مف خالؿ تمؾ التقسيمات يظير الجدكؿ8 ), نتائج الييكؿ الميني المكزعة عمى القكل العا ممة في محافظة االنبار لعاـ7991 :, كفؽ األتي ( 774 ( ( 774 ( ر ) 779 ) ) 779 ) (العددذ4ذ )ذ (كانونذاألول)ذ3102ذ اجمللدذالثاني ذ (العددذ4ذ )ذ (كانونذاألول)ذ3102ذذ اجمللدذالثاني ذ جملةذجامعةذاألنبارذللعلومذاإلندانوة جملةذجامعةذاألنبارذللعلومذاإلندانوة أ - ( احتمت المينة1), المرتبة األكلى مف حيث عدد العامميف بيا( , كالبالغ17177 )نسمة( , كبنسبة79,8 )% , مف مجمكع قكة العمؿ في المحافظة, اذ تػػشكؿ الفئة ( العمرية1 - 74 ( سنة), نسبة40,7 ( ), كالفئة العمرية% 79 - 99 سنة), نسبة ( 72,7 ( ), في حيف شكمت الفئة العمرية% 10 - ( فأكثر) نسبة92,7 .)% ب - ( جاءت المػػينة1 ) ( , بالػػمرتبة الثانية مف حيث عددىـ البالغ41799 نسمة), كشكمػػكا ن( سبة79,4 )% ( , مف إجمالي قكة العمؿ البالغة729708 نسمة) كاحتمت فئة صغار ( السف1 - 74 ,)سنة ( نسبة9,8 ( ), بينػػػػما الفئة الػػػعمرية% 79 - 99 سنة), شكػػمت ( نسػػبة77,0 ( ), امػػا فئة% 10 ( فاكثر), فشكمت نسبة قميمة بمغت77,4 .)% ت - ( شغمت الميف2), المرت( بة الثالثة, بعػػػدد العامميف البالغ20789 نسمة), شكمػػػػت ( نسبػػتيـ77,1), مف حجـ القكة العاممة في محافظة االنبار, إذ بمػػػغت نسبة الف% ئة ( العػػػمرية1 - 74 ( )سنة0,7 )% ( , كالفئة العمرية79 - 99 سنة), استحكذت عمى ( نسبة- 74,2 ( ), بينما الفئة العمرية% 10 فاكثر), ب( مغت نسبتيا مقدارىا7,27 )% .مف إجمالي القكل العاممة في محافظة االنبار ث - ( جاءت المينة4) في المرتبة الرابعة( , اذ بمغ عدد العامميف78989 ) نسمة كشكمكا ( 77.7 ( ) ككاف نصيب الفئة العمرية% 1 - 74 ( سنة) بنسبة2.4 ), كبينما الفئة% ( العمرية79 - 99 سنة) كاف نسبتيا النصيب( األكثر77.9 ), بينما نصيب الفئة% ( العمرية10 ( سنة فأكثر) كاف بنسبة72.2 .)% ث - ( جاءت المينة4) في المرتبة الرابعة( , اذ بمغ عدد العامميف78989 ) نسمة كشكمكا ( 77.7 ( ) ككاف نصيب الفئة العمرية% 1 - 74 ( سنة) بنسبة2.4 ), كبينما الفئة% ( العمرية79 - 99 سنة) كاف نسبتيا النصيب( األكثر77.9 ), بينما نصيب الفئة% ( العمرية10 ( سنة فأكثر) كاف بنسبة72.2 .)% ج - ( احتمت المينة7 ) المرتبة الخامسة التي بمغ عدد العامميف( فييا70799 ) نسمة ( كبنسبة8.4 )% ( , كاف نصيب الفئة العمرية1 - 74 سنة( ) بنسبة0.7 ) كالفئة% ( العمرية79 - 99 ( سنة) بمغت نسبتيا9.9 )% , ( بينما الفئة العمرية10 سنة فأكثر ) ( بمغت نسبتيا7.9 .) مف السكاف النشطيف في محافظة االنبار% ح - ظ( يرت المينة9) بالمرتبة السادسة( , اذ بمغ مجمكع العامميف9909 ) نسمة مشكميف ( نسبة7.4 ( ) مف إجمالي القكة العاممة في المحافظة, منيا% 0.2 ) لفئة صغار% ( السف1 - 74 سنة) بي( نما كاف نصيب الفئة العمرية79 - 99 ( )سنة7.4 ) في% ( حيف بمغت النسبة1.1 ( ) لفئة كبار السف% 10 .)سنة فأكثر ( 771 ( أ.م.د.ذحدنيذعليذعبدذ الراوي خ - ( أما بالمرتبة األخيرة جاءت المينة7) التي تشمؿ اإل دارييف كالمديريف كرجاؿ ( اإلعماؿ, إذ بمغ عددىـ4127) نسمة( , مشكميف نسبة قدرىا7.0 ) مف إجمالي% القكل ال عاممة في محافظة االنبار( , في حيف شممت الفئة العمرية79 - 99 سنة) نسبة ( قدرىا7.7) فقط% . (العددذ4ذ )ذ (كانونذاألول)ذ3102ذ اجمللدذالثاني ذ كمما تقدـ عف الكضع الميني لمقكل العاممة يتبيف بأف ىناؾ ثالث ميف رئيسية تستقطب أعداد العامميف فييا, كىي الذيف يعممكف بالزراعة كصيد األسماؾ كالعاممكف في اإلنتاج ككسائؿ النق ؿ كالعماؿ غير الكفك ءيف الذيف ال يممككف تحصيؿ دراسي , فضال عف ( المكظفيف كالتنفيذييف كعماؿ المكتبة إذ يبمغ عددىـ748209 ( ) نسمة مشكميف نسبة17 )% مف مجمكع القكل العاممة في ( المحافظة كالبالغ729708) نسمة , في حيف النسبة الباقية تتكزع عمى بقية الميف كبدرجات متباين ة. تعد دراسة مؤشرات قكة العمؿ مف المؤشرات الميمة في دراسة القكل العاممة؛ ألف مف خاللو يمكف معرفة, ما إذا كاف ىناؾ تحقيؽ لمتكازف بيف العرض كالطمب, كالكقكؼ عمى جكانب الضعؼ سكاء أكانت طبيعية أـ بشرية, إذ إف أم ضعؼ تسجمو قكة العمؿ, يؤثر بشكؿ مباشر عمى اقتصاد ذلؾ البمد المدركس, ككف العنصر البشرم ىك المحرؾ األساس القتصاد محافظة االنبار مكضكعة البحث بشكؿ خاص, فضالن عف مكاردىا الطبيعية, لذ يمكف قياس قكة العمؿ مف خالؿ األتي( 77 ). أا عدد القكل العاممة لسنة7991 عدد القكل العاممة لسنة7991 عدد القكل العاممة لسنة7991 نسبة قكة العمؿ = ػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػ ػػػػ ػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػ ػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػ× 700 عدد السكاف لنفس السف 729708 = ػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػ× 700 = 72,2 % 7072121 تعد ىذه النسبة ضئيمة بعدد السكاف المحافظة لعاـ7991 , كما يمكف قياس قكة العمؿ العمرية لفئة معينة عم ى عدد السكاف لنفس الفئة العمرية , التي تعتمد باألساس عمى ( الفئػػة العمرية79 - 99 سنة) :كعمى األتي ) 771 ) جملةذجامعةذاألنبارذللعلومذاإلندانوة ذ ذ (العددذ4ذ )ذ (كانونذاألول)ذ3102ذذذذذذذذ اجمللدذالثاني ذ 709712 نسبة قكة العمؿ العمرية = ػػػػػػػػػػػػػػػػػػػػػ ػػػػػػػػػػػػػػػػػػػػػػػػ ػػػػػػػػػػػػػػػػػػػػػ× 700 = 42 % 481972 جملةذجامعةذاألنبارذللعلومذاإلندانوة ذ ذ (العددذ4ذ )ذ (كانونذاألول)ذ3102ذذذذذذذذ اجمللدذالثاني ذ 709712 نسبة قكة العمؿ العمرية = ػػػػػػػػػػػػػػػػػػػػػ ػػػػػػػػػػػػػػػػػػػػػػػػ ػػػػػػػػػػػػػػػػػػػػػ× 700 = 42 % 481972 (العددذ4ذ )ذ (كانونذاألول)ذ3102ذذ اجمللدذالثاني ذ ك ىذه النسبة, تمثؿ الذككر كاإلناث , كمف الطبيعي أف تتبايف نسبة قكة العمؿ بينيما لعاـ7991 كتعد مساىمة المرأة في العمؿ في محافظة االنبار قميمة بحكـ الظركؼ االجتماعية التي غالبا ما تتصؼ بالطابع العشائرم, كتعطي نسبة قكة العمؿ ضمف الف ئة ( العمرية الكسطية79 - 99 )سنة , دليالن عمى ارتفاع نسبة البط الة في اغمب سكاف محافظة االنبار( , إذ تمثؿ النسبة الباقية كالبالغة91 ) العاطميف عف العمؿ, كا ذا ما قكرنت% ( بالمستكيات العالمية في الدكؿ المتقدمة التي تبمغ19 )% ( 77 ) . لذا فإنيا تعد متدنية, تؤثر بدكرىا عمى التنمية االقتصادية لممنطقة مكضكعة البحث. (العددذ4ذ )ذ (كانونذاألول)ذ3102ذ اجمللدذالثاني ذ كنظرا لعدـ تكافر إحصاءات دقيقة عف حجـ قكة العمؿ لعاـ7001 , يمكف االعتماد عمى التك قعات لمقدار حجيـ , كذلؾ مف خالؿ حساب معدؿ النمك لمقكل العاممة في محافظة الؿ األ اال ا كنظرا لعدـ تكافر إحصاءات دقيقة عف حجـ قكة العمؿ لعاـ7001 , يمكف االعتماد عمى التك قعات لمقدار حجيـ , كذلؾ مف خالؿ حساب معدؿ النمك لمقكل العاممة في محافظة االنبار مف خالؿ األتي: القكل العاممة لعاـ7981 ( كالبالغة788449 )نسمة( 72 ). االنبار مف خالؿ األتي: القكل العاممة لعاـ7981 ( كالبالغة788449 )نسمة( 72 ). القكل العاممة لعاـ7991 ( كالبالغة729708 نسمة) , كبما أف معدؿ النمك يعتمد باألساس عمى مقدار الزيادة السنكية التي تحسب عمى كفؽ اآلتي( 74 ). (العددذ4ذ )ذ (كانونذاألول)ذ3102ذ اجمللدذالثاني ذ عادة ما يتـ تكزيع السكاف الذيف بمغكا سف العاشرة فاكث ر حسب درجة اإللماـ بالقراءة كالكتابة (امي- ممـ بالقراءة كالكتابة– متعمـ ), كغمبا ما تككف تمؾ البيانات مكزعة حسب العمر كالنكع , كيعطي ذلؾ التقسيـ داللة كبيرة كىامة لقدرة البمد عمى تحقيؽ التنمية االقتصادية كاالجتماعية , كما يساعد المخططيف كراسمي السياسة االقتص ادية في البمد عمى تحديد االحتياجات المستقبمية مف المتعمميف بحسب االنشطة االقتصادية المختمفة( 79 ) . كتستعمؿ ىذه البيانات كمقاـ لمعمميات الحسابية المرتبطة بالمعدالت الحيكية النكعية حسب االلماـ بالقراءة كالكتابة كمعدالت الزكاج كالطالؽ تبعا لممستكل التعميـ لمزكج ,كالزكجة كالمستكل التعميمي اثر كبير في مجاؿ رفع إنتاجية العمؿ كزيادة االنتاج( 20 ). ( فمف خالؿ معطيات جدكؿ9 ), يتضح أفَّ ىناؾ انخفاضان في بعض المستكيات التعميمية لمعراؽ , يقابمو ارتفاع في مستكيا ت تعميمية اخرل . اذ يظير انخفاض في نسبة السكاف األمييف في العراؽ م( ف4, 72 ) عاـ% 7991 ( ليصؿ الى7, 79 ) عاـ% 7001 , كيرجع ذلؾ الى اإلجراءات التي اتخ ذتيا الدكلة آنذاؾ في ىذا المجاؿ , كمنيا انطالؽ الحممة الكبيرة مف اجؿ محك االمية كاستمرارىا حتى بعد عاـ7002 رغـ تعرض العراؽ لحرب مدمرة مف ق بؿ القكات االمريكية كمف حالفيا . الشؾ اف لمشيادة مدلكل يا الثقافي اكالن كالميني ثانيان , ك لكميي ما تأثير عمى السمكؾ الديمكغرافي , كالسيما فيما يتعمؽ بحجـ االسرة كالزكاج المبكر اك المتأخر, إذ إف سنكات الدراسة ليا تأ ثيرىا عمى تأخر سف الزكاج أحيانا , كمف ثـّ قصر العمر اإلنجابي لممرأة بشكؿ خاص( 78 ). عادة ما يتـ تكزيع السكاف الذيف بمغكا سف العاشرة فاكث ر حسب درجة اإللماـ بالقراءة كالكتابة (امي- ممـ بالقراءة كالكتابة– متعمـ ), كغمبا ما تككف تمؾ البيانات مكزعة حسب العمر كالنكع , كيعطي ذلؾ التقسيـ داللة كبيرة كىامة لقدرة البمد عمى تحقيؽ التنمية االقتصادية كاالجتماعية , كما يساعد المخططيف كراسمي السياسة االقتص ادية في البمد عمى تحديد االحتياجات المستقبمية مف المتعمميف بحسب االنشطة االقتصادية المختمفة( 79 ) . كتستعمؿ ىذه البيانات كمقاـ لمعمميات الحسابية المرتبطة بالمعدالت الحيكية النكعية حسب االلماـ بالقراءة كالكتابة كمعدالت الزكاج كالطالؽ تبعا لممستكل التعميـ لمزكج ,كالزكجة كالمستكل التعميمي اثر كبير في مجاؿ رفع إنتاجية العمؿ كزيادة االنتاج( 20 ). كالمستكل التعميمي اثر كبير في مجاؿ رفع إنتاجية العمؿ كزيادة االنتاج. ( فمف خالؿ معطيات جدكؿ9 ), يتضح أفَّ ىناؾ انخفاضان في بعض المستكيات التعميمية لمعراؽ , يقابمو ارتفاع في مستكيا ت تعميمية اخرل . (العددذ4ذ )ذ (كانونذاألول)ذ3102ذ اجمللدذالثاني ذ مقدار الزيادة السنكية = عدد القكل العاممة في التعداد الالحؽ– عدد القكل العاممة في التعداد :السابؽ مقسكما عمى عدد السنكات بيف التعداديف, كبتطبيؽ المعادلة نحصؿ عمى مقدار الزياد = ة السنكية729708 – 788449 ÷ 700 / 70 = 9011 = معدؿ النمك / ( مقدار الزيادة السنكية /مجمكع عدد القكل العاممة لمتعداديف7 ) × 700 :كمف خالؿ المعادلة نحصؿ عمى = معدؿ نمك القكل العاممة9011 ÷ 772111 × 700 = 7,2 % ( إذف معدؿ النمك لمقكل العاممة بمغ7,2), كالستخراج% إسقاطات القكل العاممة لعاـ 7001 يتطمب تطبيؽ المعادلة اآلتية( 79 ): = معدؿ نمك القكل العاممة9011 ÷ 772111 × 700 = 7,2 % ( إذف معدؿ النمك لمقكل العاممة بمغ7,2), كالستخراج% إسقاطات القكل العاممة لعاـ 7001 يتطمب تطبيؽ المعادلة اآلتية( 79 ): ( إذف معدؿ النمك لمقكل العاممة بمغ7,2), كالستخراج% إسقاطات القكل العاممة لعاـ 7001 يتطمب تطبيؽ المعادلة اآلتية( 79 ): ( 778 ( 1 + r PN = Po ( ػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػ )n 100 1 + r PN = Po ( ػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػػ )n 100 100 إذ:إف إذ:إف PN = سنة اليدؼ Po = التعداد السابؽ r = معدؿ النمك n = المدة الزمنية بيف التعداديف كمف خالؿ تطبيؽ المعادلة أعاله: القكل العاممة لعاـ 7001 = 729708 ( 7 + 7,2 / 700 ) 70 ( ) 70 إذ:إف القكل العاممة لعاـ 7001 = 729708 ( 7 + 7,2 / 700 ) = 729708 ( 7,072 ) 70 = 200799 نسمة مف خالؿ تطبيؽ المعادلة لمحصكؿ عمى مستكل ا لقكل العاممة المتكقعة لعاـ7001 , ( بمغت200799 نسمة), كىي تشكؿ( نسبة70,7 ,), مف مجمكع سكاف المحافظة% بحسب تقديرات عاـ7001 ( لسكاف المحافظة كالبالغ7489989 نسمة), كتعد ىذه النسبة متدنية إذا ما ثبت صحتيا, كال تتكافؽ مع إمكانيات محافظة االنبار الطبيعية كالبشرية, كمع ما ىك متاح مف مكاردىا االقتصادية, مما يؤثر سمبان عمى إ مكانية المحافظة االقتصادية آبالنسبة لمعراؽ, عند قياس قكتيا االقتصادية كعنػػد تطبيؽ نسبة قكة العمؿ الػػعمرية لعاـ ( نسبة قكة العمؿ العمرية لفئة79 - 99 سنة) لعاـ7001 =ػػػػػػػػػػػػػػػػػػػ ػػػػػػػػػػػػػػػػػػػػػػػػػػػػ ػػػػػػػػػػػػػػ× 700 118772 = 29,7 % ( نسبة قكة العمؿ العمرية لفئة79 - 99 سنة) لعاـ7001 =ػػػػػػػػ ( تعد النسبة29,7 ), منخفضة قياسان% بعاـ7991 بسبب ظركؼ الحرب بعد عاـ 7002 بالحرب األمريكية عمى العراؽ, كمنخفضة أيضا عما ىك عميو قياسان في الدكؿ :المتقدمة كالمشار إلييا أنفا :تركيب السكان التعميمي في محافظة االنبار يعد التعميـ مف الخصائص الديمكغرافية الميمة في تركيب السكاف, كأكثرىا تأثيران في التغيرات اال قتصادية كاالجتماعية ,كلو دكر فعاؿ في مجاؿ اإلبداع كالتنمية( 71 ) , ألنو يمثؿ ( تعد النسبة29,7 ), منخفضة قياسان% بعاـ7991 بسبب ظركؼ الحرب بعد عاـ 7002 بالحرب األمريكية عمى العراؽ, كمنخفضة أيضا عما ىك عميو قياسان في الدكؿ :المتقدمة كالمشار إلييا أنفا :تركيب السكان التعميمي في محافظة االنبار :تركيب السكان التعميمي في محافظة االنبار ا يعد التعميـ مف الخصائص الديمكغرافية الميمة في تركيب السكاف, كأكثرىا تأثيران في التغيرات اال قتصادية كاالجتماعية ,كلو دكر فعاؿ في مجاؿ اإلبداع كالتنمية( 71 ) , ألنو يمثؿ ) 779 ) يتـ التعرؼ عمى المستكل التعميمي لمسكاف, مف خالؿ معرفة نسبة السكاف األمييف كالسكاف المتع مميف نسبة الى مجمكعة السكاف الذيف ىـ في سف الدراسة كالتعميـ, كالمستكل التعميمي لسكاف ام منطقة جغرافية, يعد مف المؤشرات الدالة عمى تقدـ المنطقة اك تخمفيا, إذ إنو كمما سادت االمية في مجتمع معيف , كاف ذلؾ دليالن عمى تخمف يـ االقتصادم كاالجتماعي , فتصبح ىذه البيا نات التي يتـ الحصكؿ عمييا مف التعدادات السكانية العامة كالتقديرات عف الحالة التعميمية ذات فائدة مباشرة في التخطيط لمحك ا المية في المنطقة المراد دراستيا , سكاءن .أكانت عمى مستكل محافظة االنبار اـ العراؽ ـا الشؾ اف لمشيادة مدلكل يا الثقافي اكالن كالميني ثانيان , ك لكميي ما تأثير عمى السمكؾ الديمكغرافي , كالسيما فيما يتعمؽ بحجـ االسرة كالزكاج المبكر اك المتأخر, إذ إف سنكات الدراسة ليا تأ ثيرىا عمى تأخر سف الزكاج أحيانا , كمف ثـّ قصر العمر اإلنجابي لممرأة بشكؿ خاص( 78 ). (العددذ4ذ )ذ (كانونذاألول)ذ3102ذ اجمللدذالثاني ذ اذ يظير انخفاض في نسبة السكاف األمييف في العراؽ م( ف4, 72 ) عاـ% 7991 ( ليصؿ الى7, 79 ) عاـ% 7001 , كيرجع ذلؾ الى اإلجراءات التي اتخ ذتيا الدكلة آنذاؾ في ىذا المجاؿ , كمنيا انطالؽ الحممة الكبيرة مف اجؿ محك االمية كاستمرارىا حتى بعد عاـ7002 رغـ تعرض العراؽ لحرب مدمرة مف ق بؿ القكات االمريكية كمف حالفيا . ( 770 ( أ.م.د.ذحدنيذعليذعبدذ الراوي اما المستكيات التعميمية االخرل , فنسبة ا لسكاف في المستكل دكف االبتدائية , نجد ىناؾ انخفاض كاضح في ىذه النسبة كعمى طكؿ مدة الدراسة , كفيما يخص السكاف في مستكل االبتدائية , فقد ازداد عدد ىـ , اذ كانت نسبتيـ عاـ7991 ( 2, 78 )% , ارتفعت لتصؿ ( الى7, 79 ) عاـ% 7001 , كيعزل ىذا االرتفا ع الى فرض التعميـ كجعمو إلزاميا , كفؽ القرار ( المرقـ778 ) لسنة7918 ( كخاصة لمفئة العمرية1 - 77 )سنة بالرغـ مف ظركؼ الحرب ال تي تعرض ليا العراؽ بكؿ عاـ7002 . (جدكؿ9 ) التكزيع النسبي لمسكاف بحسب الحالة العممية في محافظة االنبار كالعراؽ لممدة 7991 - 7001 (جدكؿ9 ) التكزيع النسبي لمسكاف بحسب الحالة العممية في محافظة االنبار كالعراؽ لممدة 7991 - 7001 :المصدر جميكرية العراؽ, كزارة التخطيط, الجياز المركزم لإلحصاء, نتائج التعداد العاـ لمسكاف لعاـ 7991 ( , جدكؿ79 ), كتقديرات السكاف لسنة7001 ., محافظة االنبار الحبلت الؼلويت 7991 7001 الوحبفظت الؼشاق الوحبفظت الؼشاق اهي 7 , 74 4 , 75 4 , 75 7 , 79 دّى االبخذائيت 8 , 78 2 , 74 8 , 74 9 , 75 ابخذائيت 5 , 78 9 , 78 2 , 71 7 , 79 هخْعطت 2 , 1 4 , 9 9 2 , 77 ّ ثبًْيت هٌِيت 7 , 2 5 , 1 7 , 1 9 , 2 ْدبلْم ّبكبلْسي ط 1 , 4 5 , 2 2 , 1 2 , 8 شِبداث ػليب 7 , 0 9 , 0 4 , 0 1 , 0 الوجوْع 700 % 700 % 700 % 700 % :المصدر جميكرية العراؽ, كزارة التخطيط, الجياز المركزم لإلحصاء, نتائج التعداد العاـ لمسكاف لعاـ 7991 ( , جدكؿ79 ), كتقديرات السكاف لسنة7001 ., محافظة االنبار ,كىذا بدكره أدل الى انخفاض نسبة مف ىـ دكف االبتدائية كارتفاع نسبة مف ىـ ضمف ( مستكل االبتدائية, خاصة مع ازدياد عدد المدارس االبتدائية مف8222 ) مدرسة عاـ 7991 ( , الى77747 ) مدرسة عاـ7001 ( 27 ) عمى مستكل العراؽ ضمف برنامج حممة .االعمار ( جدكؿ70 ). (العددذ4ذ )ذ (كانونذاألول)ذ3102ذ اجمللدذالثاني ذ الْحذة اإلداسيت االػْام هذاسط االبخذائيت هذاسط ثبًْيت بٌيي بٌبث هخخلظ بٌيي بٌبث هخخلظ االًببس 7992 / 7991 710 792 787 99 99 41 7002 / 7001 782 722 729 795 89 15 الؼشاق 7992 / 7991 7754 7001 2097 7472 994 494 7002 / 7001 5759 7242 2720 7979 7479 129 :المصدر7 - جميكرم العراؽ, مجمس الكزراء, ىيئة التخطيط, الجياز المركزم لإلحصاء مديرية اإلحصاء االجتماعي كالتربكم, التعميـ الثانكم في العراؽ لمعاـ7991 / 7991 ص 72 - 71 . 7 - جميكرية العراؽ, كزارة التخطيط كالتعاكف االنمائي, الجياز المركزم لإلحصاء كتكنكلكجي ا المعمكمات, مديرية االحص اء االجتماعي كالتربكم في العراؽ, لمعاـ7001 / 7001, ص 8 - 70 . عاـ7001 ( 717) معيدا في العراؽ( ). ام ا بالنسبة لحممة الشيادات العميا , فبعد اف ازداد نسبة حممة شيادة البكالكريك س , ازداد عدد المتقدميف الى الدراسات العميا, اذ ارتفعت نسبتيـ, سكاء الماجستير اك الدكتكراه , نتيجة الحاجة الى ىذه الككادر العممية المختمفة, السيما بعد ( ازدياد عدد الجامعات كالمعاىد, اذ كانت نسبتيـ9,0 ) عاـ% 7991 ( , ارتفعت الى1,0 )% عاـ7001 .في العراؽ ( جدكؿ70 ) كاقع المدارس االبتدائية كالثانكية في محافظة االنبار كالعراؽ لألعكاـ 7991 / 7991 / 7001 / 7001 . الْحذة اإلداسيت االػْام هذاسط االبخذائيت هذاسط ثبًْيت بٌيي بٌبث هخخلظ بٌيي بٌبث هخخلظ االًببس 7992 / 7991 710 792 787 99 99 41 7002 / 7001 782 722 729 795 89 15 الؼشاق 7992 / 7991 7754 7001 2097 7472 994 494 7002 / 7001 5759 7242 2720 7979 7479 129 :المصدر7 - جميكرم العراؽ, مجمس الكزراء, ىيئة التخطيط, الجياز المركزم لإلحصاء مديرية اإلحصاء االجتماعي كالتربكم, التعميـ الثانكم في العراؽ لمعاـ7991 / 7991 ص 72 - 71 . 7 - جميكرية العراؽ, كزارة التخطيط كالتعاكف االنمائي, الجياز المركزم لإلحصاء كتكنكلكجي ا المعمكمات, مديرية االحص اء االجتماعي كالتربكم في العراؽ, لمعاـ7001 / 7001, ص 8 - 70 . ( جدكؿ70 ) كاقع المدارس االبتدائية كالثانكية في محافظة االنبار كالعراؽ لألعكاـ 7991 / 7991 / 7001 / 7001 . الْحذة اإلداسيت االػْام هذاسط االبخذائيت هذاسط ثبًْيت بٌيي بٌبث هخخلظ بٌيي بٌبث هخخلظ االًببس 7992 / 7991 710 792 787 99 99 41 7002 / 7001 782 722 729 795 89 15 الؼشاق 7992 / 7991 7754 7001 2097 7472 994 494 7002 / 7001 5759 7242 2720 7979 7479 129 :المصدر7 - جميكرم العراؽ, مجمس الكزراء, ىيئة التخطيط, الجياز المركزم لإلحصاء مديرية اإلحصاء االجتماعي كالتربكم, التعميـ الثانكم في العراؽ لمعاـ7991 / 7991 ص 72 - 71 . (العددذ4ذ )ذ (كانونذاألول)ذ3102ذ اجمللدذالثاني ذ اما بالنسبة لحممة الشيادة المتكسطة فقد ازداد عددىـ ايضا حتى أصبحت( نسبتيـ1, 77 ) عاـ% 7001 ( بعد اف كانت تشكؿ4,9 ) عاـ% 7991 اف ىذه الزيادة تتناسب مع ازدياد عدد المدارس المتكسطة, فضال عف زيادة اقباؿ الطمبة الى المدارس ( المتكسطة التي اصبح عددىا4709 ) مدرسة عاـ7001, بع د اف كانت عاـ7991 ( 7490) مدرسة , اما فيما يخص الحاصميف عمى شيادة الثانكية كالمينية, كانت نسبتيـ ( 2,1 ) عاـ% 7991 ( , انخفضت الى9,1 ) عاـ% 7001 في العراؽ. اما فيما يتعمؽ بحممة ) 777 ) جملةذجامعةذاألنبارذللعلومذاإلندانوة ذ ذ (العددذ4ذ )ذ (كانونذاألول)ذ3102ذذذذذذذذذذ اجمللدذالثاني ذ الشيادات الدبمكـ كالبكالكريك س , فقد ارتفعت نسبة الحاصميف عمى ىذه الشيادات, فبمغت ( نسبتيـ2,1 ) عاـ% 7991 , أصبحت عاـ7001 ( 1,8), كيعزل ذلؾ ال% ى فتح العديد مف الجامعات كالمعاىد, خاصة بعد عاـ7981 ,, التي أصبحت تضـ اقساـ عممية عديدة كفتح العديد مف المعاىد المختمفة, فقد كاف عدد المعاىد عاـ7991 ( 77 ) معيد, أصبحت عاـ7001 ( 717) معيدا في العراؽ( 27 ). ام ا بالنسبة لحممة الشيادات العميا , فبعد اف ازداد نسبة حممة شيادة البكالكريك س , ازداد عدد المتقدميف الى الدراسات العميا, اذ ارتفعت نسبتيـ , سكاء الماجستير اك الدكتكراه , نتيجة الحاجة الى ىذه الككادر العممية المختمفة, السيما بعد ( ازدياد عدد الجامعات كالمعاىد, اذ كانت نسبتيـ9,0 ) عاـ% 7991 ( , ارتفعت الى1,0 )% عاـ7001 .في العراؽ (العددذ4ذ )ذ (كانونذاألول)ذ3102ذذ اجمللدذالثاني ذ جملةذجامعةذاألنبارذللعلومذاإلندانوة و وم إل أل ب ر ج م مج ذ اجمللدذالثاني ذ الشيادات الدبمكـ كالبكالكريك س , فقد ارتفعت نسبة الحاصميف عمى ىذه الشيادات, فبمغت ( نسبتيـ2,1 ) عاـ% 7991 , أصبحت عاـ7001 ( 1,8), كيعزل ذلؾ ال% ى فتح العديد مف الجامعات كالمعاىد, خاصة بعد عاـ7981 ,, التي أصبحت تضـ اقساـ عممية عديدة كفتح العديد مف المعاىد المختمفة, فقد كاف عدد المعاىد عاـ7991 ( 77) معيد, أصبحت عاـ7001 ( 717) معيدا في العراؽ( 27 ). ام ا بالنسبة لحممة الشيادات العميا , فبعد اف ازداد نسبة حممة شيادة البكالكريك س , ازداد عدد المتقدميف الى الدراسات العميا, اذ ارتفعت نسبتيـ, سكاء الماجستير اك الدكتكراه , نتيجة الحاجة الى ىذه الككادر العممية المختمفة, السيما بعد ( ازدياد عدد الجامعات كالمعاىد, اذ كانت نسبتيـ9,0 ) عاـ% 7991 ( , ارتفعت الى1,0 )% عاـ7001 .في العراؽ ( جدكؿ70 ) كاقع المدارس االبتدائية كالثانكية في محافظة االنبار كالعراؽ لألعكاـ 7991 / 7991 / 7001 / 7001 . (العددذ4ذ )ذ (كانونذاألول)ذ3102ذ اجمللدذالثاني ذ 7 - جميكرية العراؽ, كزارة التخطيط كالتعاكف االنمائي, الجياز المركزم لإلحصاء كتكنكلكجي ا المعمكمات, مديرية االحص اء االجتماعي كالتربكم في العراؽ, لمعاـ7001 / 7001, ص 8 - 70 . اما فيما يتعمؽ بمحافظة االن بار, فمف خالؿ الجدك( ؿ70 ) أيضان , يتضح اف المستكل التعميمي يتجو تقريبا باالتج اه نفسو لممستكل التعميمي لمعراؽ , اذ يشير الجدكؿ الى اف نسبة األمية تشكؿ اكثر مف ثمث سكاف منطقة الدراسة( , كبنسبة7, 74 ) عاـ% 7991 , انخفضت ( لتصؿ الى4, 72 ) عاـ% 7001, كعمى الرغـ مف ىذا االنخفاض , فتعد ,نسبة مرتفعة ( كالسبب في ذلؾ يعكد الى ارتفاع نسبة السكاف الريؼ في محافظة االنبار, اذ بمغت2, 41 % ك9, 41 ) لمعاميف% 7991 ك7001 , مف مجمكع سكاف المحافظة, كالمعركؼ عف المجتمع الريفي بانو يتصؼ بعادات كتقاليد تختمؼ عف ما مكجكد لدل سكاف الحضر, إذ تعمؿ ( 777 ( أ.م.د.ذحدنيذعليذعبدذ الراوي األعراؼ االجتما عية عمى الحد مف تعميـ اإلناث, فضال عف عزكؼ المرأة عف التعميـ, كيعد الزكاج المبكر احد عكامؿ ارتفاع نسبة األمية. ام ا بالنسبة لمف ىـ دكف االبتدائية, فاف نسبتيـ في انخفاض مستمر( , فقد بمغ8, 78 ) عاـ% 7991 ( , انخفضت الى8, 74 ) عاـ% 7001 , فيي تتجو باتجاه نسبتيـ في العراؽ, بينما حصؿ ارتفاع كاضح طفيؼ في نسبة الحاصميف عمى شيادة االبتدائية مف عاـ7991 ( 2, 78 ( ) ليصؿ الى% 1, 78 ) عاـ% 7001 , كىذا ( يتناسب مع زيادة عدد المدارس االبتدائية فبعد أف كاف عددىا108 ) مدرسة عاـ7991 , ( ازدادت الى877 ) مدرسة عاـ7001. (العددذ4ذ )ذ (كانونذاألول)ذ3102ذ اجمللدذالثاني ذ كىي كاألتي ) 772 ) ) 772 ) جملةذجامعةذاألنبارذللعلومذاإلندانوة ذ ذ (العددذ4ذ )ذ (كانونذاألول)ذ3102ذذذذذذذذذذذذذ اجمللدذالثاني ذ اوال): القومية (العرق: القكمية مصطمح ذك مدلكؿ اجتماعي كسياسي, كيعني الشعكر المتبادؿ بيف افراد المجتمع السكان ي في عكاطفيـ كسمككيـ تجاه كطنيـ , التي تعد مف االسس اليامة في بناء العالقات الداخمية ألبناء المجتمع , ينحدر سكاف المحافظة مف شبو الجزيرة العربية, كبالد الشاـ الذيف نزحكا عمى شكؿ ىجرات سكانية الى اقميـ المحافظة عبر االكدية الص ,حراكية التي كانت تشكؿ مسالؾ كطرؽ طبيعية ليتخذكا مف المناطؽ السيمية المحاذية لنير الفرات مستقرا ليـ, كيتسـ سكاف المحافظة بالطابع العشائرم القبمي, متمسكيف بعاداتيـ كتقاليدىـ القديمة, التي تأخذ بصفتيا طابع البداكة بعض الشيء, كبما أف محافظة االنبار مف المحافظ,ات الحدكدية المجاكرة لعدد مف الدكؿ العربية مف جية الغرب كألف سكاف اليضبة الغربية يتسمكف بصفة البداكة كالترحاؿ متخذيف مف حرفة الرعي نشاطا اقتصاديا ليـ, كينتقمكف ,عبر الحدكد السعكدية كاألردنية كالسكرية, لذا فاف البعض منيـ تربطيـ ركابط اجتماعية تتمثؿ بتبادؿ ,األنساب قبائؿ الدكؿ المجاكرة انفة الذكر, االمر الذم يزيد مف الركابط القكمية كالى كقت قريب كانت لمقكمية الكردية كجكد في محافظة االنبار, قب ؿ دخكؿ القكات األمريكية المحتمة , لكف أعدادىـ قمت كثيران بسبب سكء األكضاع االمنية بعد االحتالؿ االمريكي عاـ 7002 مما ا دل ذلؾ الى ان تقاليـ الى اقميـ كردستاف العراؽ. (العددذ4ذ )ذ (كانونذاألول)ذ3102ذ اجمللدذالثاني ذ اما بالنسب ة لمحاصمي ف عمى الشيادة المتكسطة , فقد ( ارتفعت نسبتيـ مف1,1 ) عاـ% 7991 ( الى9 ) عاـ% 7001 , ك كذلؾ الحاؿ بالنسبة ( لمحاصميف عمى الشيادة الثانكية كالمينية لتصؿ الى7,1 ) عاـ% 7001 , بعد اف كانت ( نسبتيـ7,1 ) عاـ% 7991, كىذا يعكد الى زيادة عدد المدارس الثانكي( ة كالمينية مف707 مدرسة ثانكية) عاـ7991 ( الى279 مدرسة ثانكية) عاـ7001 , كىذا االرتفاع في نسبة الحاصميف عمى الشيادات االبتدائ ية كالمتكسطة كالثانكية كالمينية , يتجو باالتجاه نفسو الذم اتخ ذه حاممي ىذه الشيادات في العراؽ , مف خالؿ زيادة نسبة الح اصميف عمى ىذه المستكيات العممي ة( , جدكؿ9 .) اما عف حممة الشيادات الدبمكـ كالبكالكريك س( , فقد ازدادت نسبتيـ مف1,4) عاـ% 7991 ( , الى1,1 ) عاـ% 7001 , كىذا يعكد الى تكجو الدكلة بشكؿ عاـ كالمحافظة بشكؿ خاص الى فتح العديد مف الكميات منذ عاـ7989 , عندما استحدثت جامعة االنبار لتضـ ( تمؾ الكميات حتى بمغ عددىا79 ) كمية بمختمؼ االختصاصات عاـ7001 , كىذه الكميات ليا دكر كبير في زيادة نسبة الحاصميف عمى شيادة البكالكريك س , كعمى اثر ذلؾ تـ فتح ابكاب الدراسات العميا في الجا معة لدراسة الماجستير كالدكتكراه, مما زاد نسبة الحاصميف عمى ( الشيادات العميا مف7,0 ) عاـ% 7991 ( الى4,0 ) عاـ% 7001 ( 22 ) . اا :التكوين أالثني لسكان محافظة االنبار يعبر عف التركيب االثني بالقكميات, الديف كالمغة التي تكجد داخؿ اطار الكحدة االدارية, سكاءن أكانت دكلة اك محافظة, كىي مف اىـ الخصائص السك انية المؤثرة في البناء .الداخمي لممجتمع السكاني لذا فاف الكشؼ عف تفاصيؿ الخصائص السكانية االثنية مف حيث :تحديد قكميتيـ كدينيـ كلغتيـ ليا تأثير كبير في الدراسات السكانية. (العددذ4ذ )ذ (كانونذاألول)ذ3102ذ اجمللدذالثاني ذ :ثانيا: الدين يعد الديف عامال ميما في تماسؾ ابناء المجتمع السكاني, عمى عكس التنكع الديني جملةذجامعةذاألنبارذللعلومذاإلندانوة ذ ذ (العددذ4ذ )ذ (كانونذاألول)ذ3102ذذذذذذذ اجمللدذالثاني ذ ا (العددذ4ذ )ذ (كانونذاألول)ذ3102ذذ اجمللدذالثاني ذ (العددذ4ذ )ذ (كانونذاألول)ذ3102ذذذ اجمللدذالثاني ذ ا)ق ( القكمية مصطمح ذك مدلكؿ اجتماعي كسياسي, كيعني الشعكر المتبادؿ بيف افراد المجتمع السكان ي في عكاطفيـ كسمككيـ تجاه كطنيـ , التي تعد مف االسس اليامة في بناء العالقات الداخمية ألبناء المجتمع , ينحدر سكاف المحافظة مف شبو الجزيرة العربية, كبالد الشاـ الذيف نزحكا عمى شكؿ ىجرات سكانية الى اقميـ المحافظة عبر االكدية الص ,حراكية التي كانت تشكؿ مسالؾ كطرؽ طبيعية ليتخذكا مف المناطؽ السيمية المحاذية لنير الفرات مستقرا ليـ, كيتسـ سكاف المحافظة بالطابع العشائرم القبمي, متمسكيف بعاداتيـ كتقاليدىـ القديمة, التي تأخذ بصفتيا طابع البداكة بعض الشيء, كبما أف محافظة االنبار مف المحافظ,ات الحدكدية المجاكرة لعدد مف الدكؿ العربية مف جية الغرب كألف سكاف اليضبة الغربية يتسمكف بصفة البداكة كالترحاؿ متخذيف مف حرفة الرعي نشاطا اقتصاديا ليـ, كينتقمكف ,عبر الحدكد السعكدية كاألردنية كالسكرية, لذا فاف البعض منيـ تربطيـ ركابط اجتماعية تتمثؿ بتبادؿ ,األنساب قبائؿ الدكؿ المجاكرة انفة الذكر, االمر الذم يزيد مف الركابط القكمية كالى كقت قريب كانت لمقكمية الكردية كجكد في محافظة االنبار, قب ؿ دخكؿ القكات األمريكية المحتمة , لكف أعدادىـ قمت كثيران بسبب سكء األكضاع االمنية بعد االحتالؿ االمريكي عاـ 7002 مما ا دل ذلؾ الى ان تقاليـ الى اقميـ كردستاف العراؽ. :ثانيا: الدين يعد الديف عامال ميما في تماسؾ ابناء المجتمع السكاني, عمى عكس التنكع الديني ,كالمذىبي الذم يخمؽ تنازعات بيف السكاف, كفي منطقة الدراسات ال يكجد تنكع ديني كبير :كيمكف ابراز ذلؾ عمى النحك االتي 1 - :المسممون 2 - : المسيحيون يعد ثاني ديانة في العراؽ بعد المسمميف, ظيرت في القرف االكؿ الميالدم, اذ كاف معظـ العراؽ يعتنقكف المسيحية قبؿ دخكؿ االسالـ, تضاءلت اعدادىـ بعد اسالـ العديد ,منيـ يتكزع مسيحيك العراؽ عمى محافظات العراؽ كافة, اال أف أغمبيـ يتركز في العاصمة بغداد كمحافظة نينكل, كتكجد اعداد منيـ في ناحية الحبانية التابعة لقضاء الرمادم احد اقضية محافظة االنبار, كما اف غالبية مسيحيي العراؽ ىـ مف اتباع الكنيسة الكمدانية الكاثكليكية, كصمت ( اعدادىـ في العراؽ124727) نسمة( 24 ). ( شكمكا نسبة2,2 ) مف% مجمكع سكاف العراؽ عاـ7991 ( , منيـ7019 ) نسمة يسكنكف في منطقة الدراسة, مشكميف ( نسبة7,0 ) مف مجمكع سكاف محافظة االنبار لعاـ% 7991 , انخفضت اعدادىـ بعد عاـ 7991 , بسبب ظركؼ الحصار االقتصادم, اذ ىاجر العديد منيـ مف منطقة الدراسة الى خارج العراؽ, اما بعد االحتالؿ االمريكي عاـ7002 , كما تبع ذلؾ مف فقداف االمف كاالستقرار, الذم نتج عنو انخفاض في حجـ المسيحييف في محافظة االنبار, اذ كصمكا ( 210 ( ) نسمة, شكمكا نسبة02 ,0 )% ( 24 ). متمركزيف في مدينة الحبانية ضمف محا فظة .االنبار 1 - :المسممون ( يشكؿ المسممكف نسبة91 ) مف سكاف العراؽ حسب تعداد عاـ% 7991 , اما في ( منطقة الدراسة فتبمغ نسبتيـ91 , 99 ) مف مجمكع سكاف المحافظة عاـ% 7991 , مع اف الديف اإلسالمي يستحكذ عمى اكبر نسبة مف سكاف المحافظة, الى اف المسمميف انتيجكا عقائد دينية, تدعى المذىبية تنتمي لديف اإلسالـ, اذ يبرز مذىباف مف أصؿ اربعة مذاىب, كىما المذىب السني كالمذىب الشيعي, كينيج غالبية سكاف المحافظة المذىب السني, ألسباب مكقعيو كتاريخية, فمف حيث المكقع قربيا مف المممكة العربية السعكدية كالمممكة االردنية ( 774 ( أ.م.د.ذحدنيذعليذعبدذ الراوي ذ ذ حتلولذجغرايفذلدميوغراػوةذاحلربذوأثرهاذ علىذالرتكوبذالدكاني ذ ذ الياشمية, المتاف تنتيجاف المذىب اال سالمي نفسو, اما التاريخي فاف سكاف منطقة الدراسة .ينحدركف مف شبو الجزيرة العربية 2ن ال أ.م.د.ذحدنيذعليذعبدذ الراوي الياشمية, المتاف تنتيجاف المذىب اال سالمي نفسو, اما التاريخي فاف سكاف منطقة الدراسة .ينحدركف مف شبو الجزيرة العربية 3 - : الصابئة المندائيون تمثؿ عقيدة دينية اتبعيا سكاف الجزيرة العربية في مدة ما قبؿ االسالـ, كما انيا ازدىرت في كادم الرافديف كيتحدثكف المغة االرمنية, كتحتؿ المياه مكقعا متميزا في الطقكس الزكاج كتعميد االطفاؿ كاالحتفاؿ بالمناسبات الدينية, يتضمف االرتماس بالمياه الجارية, لذا لؾ حرص الصابئة عمى السكف عمى ضفاؼ االنيار, كيتحدث الصابئة المندائيكف العربية بميجتيا العراقية اك االحكازية, كلدييـ لغتيـ الخاصة ايضا المغة المندائية, اذ يسكف عدد منيـ في المحافظات الجنكبية مف العراؽ, مثؿ البصرة كميساف كالناصر ية ككذلؾ في منطقة الدراسة, كال تكجد إحصائية بأعدادىـ في العراؽ, الى اف بعض التقديرات ترجح باف عددىـ ( بمغ10000 ) نسمة7991 ( , كشكمكا نسبة2,0 ,) مف مجمكع سكاف العراؽ لمعاـ نفسو% كانخفضت إعدادىـ بعد االحتالؿ األمريكي لمعراؽ عاـ7002 , بسبب عمميات التيجير كعدـ ) 779 ) جملةذجامعةذاألنبارذللعلومذاإلندانوة ذ ذ (العددذ4ذ )ذ (كانونذاألول)ذ3102ذذذذذذذذذ اجمللدذالثاني ذ ا( الستقرار االمني, لذا ال يتجاكز عددىـ اكثر مف790) نسمة( 29 ) اك اقؿ, كىـ يتعايشكف بشكؿ سممي مع سكاف محافظة االنبار كيسكنكف بالقرب مف نير الفرات في مركز قضاء .الرمادم ثالثا المغة (العددذ4ذ )ذ (كانونذاألول)ذ3102 اجمللدذالثاني ذ :ثالثا: المغة ,تعد المغة مف الخصائص السكانية الميمة التي تكحد المجتمع السكاني في لغة كاحدة كىي كسيمة التفاىـ بيف الشعكب, كتشكؿ احدل الركابط القكمية ألم مجتمع سكاني, كيتحدث سكاف محافظة االنبار المغة العربي ة الى جانب تعدد الميجات المحمية , كمع كجكد بعض االقميات القكمية كالدينية. اال انيـ يجيدكف المغة العربية, كىذا يدؿ عمى االندماج بيف تمؾ االقميات كسكاف منطقة الدراسة, كبيذا التجانس المغكم تكتمؿ الصكرة النيائية لسكاف محافظة .االنبار, بأبعادىا الثالث القكمية كالدينية كالمغكية أنواع اخرى من خصائص التركيب السكاني: يعد الزكاج مف اىـ الخصائص الديمكغ رافية في جميع المجتمعات البشرية , اذ عف طريقو يمكف احالؿ السكاف ألنفسيـ عف طريؽ االنجاب كالميالد الشرعي . الذم يمثؿ الغالبية العظمى مف عدد المكاليد االحياء, كىك ما مكجكد في منطقة البحث , االمر الذم ينعكس ع مى الزيادة السكانية في عدد سكاف( , الذم تمثؿ في معدؿ النمك السكاني البالغ1 . 2 )% , فضال عف تشجيع الزكاج المبكر , كتعدد الزكجات التي أصبحتا ظاىرة شائعة في مجتمع منطقة البحث بشكؿ خاص كالعراؽ بشكؿ عاـ كال سيما بعد احداث عاـ7002 . 3 - : الصابئة المندائيون كما اف لمزكاج اىمية كبيرة في تككيف االسر عف طريؽ الزكاج, كتفككيا اك انحالليا عف طريؽ الترمؿ التي بمغت اعداد ظاىرة التر( مؿ الى اكثر مف790) الؼ ارممة)*( , بسبب الظركؼ التي يمر بو العراؽ بشكؿ عاـ كمحافظة االنبار بشكؿ خاص بعد احداث عاـ 7002 ( عمى اثر االحتالؿ االمريكي لمعراؽ, اك الطالؽ التي بمغت اعداده اكثر مف900 ) الؼ حالة طالؽ منذ عاـ7002 / 7070 نتيجة عدـ التكافؽ بيف الزكجي ف كتدخؿ اىؿ الزكجيف, اك نتيجة االطالع عمى ما تنشره كسائؿ االعالـ المرئية مف مسمسالت كافالـ مختمفة التي ليا تأثير كبير عمى سمكؾ الشباب ذككران كاناثان كظركؼ الحرب عاـ7002 كما .تبعيا يعد الزكاج مف اىـ الخصائص الديمكغ رافية في جميع المجتمعات البشرية , اذ عف طريقو يمكف احالؿ السكاف ألنفسيـ عف طريؽ االنجاب كالميالد الشرعي . الذم يمثؿ الغالبية العظمى مف عدد المكاليد االحياء, كىك ما مكجكد في منطقة البحث , االمر الذم ينعكس ع مى الزيادة السكانية في عدد سكاف( , الذم تمثؿ في معدؿ النمك السكاني البالغ1 . 2 )% , فضال عف تشجيع الزكاج المبكر , كتعدد الزكجات التي أصبحتا ظاىرة شائعة في مجتمع منطقة البحث بشكؿ خاص كالعراؽ بشكؿ عاـ كال سيما بعد احداث عاـ7002 . ـا ـ كما اف لمزكاج اىمية كبيرة في تككيف االسر عف طريؽ الزكاج, كتفككيا اك انحالليا عف طريؽ الترمؿ التي بمغت اعداد ظاىرة التر( مؿ الى اكثر مف790) الؼ ارممة)*( , بسبب الظركؼ التي يمر بو العراؽ بشكؿ عاـ كمحافظة االنبار بشكؿ خاص بعد احداث عاـ 7002 ( عمى اثر االحتالؿ االمريكي لمعراؽ, اك الطالؽ التي بمغت اعداده اكثر مف900 ) الؼ حالة طالؽ منذ عاـ7002 / 7070 نتيجة عدـ التكافؽ بيف الزكجي ف كتدخؿ اىؿ الزكجيف, اك نتيجة االطالع عمى ما تنشره كسائؿ االعالـ المرئية مف مسمسالت كافالـ مختمفة التي ليا تأثير كبير عمى سمكؾ الشباب ذككران كاناثان كظركؼ الحرب عاـ7002 كما .تبعيا ( 771 ( أ.م.د.ذحدنيذعليذعبدذ الراوي ذ ذ حتلولذجغرايفذلدميوغراػوةذاحلربذوأثرهاذ علىذالرتكوبذالدكاني ذ ذ كاخيرا يمكف القكؿ اف الغزك األمريكي لمعراؽ بشكؿ عاـ كالمحافظة بشك ؿ خاص , سبب الكثير مف النتائج السمبية في مجاالت الحياة المختمفة لمعراقييف, كىي اكصاؼ اليزاؿ يطمقيا بعضيـ عمى الكضع االمني في العراؽ بش كؿ عاـ كمحافظة االنبار بشكؿ خاص , إذ ( بمغ عدد القتمى بحسب احصائيات مختمفة تجاكزت790 ) الؼ شييد بحسب إحصائيات لمنظمة الصح ة العالمية , ك في اكاخر عاـ7070 ( سقط نحك7909 ) مدني عراقي في اعماؿ عنؼ كاعداد الجرحى امر مختمؼ لتحصيؿ حاصؿ تجاكز مئات االالؼ( 21 ). 3 - : الصابئة المندائيون ككاف لمحافظة االنبار النصيب االكبر بسبب االحداث االخيرة التي خمفيا االحتالؿ االمريكي بعد عاـ 7002 . ا أ.م.د.ذحدنيذعليذعبدذ الراوي كاخيرا يمكف القكؿ اف الغزك األمريكي لمعراؽ بشكؿ عاـ كالمحافظة بشك ؿ خاص , سبب الكثير مف النتائج السمبية في مجاالت الحياة المختمفة لمعراقييف, كىي اكصاؼ اليزاؿ يطمقيا بعضيـ عمى الكضع االمني في العراؽ بش كؿ عاـ كمحافظة االنبار بشكؿ خاص , إذ ( بمغ عدد القتمى بحسب احصائيات مختمفة تجاكزت790 ) الؼ شييد بحسب إحصائيات لمنظمة الصح ة العالمية , ك في اكاخر عاـ7070 ( سقط نحك7909 ) مدني عراقي في اعماؿ عنؼ كاعداد الجرحى امر مختمؼ لتحصيؿ حاصؿ تجاكز مئات االالؼ( 21 ). ككاف لمحافظة االنبار النصيب االكبر بسبب االحداث االخيرة التي خمفيا االحتالؿ االمريكي بعد عاـ 7002 . جا 7 - أىمية المكقع الجغرافي لمحافظة االنبار, سمح ذلؾ بتفعيؿ أثرىا اإلقميمي عمى مستكل العراؽ كخاصة في حقبة التسعينات , خاصة إنيا تجاكر كؿ مف الجميكرية العربية السكرية كالمممكة األردنية الياشمية, مما تجعميا قريبة مف البحر المتكسط كالبحر األحمر , مما ىيأ ليا ا لتبادؿ التجار م لمسمع كالخدمات , التي أعانت العراؽ خالؿ مدة الحصار االقتصادم بشكؿ عاـ كالمحافظة بشكؿ خاص بعد احداث عاـ7002 . 7 - اظير البحث أف ىناؾ إشكالية, يجب التنبو إلييا, متمثمة بكجكد عبء ديمكغرافي , يتركز في مناطؽ دكف أخرل ىذا العبء ناتج عف الزيادة الطب يعية لمسكاف , مما ق د يؤدم إلى زي ادة معدالت البطالة كىذه الظاىرة , قد تككف سببان في جعؿ محافظة االنبار طاردة لمسكاف في المستقبؿ نتيجة اعماؿ عنؼ التي خمفتيا الحرب االمريكية بعد عاـ7002 . 2 - إف الكاقع االقتصادم لمحافظة االنبار, شيد ضعفا في القطاع الزراعي كالص ناعي بسبب األحداث بعد ع اـ7002 , فضال عف االنقطاع المستمر لمتيار الكيربائي الذم .اضعؼ اإلنتاج في اغمب الصناعات, الذم يؤثر بدكرة عمى قيمة اإلنتاج المحمي 4 - اظير البحث إف ىناؾ تركزان كاضحان في مستكيات القطاع االقتصادم كالكثافة السكانية العامة في قضاء الفمكجة ب درجة األساس, يميو قضاء ا لرمادم , مما يكلد دكافع ىجرة السكاف مما انعكس ذلؾ عمى ارتفاع معدؿ النمك السكاني, أع مى مما ىك عميو عمى مستكل العراؽ. 3 - : الصابئة المندائيون 2 - إف الكاقع االقتصادم لمحافظة االنبار, شيد ضعفا في القطاع الزراعي كالص ناعي بسبب األحداث بعد ع اـ7002 , فضال عف االنقطاع المستمر لمتيار الكيربائي الذم .اضعؼ اإلنتاج في اغمب الصناعات, الذم يؤثر بدكرة عمى قيمة اإلنتاج المحمي ) 771 ) ) 771 ) جملةذجامعةذاألنبارذللعلومذاإلندانوة ذ ذ (العددذ4ذ )ذ (كانونذاألول)ذ3102ذذذذذذذذذذ اجمللدذالثاني ذ 9 - ىناؾ تبايف كاضح في تركيب السكاف العمر م كالنكعي كحتى النشاط االقتصادم , مما ( دفع الكثير مف األطفاؿ الذيف ىـ في سف1 - 74 )سنة , العمؿ في القطاعات المختمفة كال سيما بعد ارتفاع نسبة البطالة بعد احداث الح رب االمريكية عاـ7002 كما تبعيا. رد (العددذ4ذ )ذ (كانونذاألول)ذ3102 اجمللدذالثاني ذ جملةذجامعةذاألنبارذللعلومذاإلندانوة ذ ذ (العددذ4ذ )ذ (كانونذاألول)ذ3102ذذذذذذذذذ اجمللدذالثاني ذ 9 - ىناؾ تبايف كاضح في تركيب السكاف العمر م كالنكعي كحتى النشاط االقتصادم , مما ( دفع الكثير مف األطفاؿ الذيف ىـ في سف1 - 74 )سنة , العمؿ في القطاعات المختمفة كال سيما بعد ارتفاع نسبة البطالة بعد احداث الح رب االمريكية عاـ7002 كما تبعيا. 7 - بما اف معدؿ النمك السكاني في محافظة االنبار اكبر مف العراؽ, إال أف السكاف ظمك ا متمركزيف في ثالثة أقضية فقط, كىي أقضية الفمكجة كالرمادم كىيت, بسبب تركز األنشطة االقتصادية, لذا ينبغي العمؿ عمى اعادة تكزيع األنشطة االقتصادية , بما يتناسب كحػػجـ السكاف , كتكزيػػػػػعيـ عمى الرقعة الجغرافية غير المأىكلة اك القميمة السكاف ضمف حدكد محافظة اال نبار, الستقطاب أعدادىـ كتخفيؼ االزدحاـ السك اني داخؿ االقضية المذككرة أعاله .كخاصة ضمف المنطقة الصحراكية اا 7 - المحافظة مكضكعة البحث تفتقر الى العمالة النسكية, األمر الذم يدعك إلى تشجيع ,العمػػػػػػػالة النسكية الف مشػػػاركة المرأة في األعماؿ كاالنػػػػشطة االقتص ادية دلػػػيؿ عمى .رقي المجتمع كرفاىيتو اا 7 - المحافظة مكضكعة البحث تفتقر الى العمالة النسكية, األمر الذم يدعك إلى تشجيع ,العمػػػػػػػالة النسكية الف مشػػػاركة المرأة في األعماؿ كاالنػػػػشطة االقتص ادية دلػػػيؿ عمى .رقي المجتمع كرفاىيتو 2 - أصبح مف الضركرم البدء في تكزيع االستثمارات في مجاؿ القطاع الصناعي كالخدمي , فضال عف االىتماـ بالجانب الزراعي عمى مستكل متكازف في جميع الكحدات اإلدارية لمحافظة االنب ار كذلؾ لتقميؿ الفكارؽ المكانية , سكاء أكانت سكانية أـ اقتصادية عمى كفؽ خطط تنمكية شاممة مدركسة كضمف مراحؿ. 3 - : الصابئة المندائيون 4 - يمكف تحكيؿ العبء االقتصادم , الناتج عف الزيادة الطبيعية كعامؿ حركة ىجرة السكاف الداخمية إلى أىمية ديمكغرافية عف طريؽ زيادة المساىمة في األنشطة .االقتصادية لمف ىـ في سف العمؿ كالكفكءيف عمميا كعمميا 2 - أصبح مف الضركرم البدء في تكزيع االستثمارات في مجاؿ القطاع الصناعي كالخدمي , فضال عف االىتماـ بالجانب الزراعي عمى مستكل متكازف في جميع الكحدات اإلدارية لمحافظة االنب ار كذلؾ لتقميؿ الفكارؽ المكانية , سكاء أكانت سكانية أـ اقتصادية عمى كفؽ خطط تنمكية شاممة مدركسة كضمف مراحؿ. 4 - يمكف تحكيؿ العبء االقتصادم , الناتج عف الزيادة الطبيعية كعامؿ حركة ىجرة السكاف الداخمية إلى أىمية ديمكغرافية عف طريؽ زيادة المساىمة في األنشطة .االقتصادية لمف ىـ في سف العمؿ كالكفكءيف عمميا كعمميا 9 - تشجيع إ قامة الصناعات المتكسطة كالكبيرة في محافظة االنبار الت ي ال تحتاج إلى رؤكس أمكاؿ كبيرة , كما إنيا بد كرىا تحتاج إلى أيدم عاممة كثيفة , كبذلؾ يمكف أف يككف ليا دكر في سيكلة إقامة تمؾ المشاريع الصناعية كثانيا امتصاص البطالة التي بدأت تتفاقـ إعدادىا في اآلكنة األخ.يرة اـ ي 9 - تشجيع إ قامة الصناعات المتكسطة كالكبيرة في محافظة االنبار الت ي ال تحتاج إلى رؤكس أمكاؿ كبيرة , كما إنيا بد كرىا تحتاج إلى أيدم عاممة كثيفة , كبذلؾ يمكف أف يككف ليا دكر في سيكلة إقامة تمؾ المشاريع الصناعية كثانيا امتصاص البطالة التي بدأت تتفاقـ إعدادىا في اآلكنة األخ.يرة ( 778 ( أ.م.د.ذحدنيذعليذعبدذ الراوي ذ ذ حتلولذجغرايفذلدميوغراػوةذاحلربذوأثرهاذ علىذالرتكوبذالدكاني ذ أ.م.د.ذحدنيذعليذعبدذ الراوي 7 - ىادم احمد مخمؼ , عمـ ا لجغرافية السياسية كالجيكبكليتيؾ, بغداد, 7984 , ص772 . 7 - محمد أزىر السماؾ ك عبد الحميـ القيسي, التركيب السكاني كاإلبعاد المستقبمية لخطط التنمية في العراؽ ,مجمة النفط كالتنمية العدد1 , 7981 , ص9 . 2 - ,جكف كالرؾ ترجمة محػػػػمد شكقي إبراىيـ, دار المريخ لمطبع كالنشر , الػػػرياض, 7987 , ص777 . 4 - محمد صفكح األخرس , عمـ السكاف كقضا يا التنمية كالتخطيط ليا, ص777 . 9 - ,فتحي محمد ابك عيانة, الجغرافية السكاف ط 7 , ,دار النيضة العربية ,بيركت 7980 , ص81 . 1 - 1 - جكف كالرؾ , مصدر سابؽ , ص772 . 7 Year book of the lobour staristics , Geneva , 1975 , P.3 . 8 - طو حمادم الحديثي, جغرافية السكاف, دار الكتب لمطباعة كالنشر, المكصؿ , ط 7 , 7000 , ص127 . 9 - عبد الحسيف جكاد سريح, بعض خصائص السكاف في الككيت, مجمة الخميج العربي, مجمد771 , العدد 2 - 4 / 7989 , ص17 . 3 - : الصابئة المندائيون 70 - منصكر الراكم, سكاف الكطف العربي, الجزء األكؿ, بيت الحكمة لمنشر , بغداد, 7007 , ص471 . 77 - فتحي محمد ابك عيانة, جغرافية السكاف, مصدر سابؽ, ص289 . 77 - عماد مطير خميؼ ألشمرم , جغرافية السكاف األسس كاألركاف في ا لتطبيؽ, مطبعة العماد لمطباعة المحدكدة, الجماىيرية العظمى , 7077 , ص799 . 72 - ,يكنس حمادم عمي, مبادئ عمـ الديمكغرافية, مطابع جامعة المكصؿ7989, ص 277 .أ 74 - رياض إبراىيـ السعدم ,, اليجرة كالحركب كدكر القكل العاممة األنثكية في التنمية الريفية في العراؽ مجمة الجمعية الج ,غرافية العراقية, العدد الثاني7981 , ص779 . 71 - األمـ المتحدة , المج نة االقتصادية , الكرقة المقدمة في الندكة العممية حكؿ التعداد ال عاـ لمسكاف كالمساكف في غرب أسيا, بغداد, 7989 , ص78 . 71 - :سالـ إبراىيـ عطكؼ كبو, اقتصاديات عراؽ التنمية البشرية المستدامة عمى شبكة االنترنت http\\www.rezgar.com\\asrp? =570 71 - :سالـ إبراىيـ عطكؼ كبو, اقتصاديات عراؽ التنمية البشرية المستدامة عمى شبكة االنترنت http\\www.rezgar.com\\asrp? =570 78 - منذر جابر م حمد, الخصخصة كاالقتصاد العراقي , مجمة القاد سية لمعمكـ االدارية كاالقتصادية, المجمد ,الحادم عشر, العدد الثالث7009, ص 770 . 79 - سالـ إبراىيـ عطكؼ كبو:, الفقر كالبطالة في عراؽ التنمية البشرية المستدامة عمى المكقع www.babil-ml.org11951×016htm. 70 - محم د عدناف كديع, سكؽ العمؿ كتخطيط القكة العاممة, المعيد العربي لمتخطيط, الككيت , 7007 , ص 99 . ) 779 ) ) 779 ) جملةذجامعةذاألنبارذللعلومذاإلندانوة ذ ذ (العددذ4ذ )ذ (كانونذاألول)ذ3102ذذذذذذذذذ اجمللدذالثاني ذ 77 - أيفنيد ىكفماف , تجربة منظمة العمؿ الدكلية في مجاؿ أعداد التصنيؼ الدكلي المكحد النم ,كذجي لمميف منظمة العمؿ الدكلية, جنينؼ, مكقع عمى شبكة االنترنت: www.arb.org\ develop-1.htm. 72 - جميكرية العراؽ, كزارة التخطيط , الجياز ال مركزم لإلحصاء, اإل حصاء السكاني , نتائج التعداد العاـ لمسكاف7981 لمحافظة االنبار, جدكؿ24 , ص744 . 74 - عبد عمي حسف الخفاؼ عك بد مخكر الريحاني, جغرافية السكاف, مطبعة جامعة البصرة , 7981 , ص 741 . جملةذجامعةذاألنبارذللعلومذاإلندانوة ذ ذ (العددذ4ذ )ذ (كانونذاألول)ذ3102ذذذذذذذ اجمللدذالثاني ذ (العددذ4ذ )ذ (كانونذاألول)ذ3102ذ اجمللدذالثاني ذ 77 - أيفنيد ىكفماف , تجربة منظمة العمؿ الدكلية في مجاؿ أعداد التصنيؼ الدكلي المكحد النم ,كذجي لمميف منظمة العمؿ الدكلية, جنينؼ, مكقع عمى شبكة االنترنت: www.arb.org\ develop-1.htm. 72 - جميكرية العراؽ, كزارة التخطيط , الجياز ال مركزم لإلحصاء, اإل حصاء السكاني , نتائج التعداد العاـ لمسكاف7981 لمحافظة االنبار, جدكؿ24 , ص744 . 74 - عبد عمي حسف الخفاؼ عك بد مخكر الريحاني, جغرافية السكاف, مطبعة جامعة البصرة , 7981 , ص 741 . 3 - : الصابئة المندائيون 77 - أيفنيد ىكفماف , تجربة منظمة العمؿ الدكلية في مجاؿ أعداد التصنيؼ الدكلي المكحد النم ,كذجي لمميف منظمة العمؿ الدكلية, جنينؼ, مكقع عمى شبكة االنترنت: www.arb.org\ develop-1.htm. 72 - جميكرية العراؽ, كزارة التخطيط , الجياز ال مركزم لإلحصاء, اإل حصاء السكاني , نتائج التعداد العاـ لمسكاف7981 لمحافظة االنبار, جدكؿ24 , ص744 . ا 74 - عبد عمي حسف الخفاؼ عك بد مخكر الريحاني, جغرافية السكاف, مطبعة جامعة البصرة , 7981 , ص 741 . 79 - محمد فتحي ابك عيانو , مدخؿ إلى التحمي ؿ اإلحصائي في الجغرافية البشرية, دار المعرفة ,الجامعية ,اإلسكندرية7981 , ص721 . إ 71 - جميكرية العراؽ, كزارة التجارة , البطاقة التمكينية ,, (ب. غ. ـ) فرع االنبار7001 . 71 - سعد إبراىيـ, المجتمع كالدكلة في الكطف العربي, مركز دراسات الكحدة العربية, الطبعة الثانية, بيركت 71 - جميكرية العراؽ, كزارة التجارة , البطاقة التمكينية ,, (ب. غ. ـ) فرع االنبار7001 . 71 - جميكرية العراؽ, كزارة التجارة , البطاقة التمكينية ,, (ب. غ. ـ) فرع االنبار7001 . 71 - سعد إبراىيـ, المجتمع كالدكلة في الكطف العربي, مركز دراسات الكحدة العربية, الطبعة الثانية, بيركت 7981 , ص271 . 71 - سعد إبراىيـ, المجتمع كالدكلة في الكطف العربي, مركز دراسات الكحدة العربية, الطبعة الثانية, بيركت 7981 , ص271 . 78 - ,محمد جكاد الرضا العرب كالتربية ك الحضارة دراسة في الفكر المقارف, منتدل الفكر العربي, عماف , 7981 , ص87 . 79 - عبد عمي الخفاؼ, جغرافية السكاف اسس عامة, الطبعة االكلى , دا ر الفكر لمطباعة كالنشر كالتكزيع , عماف, 7999, ص 792 . 79 - عبد عمي الخفاؼ, جغرافية السكاف اسس عامة, الطبعة االكلى , دا ر الفكر لمطباعة كالنشر كالتكزيع , عماف, 7999, ص 792 . 20 - عبد الرحيـ البكادقجي كمحمد خالد الحريرم, عمـ السكاف, منشكرات جامعة دمشؽ, الط بعة االك لى , مكتبة االقتصاد, 7001 , ص792 . 27 منصكر الراكم , دراسات في السكاف كالتنمية في العراؽ, بيت الحكمة, بغداد7989, ص 11 . 27 - م نصكر الراكم, سكاف الكطف العربي , دراسة ت حميمية في المشكالت الديمكغرافية, الجزء االكؿ, بيت الحكمة , بغداد, 7007, ص 727 . 3 - : الصابئة المندائيون 22 - ,جميكرية العراؽ ,كزارة التعميـ العالي كالبحث العممي, رئاسة جامعة االنبار, قسـ التخطيط كالمتابعة .بيانات غير منشكرة 24 - الرائد, العدد14 , السنة السادسة, www.al-raeed.net 29 - لقاء مع احدل ع كائؿ الصابئة (احمد خميؿ فميفؿ.) التي تسكف في مركز قضاء الرمادم / حي العزيزية .(*) لقاء مع المحامي (محمد عبد حمادم) دائرة عدؿ االنبار 22 - ,جميكرية العراؽ ,كزارة التعميـ العالي كالبحث العممي, رئاسة جامعة االنبار, قسـ التخطيط كالمتابعة .بيانات غير منشكرة 24 - الرائد, العدد14 , السنة السادسة, www.al-raeed.net 29 - لقاء مع احدل ع كائؿ الصابئة (احمد خميؿ فميفؿ.) التي تسكف في مركز قضاء الرمادم / حي العزيزية .(*) لقاء مع المحامي (محمد عبد حمادم) دائرة عدؿ االنبار ( 720 (

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Outpatient Management of Patients With Angina With No Obstructive Coronary Arteries: How to Come to a Proper Diagnosis and Therapy

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Outpatient Management of Patients With Angina With No Obstructive Coronary Arteries: How to Come to a Proper Diagnosis and Therapy Meeder, J.G.; Hartzema-Meijer, M.J.; Jansen, T.P.J.; Konst, R.E.; Damman, P.; Elias-Smale, S.E. 2021, Article / Letter to editor (Frontiers in Cardiovascular Medicine, 8, (2021), article 716319) Doi link to publisher: https://doi.org/10.3389/fcvm.2021.716319 Note: To cite this publication please use the final published version (if applicable). REVIEW published: 02 November 2021 doi: 10.3389/fcvm.2021.716319 REVIEW published: 02 November 2021 doi: 10.3389/fcvm.2021.716319 Citation: Meeder JG, Hartzema-Meijer MJ, Jansen TPJ, Konst RE, Damman P and Elias-Smale SE (2021) Outpatient Management of Patients With Angina With No Obstructive Coronary Arteries: How to Come to a Proper Diagnosis and Therapy. Front. Cardiovasc. Med. 8:716319. doi: 10.3389/fcvm.2021.716319 Outpatient Management of Patients With Angina With No Obstructive Coronary Arteries: How to Come to a Proper Diagnosis and Therapy Joan G. Meeder 1, Mariëlle J. Hartzema-Meijer 2, Tijn P. J. Jansen 2, Regina E. Konst 2, Peter Damman 2 and Suzette E. Elias-Smale 2* 1 Department of Cardiology, VieCuri Medical Center, Venlo, Netherlands, 2 Department of Cardiology, Radboud University Medical Center, Nijmegen, Netherlands Two-thirds of women and one-third of men who undergo a clinically indicated coronary angiography for stable angina, have no obstructive coronary artery disease (CAD). Coronary vascular dysfunction is a highly prevalent underlying cause of angina in these so called “Angina with No Obstructive Coronary Arteries (ANOCA)” patients, foremost in middle aged women. Coronary vascular dysfunction encompasses various endotypes, namely epicardial and microvascular coronary spasms, impaired vasodilatation, and increased microvascular resistance. ANOCA patients, especially those with underlying coronary vascular dysfunction, have an adverse cardiovascular prognosis, poor physical functioning, and a reduced quality of life. Since standard ischemia detection tests and coronary angiograms are not designed to diagnose coronary vascular dysfunction, this ischemic heart disease is often overlooked and hence undertreated. But adequate diagnosis is vital, so that treatment can be started to reduce symptoms, reduce healthcare costs and improve quality of life and cardiovascular prognosis. The purpose of this review is to give a contemporary overview of ANOCA with focus on coronary vascular dysfunction. We will provide a possible work-up of patients suspected of coronary vascular dysfunction in the outpatient clinical setting, based on the latest scientific insights and international consensus documents. We will discuss the value of ischemia detection testing, and non-invasive and invasive methods to diagnose coronary vascular dysfunction. Furthermore, we will go into pharmacological and non-pharmacological therapeutic options including anti-anginal regimens and lifestyle interventions. Keywords: coronary vascular dysfunction, angina pectoris, ANOCA, INOCA, microvascular, vasospasm, vasomotor disorders Edited by: Tim van de Hoef, Academic Medical Center, Netherlands Reviewed by: Anastasia Susie Mihailidou, Northern Sydney Local Health District, Australia Jan Piek, Amsterdam University Medical Center, Netherlands Edited by: Tim van de Hoef, Academic Medical Center, Netherlands Reviewed by: Anastasia Susie Mihailidou, Northern Sydney Local Health District, Australia Jan Piek, Amsterdam University Medical Center, Netherlands *Correspondence: Suzette E. Elias-Smale [email protected] Specialty section: This article was submitted to Sex and Gender in Cardiovascular Medicine, a section of the journal Frontiers in Cardiovascular Medicine Received: 28 May 2021 Accepted: 22 September 2021 Published: 02 November 2021 Specialty section: This article was submitted to Sex and Gender in Cardiovascular Medicine, a section of the journal Frontiers in Cardiovascular Medicine Specialty section: This article was submitted to Sex and Gender in Cardiovascular Medicine, a section of the journal Frontiers in Cardiovascular Medicine a section of the journal Frontiers in Cardiovascular Medicine Received: 28 May 2021 Accepted: 22 September 2021 Published: 02 November 2021 Keywords: coronary vascular dysfunction, angina pectoris, ANOCA, INOCA, microvascular, vasospasm, vasomotor disorders UNDERLYING CORONARY PATHOPHYSIOLOGY The coronary microcirculation is an adaptive system that regulates myocardial perfusion (18). While the epicardial coronaries (diameter > 400 µm) act as transport vessels, the pre- arterioles and arterioles in the coronary microcirculation mainly determine resistance. The autoregulation of this resistance makes it possible to maintain a constant blood flow over a wide range of coronary perfusion pressures, delivering oxygen and nutrients to the tissue and removing waste products. Several mechanisms play a role in this autoregulation, namely: myogenic regulation by the vascular smooth muscle cells, metabolic control by metabolites from adjacent myocardial cells, endothelial function responsive to changes in vascular wall tension, autonomic innervation, and circulating hormones including endothelium dependent relaxation factors such as nitric oxide (NO) and prostaglandins (18, 19) and vasoconstrictor agents such as histamine, norepinephrine, and serotonin (20). Coronary vascular dysfunction can be caused by functional and structural abnormalities. Functional abnormalities include endothelial and non-endothelial related pathology (21). Endothelial function can be tested by evaluating the response of the coronary arteries to acetylcholine. If coronary vasodilation occurs after administration of acetylcholine, this indicates well-functioning endothelium. When vasoconstriction occurs, it indicates endothelial dysfunction. The latter is often found in the early phase of atherosclerosis (18, 22). Non-endothelial mediated functional abnormalities are related to decreased diastolic time, increased intramyocardial pressure, increased intracavitary pressure, and/or tissue edema. Structural abnormalities include microvascular remodeling in arterioles (intimal thickening, smooth muscle cell proliferation and perivascular fibrosis) and decreased capillary density resulting in increased microvascular resistance. This is found, among other things, in left ventricular hypertrophy, calcium surplus and amyloidosis (21). In general. any dysfunction based on non-endothelial mediated functional and/or structural abnormalities of the coronary microcirculation can be demonstrated by CFR and microvascular resistance measurements. INTRODUCTION Accruing evidence shows that coronary vascular dysfunction can be properly diagnosed with techniques such as an invasive coronary vasomotor test and that treatment based on the results of such a test leads to reduction of symptoms and improvement of quality of life (12). This review is intended to give tips and tricks to adequately recognize, diagnose and treat patients with (suspected) coronary vascular dysfunction in the outpatient clinical setting. and prognosis. Timely recognition of this disease, and thus the start of treatment, is therefore essential. and prognosis. Timely recognition of this disease, and thus the start of treatment, is therefore essential. INTRODUCTION Angina pectoris affects more than 100 million people worldwide, and is the most common symptom of myocardial ischemia (1). Two-thirds of women and one-third of men who undergo a coronary angiogram (CAG) for symptoms of cardiac ischemia do not have obstructive coronary artery disease (CAD) (2–6). In the majority (59–89%) of these so called ANOCA (Angina with November 2021 | Volume 8 | Article 716319 Frontiers in Cardiovascular Medicine | www.frontiersin.org Meeder et al. ANOCA: Outpatient Management No Obstructive Coronary Arteries) patients, the symptoms are caused by coronary vascular dysfunction (7, 8), comprising epicardial vasospasm and/or coronary microvascular dysfunction (CMD) that includes microvascular coronary spasm, increased microvascular resistance and/or decreased vasodilatory capacity as measured by coronary flow reserve (CFR) (9, 10). This condition is more common in women than in men. A recent study in 1,379 patients with ANOCA showed coronary artery dysfunction in 70% of women vs. 43% of men (8). Women are more likely to have coronary vascular dysfunction without a history of obstructive CAD, while men with this condition are more likely to have a history of coronary revascularization (11). Because standard diagnostic tests for anginal symptoms are aimed at evaluating obstructive CAD, coronary vascular dysfunction is often overlooked. Accruing evidence shows that coronary vascular dysfunction can be properly diagnosed with techniques such as an invasive coronary vasomotor test and that treatment based on the results of such a test leads to reduction of symptoms and improvement of quality of life (12). This review is intended to give tips and tricks to adequately recognize, diagnose and treat patients with (suspected) coronary vascular dysfunction in the outpatient clinical setting. No Obstructive Coronary Arteries) patients, the symptoms are caused by coronary vascular dysfunction (7, 8), comprising epicardial vasospasm and/or coronary microvascular dysfunction (CMD) that includes microvascular coronary spasm, increased microvascular resistance and/or decreased vasodilatory capacity as measured by coronary flow reserve (CFR) (9, 10). This condition is more common in women than in men. A recent study in 1,379 patients with ANOCA showed coronary artery dysfunction in 70% of women vs. 43% of men (8). Women are more likely to have coronary vascular dysfunction without a history of obstructive CAD, while men with this condition are more likely to have a history of coronary revascularization (11). Because standard diagnostic tests for anginal symptoms are aimed at evaluating obstructive CAD, coronary vascular dysfunction is often overlooked. WHY IS IT IMPORTANT TO IDENTIFY PATIENTS WITH CORONARY VASCULAR DYSFUNCTION? Patients with symptoms and signs suggestive of myocardial ischemia without obstructive CAD have an increased cardiovascular risk compared to persons without anginal symptoms. In 917 female patients with chest pain, the composite endpoint of myocardial infarction or cardiovascular death after 10 years of follow-up occurred in 6.7% of women without coronary artery disease, 12.8% of women with non-obstructive CAD, and 25.9% of women with obstructive CAD, respectively (13). A meta-analysis of more than 35,000 patients with ANOCA confirmed the increased risk of myocardial infarction and death. A worse prognosis was seen in patients with proven ischemia vs. no ischemia and patients with non-obstructive coronary disease vs. “normal” coronaries (14). Patients with proven coronary vascular dysfunction also have an increased risk of myocardial infarction, cerebral infarction, heart failure and (cardiovascular) death (15, 16). In addition to a worse cardiovascular prognosis, the disease is often associated with persistent symptoms, leading to unnecessarily repeated diagnostic examinations targeting obstructive CAD, first aid visits, hospital admissions, a reduced quality of life, and reduced labor participation (4, 14, 17). There are several treatment options for coronary vascular dysfunction. A recent randomized trial comprising 150 ANOCA patients, the Stratified Medical Therapy Using Invasive Coronary Function Testing in Angina (CorMiCa) trial, shows that targeted treatment after demonstration of coronary vascular dysfunction significantly reduces anginal complaints and improves quality of life (12). Treatment options focus on symptom relief, improvement of the cardiovascular risk profile, quality of life, CLINICAL PRESENTATION Coronary vascular dysfunction includes CMD and/or epicardial spasm. Since both conditions may differ in clinical symptoms and risk factors, the following sections differentiate between them where relevant. Coronary Microvascular Dysfunction Classic cardiovascular risk factors such as age, hypertension, diabetes, smoking, and especially dyslipidemia and obesity are also involved in CMD (29). But these risk factors only explain part of the occurrence of this disease (30). Specific risk factors for CMD (Table 1) include (premenopausal) migraine, rheumatic diseases or inflammatory bowel diseases (31–33). Female-specific risk factors, such as pre-eclampsia, Hemolysis Elevated Liver enzymes and Low Platelets (HELLP) syndrome, gestational hypertension and diabetes, recurrent spontaneous abortion, and menopausal status, may also play a role (34). It is therefore important to not only concentrate on classical risk factors, but also on migraine, inflammatory disease and female specific risk factors when asking the patient his or her medical history. DIAGNOSTIC CRITERIA FOR CORONARY VASCULAR DYSFUNCTION (CMD AND EPICARDIAL SPASM) In 2017, diagnostic criteria for epicardial spasms were published by the international Coronary Vasomotor Disorders (COVADIS) study group (Table 2) (9). In 2018, this group drew up diagnostic criteria for CMD (Table 3) (10). In 2018, this group drew up diagnostic criteria for CMD (Table 3) (10). Symptoms Angina pectoris is an important symptom of coronary vascular dysfunction. Angina equivalents such as exertional dyspnea and fatigue may also be an expression of this condition. Microvascular angina (MVA)—due to CMD—is difficult to Frontiers in Cardiovascular Medicine | www.frontiersin.org Frontiers in Cardiovascular Medicine | www.frontiersin.org November 2021 | Volume 8 | Article 716319 2 ANOCA: Outpatient Management Meeder et al. TABLE 1 | Non-classical risk factors and triggers playing a role in ANOCA. Non-classical risk factors for CMD Triggers for epicardial spasm (premenopausal) Migraine Alcohol (especially withdrawal) Rheumatic diseases Smoking Inflammatory bowel diseases Hyperventilation Pre-eclampsia/HELLP syndrome Mental stress Gestational hypertension and diabetes Drug use (cocaine, amphetamines) Recurrent spontaneous abortion Allergic reaction Menopausal status Exposure to cold Vasoconstrictor agents (e.g., propranolol, anti-migraine medication) Chemotherapy TABLE 1 | Non-classical risk factors and triggers playing a role in ANOCA. Non-classical risk factors for CMD Triggers for epicardial spasm (premenopausal) Migraine Alcohol (especially withdrawal) Rheumatic diseases Smoking Inflammatory bowel diseases Hyperventilation Pre-eclampsia/HELLP syndrome Mental stress Gestational hypertension and diabetes Drug use (cocaine, amphetamines) Recurrent spontaneous abortion Allergic reaction Menopausal status Exposure to cold Vasoconstrictor agents (e.g., propranolol, anti-migraine medication) Chemotherapy TABLE 1 | Non-classical risk factors and triggers playing a role in ANOCA. Non-classical risk factors for CMD Triggers for epicardial spasm (premenopausal) Migraine Alcohol (especially withdrawal) Rheumatic diseases Smoking Inflammatory bowel diseases Hyperventilation Pre-eclampsia/HELLP syndrome Mental stress Gestational hypertension and diabetes Drug use (cocaine, amphetamines) Recurrent spontaneous abortion Allergic reaction Menopausal status Exposure to cold Vasoconstrictor agents (e.g., propranolol, anti-migraine medication) Chemotherapy cocaine, amphetamines), allergic reactions, exposure to cold (9, 35–39) and vasoconstrictive medications (e.g., propranolol, anti-migraine medication) and chemotherapy (Table 1) (39– 41). Vasospastic angina is likely related to other vasospastic conditions such as migraine and Raynaud’s phenomenon (42, 43). DIAGNOSIS OF CORONARY VASCULAR DYSFUNCTION In the diagnostic work-up of angina (equivalents), in patients with and without a history of obstructive CAD, obstructive CAD as underlying cause of symptoms will have to be ruled out by coronary CT or CAG before considering coronary vascular dysfunction. Consideration should also be given to possible alternative diagnoses. distinguish from classic angina due to obstructive CAD because both are often exercise-related. MVA is more likely if the angina continues after exercise cessation, starts after exercise and/or has limited response to nitroglycerin administration (23). In addition, MVA is more often triggered by palpitations or mental stress (24). The intensity of symptoms can vary over time and can be so severe that patients are limited in daily life activities. Angina at rest often occur in addition to exercise-related complaints (25). This is likely due to a vasospastic component of coronary vascular dysfunction (26). Pure vasospastic (Prinzmetal) angina usually occurs at rest, mainly at night and/or early morning. However, exercise-related symptoms can also be due to vasospasm (27, 28). Based on symptoms only, it is generally not possible to make a good distinction between coronary vascular dysfunction and obstructive CAD. In clinical practice, a significant obstruction must therefore be ruled out by a CAG or coronary computed tomography (CT) scan. Alternative Diagnoses g Chest pain without obstructive CAD can be due to other conditions than coronary vascular dysfunction. Differential diagnoses can be divided into three groups: (1) non-cardiac, (2) cardiac non-ischemic, and (3) cardiac ischemic (44, 45). There are multiple causes of non-cardiac chest pain, including gastrointestinal causes such as gastroesophageal reflux, pulmonary disorders like asthma, musculoskeletal complaints like costochondritis, and psychiatric causes such as anxiety/panic disorders (46, 47). Cardiac non-ischemic pain can be the result of pericarditis, increased intraventricular pressure in systolic or diastolic heart failure or valvular heart disease or altered pain perception (nociception) (48). Cardiac ischemic etiologies include, in addition to coronary vascular dysfunction, myocardial bridging (49). Hypertension deserves special attention. Hypertensive patients often experience chest pain despite the absence of obstructive CAD (50). Strict treatment of blood pressure (target tension 120/70 mmHg) often improves symptoms of chest pain and/or exertional dyspnea. Risk Factors Coronary Microvascular Dysfunction Non-invasive Diagnostic Options for Coronary Vascular Dysfunction CFR can be measured non-invasively with various imaging techniques. All methods evaluate coronary flow (velocity) or perfusion during hyperemia using adenosine vs. rest. g y g Cardiac Positron Emission Tomography (PET), a radionuclide imaging technique, is considered the most reliable method using 15O-water, 13N-ammonia, or 82rubidium tracers (53). It has been well validated for accurate and reproducible quantification of regional myocardial blood flow (MBF) and CFR in the myocardium (54, 55). PET is considered the gold standard for the non-invasive assessment of CFR and correlates well with invasive assessment of CFR (56). A CFR < 2 is diagnostic for ischemia and thus CMD and related to a worse cardiovascular prognosis (53, 57). Despite, PET is not widely used due to some major limitations, namely, high expense, the necessity of an on-site cyclotron when using 15O-water and 13N-ammonia, and the involvement of radiation (58–60). 3. Objective evidence of myocardial ischemia a) Ischemic ECG changes during an episode of chest pain b) Stress-induced chest pain and/or ischemic ECG changes in the presence or absence of transient/reversible abnormal myocardial perfusion and/or wall motion abnormality In CMR, a technique has been developed to determine the Myocardial Perfusion Reserve Index (MPRI). Using a contrast medium (gadolinium), diffusing from the microvasculature into the interstitial space, perfusion signal intensity upslopes are evaluated in stress (induced with adenosine) vs. rest, the ratio being the MPRI, which is considered a surrogate for the CFR (61). CMR is more widely available than PET, less expensive and involves no radiation However, further validation studies of MPRI in relation to the results of coronary vasomotor testing are needed before this technique is ready to be clinically used. vasodilatory capacity and microvascular resistance. As can be appreciated in Table 3, having symptoms without obstructive CAD together with objective ischemia leads to a likely diagnosis of CMD. However, “objective ischemia” is a broad concept in the criteria, and it should be noted that several studies have shown no good correlation between demonstrated ischemia by a non-invasive ischemia detection test [exercise testing, stress Cardiac Magnetic Resonance (CMR), Single-Photon Emission Computed Tomography (SPECT), stress echocardiogram] and invasively determined coronary vascular dysfunction. This could be related to the fact that coronary vascular dysfunction causes a heterogeneous pattern of non-transmural ischemia, which is not visible as a regional perfusion defect (51, 52). TABLE 3 | Diagnostic criteria for coronary microvascular dysfunction (CMD). | g y y ( ) 1. Symptoms of myocardial ischemia a) Effort and/or rest angina b) Angina equivalents (i.e., shortness of breath) 2. Absence of obstructive CAD (<50% diameter reduction or FFR by >0.80) by: a) Coronary CTA b) Invasive coronary angiography 3. Objective evidence of myocardial ischemia a) Ischemic ECG changes during an episode of chest pain b) Stress-induced chest pain and/or ischemic ECG changes in the presence or absence of transient/reversible abnormal myocardial perfusion and/or wall motion abnormality 4. Evidence of impaired coronary microvascular function a) Impaired coronary flow reserve (cut-off values depending on methodology used: ≤2.0 and ≤2.5) b) Abnormal coronary microvascular resistance indices (e.g., IMR > 25) c) Coronary microvascular spasm, defined as reproduction of symptoms, ischemic ECG shifts but no epicardial spasm during acetylcholine testing. d) Coronary slow flow phenomenon, defined as TIMI frame count >25 | g y y ( ) 1. Symptoms of myocardial ischemia a) Effort and/or rest angina b) Angina equivalents (i.e., shortness of breath) 2. Absence of obstructive CAD (<50% diameter reduction or FFR by >0.80) by: a) Coronary CTA b) Invasive coronary angiography 3. Objective evidence of myocardial ischemia a) Ischemic ECG changes during an episode of chest pain b) Stress-induced chest pain and/or ischemic ECG changes in the presence or absence of transient/reversible abnormal myocardial perfusion and/or wall motion abnormality 4. Evidence of impaired coronary microvascular function a) Impaired coronary flow reserve (cut-off values depending on methodology used: ≤2.0 and ≤2.5) b) Abnormal coronary microvascular resistance indices (e.g., IMR > 25) c) Coronary microvascular spasm, defined as reproduction of symptoms, ischemic ECG shifts but no epicardial spasm during acetylcholine testing. d) Coronary slow flow phenomenon, defined as TIMI frame count >25 DEMONSTRATION OF CORONARY VASCULAR DYSFUNCTION (3) Coronary artery spasm—defined as transient total or subtotal coronary artery occlusion (>90% constriction) with angina and ischemic ECG changes either spontaneously or in response to a provocative stimulus (typically acetylcholine, ergonovine, or hyperventilation) As mentioned above, the gold standard to diagnose coronary vascular dysfunction, the invasive coronary vasomotor test, is currently scarce. Hence, for outpatient clinical management it is important to know the value of non-invasive alternatives that can be used to evaluate of coronary vascular dysfunction. TABLE 3 | Diagnostic criteria for coronary microvascular dysfunction (CMD). TABLE 2 | Diagnostic criteria for epicardial spasm. currently, a clear diagnostic work-up is lacking, in our opinion, it is worthwhile to do a non-invasive stress test in patients for which this test is not accessible, especially in patients with exercise- related symptoms. If this ischemia detection test is positive, the suspicion of coronary vascular dysfunction is reinforced. The stress test can be a “simple” exercise test with the advantage that the effort is physiological, and the test is readily available and cheap. TABLE 2 | Diagnostic criteria for epicardial spasm. (1) Nitrate-responsive angina—during spontaneous episode, with at least one of the following: (a) Rest angina—especially between night and early morning (b) Marked diurnal variation in exercise tolerance—reduced in morning (c) Hyperventilation can precipitate an episode (d) Calcium channel blockers (but not β-blockers) suppress episodes (2) Transient ischemic ECG changes—during spontaneous episode, including any of the following in at least two contiguous leads: (a) ST segment elevation ≥0.1 mV (b) ST segment depression ≥0.1 mV (c) New negative U waves (3) Coronary artery spasm—defined as transient total or subtotal coronary artery occlusion (>90% constriction) with angina and ischemic ECG changes either spontaneously or in response to a provocative stimulus (typically acetylcholine, ergonovine, or hyperventilation) (1) Nitrate-responsive angina—during spontaneous episode, with at least one of the following: (a) Rest angina—especially between night and early morning (b) Marked diurnal variation in exercise tolerance—reduced in morning (c) Hyperventilation can precipitate an episode (d) Calcium channel blockers (but not β-blockers) suppress episodes (2) Transient ischemic ECG changes—during spontaneous episode, including any of the following in at least two contiguous leads: (a) ST segment elevation ≥0.1 mV (b) ST segment depression ≥0.1 mV (c) New negative U waves (3) Coronary artery spasm—defined as transient total or subtotal coronary artery occlusion (>90% constriction) with angina and ischemic ECG changes either spontaneously or in response to a provocative stimulus (typically acetylcholine, ergonovine, or hyperventilation) (1) Nitrate-responsive angina—during spontaneous episode, with at least one of the following: Frontiers in Cardiovascular Medicine | www.frontiersin.org Epicardial Vasospasm If 3 of the 4 COVADIS criteria are met (Table 3), a diagnosis of CMD is likely. If all criteria are met, a definite diagnosis of CMD can be made. The gold standard to diagnose coronary vascular dysfunction is an invasive coronary vasomotor test that can comprehensively test all 4 endotypes of coronary vascular function: epicardial and microvascular coronary vasospasms, With the exception of smoking, classical risk factors such as hypertension, dyslipidemia and diabetes are not clearly related to vasospastic angina. Smoking is not only a risk factor but can also trigger vasospasm attacks (9). Anginal episodes can also be triggered by (withdrawal of) alcohol, hyperventilation, mental stress and daily hassles, stimulant drug use (e.g., November 2021 | Volume 8 | Article 716319 Frontiers in Cardiovascular Medicine | www.frontiersin.org 3 ANOCA: Outpatient Management Meeder et al. TABLE 2 | Diagnostic criteria for epicardial spasm. TABLE 2 | Diagnostic criteria for epicardial spasm. Non-invasive Diagnostic Options for Coronary Vascular Dysfunction This has the important implication that a negative ischemia detection test does not rule out coronary vascular dysfunction. Currently, the invasive coronary vasomotor test is very limited available, and therefore not feasible in the majority of patients. Although With TransThoracic Doppler Echocardiography (TTDE), the Coronary Flow Velocity Reserve (CFVR) can be determined in the Left Anterior Descending coronary (LAD). The CFVR is the ratio of the peak velocity in hyperemia (using systemic adenosine) vs. the peak velocity in rest in the LAD and a surrogate for CFR. A CFR < 2.5 is considered diagnostic for CMD. Although echocardiography is readily available and inexpensive, this method has limited application because it requires expertise from the echocardiographer and not all patients have a suitable ultrasound window (62, 63). November 2021 | Volume 8 | Article 716319 4 ANOCA: Outpatient Management Meeder et al. PET, CMR and TTDE have an ESC IIb recommendation (i.e., may be considered) for the detection of coronary vascular dysfunction (64). However, the described methods only assess CFR, which is reflecting just 1 of the 4 endotypes of coronary vascular dysfunction. Although research is being done on non- invasive measures of microvascular resistance, (65) currently, this endotype cannot be assessed non-invasively in clinical practice. Furthermore, vasospastic disease cannot be evaluated adequately by the contemporary non-invasive techniques such as cold-pressor PET. It cannot distinguish between epicardial and microvascular vasospasm and, most essential, does not correlate well with invasively assessed vasospasm (15). This is an important limitation since vasospasm is the most prevalent endotype in patients with coronary vascular dysfunction, occurring in 81–97% of patients diagnosed with coronary vascular dysfunction, while an abnormal CFR or microvascular resistance without vasospasms occurred in only 3–19% (52, 66, 67). Therefore, one should realize that the diagnosis of coronary vascular dysfunction is easily missed with non- invasive diagnostics. left coronary artery with continuous monitoring of symptoms and 12-channel ElectroCardioGram (ECG). An alternative to acetylcholine is ergonovine, but it is less effective, especially in women (68). The acetylcholine test, as mentioned in Tables 2, 3, is positive for epicardial spasms if recognizable symptoms occur, accompanied by ischemic ECG changes and a ≥90% reduction of the coronary lumen: an example of a positive acetylcholine test for epicardial spasm can be appreciated in Figure 1. If there are recognizable symptoms and ischemic ECG changes, but <90% lumen reduction, the diagnosis of microvascular spasm is made. Non-invasive Diagnostic Options for Coronary Vascular Dysfunction The acetylcholine test ends with the administration of nitroglycerin to ensure that there is complete vasorelaxation of the coronaries and that the patient is free of complaints. Using systemic infusion of adenosine, the coronary flow reserve (CFR) is determined, defined as the ratio between hyperemic coronary flow vs resting flow. This can be done by means of thermodilution or Doppler (56). An invasively measured CFR < 2 is generally used as a cut-offvalue for the detection of ischemia in CMD. The basis for this is the finding that CFR < 2, measured by PET, is related to a clearly worse cardiovascular prognosis (53, 57). CFR > 2.5 is considered normal. CFR values between 2 and 2.5 form a gray area. In the same session, the microvascular resistance is measured: if thermodilution is used, the index of microvascular resistance (IMR) is determined, if the evaluation is done with Doppler, the hyperemic microvascular resistance (HMR) is determined (69). IMR of 25 U is generally PROPOSED DIAGNOSTIC ALGORITHM used as cut-off, with values above 25 being diagnostic for CMD (Table 3). For HMR, 2.5 mmHg/cm/s is often used as a cut-offvalue for the diagnosis of microvascular disease (57, 70). PROPOSED DIAGNOSTIC ALGORITHM Coronary vascular dysfunction plays a substantial pathogenic role across the spectrum of ischemic heart disease including patients with no obstructive CAD and individuals with obstructive CAD, as well as those with persisting angina after anatomically successful coronary recanalization/revascularization. So, what to do in clinical practice with patients suspected of coronary vascular dysfunction? In current clinical practice, we still lack a generally used work-up for patients with ANOCA. In Figure 2, we propose a possible diagnostic algorithm. In patients with angina (equivalents) lasting at least 3 months, first of all, obstructive CAD as underlying cause of symptoms should be ruled out with a coronary CT scan or CAG. Also, alternative diagnoses should be considered, and if likely, ruled out. If an invasive coronary vasomotor test is available, the next step would be to perform such a test. We propose to do a comprehensive test using both acetylcholine and adenosine to investigate all endotypes of coronary vascular dysfunction to get a definite diagnosis, including the endotype of dysfunction on which treatment can be based (57). In patients who do not wish to undergo a vasomotor test or in centers where this test is not available, clinicians could consider a non-invasive stress test to evaluate ischemia. We would recommend a “simple” but As mentioned in the introduction, in the majority of patients with ANOCA, coronary vascular dysfunction is found on invasive coronary vasomotor test (59–89%) (7, 8). Most of the patients with coronary vascular dysfunction have an abnormal acetylcholine test (52, 66, 67). In addition, patients may have an abnormal CFR or microvascular resistance which might influence prognosis (67). Therefore, we recommend that in all patients undergoing an invasive test a complete coronary vasomotor test with administration of both acetylcholine and adenosine should be performed. The Invasive Coronary Vasomotor Test As mentioned above, the coronary vasomotor test is currently the only test that can comprehensively evaluate all endotypes of coronary vascular dysfunction. During a CAG, obstructive CAD is ruled out, after which vasomotor tests are performed. To evaluate coronary vasospasm, ascending doses (usually 2, 20, 100, and 200 µg) of acetylcholine are given in the FIGURE 1 | Epicardial spasm provoked by acetylcholine during coronary vasomotor test. Case of a 46-year-old female with severe chronic angina. At 2 mcg of acethylcholine a severe spasm of the LAD occurs with concurrent severe angina and ST elevation at the ECG. ACH, Acetylcholine; ECG, electrocardiogram. FIGURE 1 | Epicardial spasm provoked by acetylcholine during coronary vasomotor test. Case of a 46-year-old female with severe chronic angina. At 2 mcg of acethylcholine a severe spasm of the LAD occurs with concurrent severe angina and ST elevation at the ECG. ACH, Acetylcholine; ECG, electrocardiogram. Frontiers in Cardiovascular Medicine | www.frontiersin.org Frontiers in Cardiovascular Medicine | www.frontiersin.org November 2021 | Volume 8 | Article 716319 Meeder et al. ANOCA: Outpatient Management FIGURE 2 | Possible diagnostic algorithm. FIGURE 2 | Possible diagnostic algorithm. FIGURE 2 | Possible diagnostic algorithm. PROPOSED DIAGNOSTIC ALGORITHM ANTI-ANGINAL TREATMENT As can be appreciated in Table 4, several anti-anginal treatment options are available for CMD and epicardial spasm. ASPIRIN The role of aspirin in the treatment of CMD is unclear. The 2013 ESC guideline for stable angina pectoris recommends aspirin for all patients with microvascular angina pectoris (23). The current ESC guideline for chronic coronary syndromes (2019) does not provide clear guidance on this (74). Obviously, aspirin is indicated in patients with a previous cardiovascular event (80). However, for patients without a cardiovascular event, this recommendation is not based on scientific studies. In clinical practice, aspirin is given at the discretion of the treating cardiologist. Some experts in coronary vascular dysfunction do not give aspirin because 3 recent large clinical trials have shown that primary prevention with aspirin is not useful, even in high-risk populations (81). Others are more liberal in giving aspirin, especially in patients with evident but non-obstructive atherosclerosis on CAG, a high calcium score and/or non- calcified plaque on CT coronary angiography and/or a positive ischemia detection test. In epicardial coronary spasm, high-dose aspirin (as given for pericarditis) is not recommended because the blockade of prostacyclin production can aggravate the spasm (82). The use of low-dose aspirin (80–100 mg per day) in patients without concomitant obstructive CAD is still under discussion due to the lack of robust outcome data. Therefore, aspirin is not routinely given in patients with coronary spasm without obstructive CAD (77). Of course, one could deviate from this routine in individual cases, for example in patients with non- obstructive atherosclerosis with focal epicardial spasm at the location of coronary plaques. If it is possible to refer patients for coronary vasomotor testing in another hospital, we advise that an invasive test should be considered in the following cases in particular: (1) If having a definite diagnosis is important to the patient, e.g., to promote acceptance of the disease or to have a clear diagnosis in case of a working dispute. (2) If having a definite diagnosis is important to the cardiologist, e.g., when medical therapy is not effective and doubts raise about the likelihood of the diagnosis. We recommend to start treatment in all patients with suspected coronary vascular dysfunction and adjust it according to effectiveness, side effects, and, if possible, the definite diagnosis. Recommendations for therapy are given in the section below. TREATMENT OPTIONS Recommendations for drug treatment of coronary vascular dysfunction are mainly based on small studies because large outcome trials are lacking to date (72). In the literature, recommendations exist for both CMD and epicardial spasm. Since the exact underlying mechanism is usually unknown in patients with suspected coronary vascular dysfunction, this distinction is not so strict in clinical practice. The choice of medication and dosage will differ per patient, not only based on the possible underlying pathophysiological mechanism, but also on parameters such as heart rate and blood pressure, effectiveness, and the occurrence of side effects. SAFETY OF THE INVASIVE CORONARY VASOMOTOR TEST Recent large studies have shown that coronary vasomotor tests can be performed safely. Complication risks of 0–0.7% are found for the occurrence of serious complications such as myocardial infarction, ventricular fibrillation or death. This is comparable to a CAG with FFR measurement (22, 71). November 2021 | Volume 8 | Article 716319 Frontiers in Cardiovascular Medicine | www.frontiersin.org 6 ANOCA: Outpatient Management Meeder et al. physiological exercise test, especially in patients with exercise related symptoms. When this exercise test indicates cardiac ischemia, the diagnosis of coronary vascular dysfunction is strengthened. If it is negative for ischemia, the diagnosis vascular dysfunction cannot be ruled out. In hospitals with expertise in TTDE or PET, non-invasive evaluation of the CFR could be performed. If CFR is <2–2.5 (depending on the method used), CMD is diagnosed. If the CFR is normal, coronary vascular dysfunction is still not ruled out. So, non-invasive ischemia testing and CFR measurements can only be used to rule in, or strengthen the diagnosis, but cannot be used to rule out coronary vascular dysfunction. epicardial spasm is suspected, triggers such as smoking/co- smoking and drug use (including cocaine or amphetamines) should be strongly discouraged (23). Angiotensin-Converting- Enzyme (ACE) inhibitors and angiotensin II antagonists are not only effective in lowering blood pressure, but also improve endothelial function and have a beneficial effect on both CMD and epicardial vasospasm (40, 78, 79). Frontiers in Cardiovascular Medicine | www.frontiersin.org November 2021 | Volume 8 | Article 716319 Second-Line Therapy If the effect on the complaints of first-line treatment is insufficient, a long-acting nitrate can be added (23). It can also be considered to combine a non-dihydropyridine with a dihydropyridine calcium antagonist, although this combination frequently causes side effects such as edema formation (93). Nicorandil has also been shown to be an effective agent for epicardial spasms and can be added if symptoms are insufficiently controlled (83). Third-Line Therapy In case of insufficient relief of symptoms on first- and second- line therapy, consideration should be given to establish or dismiss the diagnosis coronary vascular dysfunction with a coronary vasomotor test if this has not yet been done. Third-line medication includes Trimetazidine, an anti-ischemic metabolic agent that improves myocardial glucose utilization through inhibition of fatty acid metabolism. It improves angina and stress testing results when compared to conventional therapy (92). Also, a low dose of imipramine, a tricyclic antidepressant can be considered, acting as a pain reliever (40). Besides medical therapy, non-medical therapy, as will be discussed below, can be very helpful in patients with refractory symptoms. NON-MEDICAL THERAPY FOR CORONARY VASCULAR DYSFUNCTION Non-medical anti-anginal therapy such as a Transcutaneous Electrical Nerve Stimulation) (TENS) can be considered for severe refractory symptoms (31). However, studies report varying results of this therapy on angina pectoris in CMD patients (100). Another option to relieve symptoms and improve quality of life is spinal cord stimulation, which modulates nociceptive signals and reduces ischemia through its anti-adrenergic effect. In addition, symptom relief has also been reported with “enhanced external counter pulsation (EECP),” a treatment in which inflatable cuffs are placed around the legs and buttocks which stimulate the EXPERIMENTAL THERAPY FOR CORONARY VASCULAR DYSFUNCTION A number of novel drugs are promising, such as the rho kinase inhibitor Fasudil, which has been shown to be effective in preventing acetylcholine-induced vasospasm (94). Other potentially effective agents include type 3 and type 5 phosphodiesterase inhibitors (cilostazol and sildenafil, respectively). In a multicenter randomized trial of vasospastic angina patients refractory to amlodipine, cilostazol reduced angina frequency and intensity without serious adverse effects (95). In women with ANOCA, sildenafil acutely improved CFR among women with CFR <2.5 (i.e., CMD) (96). Endothelin 1 (ET-1) contributes to coronary endothelial dysfunction and its tonic inhibitory effect on myocardial perfusion is related to atherosclerosis risk factor burden (97). Two small randomized trials of an endothelin-1 (ET-1) receptor antagonist in MVA suggested a beneficial effect (98, 99). Currently, the Precision Medicine With Zibotentan in Microvascular Angina (PRIZE) trial is investigating the effect of zibotentan, an oral endothelin A receptor-selective antagonist, on symptoms, exercise duration and myocardial blood flow in patients with microvascular angina (ClinicalTrials.gov Identifier: NCT04097314). A comprehensive, contemporary overview of potential novel drugs is given by Bairey Merz et al. (92). Second-Line Therapy In patients with refractory symptoms and/or intolerance to the first-line medication, other anti-anginal medications such as long-acting nitrates or ranolazine may be given (23). These can be used in addition to first-line therapy (40). In practice, nicorandil often works better than other long-acting nitrates because, in addition to its effect on nitric oxide production, it has a beneficial effect on the smooth muscle cells around the vessel wall (40, 89). Long-acting nitrates relieve symptoms in about half of patients with suspected CMD (23, 40, 90). Ranolazine is a sodium channel blocker that reduces intracellular calcium in cardiomyocytes leading to improved intraventricular relaxation, potentially improving microcirculation. Studies are unclear about the effect of this drug, although a recent randomized trial in 81 patients showed that ranolazine improves symptoms and myocardial perfusion in patients with a CFR < 2.5 (91). First-Line Therapy y In patients with predominantly resting symptoms, calcium channel blockers are recommended because they have been shown to be effective in both epicardial spasms and CMD (40, 72, 83). Both non-dihydropiridines (e.g., diltiazem) and dihydropiridines (e.g., nifedipine retard) calcium antagonists can be given. β-blockers are recommended in patients with predominantly exercise-related complaints (23). In terms of choice of beta-blocker, atenolol and nebivolol are particularly recommended (40). Atenolol improves exercise capacity and anginal symptoms (84). Nebivolol is not only a selective β-1 receptor blocker, but also has vasodilatory effects through NO production, which is likely to be beneficial in case of a vasospastic component (85). Several small studies showed a better effect of nebivolol than metoprolol (86, 87). In addition to nebivolol, carvedilol and labetalol (β-blockers with both alpha-1 and β- adrenergic receptor antagonist properties) are recommended for CMD due to their vasodilatory effect (88). Short-acting nitrates are recommended for stopping attacks of angina, although this will not relieve symptoms in all patients (23, 40, 76). CARDIOVASCULAR RISK MANAGEMENT Since classical cardiovascular risk factors also play a role in CMD and epicardial spasms, it is recommended that these be strictly controlled with statins, antihypertensive drugs, anti- diabetic therapy, and lifestyle modifications such as weight reduction and smoking cessation (23, 73, 74). It should be emphasized that it is important to control blood pressure very tightly (target tension 120/70 mmHg). This is not only important in the context of lowering the cardiovascular risk, but in practice it has been found that strict blood pressure regulation often provides clear complaints relief. Statins, in addition to their cholesterol-lowering effect (75), are recommended because of their beneficial effect on endothelial function (76). In addition, several studies have shown a beneficial effect of statins in the prevention of coronary spasm attacks (77). When TABLE 4 | Anti-anginal treatment for CMD and epicardial spasm. Medication Coronary microvascular dysfunction Epicardial spasm First-line Calcium channel blocker (Diltiazem, Nifedipine) ß-blocker (Nebivolol, Atenolol, Carvedilol, Labetalol) Short acting nitrate Calcium channel blocker (Diltiazem, Nifedipine) Short acting nitrate Second-line Long acting nitrate Nicorandil Ranolazine Long acting nitrate Nicorandil Third-line Imipramine Trimetazidine November 2021 | Volume 8 | Article 716319 TABLE 4 | Anti-anginal treatment for CMD and epicardial spasm. Frontiers in Cardiovascular Medicine | www.frontiersin.org November 2021 | Volume 8 | Article 716319 7 ANOCA: Outpatient Management Meeder et al. CMD attacks (23, 74). Non-selective β-blockers, such as propranolol, should be avoided if there is (suspected) coronary artery spasm, as they can trigger spasm (40, 41). However, as mentioned above, nebivolol was shown to reduce coronary vasospasm, although not as effective as diltiazem (85). MENOPAUSE PRACTITIONER If the symptoms appear to be related to the menopause, for example, anginal symptoms provoked by menopause-related increase in palpitations, it can be considered to refer patients to a menopause practitioner for additional tips and interventions. Epicardial Spasm First-Line Therapy First-line therapy consists of a (non-) dihydropyridine calcium channel blocker and a short-acting nitrate to stop vasospasm November 2021 | Volume 8 | Article 716319 Frontiers in Cardiovascular Medicine | www.frontiersin.org 8 ANOCA: Outpatient Management Meeder et al. return of blood to the heart through continuous inflation and deflation (101). energy by helping the patient pacing themself and enable energy conservation. Furthermore, they can give insight in the patients own activities and how to achieve a balanced lifestyle. With this insight, patients can make decisions on how to distribute their energy throughout the day/week and thereby limit the loss of functioning. LIFESTYLE ADJUSTMENTS Many patients with coronary vascular dysfunction are limited in their daily life by chronic, severe symptoms. Because it often concerns middle-aged women who work and/or have a family with growing children, the disease often has a major impact on daily life. There is little scientific literature available on the influence of lifestyle changes on symptoms. At the Radboud University Medical Center, Nijmegen, the Netherlands, we have over 5 years of experience with the guidance of these patients by a nurse practitioner. The following advice is based on this experience. Since the majority of cardiology clinics do not have a nurse practitioner, we have formulated the recommendations in such a way that they are generally applicable. EXERCISE Exercise helps to reduce symptoms and improve exercise tolerance (102). A regular cardiac rehabilitation program is often too strenuous for patients with seriously debilitating symptoms. To date, no exercise program exists that is tailored to patients with ANOCA. We did in-depth interviews with 10 of our patients on this subject. Several important barriers to perform physical activity came up: anxiety to develop symptoms, mental pressure leading to symptoms and uncertainty of their physical limitations due to variation of symptoms over time. Regarding wishes for an exercise program, the patients found it important to exercise under supervision of a healthcare professional with knowledge of ANOCA, who knows how to train and give advice on coping with symptoms during or after physical activity, leading to a feeling of security and being taken care of. Next to fitness training they would like to train to perform their house holding activities,. The patients also stressed the importance of a minimum of stimuli (for example audio: the acoustics as well as the volume, preferably no music or loud voices) and pressure (for example setting goals with time limits) since it can trigger symptoms. Furthermore, they expressed the need for a slow start-up. Research has confirmed the importance to do a thorough warming-up (minimal 10 min on 50–60% of maximum intensity) to avoid “warm-up angina” (103, 104). Furthermore, experience learns that when patients exercise too intensively, it aggravates symptoms and often causes excessive fatigue the next day. Learning to listen to their body signals and take adequate actions, is difficult but essential to avoid these symptoms. MENTAL STRESS Mental stress and/or overstimulation can trigger symptoms, probably related to vasospasm (36). Patients with severe symptoms often report concentration problems and symptoms triggered by work deadlines or outside stimuli like traffic noise and social events. It is therefore important to teach patients how to deal with stress/outside stimuli. Possible interventions include mindfulness, yoga, Tai Chi, or walking in nature. A lot of patients also experience considerable mental stress during a disability trajectory. Occupational physicians or disability experts should therefore be well informed of the disease so that they can support the patient as good as possible. Another important cause of mental stress is having to deal with a chronic disabling disease at a relatively young age. Acceptance of the disease is very difficult for patients with severely limiting symptoms and can aggravate symptoms. Psychological counseling can offer help. KNOWLEDGE GAPS AND FUTURE DIRECTIONS IN OUTPATIENT MANAGEMENT Although research on diagnostic and therapeutic strategies for coronary vascular dysfunction is rapidly expanding, many knowledge gaps still need to be filled. A prerequisite for improving healthcare for these patients is to increase awareness among cardiologists that coronary vascular dysfunction is a plausible cause underlying cardiac symptoms in patients with ANOCA. Regarding to diagnostics, many questions are still open, of which we will discuss a number. Firstly, the diagnostic value of standard non-invasive ischemia detection tests like exercise tests, SPECT, PET, CMR for coronary vascular dysfunction needs to be established as long as invasive coronary vasomotor tests are scarce. Secondly, it would be worthwhile to improve non-invasive diagnostics, especially for coronary vasospasm, which is the most common form of coronary vascular dysfunction. Thirdly, invasive methods to diagnose CMD could be improved. 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An occupational therapist can offer practical help to make household chores easier to save November 2021 | Volume 8 | Article 716319 Frontiers in Cardiovascular Medicine | www.frontiersin.org 9 Meeder et al. ANOCA: Outpatient Management and relatively high inter- and intra-observer variability (56). Furthermore, these methods require hyperemia for which intravenous adenosine is administered which can cause side effects including chest pain, dyspnea and AV-blocks. Moreover, it should be avoided in patients with severe chronic obstructive pulmonary disease (COPD) and is contraindicated in patients with asthma. and relatively high inter- and intra-observer variability (56). Furthermore, these methods require hyperemia for which intravenous adenosine is administered which can cause side effects including chest pain, dyspnea and AV-blocks. Moreover, it should be avoided in patients with severe chronic obstructive pulmonary disease (COPD) and is contraindicated in patients with asthma. 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(2019) 40:2350–2359. doi: 10.1093/eurheartj/ehz245 November 2021 | Volume 8 | Article 716319 Frontiers in Cardiovascular Medicine | www.frontiersin.org 12 Meeder et al. ANOCA: Outpatient Management The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. 107. Xaplanteris P, et al. Catheter-based measurements of absolute coronary blood flow and microvascular resistance: feasibility, safety, and reproducibility in humans. Circ Cardiovasc Interv. (2018) 11:e006194. doi: 10.1161/CIRCINTERVENTIONS.117.006194 108. van ’t Veer M, et al. Novel monorail infusion catheter for volumetric coronary blood flow measurement in humans: in vitro validation. EuroIntervention. (2016) 12:701–7. doi: 10.4244/EIJV12I6A114 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. 109. Keulards DCJ, et al. Safety of absolute coronary flow and microvascular resistance measurements by thermodilution. EuroIntervention. (2020) 17:229–32. doi: 10.4244/EIJ-D-20-00074 109. Keulards DCJ, et al. Safety of absolute coronary flow and microvascular resistance measurements by thermodilution. EuroIntervention. (2020) 17:229–32. doi: 10.4244/EIJ-D-20-00074 110. Konst RE, et al. Absolute coronary blood flow measured by continuous thermodilution in patients with ischemia and nonobstructive disease. J Am Coll Cardiol. (2021) 77:728–41. doi: 10.1016/j.jacc.2020.12.019 110. Konst RE, et al. Absolute coronary blood flow measured by continuous thermodilution in patients with ischemia and nonobstructive disease. J Am Coll Cardiol. (2021) 77:728–41. doi: 10.1016/j.jacc.2020.12.019 Copyright © 2021 Meeder, Hartzema-Meijer, Jansen, Konst, Damman and Elias-Smale. 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. Frontiers in Cardiovascular Medicine | www.frontiersin.org November 2021 | Volume 8 | Article 716319 REFERENCES No use, distribution or reproduction is permitted which does not comply with these terms. 111. Fournier S, et al. Normal values of thermodilution-derived absolute coronary blood flow and microvascular resistance in humans. EuroIntervention. (2020) 17:e309–16. doi: 10.4244/EIJ-D-20-00684 111. Fournier S, et al. Normal values of thermodilution-derived absolute coronary blood flow and microvascular resistance in humans. EuroIntervention. (2020) 17:e309–16. doi: 10.4244/EIJ-D-20-00684 Conflict of Interest: PD has received consultancy fees from Philips and Abbott, and research grants from Philips, Abbott, and AstraZeneca. PD and SE-S have received a research grant from Abbott. November 2021 | Volume 8 | Article 716319 Frontiers in Cardiovascular Medicine | www.frontiersin.org 13

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Changes in Sexual Function Following Uphold Transvaginal mesh Surgery for the Treatment of Urogenital Prolapse

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Changes in Sexual Function Following Uphold Transvaginal mesh Surgery for the Treatment of Urogenital Prolapse OPEN Tsun-Wen Hsiao1,2, Chin-Ru Ker1,2, Kun-Ling Lin   1,2, Yung-Shun Juan1,3, Ming-Ping Wu4, Yi-yin Liu1,4,5 & Cheng-Yu Long1,5* Uphold transvaginal mesh implantation is an option for treating pelvic organ prolapse (POP). This prospective cohort study aims to evaluate the effect of Uphold transvaginal mesh implantation on female sexual function. 205 women with symptomatic POP were recruited and evaluated pre- operatively and re- evaluated six months post-operatively in terms of anatomical restoration, quality of life influenced by urinary incontinence and female sexual function. 30 women eventually completed the assessments and been statistically evaluated. The main outcome focused on sexual function. In our study, we found that Uphold transvaginal mesh surgery could achieve effective anatomical restoration of POP and better sexual function regardless of concomitant sling surgery. Female pelvic organ prolapse (POP) can adversely affect the quality of life in regard to sexual function. Possible etiologic factors include nerve injury, blood supply impairment and changes in topography of pelvic floor. Pelvic surgery may also alter vaginal topography and affect subsequent sexual function. In addition, close proximity of vaginal grafts or scar tissue formation following pelvic surgery can cause stiffer vaginal wall inducing dyspare- unia1. However, there are conflicting evidences with respect to the effect of transvaginal mesh (TVM) surgery on sexual function. Some studies showed TVM surgery resulted in deteriorating sexual function, while oth- ers revealed no or positive effect of the procedure on sexual function2–11. There are kinds of transvaginal mesh implantation methods such as single incision vaginal mesh and trans-obturator vaginal mesh. Different kinds of transvaginal mesh implantation may cause different effect on sexual function. Recent study has shown that single incision vaginal mesh may cause lower rate of mesh erosion than that of trans-obturator vaginal mesh12. Whether less erosion rate may relate to less sexual dysfunction or not need further investigation.i Uphold Lite Vaginal Support System with Capio SLIM Suture Capturing Device (Boston scientific, USA) is a newer generation of transvaginal mesh TVM kit. It has been used to treat POP since 2013. It is type I single incision mesh with characteristic of small in size and monoporous texture that could decrease biomaterial load to avoid mesh-related complications, such as mesh erosion. However, Food and Drug Administration (FDA) of the United States announced a warning in 2011 questioning the long-term safety of the surgical mesh. www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports Scientific Reports | (2019) 9:17047 | https://doi.org/10.1038/s41598-019-52990-0 Changes in Sexual Function Following Uphold Transvaginal mesh Surgery for the Treatment of Urogenital Prolapse OPEN Data are given as median (range) or mean ± standard deviation. Pre-op, preoperative; Post-op, postoperative; Tvl, total vaginal length; UDI-6, Urogenital Distress Inventory; IIQ-7, Incontinence Impact Questionnaire. *Wilcoxon signed rank test; **Paired t-test. Changes in Sexual Function Following Uphold Transvaginal mesh Surgery for the Treatment of Urogenital Prolapse OPEN They have proposed on February 26, 2016 that urogynecologic surgical mesh instrumentation to be reclassified from class II to class III with special controls and be subjected to premarket notification requirements13. This warning raised the need to thoroughly evaluate the outcome of TVM. Few studies were conducted for Uphold TVM surgery including evaluation of post-treatment sexual function. Thus, we conducted this study to assess the effect of Uphold TVM surgery on sexual function. 1Graduate Institute of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan. 2Department of Obstetrics and Gynecology, Kaohsiung Medical University Hospital, Kaohsiung Medical University, Kaohsiung, Taiwan. 3Department of Urology, Kaohsiung Medical University Hospital, Kaohsiung Medical University, Kaohsiung, Taiwan. 4Department of Obstetrics and Gynecology, Chi Mei Foundation Hospital, Tainan, Taiwan. 5Department of Obstetrics and Gynecology, Kaohsiung Municipal Hsiao-Kang Hospital, Kaohsiung Medical University, Kaohsiung, Taiwan. *email: [email protected] Scientific Reports | (2019) 9:17047 | https://doi.org/10.1038/s41598-019-52990-0 www.nature.com/scientificreports/ N = 30 Mean age (years) 57.3 ± 8.6 Mean parity 2.5 ± 0.6 Mean BMI (kg/m2) 25.2 ± 3.8 Menopause 26 (86.7) Hormone therapy 6 (20.0) Diabetes Mellitus 0 Hypertension 13 (43.3) History of hysterectomy 6 (20.0) Uphold TVM 30 Concomitant surgery    Posterior repair 5 (16.7)    Vaginal hysterectomy 9 (30.0)    TVT-O 10 (33.3)    Ophira 2 (6.7) Table 1. Clinical background of all subjects. Data are given as mean ± standard deviation, or n (%). TVM, transvaginal mesh; BMI, body mass index; TVT-O, transobturator tape; TVT, tension-free vaginal tape. N = 30 Mean age (years) 57.3 ± 8.6 Mean parity 2.5 ± 0.6 Mean BMI (kg/m2) 25.2 ± 3.8 Menopause 26 (86.7) Hormone therapy 6 (20.0) Diabetes Mellitus 0 Hypertension 13 (43.3) History of hysterectomy 6 (20.0) Uphold TVM 30 Concomitant surgery    Posterior repair 5 (16.7)    Vaginal hysterectomy 9 (30.0)    TVT-O 10 (33.3)    Ophira 2 (6.7) Table 1. Clinical background of all subjects. Data are given as mean ± standard deviation, or n (%). TVM, transvaginal mesh; BMI, body mass index; TVT-O, transobturator tape; TVT, tension-free vaginal tape. Table 1. Clinical background of all subjects. Data are given as mean ± standard deviation, or n (%). TVM, transvaginal mesh; BMI, body mass index; TVT-O, transobturator tape; TVT, tension-free vaginal tape. Table 1. Clinical background of all subjects. Data are given as mean ± standard deviation, or n (%). TVM, transvaginal mesh; BMI, body mass index; TVT-O, transobturator tape; TVT, tension-free vaginal tape. Table 1. Clinical background of all subjects. Changes in Sexual Function Following Uphold Transvaginal mesh Surgery for the Treatment of Urogenital Prolapse OPEN Data are given as mean ± standard deviation, or n (%). TVM, transvaginal mesh; BMI, body mass index; TVT-O, transobturator tape; TVT, tension-free vaginal tape. POP stage N = 30  N = 205 p value number (%) number (%) Stage 2 5 (16%) 18 (8.8%) 0.18* Stage 3 22 (73%) 161 (80.5%) 0.52* Stage 4 3 (10%) 26 (12.7%) 1.0# Table 2. Pelvic organ prolapse quantification (POP-Q) stage distribution. *Chi-square test. #Fisher’s exact test. POP stage N = 30  N = 205 p value number (%) number (%) Stage 2 5 (16%) 18 (8.8%) 0.18* Stage 3 22 (73%) 161 (80.5%) 0.52* Stage 4 3 (10%) 26 (12.7%) 1.0# Table 2. Pelvic organ prolapse quantification (POP-Q) stage distribution. *Chi-square test. #Fisher’s exact test. Table 2. Pelvic organ prolapse quantification (POP-Q) stage distribution. *Chi-square test. #Fisher’s exact test. Table 2. Pelvic organ prolapse quantification (POP-Q) stage distribution. *Chi-square test. #Fisher’s exact test. Table 2. Pelvic organ prolapse quantification (POP-Q) stage distribution. *Chi-square test. #Fisher’s exact test. POP-Q parameters (cm) N = 30 P value* Pre-op Post-op Aa 1.0 (−1~3) −1.5 (−3~−1) <0.001* Ba 3.0 (0~8) −1.5 (−3~−1) <0.001* C 0 (−4~8) 8 (−9~−6) <0.001* Ap −2 (−3~3) −2 (−3~0) <0.001* Bp 0 (−2~7) −2 (−3~0) <0.001* Tvl 9 (8~10) 10 (8~11) <0.001* UDI-6 22.2 ± 14.4 6.7 ± 3.3 <0.01** IIQ-7 13.3 ± 6.2 6.2 ± 3.0 <0.01** Table 3. Pelvic organ prolapse quantification (POP-Q) values and incontinence-related quality of life before and after surgery. Data are given as median (range) or mean ± standard deviation. Pre-op, preoperative; Post-op, postoperative; Tvl, total vaginal length; UDI-6, Urogenital Distress Inventory; IIQ-7, Incontinence Impact Questionnaire. *Wilcoxon signed rank test; **Paired t-test. Table 3. Pelvic organ prolapse quantification (POP-Q) values and incontinence-related quality of life before and after surgery. Data are given as median (range) or mean ± standard deviation. Pre-op, preoperative; Post-op, postoperative; Tvl, total vaginal length; UDI-6, Urogenital Distress Inventory; IIQ-7, Incontinence Impact Questionnaire. *Wilcoxon signed rank test; **Paired t-test. Table 3. Pelvic organ prolapse quantification (POP-Q) values and incontinence-related quality of life before and after surgery. Data are given as median (range) or mean ± standard deviation. Pre-op, preoperative; Post-op, postoperative; Tvl, total vaginal length; UDI-6, Urogenital Distress Inventory; IIQ-7, Incontinence Impact Questionnaire. *Wilcoxon signed rank test; **Paired t-test. Table 3. Pelvic organ prolapse quantification (POP-Q) values and incontinence-related quality of life before and after surgery. Discussion Current trend of evaluating the outcome of POP surgery has pivoted not only towards anatomic correction but also towards patients centered quality of life (QoL) and functional outcomes. Uphold Vaginal Support System with Capio SLIM Suture Capturing Device is a new single incision TVM kit widely used for managing POP. Its efficacy and effect on POP and QoL have been investigated in recent years14. Previous studies investigating influ- ence of POP surgery on sexual function has revealed conflicting evidences2–12. These conflicting outcomes could be a result of difference in (1) various follow-up time periods between studies, (2) treatment response between premenopausal and postmenopausal women10, (3) mesh placement site in anterior, posterior or total vaginal wall11, or (4) routes and textures of mesh such as trans-obturator or single-incision mesh12. We applied this single incision transvaginal mesh in women, who had symptomatic POP. g y p In the aspect of anatomical restoration in women with POP-Q stage ≥2 apical prolapse, with or without concomitant anterior vaginal wall prolapse, Uphold TVM surgery could achieve optimal anatomical outcome at the vaginal apex. In our study, anatomical outcomes has significant improvement (96.7%) in all POP-Q points at 6-month follow-up (Aa, Ba, C, Ap, Bp, Tvl, p < 0.001). These objective outcomes also reflected in subjective outcome that UDI-6 and IIQ-7 questionnaire reported favorable results with significant improvement at 6-month follow-up (p < 0.01). These results were comparable with the previous study, in which Altman et al. reported a 94% success rate at 1-year follow-up and 83.3% at 5-year follow-up. With regards to subjective outcome, Altman, et al. also found 91% of the women at 1-year follow-up and 78.8% at 5-year follow-up experienced a significant decrease in bothersome sensation3,4. These data have shown the efficacy of Uphold transvaginal mesh surgery in anatomic restoration and associated symptoms.i Considering sexual function, our study has pointed out significant improvement in all domains of FSFI ques- tionnaire including total score except for lubrication domain at 6-months follow-up. As compared to this result of the previous study, Altman, et al. reported significant decline in PISQ-12 scores (p < 0.001) in women who underwent apical prolapse correction with Uphold TVM at 1-year follow-up. More specifically in their study, the only significant changes were partner-related domains. In their study, dyspareunia improved and this was com- patible with our result. Result ll *Paired t-test. **Statistical significance. Table 4. Changes in scores of Female Sexual Function Index before and 6 months after surgery. Data are given as mean ± standard deviation. Pre-op, preoperatively; Post-op, postoperatively. *Paired t-test. **Statistical significance. Table 4. Changes in scores of Female Sexual Function Index before and 6 months after surgery. Data are given as mean ± standard deviation. Pre-op, preoperatively; Post-op, postoperatively. *Paired t-test. **Statistical significance. Domains Uphold alone (n = 18) Uphold + sling (n = 12) Pre-op Post-op P value* Pre-op Post-op P value* Sexual desire 2.5 ± 1.0a 3.3 ± 0.6 0.007** 2.3 ± 0.6a 2.3 ± 0.7 0.34 Sexual arousal 2.8 ± 1.0b 3.4 ± 0.5 0.044** 2.8 ± 0.5b 3.0 ± 0.4 0.025** Lubrication 4.0 ± 0.9c 3.9 ± 0.6 0.59 3.1 ± 1.3c 3.6 ± 1.1 <0.001** Orgasm 4.0 ± 1.3d 4.5 ± 0.6 0.07 3.4 ± 0.8d 3.5 ± 0.7 0.039** Satisfaction 3.9 ± 1.6e 4.8 ± 1.0 0.001** 4.0 ± 0.9e 4.6 ± 0.7 <0.001** Dyspareunia 4.3 ± 1.6f 5.1 ± 1.2 0.001** 4.3 ± 1.3f 4.8 ± 0.8 0.023** Total scores 21.5 ± 6.3g 24.9 ± 3.3 0.009* 19.9 ± 3.3g 21.9 ± 2.4 <0.001** Table 5. Changes in scores of Female Sexual Function Index in both groups before and 6 months after surgery. Data are given as mean ± standard deviation. Pre-op, preoperatively; Post-op, postoperatively. p value comparing Uphold alone goupr and Uphold plus sling group: a= 0.44, b = 1.0, c = 0.054, d = 0.13, e = 0.85, f = 1.0, g = 0.37. Table 5. Changes in scores of Female Sexual Function Index in both groups before and 6 months after surgery. Data are given as mean ± standard deviation. Pre-op, preoperatively; Post-op, postoperatively. p value comparing Uphold alone goupr and Uphold plus sling group: a= 0.44, b = 1.0, c = 0.054, d = 0.13, e = 0.85, f = 1.0, g = 0.37. We assessed female sexual function with FSFI questionnaire, and the total score and all domains except for lubrication domain showed significant improvement (Table 4). Subgroup analysis of FSFI comparing women with and without concomitant sling procedures revealed that both group showed significant improvement in all domains except for sexual desire. Subgroup statistical analysis between Uphold TVM only group and Uphold TVM plus midurethral sling group showed no difference in FSFI presentation in both groups (Table 5). Scientific Reports | (2019) 9:17047 | https://doi.org/10.1038/s41598-019-52990-0 Result ll Totally, 205 POP women were screened. Only 30 of them, who had completed the evaluation before and 6 months after surgery, were included in the study. Characteristics of all these subjects were listed in Table 1. The mean age was 57.3 ± 8.6 years old, mean parity was 2.5 ± 0.6 and mean BMI (kg/m2) was 25.2 ± 3.8. 6 of them were under hormone therapy, 26 women were menopausal, 13 of them had hypertension and 6 of them had a history of hys- terectomy. All women had Uphold TVM surgery. Some of them had concomitant surgery included 5 posterior colporrhaphies, 9 vaginal hysterectomies (30%), 10 trans-obturator tapes (TVT-O) and 2 single-incision slings with Ophira (Promedon S.A, Argentina). The distribution of POP stage was listed in Table 2. All women suffered from symptomatic POP; 5 of them (16%) were POP stage 2; 22 of them (73%) POP stage 3; 3 of them (10%) POP stage 4; 25 of them (83%) advanced stage. Although only 30 out of 205 women had completed the whole evalua- tion, there were no significant difference of POP stage distribution between the 205 women group and 30 women group (Table 2).i As for the POP-Q analysis, there was a significant improvement at points Aa, Ba, C, Ap, Bp and tvl (p < 0.001) after operation, and a 96.7% of success rate for POP correction was noted (Table 3). With respect to the changes in quality of life (QoL), there were statistically significant improvement in both UDI-6 and IIQ-7 questionnaires. No mesh erosion was found in all women at 6 months follow-up. Scientific Reports | (2019) 9:17047 | https://doi.org/10.1038/s41598-019-52990-0 www.nature.com/scientificreports/ Domains N = 30 Pre-op Post-op P value* Sexual desire 2.4 ± 0.9 2.9 ± 0.8 0.009** Sexual arousal 2.8 ± 0.8 3.2 ± 0.5 0.011** Lubrication 3.7 ± 1.2 3.8 ± 0.8 0.50 Orgasm 3.8 ± 1.1 4.1 ± 0.8 0.034** Satisfaction 4.0 ±1.4 4.8 ± 0.9 <0.01** Dyspareunia 4.3 ± 1.4 5.0 ± 1.0 <0.01** Total scores 20.8 ± 5.3 23.7 ± 3.3 <0.01** Table 4. Changes in scores of Female Sexual Function Index before and 6 months after surgery. Data are given as mean ± standard deviation. Pre-op, preoperatively; Post-op, postoperatively. *Paired t-test. **Statistical significance. Table 4. Changes in scores of Female Sexual Function Index before and 6 months after surgery. Data are given as mean ± standard deviation. Pre-op, preoperatively; Post-op, postoperatively. Discussion However, the scores of other domains in PISQ-12 did not differ in the behavioral-emotive and physical sections. When it came to 5-year follow-up, the number of sexually active women became fewer but PISQ-12 including partner domain improved. These discrepancy between our study and previous studies may be Scientific Reports | (2019) 9:17047 | https://doi.org/10.1038/s41598-019-52990-0 www.nature.com/scientificreports/ explained by the difference in study subjects’ characteristics and their individual performance. FSFI questionnaire doesn’t include partner domain but only demonstrates female’s aspect of sexual function. The improvement in sexual function may mainly benefit female but not male partners in short-term period. Dyspareunia improved in both studies which can explained this interpretation as well.f p p Dyspareunia is a concern related to sexual function and transvaginal mesh. Different types of transvaginal mesh may have different results in sexual function. Uphold Vaginal Support System is designed for single incision surgery that may express less mesh extrusion thus less dyspareunia. Lo et al. reported that single incision mesh achieved no mesh exposure event and significant improvement in PISQ-12 in short term follow-up5. Long et al. compared the extrusion rate between single incision mesh and trans-obturator mesh approaches and revealed less extrusion and dyspareunia rate in single incision mesh. In their study, they reported lower score in dyspareunia domain in FSFI questionnaire following trans-obturator TVM repair, and mesh extrusion rates as 11.4% at 4 and 10 weeks postoperatively. Dyspareunia has been one of the concerns associated with the use of trans-obturator synthetic mesh, ranging from 20–36% in previous studies12. Different from trans-obturator mesh, we found significant improvements on all domains of FSFI after single-incision TVM surgery, except for the lubrication domain. No mesh extrusion was found in our study. This improvement in dyspareunia and sexual function may be due to the evolution of new TVM, that is smaller in size, lighter, less dense texture, and single-incision design. Another reason for favorable sexual function may be that only anterior compartment but not total vaginal wall was involved in this Uphold TVM procedure. In our previous study, total TVM appeared to cause a greater sexual impairment compared with anterior TVM alone11.ht p p There are some limitations in our study. We evaluated the subjects before and 6 months after treatment according to previous literatures. However, in this interval of follow-up, we could only reveal short-term effect of the intervention. Discussion Thirty of the 250 women had completed the evaluation before and 6 months after surgery. The remaining 175 women could not complete the study, mainly due to resistance of completing questionnaires because of time-consuming and being ashamed of answering personal questions. Due to relatively small number of cases, we use multiple parameter POP-Q system to evaluate the postoperative changes so to have better sta- tistical power. Although there were some characteristics of our patients, we tried not to make any change to the patient’s underlying conditions such as medication of controlling hypertension, topical use of hormone therapy for a long time. Application of topical estrogen cream in treating postmenopausal women may have an effect on sexual function10,15,16. In our series, 6 women had hormone therapy. Very low-dose topical vaginal estrogen hormone was used in 5 cases (5/6; 83.3%) and systemic low-dose hormone was used by one patient (1/6; 17.7%). They kept hormone therapy long before, during and after POP surgery, we didn’t change the patient’s usual man- agement to avoid adding another possible variable. We treat patients with only one kind of transvaginal mesh to keep consistent management. Some concomitant surgeries were inevitable because it is clinically common to find multiple problems to be solved in one patient. It is estimated 50% patient with POP may have stress incontinence at the same time. All concomitant surgeries were performed on the basis of patient’s consents after fully discus- sion. Several studies have shown that vaginal hysterectomy would not be a factor interfering sexual function3,17,18. We also did the subgroup comparison between women treated with Uphold TVM with and without concom- itant mid-urethral sling, the statistical analysis shows no difference between two groups which indicated that mid-urethral sling may not be the factor to change sexual function in our study. Considering limitation men- tioned, more rigorous study is needed in the future.fh g y In this study, focusing on the effect of Uphold TVM surgery on sexual function. The results of our study indi- cated that the Uphold TVM surgery was effective in anatomical restoration of POP and was favorable for sexual function. Women with Uphold TVM surgery reported higher scores of FSFI in most domains, except for the lubrication domain. Moreover, no mesh extrusion is found and there is no detrimental effect on sexual function in women treated with Uphold TVM with or without sling surgery. Discussion We believe these concepts are helpful when counseling women with POP before surgery arrangement about possible impacts on sexual function. However, we acknowledge that the case numbers and duration of follow-up of this study were relatively limited and short, studies with larger sample size and longer investigation are expected in the future. Material and Methods Inclusion and exclusion criteria. From September 2015 through August 2017, two hundred and five women with symptomatic POP stages ranging from II to IV defined by the POP quantification (POP-Q) stag- ing system11 were treated with TVM with Uphold TVM at our hospitals performed by two skilled and experi- enced urogynecologic physicians. These women were fully informed and discussed about choices of surgeries. Only women signed the inform consents of surgery and participation in this study before treatment started were included in the study.i y “Sexually active” was defined as having vaginal intercourse at least 1 time per month within 6 months before treatment and continuously after treatment. Inclusion criteria were women who agree to participate in the study, who were POP stage from II to IV initially and undergoing transvaginal mesh surgery with Uphold TVM, who were sexually active and who completed evaluation pre-operatively and 6 months post-operatively. Exclusion criteria were women did not agree to participate in this study, women who cannot complete the whole treatment and evaluation. These patients were interviewed using short standardized forms including Urogenital Distress Inventory (UDI-6), Incontinence Impact Questionnaire (IIQ-7) and Female sexual function index Questionnaire (FSFI) concerning their urinary and sexual symptoms before and 6 months after treatment. Primary outcome was the POP-Q, UDI-6, IIQ-7 and FSFI. Anatomical restoration was evaluated before and 6 months after treatment by pelvic examination based on POP-Q system postoperatively and surgical failure was defined as the most distal portion being POP stage II or greater, regardless of a primary or a new site19,20. Scientific Reports | (2019) 9:17047 | https://doi.org/10.1038/s41598-019-52990-0 www.nature.com/scientificreports/ Concomitant operations included posterior colporrhaphy, vaginal total hysterectomy, midurethral sling with TVT-O (Gynecare TVT-Obturator System, Ethicon, Inc., Somerville, NJ, USA) and mini-sling (Ophira Mini Sling System; Promedon, Córdoba, Argentina). Midurethral sling were performed in some women with stress incontinence or occult urodynamic stress incontinence (USI), unless they did not agree to have additional surgery. Surgical procedure. We applied Uphold Lite Vaginal Support System with Capio SLIM Suture Capturing Device (Boston scientific, USA) in the anterior vaginal wall. Diluted pitressin (Pfizer, New York, USA) with 100 ml 9% normal saline was injected to anterior vaginal wall below the bladder neck. Initial incision site was made 3 cm below urethral orifice till 2 cm above uterine cervix. Compartment between anterior vaginal wall and bladder visceral layer was dissected by metzenbaum scissors bilaterally. Material and Methods Check bilateral ischial spine manually with index finger. Capio SLIM Suture Capturing Device was assembled with mesh. Insert the tip of assembled device with mesh guided by index finger to ischial spine. The surgeon passed the special designed needle through the sacros- pinous ligament at a level of 2 cm medial to the ischial spine, anchoring the mesh on the sacrospinous ligament. Repeated the procedure with the same manner on the other site. Then we sutured the mesh with the cervical stroma, bilateral cardinal ligaments, and pubocervical fascia beneath bladder neck with PDS 2.0 (Ethicon, New Jersey, USA). Finally, closure of the vaginal incision wound was made with vicryl 2.0 (Ethicon, New Jersey, USA) continuously. Vaginal packing was placed for 48 hours after surgery. y g p g pt g y Ethics approval by the Institutional Review Board of Kaohsiung medical university hospitals had been obtained for collecting data and further analysis. All participants were requested to sign an informed consent approved by the Institutional Review Board of the Kaohsiung Medical University before the treatment. Surgery was performed on the basis of official surgery consent in Kaohsiung Medical University hospital. Statistical anal- ysis was performed using Paired t-test or Wilcoxon signed-rank test for continuous variables. A difference was considered statistically significant when  p< 0.05. The study protocols were approved by the Institutional Review Board of Kaohsiung Medical University Hospital, by which relevant guidelines and regulations were followed accordingly.f g y We assessed the power of tests for differentiating the surgical outcome in this study. Power analysis showed that around 40 to 50 cases would have a power of 80%. Some comparisons could not reach sufficient statistical power with limited number of cases in our series. We used multiple parameters of POP-Q system to evaluate the postoperative changes and found that more than 30 women included in this study, there would be a power of more than 85% for discrimination. Ethical approval and informed consent. Ethics approval by the Institutional Review Board of Kaohsiung Medical University Hospital had been obtained for data analysis. Data availability The datasets analyzed for the current study are available from the corresponding author upon reasonable request. Received: 12 December 2018; Accepted: 22 October 2019; Published: xx xx xxxx Received: 12 December 2018; Accepted: 22 October 2019 Published: xx xx xxxx References References 1. Shatkin-Margolis, A. & Pauls, R. Sexual function after prolapse repair. Curr Opin Obstet Gynecol. 29, 343–348 (2017) l h f l l f f l fl h h l ft d References 1. Shatkin-Margolis, A. & Pauls, R. Sexual function after prolapse repair. Curr Opin Obstet Gynecol. 29, 343–348 (2017).lt 1. Shatkin-Margolis, A. & Pauls, R. Sexual function after prolapse repair. Curr Opin Obstet Gynecol. 29, 343–348 (2017). 2. Su, T. H. et al. Short term impact on female sexual function of pelvic floor reconstruction with the prolift procedure. J Sex Med. 6, 3201–3207 (2009). gt p p p p y ( ) 2. Su, T. H. et al. Short term impact on female sexual function of pelvic floor reconstruction with the prolift procedure. J Sex Med. 6, 3201–3207 (2009). 3. Rahkola-Soisalo, P., Mikkola, T., Altman, D. & Falconer, C. Pelvic organ prolapse repair using the Uphold vaginal Support System Female Pelvic Med Reconstr Surg., https://doi.org/10.1097/SPV.0000000000000530 (2017) ™ 4. Altman, D. et al. Pelvic organ prolapse repair using the Uphold™ vaginal support system: a 1-year multicenter study. Int Urogynecol J. 27, 1337–1345 (2016). 5. Lo, T. S. et al. Anterior-apical single-incision mesh surgery (SIMS): surgical and functional outcomes at 1 year. J Minim Invasive Gynecol. 22, 50–56 (2015).t y 6. Hoda, M., Wagner, S., Greco, F., Heynemann, H. & Fornara, P. Prospective follow‐up of female sexual function after vaginal surgery for pelvic organ prolapse using transobturator mesh implants. J Sex Med. 8, 914–922 (2011).lt 6. Hoda, M., Wagner, S., Greco, F., Heynemann, H. & Fornara, P. Prospective follow‐up of female sexu for pelvic organ prolapse using transobturator mesh implants. J Sex Med. 8, 914–922 (2011).lt p g p p g p 7. Farthmann, J. et al. Improvement of pelvic floor-related quality of life and sexual function after vaginal mesh implantation for cystocele: primary endpoint of a prospective multicentre trial. Arch Gynecol Obstet. 294, 115–21 (2016). 7. Farthmann, J. et al. Improvement of pelvic floor-related quality of life and sexual function after vaginal mesh implantation for cystocele: primary endpoint of a prospective multicentre trial. Arch Gynecol Obstet. 294, 115–21 (2016). y p y p p p y 8. Hugele, F. et al. Two years follow up of 270 patients treated by transvaginal mesh for anterior and/or apical prolapse. Eur J Obstet Gynecol Reprod Biol. 208, 16–22 (2017). 8. Hugele, F. et al. Scientific Reports | (2019) 9:17047 | https://doi.org/10.1038/s41598-019-52990-0 Data availabilityh Data availability The datasets analyzed for the current study are available from the corresponding author upon reasonable request Competing interestsh p g The authors declare no competing interests. References & Seckiner, I. Comparison of the Kelly’s plication and TOT simultaneously with vagina hysterectomy, on the incontinence, and sexual functions. International braz j urol. 44, 779–784 (2018). 17. Tepe, N., Bayrak, O., Ozcan, H., Ugur, M. & Seckiner, I. Comparison of the Kelly’s plication and TOT simultaneously with vaginal hysterectomy, on the incontinence, and sexual functions. International braz j urol. 44, 779–784 (2018). y y j 18. Detollenaere, R. et al. Sacrospinous hysteropexy versus vaginal hysterectomy with suspension of the uterosacral ligaments in wo with uterine prolapse stage 2 or higher: multicentre randomised non-inferiority trial. BMJ. 351, h3717 (2015).hl p p g g y J ( ) 19. Bump, R. C. et al. The standardization of terminology of female pelvic organ prolapse and pelvic floor dysfunction. Am J Obstet Gynecol. 175, 10–17 (1996).h p p g g y 19. Bump, R. C. et al. The standardization of terminology of female pelvic organ prolapse and pelvic floor dysfunction. Am J Obstet Gynecol. 175, 10–17 (1996).h g g y 19. Bump, R. C. et al. The standardization of terminology of female pelvic organ prolapse and pelvic floor dysfunction. Am J O Gynecol. 175, 10–17 (1996).h 20. Rosen, R. et al. The Female Sexual Function Index (FSFI): A multidimensional self-report instrument for the assessment of female sexual function. J Sex Marital Ther. 26, 191–208 (2000). 0. Rosen, R. et al. The Female Sexual Function Index (FSFI): A multidimensional self-report instrument for the assessment of female sexual function. J Sex Marital Ther. 26, 191–208 (2000). Author contributions Dr. Tsun-Wen Hsiao and Chin-Ru Ker were responsible for the writing of the manuscript. Dr. Kun-Ling Lin participated in patient recruitment and data acquirement. Dr. Ming-Ping Wu, Dr. Yi-Yin Liu Dr. and Yung-Shun Juan conducted statistical analysis; while Dr. Cheng-Yu Long, the corresponding author, designed and directed the study. References Two years follow up of 270 patients treated by transvaginal mesh for anterior and/or apical prolapse. Eur J Obstet Gynecol Reprod Biol. 208, 16–22 (2017). y p 9. Geynisman-Tan, J. et al. Recovering sexual satisfaction after prolapse surgery: a secondary analysis of surgical recovery. In Urogynecol J. 29, 1675–1680 (2018). gy ( ) 0. Long, C. Y. et al. Comparison of the changes in sexual function of premenopausal and postmenopausal women following transvaginal mesh surgery. J Sex Med. 8, 2009–2016 (2011). gy 10. Long, C. Y. et al. Comparison of the changes in sexual function of premenopausal and postmenopausal women following transvaginal mesh surgery. J Sex Med. 8, 2009–2016 (2011). g g y 11. Long, C. Y. et al. Changes in female sexual function following anterior with and without posterior vaginal mesh surgery for the treatment of pelvic organ prolapse. J Sex Med. 9, 2167–2174 (2012). 11. Long, C. Y. et al. Changes in female sexual function following anterior with and without posterior vaginal mesh surgery for the treatment of pelvic organ prolapse. J Sex Med. 9, 2167–2174 (2012). p g p p 2. Long, C. Y. et al. Comparison of clinical outcomes using “Elevate anterior” versus “Perigee” system devices for the treatment of pelvic organ prolapse. BioMed Research International. 3, 479610, https://doi.org/10.1155/2015/479610 (2015). g p p p g 13. Reclassification of urogynecologic surgical mesh instrumentation, https://www.fda.gov/downloads/AdvisoryCommittees/ CommitteesMeetingMaterials/MedicalDevices/MedicalDevicesAdvisoryCommittee/Gastroenterology-UrologyDevicesPanel/ UCM490205.pdf, (Accessed February 26, 2016). p ( y ) 4. Ker, C. R., Lin, K. L., Loo, Z. X., Juan, Y. S. & Long, C. Y. Comparison of UpholdTM vaginal mesh procedure with hysterectomy or uterine preservation for the treatment of pelvic organ prolapse. Sci Rep. 8(1), 9438 (2018). 5. Da Silva Lara, L. et al. Menopause leading to increased vaginal wall thickness in women with genital prolapse: impact on sexua response. J Sex Med. 6, 3097–3110 (2009).f response. J Sex Med. 6, 3097 3110 (2009). 16. Long, C. Y. et al. A randomized comparative study of the effects of oral and topical estrogen therapy on the vaginal vascularization and sexual function in hysterectomized postmenopausal women. Menopause. 13(5), 737–743 (2006). p 6. Long, C. Y. et al. A randomized comparative study of the effects of oral and topical estrogen therapy on the vaginal vascularization and sexual function in hysterectomized postmenopausal women. Menopause. 13(5), 737–743 (2006). Scientific Reports | (2019) 9:17047 | https://doi.org/10.1038/s41598-019-52990-0 www.nature.com/scientificreports/ 7. Tepe, N., Bayrak, O., Ozcan, H., Ugur, M. Additional information Correspondence and requests for materials should be addressed to C.-Y.L. Correspondence and requests for materials should be addressed to C.-Y.L. Reprints and permissions information is available at www.nature.com/reprints. Reprints and permissions information is available at www.nature.com/reprints. Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the 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/. 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English

Comparison of Vacuum-Assisted Sheath and Normal Sheath in Minimally Invasive Percutaneous Nephrolithotomy: A Systematic Review and Meta-Analysis

Research Square (Research Square)

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Abstract Background:  A systematic review and meta-analysis was conducted to compare the safety and efficacy of vacuum- assisted sheaths and conventional sheaths in minimally invasive percutaneous nephrolithotomy (MPCNL) in the treat- ment of nephrolithiasis. Methods:  PubMed, Web of Science, Embase, EBSCO, and Cochrane Library databases (updated March 2021) were used to search for studies assessing the effect of vacuum-assisted sheaths in patients who underwent MPCNL. The search strategy and study selection processes were implemented in accordance with the PRISMA statement. Methods:  PubMed, Web of Science, Embase, EBSCO, and Cochrane Library databases (updated March 2021) were used to search for studies assessing the effect of vacuum-assisted sheaths in patients who underwent MPCNL. The search strategy and study selection processes were implemented in accordance with the PRISMA statement. Result:  Three randomized controlled trials and two case-controlled trials that satisfied the inclusion criteria were enrolled in this meta-analysis. Overall, the stone-free rate (SFR) in patients who underwent vacuum-assisted sheaths was significantly higher than that in patients who underwent conventional sheaths (RR 1.23, 95% CI 1.04, 1.46, P = 0.02), with significant heterogeneity among the studies ­(I2 = 72%, P = 0.03). In terms of the outcome of complica- tions, vacuum-assisted sheath could bring a benefit to the postoperative infection rate (RR 0.48, 95% CI 0.33, 0.70, P < 0.00001) with insignificant heterogeneity among the studies ­(I2 = 0%, P = 0.68). There was no significant difference in the blood transfusion rate (RR 0.35, 95% CI 0.07, 1.73, P = 0.17), with significant heterogeneity ­(I2 = 66%, P = 0.35). Three studies contained operative time data, and the results indicated that the vacuum-assisted sheath led to a shorter operative time (MD = − 15.74; 95% CI − 1944, − 12.04, P < 0.00001) with insignificant heterogeneity ­(I2 = 0%, P = 0.91). Conclusion:  The application of a vacuum-assisted sheath in MPCNL improves the safety and efficiency compared to the conventional sheath. A vacuum-assisted sheath significantly increases the SFR while reducing operative time and postoperative infection. Keywords:  Nephrolithiasis, Minimally invasive percutaneous nephrolithotomy, Vacuum-assisted sheath, Conventional sheath, Meta-analysis © The Author(s) 2021. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://​creat​iveco​mmons.​org/​licen​ses/​by/4.​0/. The Creative Commons Public Domain Dedication waiver (http://​creat​iveco​ mmons.​org/​publi​cdoma​in/​zero/1.​0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Background Urolithiasis is the third most common disease of the uri- nary tract, and its prevalence has increased over the past decades [1]. The prevalence rate of kidney stones world- wide is approximately 1.7% to 8.8%, and kidney stones cost approximately $2.1 billion in 2020 alone [2]. Patients *Correspondence: [email protected]; [email protected] 1 Health Management Center, West China Hospital, Sichuan University, Chengdu, China g 2 Department of Endocrinology and Metabolism, Center for Diabetes and Metabolism Research, West China Hospital, Sichuan University, Chengdu, China Zhu et al. BMC Urology (2021) 21:158 https://doi.org/10.1186/s12894-021-00925-1 Zhu et al. BMC Urology (2021) 21:158 https://doi.org/10.1186/s12894-021-00925-1 © The Author(s) 2021. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://​creat​iveco​mmons.​org/​licen​ses/​by/4.​0/. The Creative Commons Public Domain Dedication waiver (http://​creat​iveco​ mmons.​org/​publi​cdoma​in/​zero/1.​0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Quality assessment of individual studies All assessments were performed independently by two researchers, with differences resolved by discussion. The methodological quality of each RCT was assessed accord- ing to the Jadad Score, which comprises the following three evaluation elements: randomization (0–2 points), blinding (0–2 points), and dropouts and withdrawals (0–1 points) [11]. One point was awarded for each ele- ment that was conducted and appropriately described in the original article. The total score varies from 0 to 5 points. An article with a Jadad score of ≤ 2 is considered to be of low quality, while a Jadad score of ≥ 3 indicates a high-quality study [12]. To date, there is still a lack of high-level evidence. The present write-up, therefore, aims to systematically review and perform a meta-analysis of the current stud- ies to investigate the effectiveness and safety of vacuum- assisted sheaths for the treatment of nephrolithiasis. Statistical analysis k ( ) Risk ratios (RRs) with 95% confidence intervals (CIs) were calculated for dichotomous outcomes. Heterogene- ity was evaluated using the ­I2 statistic, with ­I2 > 50% indi- cating significant heterogeneity [13]. Sensitivity analysis was performed to evaluate the influence of a single study on the overall estimate by omitting one study in turn or performing subgroup analysis. The random effects model was used for meta-analysis. Owing to the limited num- ber of included studies (< 10), publication bias was not assessed. Statistical significance was accepted at P < 0.05. All statistical analyses were performed using Review Manager Software Version 5.3 (The Cochrane Collabora- tion, Software Update, Oxford, UK). Data extraction and outcome measures Baseline information extracted from the original studies included the first author, year of publication, number of patients, patient age and sex distributions, type of cal- cium stone, detailed methods for the two groups, and the evaluation of evidence level. Data were independently extracted by two investigators, and discrepancies were resolved by consensus. p p p In 2001, minimally invasive percutaneous nephroli- thotomy (MPCNL), which involves the use of a small access sheath (i.e., 20F or less) was introduced in clinical practice [7, 8]. Despite the popularity of MPCL due to the lower risk of trauma and morbidities, it suffers from certain drawbacks. The efficiency of extraction of stone fragments and dust is lower than that of standard PCNL. Furthermore, the size of the sheath and the force of irri- gation can lead to a higher incidence of infections due to the rise in renal intraluminal pressure and limitations in lithotripsy equipment [9]. With the development of new technologies, vacuum-assisted sheaths have emerged. Stone fragments and irrigation fluid can be sucked out continuously and contemporarily in the gap between the scope and sheath. Some studies on this issue have been conducted recently, but the outcomes of the effect and efficiency of MPCNL with a vacuum-assisted sheath are unsettled. The primary outcomes were stone-free rate (SFR) and perioperative complications (including postopera- tive infection rate, blood transfusion rate, and perfora- tion rate). Secondary outcomes were operative time and hospitalization. Materials and methodsh The systematic review and meta-analysis were carried out following the guidelines of the Preferred Reporting Items for Systematic Reviews and Meta-analysis (PRISMA) statement and the Cochrane Handbook for System- atic Reviews of Interventions [10]. Ethical approval and patient consent were not required, as all the analyses were performed in previously published studies. Open Access T Zhu et al. BMC Urology (2021) 21:158 Zhu et al. BMC Urology (2021) 21:158 Page 2 of 8 searched, and the above process was repeated to ensure that all eligible studies were identified.h with nephrolithiasis often suffer from short-term compli- cations such as acute renal colic, nausea, vomiting, and hematuria and long-term complications such as chronic renal failure and hydronephrosis [3]. Therefore, treat- ment of calculi has always been the focus of surgeons. For renal stones > 2 cm in size, the American Urological Association recommends percutaneous nephrolithotomy (PCNL) as the primary treatment [4]. Previous stud- ies have shown that standard PCNL is a highly effective approach [5]. However, it is often associated with major complications such as bleeding with the need for blood transfusion, postoperative fever, and pneumothorax [6]. i The inclusion criteria were as follows: (1) the study design was a randomized controlled trial, (2) the patient had a history of kidney stones and underwent minimally invasive PCNL, (3) the intervention approach was a suc- tioning sheath versus a normal sheath, and (4) the entire text was available. Studies satisfying these inclusion crite- ria in all languages were included. Literature search, study characteristics, and quality assessment A total of 126 articles were initially identified from the databases. After removing duplicates, 91 articles were retained. Then, 83 studies were excluded from the study due to unrelated abstracts and titles. One article with insufficient data, one article without full text, and three for not were also excluded from our analysis due to the study design. Finally, three randomized controlled trials (RCTs) with 857 patients who satisfied all inclusion crite- ria were subjected to the meta-analysis [14–16]. The arti- cle selection process was performed in accordance with Result the PRISMA statement (Fig. 1). The baseline character- istics of the three included studies are shown in Table 1. The studies included in this meta-analysis were published between 2016 and 2020. The JADAD score for three stud- ies was 2, 3, and 4. One study was of low quality, as no blinding was used and the specific method of randomiza- tion was explained [14]. the PRISMA statement (Fig. 1). The baseline character- istics of the three included studies are shown in Table 1. The studies included in this meta-analysis were published between 2016 and 2020. The JADAD score for three stud- ies was 2, 3, and 4. One study was of low quality, as no blinding was used and the specific method of randomiza- tion was explained [14]. Literature search and selection criteria We systematically searched relevant published articles in several databases, including PubMed, Embase, Web of Science, EBSCO, and the Cochrane Library, from inception to March 2021 with the following keywords: “percutaneous nephrolithotomy,” “minimally,” “sheath,” “evacuation,” and “suction.” The reference lists in the retrieved studies and relevant reviews were manually Zhu et al. BMC Urology (2021) 21:158 Zhu et al. BMC Urology (2021) 21:158 Page 3 of 8 Primary outcomes Stone‑free rate All studies included for the analysis reported the SFR, where stone-free was defined as stone fragments ≤ 4 mm (1 study using computed tomography (CT) scan and 2 Fig. 1  Flow diagram of the study search and selection process Fig. 1  Flow diagram of the study search and selection process Zhu et al. BMC Urology (2021) 21:158 Page 4 of 8 Table 1  Characteristics of included studies No Author Year Experimental group Control group Study design Quality assessment Number (n) Age (Mean ± SD) Male (n) Stone Access sheath and lithotripsy energy Number (n) Age (Mean ± SD) Male (n) Stone size Access sheath and lithotripsy energy 1 Huang 2016 91 43.5 ± 2.9 53 Stone dimension 16.7 ± 5.8 mm 16F vacuum sheath/hol- mium laser 91 44.1 ± 3.2 51 15.1 ± 6.3 mm 16F peel-away sheath/hol- mium laser RCT​ 3a 2 Du 2018 311 43.6 ± 17.4 187 Stone size 13.6 ± 5.2 ­cm2 16-18F vac- uum sheath/ ultrasound 304 41.2 ± 16.9 181 13.9 ± 4.7 ­cm2 16-18F peel- away sheath/ ultrasound RCT​ 2a 3 Xu 2020 30 52.1 ± 11.5 18 Stone diameter 4.2 ± 1.0 cm 20F vacuum sheath/pneu- matic and holmium laser 30 51.8 ± 9.6 14 3.8 ± 1.4 cm 20F conven- tional sheath/ pneumatic and holmium laser RCT​ 4a Zhu et al. BMC Urology (2021) 21:158 Page 5 of 8 95% CI 0.07, 1.73, P = 0.17), with significant heterogene- ity ­(I2 = 66%, P = 0.35) (Fig. 4) reported in the three stud- ies [14–16]. Only one study reported perforation, and the vacuum-assisted sheath had a higher incidence of perfo- ration (7/91 vs. 1/91; P < 0.001). studies using X-ray). Our results indicated that the SFR of the vacuum-assisted sheath group was significantly higher than that of the conventional sheath group (RR 1.23, 95% CI 1.04, 1.46, P = 0.02), with significant hetero- geneity among the studies ­(I2 = 72%, P = 0.03) (Fig. 2). Discussion PCNL has been accepted as the gold standard for the treatment of large renal stones and is widely used in clinical practice [3, 17]. Although technological advances have ensured much progress in this field, many complica- tions still exist [18]. To improve the safety and efficacy of this procedure, a small-size sheath was invented with the advent of mini-perc technology [19]. Due to the smaller size of the sheaths, MPCNL is associated with flaws such as longer operative time and infectious complications [20]. Recently, a sheath with irrigation and suctioning sys- tems has been developed that can allow continuous infu- sion with saline intraoperatively [21]. Vacuum aspiration can be regulated manually or mechanically to keep the collecting system under negative pressure. Additionally, the nephroscope moves in and out conveniently through the movable sealing lid while preventing extremely high or low pressure [22]. To evaluate the effect and efficacy of the vacuum-assisted sheath, this meta-analysis was performed. Prolonged operative time is another independent risk factor for infectious complications [27]. The smaller size of access causing a limitation for more options for lithotripsy is a major inherent limitation of MPCNL. Another limitation is the small visual field in miniature endoscopes, which leads to a prolonged time to break the stones into smaller fragments [28]. Despite the dif- ferent definitions of the operation time, present evidence indicates that vacuum-assisted sheaths can significantly shorten the operation time. The vacuum-assisted sheath could simultaneously suck out the small clots and frag- ments of stone in the gap. Furthermore, clearer vision was achieved during the procedure to shorten the sur- gery time [29]. Although a shorter operation time may decrease blood loss, this was not proven to be the case in our study, as it depends on many factors, such as punc- ture site, number, and size of the sheath [30]. SFR is the main parameter for judging the efficacy of minimally invasive stone removal surgery [23]. Despite the difference in imaging modalities and follow-up time in the definition of SFR, our results show that vacuum- assisted sheaths have an improved stone clearance com- pared to normal sheaths. One possible explanation may be the low positive or low negative state of intrapelvic pressure controlled by the sheath while flushing and irri- gation. In this situation, the kidney parenchyma shrank, tension in the renal pelvis decreased, and renal parenchy- mal compliance improved. Hospitalization clear surgical field to avoid missing stone fragments, and therefore, a higher SFR is reached [14]. Only studies reported hospitalization data, and the results showed that vacuum-assisted sheaths might prolong the hospitalization time compared to normal sheaths (10.6 ± 3.7 vs. 6.4 ± 2.3; P < 0.001). Xu et  al. reported a higher incidence of postopera- tive fever [15]. Due to the small size of the sheath, high- pressure perfusion is very often performed. It is known that the limit of renal intrapelvic pressure is 30 mmHg [24]. Higher pressure can injure the integrity of the pel- vic wall epithelium, leading to exposure of the lymphatic and venous systems [25]. In addition, tissue edema and congestion caused by urinary tract infection and stones are more likely to cause pelvic fluid absorption. When the bacteria along with the associated toxin reflux are absorbed, it may lead to infectious complications such as postoperative fever or sepsis [26]. By using the suc- tion sheath, the renal pelvis is kept in a negative pres- sure state. Therefore, the infectious fluid flows smoothly, and the absorption of irrigation, bacteria, and toxin reflux is reduced. Du et  al. found that MPCNL with a vacuum sheath has a low intrapelvic pressure compared to standard PCNL and MPCNL [14]. Xu et al. found that intrapelvic pressure ≥ 30 mmHg was achieved in the non- vacuum-sheath group [15]. Secondary outcomes Operative timeh All the studies reported infection-related complica- tions and blood transfusion rates. Three studies reported postoperative fever [14–16]. These results indicated that vacuum-assisted sheaths provided a benefit to the postoperative infection rate (RR 0.48, 95% CI 0.33, 0.70, P < 0.00001), with insignificant heterogeneity among the studies ­(I2 = 0%, P = 0.68) (Fig. 3). There was no sig- nificant difference in the blood transfusion rate (RR 0.35, Three studies containing operative time data [15, 16] were analyzed, and the results indicated that the opera- tive time of the vacuum-assisted sheath group was sig- nificantly reduced compared to that of the conventional sheath group (MD = − 15.74; 95% CI − 1944, − 12.04, P < 0.00001), with insignificant heterogeneity ­(I2 = 0%, P = 0.91) (Fig. 5). Fig. 2  Forest plot for the meta-analysis of stone free rate Fig. 3  Forest plot for the meta-analysis of postoperative infection Fig. 4  Forest plot for the meta-analysis of transfusion Fig. 2  Forest plot for the meta-analysis of stone free rate Fig. 2  Forest plot for the meta-analysis of stone free rate Fig. 3  Forest plot for the meta-analysis of postoperative infection Fig. 3  Forest plot for the meta-analysis of postoperative infection Fig. 4  Forest plot for the meta-analysis of transfusion Zhu et al. BMC Urology (2021) 21:158 Zhu et al. BMC Urology (2021) 21:158 Page 6 of 8 Discussion Thus, the nephroscope can reach more renal calyces. Furthermore, when the calyceal neck is narrow or the angle is hard to reach, this sheath can perform lithotripsy and simultaneously suction out the fragments, making it a one-step procedure. Addi- tionally, continuous negative pressure suction ensures a Admittedly, there are a few limitations to this study. First, only three randomized controlled trials were included in our study, and the geographical region was single. Second, the impact of differences in puncture Fig. 5  Forest plot for the meta-analysis of operative time Fig. 5  Forest plot for the meta-analysis of operative time Zhu et al. BMC Urology (2021) 21:158 Page 7 of 8 3. Torricelli FCM, Monga M. Staghorn renal stones: what the urologist needs to know. Int Braz J. 2020;46(6):927–33. https://​doi.​org/​10.​1590/​s1677-​ 5538.​ibju.​2020.​99.​07. kidney calices and depth in sheath placement was not assessed in the included studies. Finally, there are some unpublished data and missing negative data in the origi- nal reports, and because of this publication bias, our con- clusion may be skewed. 3. Torricelli FCM, Monga M. Staghorn renal stones: what the urologist needs to know. Int Braz J. 2020;46(6):927–33. https://​doi.​org/​10.​1590/​s1677-​ 5538.​ibju.​2020.​99.​07. 4. Pradère B, Doizi S, Proietti S, Brachlow J, Traxer O. Evaluation of guidelines for surgical management of urolithiasis. J Urol. 2018;199(5):1267–71. https://​doi.​org/​10.​1016/j.​juro.​2017.​11.​111. 5. Ghani KR, Andonian S, Bultitude M, Desai M, Giusti G, Okhunov Z, et al. Percutaneous nephrolithotomy: update, trends, and future directions. Eur Urol. 2016;70(2):382–96. https://​doi.​org/​10.​1016/j.​eururo.​2016.​01.​047. The application of a vacuum-assisted sheath in MPCNL improves the safety and efficiency compared to the con- ventional sheath. A vacuum-assisted sheath significantly increases the SFR while reducing operative time and postoperative infection. Due to the inherent limitations of the included studies, large-scale, multiregion, mul- ticenter, prospective RCTs should be performed in the future to validate our results. 6. Seitz C, Desai M, Häcker A, Hakenberg OW, Liatsikos E, Nagele U, et al. Incidence, prevention, and management of complications following percutaneous nephrolitholapaxy. Eur Urol. 2012;61(1):146–58. https://​doi.​ org/​10.​1016/j.​eururo.​2011.​09.​016. g j 7. Ruhayel Y, Tepeler A, Dabestani S, MacLennan S, Petřík A, Sarica K, et al. Tract sizes in miniaturized percutaneous nephrolithotomy: a systematic review from the European Association of Urology Urolithiasis Guidelines Panel. Eur Urol. 2017;72(2):220–35. https://​doi.​org/​10.​1016/j.​eururo.​2017.​ 01.​046. 7. Ruhayel Y, Tepeler A, Dabestani S, MacLennan S, Petřík A, Sarica K, et al. Tract sizes in miniaturized percutaneous nephrolithotomy: a systematic review from the European Association of Urology Urolithiasis Guidelines Panel. Availability of data and materials 14. Du C, Song L, Wu X, Fan D, Zhu L, Liu S, et al. Suctioning minimally inva- sive percutaneous nephrolithotomy with a patented system is effective to treat renal staghorn calculi: a prospective multicenter study. Urol Int. 2018;101(2):143–9. https://​doi.​org/​10.​1159/​00048​8399. The data and materials can be obtained by contacting the corresponding author. Acknowledgeme Not applicable. 10. Liberati A, Altman DG, Tetzlaff J, Mulrow C, Gøtzsche PC, Ioannidis JP, et al. The PRISMA statement for reporting systematic reviews and meta- analyses of studies that evaluate health care interventions: explanation and elaboration. PLoS Med. 2009;6(7): e1000100. https://​doi.​org/​10.​1371/​ journ​al.​pmed.​10001​00. Authors’ contributions WZH, ZL, and ZXF participated in the design of this study. ZL drafted the manuscript. GLP and ZY collected and analyzed the data, and HY and ZXF critically revised the manuscript. All authors have read and approved the final manuscript. 11. Jadad AR, Moore RA, Carroll D, Jenkinson C, Reynolds DJ, Gavaghan DJ, et al. Assessing the quality of reports of randomized clinical trials: is blind- ing necessary? Control Clin Trials. 1996;17(1):1–12. https://​doi.​org/​10.​ 1016/​0197-​2456(95)​00134-4. Discussion Eur Urol. 2017;72(2):220–35. https://​doi.​org/​10.​1016/j.​eururo.​2017.​ 01.​046. 8. Feng MI, Tamaddon K, Mikhail A, Kaptein JS, Bellman GC. Prospective randomized study of various techniques of percutaneous nephrolithot- omy. Urology. 2001;58(3):345–50. https://​doi.​org/​10.​1016/​s0090-​4295(01)​ 01225-0. Competing interests g 17. Türk C, Petřík A, Sarica K, Seitz C, Skolarikos A, Straub M, et al. EAU guide- lines on interventional treatment for urolithiasis. Eur Urol. 2016;69(3):475– 82. https://​doi.​org/​10.​1016/j.​eururo.​2015.​07.​041. The authors declare no competing interests. Received: 24 May 2021 Accepted: 10 November 2021 18. Skolarikos A, Alivizatos G, de la Rosette JJ. Percutaneous nephrolithotomy and its legacy. Eur Urol. 2005;47(1):22–8. https://​doi.​org/​10.​1016/j.​eururo.​ 2004.​08.​009. 19. Sabnis RB, Ganesamoni R, Sarpal R. Miniperc: what is its current status? Curr Opin Urol. 2012;22(2):129–33. https://​doi.​org/​10.​1097/​MOU.​0b013​ e3283​502fb4. Funding 12. Kjaergard LL, Villumsen J, Gluud C. Reported methodologic quality and discrepancies between large and small randomized trials in meta-analyse. Ann Intern Med. 2001;135(11):982–9. This work is supported by the National Natural Science Foundation of China (82070846) and Program for Overseas High-Level Talents Introduction of Sichuan Province of China (21RCYJ0046). 13. Higgins JP, Thompson SG. Quantifying heterogeneity in a meta-analysis. Stat Med. 2002;21(11):1539–58. https://​doi.​org/​10.​1002/​sim.​1186. Declarations 15. Xu G, Liang J, He Y, Li X, Yang W, Lai D, et al. Comparison of two different minimally invasive percutaneous nephrostomy sheaths for the treatment of staghorn stones. BJU Int. 2020;125(6):898–904. https://​doi.​org/​10.​1111/​ bju.​15031. Abbreviations C l MPCNL: Minimally invasive percutaneous nephrolithotomy; SFR: Stone-free rate; PCNL: Percutaneous nephrolithotomy; PRISMA: Preferred Reporting Items for Systematic Reviews and Meta-analysis; MINORS: The Methodological Index for Nonrandomized Studies; CIs: Confidence intervals; RCTs: Randomized controlled trials; CCTs: Case-controlled trials; CT: Computed tomography. 9. Omar M, Noble M, Sivalingam S, El Mahdy A, Gamal A, Farag M, et al. Systemic inflammatory response syndrome after percutaneous nephroli- thotomy: a randomized single-blind clinical trial evaluating the impact of irrigation pressure. J Urol. 2016;196(1):109–14. https://​doi.​org/​10.​1016/j.​ juro.​2016.​01.​104. MPCNL: Minimally invasive percutaneous nephrolithotomy; SFR: Stone-free rate; PCNL: Percutaneous nephrolithotomy; PRISMA: Preferred Reporting Items for Systematic Reviews and Meta-analysis; MINORS: The Methodological Index for Nonrandomized Studies; CIs: Confidence intervals; RCTs: Randomized controlled trials; CCTs: Case-controlled trials; CT: Computed tomography. Ethics approval and consent to participate Not applicable. 16. Huang J, Song L, Xie D, Li M, Deng X, Hu M, et al. A Randomized Study of Minimally Invasive Percutaneous Nephrolithotomy (MPCNL) with the aid of a patented suctioning sheath in the treatment of renal calculus complicated by pyonephrosis by one surgery. BMC Urol. 2016;16(1):71. https://​doi.​org/​10.​1186/​s12894-​016-​0184-0. References 1. Bouatia M, Benramdane L, Idrissi MOB, Draoui M. An epidemiological study on the composition of urinary stones in Morocco in relation to age and sex. Afr J Urol. 2015;21(3):194–7. 1. 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Clinical comparison of minipercutaneous nephrolithotomy with vacuum cleaner effect or with a vacuum-assisted access sheath: a single center experi- ence. J Endourol. 2020. https://​doi.​org/​10.​1089/​end.​2020.​0555. 28. Cheng F, Yu W, Zhang X, Yang S, Xia Y, Ruan Y. Minimally invasive tract in percutaneous nephrolithotomy for renal stones. J Endourol. 2010;24(10):1579–82. https://​doi.​org/​10.​1089/​end.​2009.​0581. 23. De S, Autorino R, Kim FJ, Zargar H, Laydner H, Balsamo R, et al. Percutane- ous nephrolithotomy versus retrograde intrarenal surgery: a systematic review and meta-analysis. Eur Urol. 2015;67(1):125–37. https://​doi.​org/​10.​ 1016/j.​eururo.​2014.​07.​003. 29. Lai D, Chen M, Sheng M, Liu Y, Xu G, He Y, et al. Use of a novel vacuum- assisted access sheath in minimally invasive percutaneous nephrolithot- omy: a feasibility study. J Endourol. References 2020;34(3):339–44. https://​doi.​org/​10.​ 1089/​end.​2019.​0652. 24. Guohua Z, Wen Z, Xun L, Wenzhong C, Yongzhong H, Zhaohui H, et al. The influence of minimally invasive percutaneous nephrolithotomy on renal pelvic pressure in vivo. Surg Laparoscopy Endoscopy Percutaneous Tech. 2007;17(4):307–10. https://​doi.​org/​10.​1097/​SLE.​0b013​e3180​6e61f4. 30. Sahan A, Cubuk A, Ozkaptan O, Ertaş K, Canakci C, Eryildirim B, et al. Safety of upper pole puncture in percutaneous nephrolithotomy with the guid- ance of ultrasonography versus fluoroscopy: a comparative study. Urol Int. 2020;104(9–10):769–74. https://​doi.​org/​10.​1159/​00050​9448. 25. Goel P, Bajpai M, Kandasamy D. An enigmatic route to the contralateral pelvicalyceal system on antegrade pyelogram. J Indian Assoc Pediatr Surg. 2018;23(4):236–8. https://​doi.​org/​10.​4103/​jiaps.​JIAPS_​139_​18. Publisher’s Note S i N i 26. de la Rosette J, Denstedt J, Geavlete P, Keeley F, Matsuda T, Pearle M, et al. The clinical research office of the endourological society ureteros- copy global study: indications, complications, and outcomes in 11,885 patients. J Endourol. 2014;28(2):131–9. https://​doi.​org/​10.​1089/​end.​2013.​ 0436. Springer Nature remains neutral with regard to jurisdictional claims in pub- lished maps and institutional affiliations. • fast, convenient online submission • thorough peer review by experienced researchers in your field • rapid publication on acceptance • support for research data, including large and complex data types • gold Open Access which fosters wider collaboration and increased citations maximum visibility for your research: over 100M website views per year • At BMC, research is always in progress. Learn more biomedcentral.com/submissions Ready to submit your research Ready to submit your research ? Choose BMC and benefit from: ? 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Elevational Distribution and Ecology of Small Mammals on Tanzania's Second Highest Mountain

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RESEARCH ARTICLE Elevational Distribution and Ecology of Small Mammals on Tanzania's Second Highest Mountain William T. Stanley1†*, Philip M. Kihaule2 1 The Field Museum of Natural History, Department of Science and Education, Chicago, Illinois, United States of America, 2 University of Dar es Salaam, Department of Zoology, Dar es Salaam, Tanzania † Deceased. * [email protected] a11111 Abstract OPEN ACCESS Citation: Stanley WT, Kihaule PM (2016) Elevational Distribution and Ecology of Small Mammals on Tanzania's Second Highest Mountain. PLoS ONE 11 (9): e0162009. doi:10.1371/journal.pone.0162009 Editor: Mathew S. Crowther, University of Sydney, AUSTRALIA Received: January 4, 2016 Accepted: August 16, 2016 Published: September 21, 2016 Copyright: © 2016 Stanley, Kihaule. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper. Funding: Funding for this study came from the Field Museum of Natural History, including awards from The Field Museum/IDP Foundation, Inc. African Training Fund, and the Barbara Brown Fund of the Field Museum. 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. Mt. Meru is Tanzania’s second highest mountain and the ninth highest in Africa. The distribution and abundance of small mammals on this massif are poorly known. Here we document the distribution of shrews and rodents along an elevational gradient on the southeastern versant of Mt. Meru. Five sites were sampled with elevational center points of 1950, 2300, 2650, 3000, and 3600 m, using a systematic methodology of standard traps and pitfall lines, to inventory the shrews and rodents of the slope. Ten species of mammal were recorded, comprising 2 shrew and 8 rodent species with the greatest diversity for each group at 2300 m. No species previously unrecorded on Mt. Meru was observed. Two rodent genera that occur in nearby Eastern Arc Mountains (Hylomyscus and Beamys) were not recorded. The rodent Lophuromys verhageni and a recently described species of shrew, Crocidura newmarki, are the only endemic mammals on Mt. Meru, and were widespread across the elevational gradient. As in similar small mammal surveys on other mountains of Tanzania, rainfall positively influenced trap success rates for shrews, but not for rodents. This study provides new information on the local small mammal fauna of the massif, but numerous other questions remain to be explored. Comparisons are made to similar surveys of other mountains in Tanzania. Introduction The distribution of mammals along mountain slopes is of increasing interest to ecologists and mammalogists to document species turnover along environmental gradients and, as a result, the efforts to document the montane faunas of various massifs around the world have intensified over the past few decades. Climate change has recently increased this curiosity and the need for detailed investigations concerning this subject. Documenting the present elevational distribution of organisms along a given slope will facilitate the monitoring of that biota during times of climatic perturbation or habitat alteration. Biogeographic, ecological, and evolutionary studies are also advanced by a greater comprehension of montane biotic systems. Examples of PLOS ONE | DOI:10.1371/journal.pone.0162009 September 21, 2016 1 / 15 Shrews and Rodents of Mt. Meru elevational surveys of small mammals in montane localitiesusing systematic sampling protocols include Chile [1], Costa Rica [2], Madagascar [3], Malaysia [4], Philippines [5], Taiwan [6], and U.S.A. [7]. Each of these studies produces a more complete understanding of both specific and broad patterns of mammalian elevational distribution, and the mechanisms that led to such an array. [8]. The utility of such studies in monitoring impacts of environmental change through time cannot be overstated. For example, in Yosemite Valley, California, a survey along an elevational transect documented significant range shifts in various mammalian species since an identical survey was conducted along the same transect a century earlier [7]. The montane mammals of sub-Saharan Africa have been studied for over a century, and research concerning their elevational distribution on some massifs is well documented [9], [10]. Published results from detailed systematic inventories of mammals on mountains of eastern Africa include, Kerbis Peterhans et al. [11] for the Rwenzori Mountains, Stanley and Hutterer [12] for the Udzungwa Mountains, and Mulungu et al. [13] and Stanley et al. [14] for Mt. Kilimanjaro. Certain other topographically important massifs remain enigmatic with regards to the mammals occurring on them. Mt. Meru, Tanzania’s second highest mountain, and the ninth highest in Africa, is a case in point. While its neighbor, Mt. Kilimanjaro, has been the subject of several studies on the locally occurring mammal fauna [13], [14], [15], [16], Mt. Meru remains understudied. While some aspects of the ecology of the mountain have been documented [17], the mammal fauna has been largely neglected. The most complete analysis of the fauna of Mt. Meru to date is that of Demeter and Hutterer [18], but is not based on any standard trapping protocol and several different habitats on the massif are not included. The need for more detailed information of this and nearby mountains has been emphasized by the results of other studies [19], which indicate that climate change is affecting the ecology and habitat of neighboring Mt. Kilimanjaro. Baseline data for the small mammals of Mt. Meru will provide the means for future analyses of the impact of climate change on the ecology of this volcano. We used a standardized methodology that has been recently employed in myriad other montane sites of Tanzania [12], [20], [21], [22] to survey the small mammals (shrews and rodents) at five different elevations and habitats along the southeastern slope of Mt. Meru. Our study has three principal goals: 1) to conduct the first intensive survey of the presence and abundance of small mammals along an elevational gradient on the mountain; 2) to test for differences between rodents and shrews in their elevational distribution, their response to different trapping methodologies, and the relationship of captures to rainfall patterns; and 3) to compare the generated results to similar studies on other mountains Tanzania. Materials and Methods Study site Mt. Meru is in northeastern Tanzania and reaches an elevation of 4,566 m (14,977 ft), and ranks ninth among the 10 highest mountains of Africa. An active volcano (the mountain last erupted in 1910), Mt. Meru is the centerpiece of Arusha National Park The mountain is a popular destination for climbers, and there is one path that originates in the lowlands and runs up the southeastern side of the mountain [18]. Between 16 July and 19 August 2009, we sampled the small mammals (shrews and rodents) at five different elevations, ranging from roughly 1950 to 3600 m, along the climbing route on the southeastern slope of Mt. Meru (Fig 1). All sampling sites were on Mt. Meru in Arusha National Park, Arumeru District, Arusha Region, Tanzania. The specific localities, elevations, habitats (sensu Demeter and Hutterer [18]), and dates of sampling are listed below. Using a minimum-maximum thermometer and a mobile rainfall gauge, measurements of temperature and precipitation were collected on a daily PLOS ONE | DOI:10.1371/journal.pone.0162009 September 21, 2016 2 / 15 Shrews and Rodents of Mt. Meru Fig 1. Map of Mt. Meru showing routes, elevational contours, and study sites. doi:10.1371/journal.pone.0162009.g001 basis at each respective camp; these data are summarized in Table 1. The elevations given for each site are centered at the associated camp and sampling efforts spanned roughly 100–200 m above and below the camp: Site 1–1950 m)—Fig Tree Arch, 3.24406°S, 36.82845°E, 1950 m; lower montane forest; 16– 23 July 2009. Site 2–2300 m)—Site 2, 3.24725°S, 36.80066°E, 2300 m; upper montane forest; 23–30 July 2009. Site 3–2650 m)—Meru Crater, 3.242°S, 36.78736°E, 2650 m; ecotone between montane forest and ericaceous zone; 13–19 August 2009. Site 4–3000 m)—Mgongo wa Tembo, 3.2235°S, 36.78675°E, 3000 m; mix of forest, ericaceous plants, and some bamboo; 30 July-6 August 2009. PLOS ONE | DOI:10.1371/journal.pone.0162009 September 21, 2016 3 / 15 Shrews and Rodents of Mt. Meru Table 1. Climatic data registered at each camp site on Mt. Meru in July-August 2009 in the context of an elevational survey of small mammals. Totals presented as mean ± standard deviation, range, and sample size (number of days measured). Rainfall samples are given as number of days monitored and (number of days with rain). Elevation (m) 1950 2300 2650 3000 3600 Daily minimum temperature (°C) Daily maximum temperature (°C) 10.2 ± 0.4 16.7 ± 1.0 Daily rainfall (mm) 10–11 16–18 0–3 N=7 N=6 N = 7 (2) 7.9 ± 0.9 14.2 ± 1.8 0 6.5–9.0 11.5–16 0.7 ± 1.2 N=7 N=6 N=6 6.2 ± 0.7 13.1 ± 2.7 0 5.5–7.5 10–18 N=6 N=6 N=6 4.3 ± 0.9 13.8° ± 3.4 3.0 ± 3.3 3–5 9.0–16.5 0–6.2 N=7 N=6 N = 6 (3) 2.1 ± 1.4 16.3 ± 1.6 0 0–4 14.5–18.0 N=7 N=6 N=6 doi:10.1371/journal.pone.0162009.t001 Site 5–3600 m)—near Saddle Hut, 3.21609°S, 36.76897°E, 3600 m; ecotone between ericaceous and alpine zones; 6–13 August 2009. Field methodology Pitfall lines and traplines were installed to capture principally shrews and rodents, respectively. Each pitfall line were comprised of 11 buckets, placed 5 m apart, and buried in the ground so that the top of the bucket was level with the ground. Each of these 15 l buckets was 26 cm high and had an upper and lower diameter of 26 cm and 24 cm, respectively. Each pitfall line had a 50 cm high black plastic drift fence running over the center of each bucket. This technique has been used with considerable success in other mammal surveys. Trap lines utilized three different kinds of traps: Museum Specials, 14 x 7 cm; Victor Rat Traps (referred to here as Victor Trap), 17.5 x 8.5 cm; and medium-sized Sherman Traps, 23 x 9.5 x 8 cm. To maximize capture success, traps were set at sites considered likely to be frequented by small mammals, rather than at fixed distances or in a grid. Consequently, distances between consecutive traps were not constant. Bait for each trap consisted of freshly fried coconut coated in peanut butter, and traps were rebaited each day in the late afternoon. Additional details on these trapping techniques are presented by Stanley et al. [22]. Both pitfall and trap lines were checked twice each day, in the early morning and mid-afternoon, for captured animals. Not all traps or buckets were employed for equal amounts of time (some trap lines were set the first day of the survey, others were installed on a subsequent day), so we use the terms “trap night”, “bucket night” and “sample night” to quantify sampling effort. A “trap-night” refers to one trap in operation for a 24 hr period (0700 to 0700 hrs). A “bucket-night” denotes one bucket in operation for a 24 hr period (0700 to 0700 hrs). The term “sample-night” is used in reference to overall sampling effort (including the number of trap-nights and bucket-nights). We refer to the success rate of each method as either “trap success” or “bucket success”, and calculate these values by dividing the number of individuals captured by the number of trapnights or bucket-nights and multiplying by 100. In discussions involving the overall capture PLOS ONE | DOI:10.1371/journal.pone.0162009 September 21, 2016 4 / 15 Shrews and Rodents of Mt. Meru success, the term “sample success” refers to the success rate for pitfall and trap lines combined. This is calculated by dividing the number of individuals captured by the number of samplingnights and multiplying by 100. Animals were handled following the protocol approved by the American Society of Mammalogists [23]. As all of the species handled are small-bodied, animals were dispatched by cervical dislocation and following AVMA guidelines [23]. Voucher specimens were prepared as either museum study skins with associated skulls and axial skeletons or embalmed in formalin. Standard museum measurements [24] were taken by WTS, and tissues including heart, liver, kidney and/or muscle were extracted from select specimens and frozen in liquid nitrogen, or saved in dimethyl sulfoxide buffer (DMSO) at ambient temperature. All voucher specimens are deposited in the Field Museum of Natural History (FMNH), Chicago, and the University of Dar es Salaam (UDSM), Dar es Salaam, and all tissue samples are in liquid nitrogen storage in the FMNH. We follow the taxonomy presented for shrews by Hutterer [25] and rodents by Carleton and Stanley [26], [27], Holden [28], and Musser and Carleton [29]. Ethics Statement Permits for the collection and export of specimens were provided by the Tanzania Commission for Science and Technology (reference no. 2009-134-ER-90-172), the Tanzania Ministry of Natural Resources and Tourism, Wildlife Division (reference no. GD/R.40/1/), and the Tanzania National Parks (reference no. TNP/HQ/C.10/13). Import of specimens into the USA was approved by the US Fish and Wildlife Service (3177-W10414-9/04/09). Shrews and rodents were euthanized following the protocol approved by the American Society of Mammalogists [23], and the study was approved by the Field Museum of Natural History Institutional Animal Care and Use Committee (09–3). Results Over the course of the survey, we accumulated 7,111 sample-nights (4592 trap-nights and 2519 bucket-nights) and captured 751 small mammals, including 276 shrews representing 2 species, and 475 rodents representing 8 species (Tables 2, 3 and 4). Significantly more shrews were captured in buckets than in traps (X2 = 61.3, P<0.05), and significantly more rodents were caught in traps than in buckets (X2 = 232.7, P<0.05), a pattern observed in previous Tanzanian small mammal studies employing the same field protocols [12], [14], [30]. In 4592 trap-nights, 581 mammals were captured for an overall trap success of 12.6%. Of the mammals caught in traps, 465 were rodents (10.1% trap success) and 116 were shrews (2.5% trap success). In the accrued 2519 bucket-nights, 170 mammals were captureed for a total bucket success of 6.7%. Of these, 160 were shrews (6.3% success) and 10 were rodents (0.4% success). This conspicuous difference was evident both across the entire survey and at each of the five sites sampled (Table 2). Both shrew species (Crocidura allex and C. newmarki, weighing between 3.5–11 g) found during the survey were caught in traps. Ten rodents representing 3 species (Dendromus insignis, Mus triton, and Praomys taitae) were caught in buckets and ranged in weight 10–27 g. Of the 10 Dendromus captured, the lightest (10–13 g; n = 6) were captured in buckets (all adults based on fused cranial sutures), and the heaviest individuals (14–22.5 g; n = 4) were captured in traps. At each elevational site, captures (and overall sample success) ranged from 49 [3.5%] at 3600 m to 257 [18.0%] at 2300 m (Table 2). For shrews, the lowest values were recorded at the 3600 m site (20 [1.4%]) and the highest values at the 3000 m site (87 [6.1%]; Table 3). For rodents, the lowest (29 [2.1%]) and highest (208 [14.6%]) values were observed at the 3600 and 2300 m sites, respectively (Table 4). The cumulative number of species trapped at a site reached PLOS ONE | DOI:10.1371/journal.pone.0162009 September 21, 2016 5 / 15 Shrews and Rodents of Mt. Meru Table 2. Total capture rates for rodents and shrews by trap technique on the southeastern slope of Mt. Meru in July-August 2009. Elevation 1950 m 2300 m 2650 m 3000 m 3600 m Totals 506 506 506 506 495 2519 BUCKETS # bucket-nights # individuals (% bucket success) 52 24 18 63 13 170 (10.3) (4.7) (3.6) (12.3) (2.6) (6.7) # species 3 3 3 4 3 5 # shrews 51 22 17 58 12 160 (6.3) (% bucket success) (10.1) (4.3) (3.4) (11.3) (2.4) # shrew species 2 2 2 2 2 2 # rodents 1 2 1 5 1 10 (0.2) (0.4) (0.2) (1.0) (0.2) (0.4) 1 1 1 2 1 3 # trap-nights 920 920 920 920 912 4592 # individuals 115 233 104 93 36 581 (12.5) (25.5) (11.3) (9.3) (3.9) (12.6) (% bucket success) # rodent species TRAPS (% trap success) # species 5 6 8 9 6 9 # rodents 86 206 81 64 28 465 (9.3) (22.6) (8.8) (6.5) (3.1) (10.1) (% trap success) # rodent species 3 4 6 7 4 7 # shrews 29 27 23 29 8 116 (3.2) (2.9) (2.5) (2.8) (0.9) (2.5) 2 2 2 2 2 2 # sample-nights 1426 1426 1426 1426 1407 7111 # individuals 167 257 122 156 49 751 (11.7) (18.0) (8.6) (10.9) (3.5) (10.5) 5 6 9 10 6 10 (% bucket success) # shrew species TOTAL (% sample success) # species doi:10.1371/journal.pone.0162009.t002 an asymptote at the 1950, 2650, and 3600 m sites. In contrast, new species (for a given site) were captured at 2300 m, (where Graphiurus murinus was captured for the first time at that site on the last day of trapping) and at 3000 m, (where Mus triton and Otomys tropicalis were both captured on the last day). The species accumulation curves illustrate these results (Fig 2). Table 3. Elevational distribution of shrew species along the southeastern slope of Mt. Meru in July-August 2009. Only specimens caught in traps or buckets are included. Elevation 1950 m 2300 m 2650 m 3000 m 3600 m Totals Crocidura allex 31 31 18 36 16 132 C. newmarki 49 18 22 51 4 144 Total # individuals 80 49 40 87 20 276 Total # species 2 2 2 2 2 2 Total # sample-nights 1426 1426 1426 1426 1407 7111 Sample success (%) 5.6 3.4 2.8 6.1 1.4 3.9 Total # caught in buckets 51 22 17 58 12 160 Total # bucket-nights 506 506 506 506 495 2519 Bucket success (%)pitfall lines 10.1 4.3 3.3 11.5 2.4 6.3 Species doi:10.1371/journal.pone.0162009.t003 PLOS ONE | DOI:10.1371/journal.pone.0162009 September 21, 2016 6 / 15 Shrews and Rodents of Mt. Meru Table 4. Elevational distribution of rodent species along the southeastern slope of Mt. Meru in July-August 2009. Only specimens caught in traps or buckets are included. Elevation 1950 m 2300 m 2650 m 3000 m 3600 m Totals Otomys tropicalis 0 0 2 1 1 4 Dendromus insignis 0 0 1 7 2 10 Grammomys dolichurus 3 4 2 4 0 13 Lophuromys verhageni 0 18 9 30 2 59 Mus triton 0 0 0 1 0 1 Praomys taitae 79 185 38 4 0 306 Rhabdomys dilectus 0 0 24 7 24 55 Graphiurus murinus 5 1 6 15 0 31 Total # individuals 87 208 82 69 29 475 Species Total # species 3 4 7 8 4 8 Total # sample-nights 1426 1426 1426 1426 1407 7111 Sample success (%) 6.1 14.6 5.7 4.8 2.1 6.7 Total # caught in traps 86 206 81 64 28 465 Total # trap-nights 920 920 920 920 912 4592 Trap success (%) 9.3 22.4 8.8 6.9 3.1 10.1 doi:10.1371/journal.pone.0162009.t004 As in past surveys of montane mammals in Tanzania [12], [14], the relationship between the amount of rainfall and shrew capture success is more pronounced than that between rainfall and rodent capture success. During the survey of Mt. Meru, only two sites (1950 and 3000 m) received rain while buckets and traps were in place. The Product-moment correlation coefficients (r) of amount of daily rainfall with total capture success of shrews (for buckets and traps combined) are 0.60 and 0.55 for the 1950 and 3000 m sites, respectively. For rodents these values for the same two elevational zones were both negative (-0.53 and -0.30, respectively). While none of these r values are significant, Fig 3 illustrates the increase in shrew captures during or shortly after measureable rainfall, a pattern not exhibited by rodentcapturess. The correlation of four daily capture parameters (number of individuals, number of species, number of new species [i.e. previously unsampled at a given site], and cumulative number of species) with cumulative sample-nights was analyzed for both types of trapping methodology (Table 5) and mammalian order (Table 6). Because only two species of shrews were recorded during the entire survey (Tables 2 and 3), and both were caught the first day of capture and every subsequent day, correlation analysis between cumulative sampling effort and some parameters is not applicable. Other comparisons revealed differing patterns. The correlation between sampling effort and number of individuals fluctuated among elevations and the number of species captured each day was not correlated with cumulative sampling effort. For all taxa, a negative correlation existed between new species captured and cumulative sampling effort, but the correlation was significant in only five cases across the transect and not consistently based on a single parameter. Correlation analysis revealed a significant positive correlation between cumulative sample-nights and cumulative species across all sites for trap lines, bucket lines (for captures of both shrews and rodents), and both sampling methods combined. Each site exhibited the same general pattern, although not in all parameters examined. For example, there was no strong relationship between cumulative sample nights and cumulative shrew species captured in buckets at the 2650 and 3600 m sites (Table 5). Table 6 illustrates the same general pattern when the analysis is segregated by each of the two represented orders (shrews–Soricomorpha and rodents–Rodentia). Again, the low number of shrew species PLOS ONE | DOI:10.1371/journal.pone.0162009 September 21, 2016 7 / 15 Shrews and Rodents of Mt. Meru Fig 2. Species accumulation curves (for both pitfall and trap lines combined) for each site surveyed for small mammals on Mt. Meru. The dashed lines represent the number of captures each day; the solid lines represent the cumulative number of new species for the site observed each day. The graph at the lower right shows the number of specimens of shrew, rodent, and combined small mammals captured at each site. doi:10.1371/journal.pone.0162009.g002 captured on Mt. Meru influences the analysis, and the strongest relationship is that of cumulative species of rodents with cumulative sampling effort. The relationship between elevation and number of individuals or species collected, or sample success was not notable. The low and relatively constant number of shrew species was observed at all elevations, and the only prominent negative relationship (high but not significant r values) exists in the associations of the total number of individuals and total trap success with elevation (Table 7). The highest species diversity was seen at the 3000 m site and the lowest at the 1950 m site. While the lowest number of individuals collected was at the 3600 m site, PLOS ONE | DOI:10.1371/journal.pone.0162009 September 21, 2016 8 / 15 Shrews and Rodents of Mt. Meru Fig 3. The relationship between numbers of individuals of small mammals captured each day of the sampling period, and rainfall, at each site on Mt. Meru. Rodentia are on the left and Soricomorpha are on the right. doi:10.1371/journal.pone.0162009.g003 the species diversity was relatively high, with 6 species, in contrast to that of the 1950 m site, with 5 species, which had the second highest sample success of any of the sites (Table 2). The bucket success rates for all captured mammals was 6.7%, and 170 individuals (160 shrews and 10 rodents) were collected in 385 buckets (77 buckets installed at each of 5 sites), but most buckets captured no animals. Over the entire survey, 287 buckets caught nothing, and only98 buckets (25% of total installed) captured animals. When comparing the capture rate of individual buckets, the following figures were recorded (number of buckets–number of animals): 61 buckets—1 animal, 20–2, 10–3, 3–4, 3–5, and 1–11. A similar pattern was exhibited by traps although there was a 12.6% trap success in 750 traps with 581 captures; only 313 traps (42% of total employed) caught at least one animal. When comparing the capture rate of individual traps, the following figures were recorded (number of traps–number of animals): 166 traps– 1 animal, 74–2, 43–3, 18–4, 8–5, 2–6, and 1–7. These different values for trap success of individual buckets and traps do not follow a Poisson distribution (G-test for goodness of fit = 34.0 for buckets, 63.7 for traps; P<0.01) indicating a lack of trap independence. Discussion The combination of traps and pitfall lines were effective in sampling non-volant small mammal communities at different elevations on Mt. Meru, as in past studies in Tanzania [12], [14], [22], [30], [31]. However, species accumulation curves failed to reach a plateau at every site: the 2300 and 3000 m site had one and two species, respectively, captured during the last 24-hour period of trapping. While we are relatively confident that the vast majority of shrew and small PLOS ONE | DOI:10.1371/journal.pone.0162009 September 21, 2016 9 / 15 Shrews and Rodents of Mt. Meru Table 5. Product-moment correlation coefficients (r) of cumulative sample-nights with four parameters of trap/bucket captures associated with small mammal surveys on Mt. Meru. Results are given separately for rodents and shrews, and then all small mammals combined. Values in parentheses represent strong but not statistically significant correlations. Daily cumulative sample-nights correlated with (across) Number of individuals Number of Species New species added Cumulative species Total Traps (rodents only) -0.425** 0.060 (-0.317) 0.943** Traps (all captures) -0.448** 0.001 -0.388* 0.920** -0.254 (-0.311) (-0.293) - -0.226 -0.176 (-0.255) 0.910** -0.479** -0.011 -0.350* 0.962** Buckets (shrews only) Buckets (all captures) Traps and buckets combined (all captures) 1950 m Traps (rodents only) -0.403 0.119 (-0.697) (0.605) Traps (all captures) 0.071 0.671 (-0.636) 0.785* Buckets (shrews only) -0.453 (-0.611) (-0.605) - Buckets (all captures) -0.464 -0.677 -0.784* 0.605 Traps and buckets combined (all captures) -0.159 0.119 -0.802* 0.605 Traps (rodents only) -0.350 0.009 -0.491 0.796* Traps (all captures) -0.260 0.429 (-0.691) 0.846* Buckets (shrews only) -0.888** -0.774* -0.605 - Buckets (all captures) -0.851 (-0.693) (-0.677) 0.796* Traps and buckets combined (all captures) -0.385 -0.210 -0.576 0.796* 0.221 0.546 (-0.707) 0.812* 0.751* 2300 m 2650 m Traps (rodents only) Traps (all captures) 0.336 0.627 -0.633 Buckets (shrews only) -0.823* (-0.676) -0.796* 0.605 Buckets (all captures) -0.779* -0.413 -0.438 0.889** 0.081 (0.667) (-0.682) 0.866** Traps and buckets combined (all captures) 3000 m Traps (rodents only) 0.071 0.484 -0.523 0.935** Traps (all captures) 0.551 0.636 -0.831* 0.912** Buckets (shrews only) 0.129 0.200 -0.605 - Buckets (all captures) 0.090 0.293 -0.401 0.611 Traps and buckets combined (all captures) 0.453 0.255 -0.458 0.941** 3600 m Traps (rodents only) 0.285 0.588 0.144 0.975** Traps (all captures) 0.416 (0.670) 0.000 0.971** Buckets (shrews only) 0.309 0.224 -0.408 0.612 Buckets (all captures) 0.368 0.378 -0.196 0.849* Traps and buckets combined (all captures) 0.501 (0.688) -0.289 0.927** * = p0.05 ** = p0.01. doi:10.1371/journal.pone.0162009.t005 rodent species occurring at each site were documented, and are justified in making inter-site comparisons along the transect and to other elevational transects conducted in Tanzania [12], [14], we suggest additional surveys are needed on Mt. Meru to determine with certainty the complete list of small mammals in different elevation zones. Ten mammal species (2 shrews and 8 rodents) were documented along an elevational transect from roughly 1950 m to 3600 m on the eastern slope of Mt. Meru, none of which are PLOS ONE | DOI:10.1371/journal.pone.0162009 September 21, 2016 10 / 15 Shrews and Rodents of Mt. Meru Table 6. Product-moment correlation coefficients (r) of cumulative sample-nights with four parameters of trap success shrew and rodent captures associated with small mammal surveys on Mt. Meru. Values in parentheses represent strong but not significant correlations. Shrew and rodent captures correlated with (across) Number of individuals Number of species New species added Cumulative species Total, shrews (-0.290) -0.346* (-0.293) - Total, rodents -0.421** 0.141 -0.252 0.969** 1950 m, shrews 0.141 - -0.605 - 1950 m, rodents -0.414 0.119 (-0.697) (0.605)* 2300 m, shrews -0.769* -0.408 -0.605 - 2300 m, rodents -0.341 -0.009 -0.491 0.796* 2650 m, shrews -0.275 0.605 -0.796* 0.605 2650 m, rodents -0.203 0.628 -0.611 0.900** 3000 m, shrews 0.351 - -0.725* - 3000 m, rodents 0.011 0.629 -0.605 0.941** 3600 m, shrews 0.644 0.408 -0. 408 0.612 3600 m, rodents 0.339 0.784* 0.000 0.980** * = p0.05 ** = p0.01. doi:10.1371/journal.pone.0162009.t006 introduced taxa. With the exception of Crocidura newmarki and Lophuromys verhageni, which are endemic to Mt. Meru [32], [33], all species have more or less broad distributions. Crocidura allex is found on other east African mountains, which include Kilimanjaro, Ngorongoro, Kenya, and Aberdares [25]. Among the rodents, the species with the broadest distributions are Grammomys dolichurus, Rhabdomys dilectus, and Graphiurus murinus, all of which are distributed in eastern and southern Africa [28], [29]. The rodent species with the most restricted distribution, other than the local endemic Lophuromys verhageni, was Praomys taitae, which occurs from southeastern Kenya through eastern Tanzania [27]. The faunal list of Demeter and Hutterer [18] represents the heretofore most complete list of shrews and rodents known to occur on Mt. Meru, and their list contained species not observed during this survey. Some of these taxa are larger and not the subjects of the methodology employed during this study (i.e. Paraxerus, Thryonomys, and Tachyoryctes) and are not considered further here. The elevational range of the study of Demeter and Hutterer [18] was 1200 m to 2750 m and included towns and villages such as Arusha and Tengeru, and habitats other than forest, such as savanna. Some of the taxa they listed include taxa typically found at Table 7. Product-moment correlation coefficients (r) between elevation and trap success associated with small mammal surveys on Mt. Meru. Values in parentheses represent strong but not significant correlations. Elevation correlated with (r) p Total number of individual mammals collected (-0.75) > 0.05 Total trap success (-0.75) > 0.05 Total number of species collected 0.59 > 0.05 Total number of shrews collected -0.53 > 0.05 Shrew trap success -0.52 > 0.05 Total number of shrew species collected - - Total number of rodents collected -0.62 > 0.05 Rodent trap success -0.61 > 0.05 Total number of rodent species collected 0.33 > 0.05 doi:10.1371/journal.pone.0162009.t007 PLOS ONE | DOI:10.1371/journal.pone.0162009 September 21, 2016 11 / 15 Shrews and Rodents of Mt. Meru elevations and habitats lower than the forest on Meru, including Mastomys natalensis and Pelomys fallax. However, some taxa listed from forest localities that we did not observe are worthy of discussion here, including Crocidura hildegardeae, C. luna, Aethomys kaiseri, Hylomyscus denniae, Lemniscomys striatus, Mus gratus, Rattus rattus, and Otomys irroratus, The two species of Crocidura were collected at elevations of 1700 m or lower below the elevational range of the current study and we conclude that they were not present (or notably rare) at our sampled sites. The specimens of Aethomys kaiseri, Lemniscomys striatus, and Rattus rattus cited by Demeter and Hutterer [18] came from habitats below 2000 m, with the exception of specimens from”House Mgondah” at 2000 m. The authors suggest Aethomys and Rattus may have occurred at this locality because of human influence, and certainly Rattus is a known commensal [29]. While the vast majority of our efforts were in primary habitat, we did place traps around the dwellings of Saddle Hut (not included in this analysis) to collect rodents living under the buildings. At this site, we collected Crocidura allex, Lophuromys verhageni, and Rhabdomys dilectus but no Aethomys, Lemniscomys or Rattus. We saw no evidence of these species in forested habitats of the mountain, and hypothesize that all three genera were found at this locality either having been introduced by human activities, or are supported by habitat alteration associated with the dwelling. Demeter and Hutterer [18] list Mus gratus among the taxa occurring on Mt. Meru. While one locality (“Forest House”; 1700 m) associated with this species was below our study area, another (Meru East: 1550–2750 m) does overlap with our elevational transect. Given that there was no specific elevational information for the M. gratus collection sites, we cannot definitely determine if it was obtained in forest. We saw no evidence of this species, and only one specimen of M. triton was obtained during the 2009 survey. The records of Otomys irroratus listed by Demeter and Hutterer [18] (now O. angoniensis [29]) are interesting to us because there are also specimens in the Field Museum collected by B. Cooper in 1938 from the crater of Mt. Meru at roughly 2900 m (FMNH 48610–48619). We recorded O. tropicalis from the same crater and at elevations ranging from 2650–3600 m, but found no evidence of O. angoniensis. Temporal (1938 vs. 2009) or seasonal (January vs. July/ August) variation may explain these differences, but additional surveys of the mountain are needed to determine the current presence and distributional extent of both Otomys species. The records of Hylomyscus denniae on Mt. Meru [18] (and Ngorongoro [34]) are of a taxon now referred to Praomys taitae [26], [27]. Another rodent that is found on northern Eastern Arc Mountains and the Southern Highlands [29], and has been recorded from Moshi (Dieterlen [35]), is Beamys hindei, and this taxon was not recorded on Mt. Meru by us or in past work. In addition to the survey of Mt. Meru, similar detailed faunal surveys have been conducted on Mt. Kilimanjaro [14] and Ngorongoro (WT Stanley, unpubl. data) and neither Hylomyscus or Beamys have been recorded on any of these Northern Highland mountains during these transects, suggesting they do not occur in these locales. Assuming this to be correct, this supports the hypothesis that the establishment of these two rodent taxa on the mountains where they are found was via a southern route. We suspect establishment via a northern route from Kenya would have resulted in populations of both of these rodents on some, if not all of the Northern Highlands. The results of this survey differ from those on other mountains in Tanzania using identical techniques [12], [14]. The most striking difference is the low diversity of shrews on Mt. Meru, a non-Eastern Arc massif. Restricting comparisons to species lists generated by our surveys of the other Tanzanian mountains, for shrews, Mt. Meru had one genus (Crocidura) and two species (C. allex, C. newmarki); Mt. Kilimanjaro (non-Eastern Arc massif) had three genera (Crocidura, Myosorex, and Sylvisorex) and six species (C. allex, C. hildegardeae, C. monax, C. olivieri, PLOS ONE | DOI:10.1371/journal.pone.0162009 September 21, 2016 12 / 15 Shrews and Rodents of Mt. Meru M. zinki, and S. granti); and the Udzungwas (part of the Eastern Arc system) had three genera (Crocidura, Myosorex, and Sylvisorex) and nine species (C. hildegardeae, C. desperata, C. elgonius, C. munissii, C. olivieri, C. telfordi, M. kihaulei, S. lixus, and S. megalura). The Udzungwa survey included dry forest near the base of the scarp at 600 m and after removing taxa found in this zone, measures of shrew diversity drop from 9 to 7 species. Even with this reduction, the Mt. Meru shrew list stands in stark contrast to the lists of these other two massifs. The reasons for such a strikingly low shrew diversity on Mt. Meru are unknown, but given the relatively high diversity of species on Mt. Kilimanjaro and a comparable level to the Udzungwas, geological origin does not seem to explain these differences. Volcanic activity on the mountain has been recent in comparison to Mt. Kilimanjaro [36], but it seems unlikely that eruptions would have contributed to extinction of taxa once existing on Mt. Meru. Unlike Mt. Kilimanjaro and the Udzungwas, elevation was not significantly correlated with capture rates or species diversity for either shrews or rodents. In the Udzungwas, rodent diversity and abundance increased with elevation [12], and on Mt. Kilimanjaro, shrew abundance and diversity decreased with elevation [14]. Similar patterns, or any influence of elevation on abundance or diversity were not seen on Mt. Meru. As on Mt. Kilimanjaro [14], the greatest diversity of shrews and rodents on Mt. Meru was at 3000 m, and not at the top of the transect as in Udzungwa [12], and the greatest diversity was documented within forested habitats and not above tree line (Tables 2, 3 and 4). Similarities among the Mt. Meru, Mt. Kilimanjaro, and Udzungwa transects were observed in the effect rainfall had on the capture rates of shrews, but this effect was not seen on rodents. The Mt. Meru survey adds additional support to the idea that the amount of rainfall during a survey period is strongly correlated with estimates of shrew diversity or abundance. Also, there was a lack of capture independence among traps and buckets across the entire transect at each site on Meru, as was also the case on Mt. Kilimanjaro and the Udzungwas [12], [14]. Based on these parallel results, we hypothesize that the presence of a captured animal in a bucket may attract other animals to that bucket and placement of traps influences the chances of multiple captures over time in a given trap. Although this may change with future taxonomic studies, the only endemic mammals on Mt. Meru are Lophuromys verhageni [32] and Crocidura newmarki [33]. The Lophuromys was found at all sites along our transect, with the exception of the lowest (1950 m), which was unexpected, as L. aquilus is found in moist habitats on Mt. Kilimanjaro at 2000 m [14]. While the habitat at the Mt. Meru 1950 m site appeared suitable for Lophuromys based on our experience of trapping this genus on different Tanzanian mountains where they can be abundant [12], [14], [37], its absence is unexpected. No rodent species was recorded at all sites during the Mt. Meru survey, while both shrew species occurred along the complete transect. Much still needs to be learned about the ecology of small mammals on the high mountains of east Africa. For example, the upper limits of these animals on Mt. Meru, as well as on Mt. Kilimanjaro, remain unknown. Additional surveys are needed to determine these aspects and to further elucidate the natural history of the mammals of Meru. Acknowledgments We thank the Tanzania Commission for Science and Technology, the Tanzania Wildlife Research Institute, the Ministry of Natural Resources and Tanzanian National Parks for permission to conduct this research. We are grateful to Unique Safaris, the Wildlife Conservation Society (Tanzania), and Ms. Gladys Ng’umbi (Arusha National Park ecologist) for logistical support. R. Banasiak provided important assistance with the figures. An anonymous reviewer provided helpful comments on a previous version of this paper. PLOS ONE | DOI:10.1371/journal.pone.0162009 September 21, 2016 13 / 15 Shrews and Rodents of Mt. Meru Author Contributions Conceptualization: WTS PMK. Data curation: WTS. Formal analysis: WTS. Funding acquisition: WTS. Investigation: WTS PMK. Methodology: WTS PMK. Resources: WTS. Visualization: WTS. Writing – original draft: WTS. Writing – review & editing: WTS. References 1. Patterson BD, Meserve PL, Lang BK (1989) Distribution and abundance of small mammals along an elevational transect in temperate rainforests of Chile. J Mammal 70: 67–78. 2. McCain CM (2004) The mid-domain effect applied to elevational gradients: species richness of small mammals in Costa Rica. J Biogeogr 31: 19–31. 3. Goodman SM, Ganzhorn JU, Rakotondravony D (2003) Introduction to the mammals. In: Goodman SM, Benstead JP, editors. The Natural History of Madagascar. Chicago: The University of Chicago Press. Pp. 1159–1186. 4. Md Nor S (2001) Elevational diversity patterns of small mammals on Mount Kinabalu, Malaysia. Glob Ecol Biogeogr 10: 41–62. 5. Rickart EA, Heaney LR, Utzurrum RB (1991) Distribution and ecology of small mammals along an elevational transect in southeastern Luzon, Philippines. J Mammal 72: 458–469. 6. Yu HT (1994) Distribution and abundance of small mammals along a subtropical elevational gradient in central Taiwan. J Zool 234: 577–600. 7. Moritz C, Patton JL, Conroy CJ, Parra JL, White GC, Beissinger SR (2008) Impact of a century of climate change on small-mammal communities in Yosemite National Park, USA. Science 322: 261–264. doi: 10.1126/science.1163428 PMID: 18845755 8. McCain CM (2005) Elevational gradients in diversity of small mammals. Ecology 86: 366–372. 9. Happold DCD, Happold M (1989) Biogeography of small montane mammals in Malawi, Central Africa. J. Biogeogr 16: 353–367. 10. Yalden DW (1988) Small mammals of the Bale Mountains, Ethiopia. Afr J Ecol 26: 281–294. 11. Kerbis Peterhans JC, Kityo RM, Stanley WT, Austin PK (1998) Small mammals along an elevational gradient in Rwenzori Mountains National Park, Uganda. In: Osmaston H, Tukahirwa J, Basalirwa C, Nyakaana J, editors. The Rwenzori Mountains National Park, Uganda. Exploration, Environment and Biology. Conservation, Management and Community. Kampala: Makerere University. Pp. 149–171 12. Stanley WT, Hutterer R (2007) Differences in abundance and species richness between shrews and rodents along an elevational gradient in the Udzungwa Mountains, Tanzania. Acta Theriol 52: 261– 275. 13. Mulungu LS, Makundi RH, Massawe AW, Machang’u RS, Mbije NE (2008) Diversity and distribution of rodent and shrew species associated with variations in altitude on Mount Kilimanjaro, Tanzania. Mammalia 72: 178–185. 14. Stanley WT, Rogers MA, Kihaule PM, Munissi MJ. 2014. Elevational distribution and ecology of small mammals on Africa’s highest mountain. 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Stanley WT, Hutterer R, Giarla TC, Esselstyn JA (2015) Phylogeny, phylogeography and geographical variation in the Crocidura monax (Soricidae) species complex from the montane islands of Tanzania, with descriptions of three new species. Zool J Linn Soc 174: 185–215. 34. Bishop IR (1979) Notes on Praomys (Hylomyscus) in eastern Africa. Mammalia 43: 521–530. 35. Dieterlen F (1979) Der früheste Fund der afrikanischen Kleinen Hamsterratte (Beamys hindei) (Cricetomyinae; Cricetidae; Rodentia). Stuttgarter Beitr zur Naturk ser. A 330: 1–3. 36. Guest NJ, Leedal GP (1953) Volcanic activity of Mt. Meru. Records of the Geological Survey of Tanganyika 3: 40–46. 37. Stanley WT, Goodman SM (2011) Small mammal inventories in the East and West Usambara Mountains, Tanzania. 4. Rodentia. In: Stanley WT, editor. Studies of Montane Vertebrates of Tanzania. Fieldiana Life Earth Sci 4: 53–73. PLOS ONE | DOI:10.1371/journal.pone.0162009 September 21, 2016 15 / 15 

https://openalex.org/W2941756905

https://periodicos.ifgoiano.edu.br/index.php/multiscience/article/download/556/423

Portuguese

Jogo lúdico de caça palavras sobre a fauna brasileira; atividade desenvolvida no projeto de Ciências Biológicas do Pibid/IF Goiano - Campus Urutaí

Multi-Science Journal

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Jogo lúdico de caça palavras sobre a fauna brasileira; atividade desenvolvida no projeto de Ciências Biológicas do Pibid/IF Goiano - Campus Urutaí Lucas Rodrigues Guimaraes 1* Ana Paula Custodio Avelino 1, Thalita Teresinha De Sousa 1, Joice Gomes De Queiroz 1, Andressa De Souza Almeida 1, Alexia Ferreira Huntra 1, Bruna Cleyderman Gonzaga 1, Laura Meireles 1, Guilherme Malafaia 1 (Coordenador de Área do Pibid Biologia) 1 1Instituto Federal de Educação, Ciência e Tecnologia Goiano - Campus Urutaí GO.75790-000 E-mail do autor: [email protected] Conteúdo disponível em: https://www.ifgoiano.edu.br/periodicos/ ANAIS DO I WORKSHOP SOBRE TEMAS & CONTEMPORÂNEOS DA PEDAGÓGICO ENCONTRO 2018/1 EDUCAÇÃO ISSN: 2359-6902 Multi-Science Journal W e bsi te do pe ri ó di co : ht t ps: // www .i fg o ia no .e du .b r/ pe ri o di cos /i nde x .php/ mu lt isc ie n ce 12 12 Multi-Science Journal, 1(11): 2018 – Edição Suplementar R E S U M O A didática proposta além de ser uma didática lúdica, visa à interação entre os envolvidos na atividade, é um recurso visual do ambiente da aprendizagem, que, quando usado de maneira adequada, desperta a curiosidade dos envolvidos, aproxima ainda mais os alunos, e favorece o desenvolvimento da capacidade de observação e de raciocínio. Quando a informação é obtida através da leitura, escrita, ou fala, obtém a falta de atenção dos mesmos, em sua grande parte, já o que está sendo visto, apresentado, e representado desperta o interesse, contribuindo para a percepção do objetivo proposto. Neste intuito, realizou uma atividade de caça palavras com o tema da fauna onde ele tem que por meio de dicas tais como: descrição corporal, hábitos alimentares, coloração, entre outros eles teriam que desvendar qual seria o animal em questão. O tema proposto aos alunos teve como objetivo: estimular o aluno, despertar seu interesse à cerca do assunto, levar eles a pensar em variam possibilidades e por modo de exclusão chegar no animal a ser descoberto e favorecer o desenvolvimento da capacidade de observação, e ter como objetivo principal melhor ficção da matéria. Foi utilizado para a dinâmica: um jogo caça palavra que foi criado a partir revistas de jogos e joguinhos de jornal com tema já dito anteriormente. Os resultados obtidos com a realização do caça palavra são bastante positivos, tanto para os alunos que obtiveram maior aprendizado, e fixação da matéria e para o professor onde tiveram maior percepção. Além disso, a atividade proporcionou uma aproximação entre os alunos. Histórico do resumo Recebido: 30 janeiro 2018 Aceito: 03 fevereiro 2018 Histórico do resumo Recebido: 30 janeiro 2018 Aceito: 03 fevereiro 2018 Palavras chaves: Biologia Ludicidade Educação Multi-Science Journal – 1(11) – Edição Suplementar Evento voltado para estudantes, professores e técnicos administrativos I WORKSHOP PEDAGÓGICO A educação muda o mundo & 5 A 9/02 PALESTRAS GRUPOS DE ESTUDOS MESAS REDONDAS MOSTRA DE TRABALHOS CONTEMPORÂNEA EDUCAÇÃO ENCONTRO Iniciando o semestre letivo de 2018 – Planejamento e reflexões iniciais 2018/1 https://www.ifgoiano.edu.br/urutai Multi-Science Journal – 1(11) – Edição Suplementar

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http://journal.ubpkarawang.ac.id/mahasiswa/index.php/buanafarma/article/download/883/659

Indonesian

ANALISIS KEPATUHAN MINUM OBAT DAN PENGETAHUAN ANALISIS KEPATUHAN MINUM OBAT DAN PENGETAHUAN PUSKESMAS KARAWANG KOTA

Jurnal Buana Farma

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Dedy Frianto*, Resha Rosalaia, Surya Amal Fakultas Farmasi, Universitas Buana Perjuangan Karwang, Karawang, Jawa Barat, Indonesia *Penulis Korespondensi: [email protected] ABSTRAK Penyebab kematian di seluruh dunia diakibatkan penyakit tidak menular (PTM) yaitu hipertensi, yang terjadi pada beberapa dekade terakhir cenderung meningkat di Indonesia. Pengobatan berbagai macam penyakit diantaranya hipertensi di pengaruhi oleh oleh kepatuhan minum obat, Pengetahuan serta Kualitas hidup pasien. Penelitian ini bertujuan untuk mengetahui korelasi tingkat kepatuhan minum obat dengan kualitas hidup , korelasi tingkat pengetahuan dengan kualitas hidup dan korelasi tingkat kepatuhan minum obat dengan tingkat pengetahuan. Penelitian ini Menggunakan deskriptif analitik dengan pengumpulan data secara prospektif. Jumlah responden sebanyak 35 orang, pengumpulan data menggunakan kuesioner MMAS-8, EQ-5D-5L dan VAS, Pengetahuan dan rekam medik. Analisis data menggunakan uji korelasi Spearmen. Hasil penelitian korelasi kepatuhan dengan kualitas hidup (EQ-5D-5L) didapatkan nilai p=0,003 (<0,05) dengan nilai koefisien 0,491 yang artinya terdapat hubungan signifikan dan cukup kuat. Hasil penelitian korelasi kepatuhan dengan kualitas hidup (VAS) didapatkan nilai p=0,001 (<0,05) dengan nilai koefisien 0,517 yang artinya terdapat hubungan signifikan dan kuat. Hasil penelitian korelasi pengetahuan dengan kualitas hidup (EQ-5D-5L) didapatkan nilai p=0,003 (<0,05) dengan nilai koefisien 0,481 yang artinya terdapat hubungan signifikan dan cukup kuat. Hasil penelitian korelasi pengetahuan dengan kualitas hidup (VAS) didapatkan nilai p=0,004 (<0,05) dengan nilai koefisien 0,473 yang artinya terdapat hubungan signifikan dan cukup kuat. Hasil penelitian korelasi pengetahuan dengan kepatuhan minum obat didapatkan nilai p=0,000 (<0,05) dengan nilai koefisien 0,688 yang artinya terdapat hubungan signifikan yang kuat. Semakin baik kepatuhan minum obat dan pengetahuan hipertensi maka semakin baik kualitas hidup pasien hipertensi. Kata Kunci : Kepatuhan, Pengetahuan, Kualitas Hidup, Hipertensi Kata Kunci : Kepatuhan, Pengetahuan, Kualitas Hidup, Hipertensi Abstract The cause of death worldwide is non-communicable diseases (NCDs), namely hypertension, which in the last few decades has tended to increase in Indonesia. Treatment of various diseases, including hypertension, is influenced by medication adherence, knowledge and the patient's quality of life. This study aims to determine the correlation between the level of adherence to taking medication and quality of life, the correlation between the level of knowledge and the quality of life and the correlation between the level of adherence to taking medication and the level of knowledge. This research uses descriptive analytics with prospective data collection. The number of respondents was 35 people, data collection used the MMAS-8, EQ-5D-5L and VAS, Knowledge and medical record questionnaires. Data analysis used the Spearman correlation test. The results of research on the correlation between compliance and quality of life (EQ-5D-5L) showed a value of p=0.003 (<0.05) with a coefficient value of 0.491, which means there is a significant and quite strong relationship. The results of research on the correlation between compliance and quality of life (VAS) showed a value of p=0.001 (<0.05) with a coefficient value of 0.517, which means there is a significant and strong relationship. The results of research on the correlation of knowledge with quality of life (EQ-5D-5L) showed a value of p=0.003 (<0.05) with a coefficient value of 0.481, which means there is a significant and quite strong relationship. The results of research on the correlation between knowledge and quality of life (VAS) showed a value of p=0.004 (<0.05) with a coefficient value of 0.473, which means there is a significant and quite strong relationship. The results of the research on the correlation between knowledge and adherence to taking medication showed a value of p=0.000 (<0.05) with a coefficient value of 0.688, which means there is strong significant relationship. The better the adherence to taking medication and knowledge of hypertension, the better the quality of life of hypertensive patients. Keywords: Compliance, Knowledge, Quality of Life, Hypertension Keywords: Compliance, Knowledge, Quality of Life, Hypertension 129 ANALISIS KEPATUHAN MINUM OBAT DAN PENGETAHUAN TERHADAP KUALITAS HIDUP PASIEN HIPERTENSI DI PUSKESMAS KARAWANG KOTA Dedy Frianto*, Resha Rosalaia, Surya Amal PENDAHULUAN kesehatan diri nya dalam mencegah hipertensi. pentingnya pengukuran tingkat kepatuhan dan pengetahuan terhadap kualitas hidup pada pasien hipertensi perlu dilakukan analisis kualitas hidup karena penyakit hipertensi membutuhkan waktu yang panjang dalam pengobatannya. Hal ini yang mendorong peneliti untuk melakukan penelitian “Analisis Kepatuhan Minum Obat dan Pengetahuan Terhadap Kualitas Hidup Pasien Hipertensi di Puskesmas Karawang Kota” menggunakan metode prospektif dengan penyebaran kuisioner. Penyebab utama kematian di Indonesia adalah penyakit tidak menular yaitu hipertensi yang beberapa dekade terakhir ini cenderung meningkat baik morbiditas maupun mortalitas di dunia maupun di Indonesia. Penyebab utama kematian di Indonesia mencapai 37 persen dengan peringkat kedelapan (Kemenkes, 2017). Penyakit tidak menular dalam pemeriksaan hipertensi merupakan program baru sehingga dalam operasional kegiatan masih belum menunjukkan aktivitas yang optimal. Tujuan pengobatan hipertensi adalah untuk menjaga tekanan darah tetap terkendali untuk mencegah komplikasi. Riskesdas (2018) menyatakan prevalensi hipertensi berdasarkan hasil pengukuran pada penduduk usia ≥18 tahun sebesar 34,1 %, adanya peningkatan dibandingkan prevalensi hipertensi pada Riskesdas Tahun 2013 sebesar 25,8%. Pasien hipertensi yang mengikuti program rujuk balik di Karawang tercatat 1.742 orang, namun masih banyak yang tidak mengikuti program rujuk balik ini di daerah Karawang karena kurangnya pengetahuan mengenai penyakit yang diderita (BPJS,2021). Jurnal Buana Farma: Jurnal Ilmiah Farmasi, Vol. 3, No. 4, Desember 2023 Jurnal Buana Farma: Jurnal Ilmiah Farmasi, Vol. 3, No. 4, Desember 2023 METODE PENELITIAN Penelitian ini menggunakan metode Deskriptik analitik dengan pendekatan observasional. Pengumpulan data dilakukan secara prospektif. Data yang didapatkan berupa kuesioner dan rekam medik. Responden yang digunakan sebanyak 35 responden pada pasien hipertensi yang mengikuti program rujuk balik di Puskesmas Karawang Kota. Data yang diperoleh kemudian dibuat tabulasi kemudian dianalisis menggunakan uji korelasi spearmen memakai SPSS 21. Jurnal Buana Farma: Jurnal Ilmiah Farmasi, Vol. 3, No. 4, Desember 2023 130 PEMBAHASAN Pengetahuan dan komitmen penggunaan obat antihipertensi diperlukan untuk mengurangi atau menurunkan jumlah penderita hipertensi di negara ini. Namun, telah terbukti bahwa minum obat antihipertensi tidak mendukung penggunaan obat antihipertensi saja (Saepuddin, 2013). Kepatuhan minum obat sangat mempengaruhi seseorang dalam mencegah hipertensi. Semakin patuh atau rutin seseorang mengkonsumsi obat tekanan darah maka semakin paham bahwa pencegahan hipertensi sangat bermanfaat bagi Tabel 1. Distribusi Karakteristik Sosiodemografi Pasien Hipertensi di Puskesmas Karawang Kota, Januari 2023 Karakteristik Demografi N (n=35) % Usia 35-44 5 14,3 45-54 13 37,1 55-64 17 48,6 Tabel 1. Distribusi Karakteristik Sosiodemografi Pasien Hipertensi di Puskesmas Karawang Kota, Januari 2023 Jurnal Buana Farma: Jurnal Ilmiah Farmasi, Vol. 3, No. 4, Desember 2023 130 Jurnal Buana Farma: Jurnal Ilmiah Farmasi, Vol. 3, No. 4, Desember 2023 130 Jenis Kelamin Laki-laki 9 25,7 Perempuan 26 74,3 Pendidikan SD 4 11,4 SMP 8 22,9 SMA 17 48,6 PT 6 17,1 Pekerjaan Tidak Bekerja 35 100 Bekerja 0 0 Lama menderita <5 tahun 3 8,6 >5 tahun 32 91,4 Obat Yang Dipakai Amlodipine 5mg 28 80 Captopril 12,5 mg 4 11 Captopril 25 mg 1 3 Amlodipine 5mg+Ramipril 10 mg 1 3 Amlodipine 5mg+Lisinopril 1 3 penurunan tubuh. Adapun karakteristik responden berdasarkan jenis kelamin yang dapat dilihat pada tabel 1.1 diatas jumlah responden pada jenis kelamin yang terbanyak adalah perempuan sebanyak 26 orang (74,3%). Menurut riskesdas (2018) bahwa prevalensi perempuan lebih tinggi dari laki – laki, hal ini yang menyebabkan faktor resiko tingginya kejadian hipertensi pada perempuan lebih besar khususnya pada ibu rumah tangga. Berdasarkan tabel 1 diatas jumlah responden kategori pendidikan yang terbanyak adalah tingkat SMA sebanyak 17 orang (48,6%). Menurut Sutrisno (2018) tingkat Pendidikan memiliki pengaruh yang kuat terhadap perilaku pengendalian hipertensi. Penelitian ini sejalan dengan Badan Pusat Statistika Kabupaten Karawang bahwa pada kecamatan Karawang Barat 43.572 orang dengan lulusan SMA terbanyak. Berdasarkan tabel 1.1 diatas jumlah responden kategori pekerjaan kebanyakan tidak bekerja karena sudah pensiun dari tempat kerjaan sebelumnya, berjumlah 35 orang (100%). Pekerjaan seseorang dapat mempengaruhi tingginya aktivitas fisik. Pada pekerja membuktijan faktor – faktor kerja yang dapat mempengaruhi meningkatnya hipertensi. Jenis pekerjaan yang terkait dengan risiko penyakit salah satunya yaitu kurang berolahraga dan stress yang dapat menyebabkan hipertensi (WHO,2015). Maka disimpulkan dengan responden yang tidak bekerja dapat memperkecil resiko dari penyakit hipertensi yaitu dengan penatalaksanaan secara non farmakologis yaitu melakukan terapi diet garam maksimal 2 gr garam dapur per harinya serta istirahat yang cukup. PEMBAHASAN Penelitian yang dilakukan oleh Gaili (2016) menyebutkan bahwa captopril dianggap kurang aman digunakaan sebagai antihipertensi pada Sebagian besar pasien, sehingga dokter menyarankan untuk mengganti terapi dengan amlodipine. Kemudian adapula faktor ketersediaan obat yang di lakukan di Puskesmas Karawang Kota banyak nya amlodipine 5mg. Tabel 2. Distribusi Kepatuhan Pasien Hipertensi B d k MMAS 8 Total Berdasarkan tabel 1 diatas jumlah responden lamanya menderita penyakit hipertensi diatas 5 tahun sebanyak 32 tahun (91,4%). Penelitian ini dikuatkan oleh penelitian Rahmayanti (2018) Meningkatnya tekanan darah seiring bertambahnya umur seseorang. Hal ini merupakan adanya pengaruh degenerasi yang terjadi pada orang yang bertambahnya usia. Berdasarkan hal ini mungkin dapat menjelaskan jumlah penderita hipertensi yang memiliki Riwayat hipertensi diatas 5 tahun lebih banyak. Berdasarkan tabel 1 diatas, dapat diketahui bahwa dari 35 responden jenis obat yang paling banyak digunakan adalah amlodipine 5 mg sebanyak 28 responden (80%). Obat – obat yang diresepkan berdasarkan data yang didapatkan di Puskesmas Karawang Kota adalah golongan CCB (Calcium Channel Blocker) yaitu amlodipine 5 mg. Hal tersebut karena pasien mengeluhkan batuk kering yang mengganggu saat mengkonsumsi captopril dan lebih nyaman mengkonsumsi amlodipine yang digunakan cukup satu kali sehari. Penelitian yang dilakukan oleh Gaili (2016) menyebutkan bahwa captopril dianggap kurang aman digunakaan sebagai antihipertensi pada Sebagian besar pasien, sehingga dokter menyarankan untuk mengganti terapi dengan amlodipine. Kemudian adapula faktor ketersediaan obat yang di lakukan di Puskesmas Karawang Kota banyak nya amlodipine 5mg. Berdasarkan tabel 2 diatas, dapat diketahui bahwa dari 35 responden diantaranya 4 responden mempunyai tingkat kepatuhan yang patuh (11,5%), 17 responden mempunyai tingkat kepatuhan yang cukup patuh (48,5%) dan 14 responden mempunyai tingkat kepatuhan yang tidak patuh (40%). Jika tingkat kepatuhan sangat rendah, maka akan memperburuk kesehatan, bahkan jika pengobatannya sesuai dengan standar perawatan yang ada (Rahmadani and Sari, 2018). Hasil penelitian ini hampir sama dengan penelitian Alfiani dan Putra tahun 2017, menghasilkan responden patuh kategori sedikit dinyatakan sebanyak 7 responden (16,6%), responden cukup patuh sedang sebanyak 33 responden (55%) dan responden yang tidak patuh sebanyak 20 reponden (33,4%). Hal ini dapat dilakukan dengan sengaja, dengan tidak meminum obat karena merasa kondisinya semakin baik atau buruk, atau secara tidak sengaja, seperti lalai meminum obat (Alfian and Putra, 2017). Tabel 3. Distribusi Pengetahuan Pasien Hipertensi Tingkat Pengetahuan Frekuensi % Cukup 20 57,1 Baik 5 14,3 Kurang 10 28,6 Total 35 100 Tabel 3. Distribusi Pengetahuan Pasien Hipertensi Tabel 2. PEMBAHASAN Berdasarkan tabel 1 diatas jumlah responden kategori pendidikan yang terbanyak adalah tingkat SMA sebanyak 17 orang (48,6%). Menurut Sutrisno (2018) tingkat Pendidikan memiliki pengaruh yang kuat terhadap perilaku pengendalian hipertensi. Penelitian ini sejalan dengan Badan Pusat Statistika Kabupaten Karawang bahwa pada kecamatan Karawang Barat 43.572 orang dengan lulusan SMA terbanyak. Berdasarkan tabel 1.1 diatas jumlah responden kategori pekerjaan kebanyakan tidak bekerja karena sudah pensiun dari tempat kerjaan sebelumnya, berjumlah 35 orang (100%). Pekerjaan seseorang dapat mempengaruhi tingginya aktivitas fisik. Pada pekerja membuktijan faktor – faktor kerja yang dapat mempengaruhi meningkatnya hipertensi. Jenis pekerjaan yang terkait dengan risiko penyakit salah satunya yaitu kurang berolahraga dan stress yang dapat menyebabkan hipertensi (WHO,2015). Maka disimpulkan dengan responden yang tidak bekerja dapat memperkecil resiko dari penyakit hipertensi yaitu dengan penatalaksanaan secara non farmakologis yaitu melakukan terapi diet garam maksimal 2 gr garam dapur per harinya serta istirahat yang cukup. Berdasarkan tabel 1 diatas jumlah responden yang banyak menderita hipertensi di atas 55 tahun sebanyak 17 orang (48,6 %) yang dilakukan terhadap 35 penderita hipertensi yang mengikuti program prolanis. Dalam hasil penelitian ini dapat diketahui bahwa usia merupakan salah satu faktor yang mempengaruhi pasien menderita hipertensi. Hal ini sejalan dengan teori yang diungkapkan oleh Notoatmojo yaitu semakin tinggi umur seseorang maka semakin tinggi beresiko penyakit hipertensi. Bertambahnya usia maka tingkat kepatuhan rendah dikarenakan fungsi fisologis terjadi akibat Jurnal Buana Farma: Jurnal Ilmiah Farmasi, Vol. 3, No. 4, Desember 2023 131 131 Jurnal Buana Farma: Jurnal Ilmiah Farmasi, Vol. 3, No. 4, Desember 2023 Berdasarkan tabel 1 diatas jumlah responden lamanya menderita penyakit hipertensi diatas 5 tahun sebanyak 32 tahun (91,4%). Penelitian ini dikuatkan oleh penelitian Rahmayanti (2018) Meningkatnya tekanan darah seiring bertambahnya umur seseorang. Hal ini merupakan adanya pengaruh degenerasi yang terjadi pada orang yang bertambahnya usia. Berdasarkan hal ini mungkin dapat menjelaskan jumlah penderita hipertensi yang memiliki Riwayat hipertensi diatas 5 tahun lebih banyak. Berdasarkan tabel 1 diatas, dapat diketahui bahwa dari 35 responden jenis obat yang paling banyak digunakan adalah amlodipine 5 mg sebanyak 28 responden (80%). Obat – obat yang diresepkan berdasarkan data yang didapatkan di Puskesmas Karawang Kota adalah golongan CCB (Calcium Channel Blocker) yaitu amlodipine 5 mg. Hal tersebut karena pasien mengeluhkan batuk kering yang mengganggu saat mengkonsumsi captopril dan lebih nyaman mengkonsumsi amlodipine yang digunakan cukup satu kali sehari. Jurnal Buana Farma: Jurnal Ilmiah Farmasi, Vol. 3, No. 4, Desember 2023 PEMBAHASAN Distribusi Kepatuhan Pasien Hipertensi Berdasarkan MMAS-8 Berdasarkan tabel 3 diatas, dapat diketahui bahwa dari 35 responden, sebanyak 20 responden dengan tingkat pengetahuan resoponden yang cukup baik (57,1%), 5 responden dengan tingkat pengetahuan responden yang baik (14,3%) dan sebanyak 10 Tingkat Kepatuhan Frekuensi % Patuh 4 11,5 Cukup Patuh 17 48,5 Tidak Patuh 14 40 Jurnal Buana Farma: Jurnal Ilmiah Farmasi, Vol. 3, No. 4, Desember 2023 132 dirumah. Penelitian ini sejalan dengan penelitian Hiraida (2019) bahwa tingkat pengetahuan kategori baik sebanyak 8 responden (13,4%) tingkat pengetahuan cukup baik 33 responden (55%) dan tingkat pengetahuan kurang baik sebanyak 19 responden (31,6%). responden dengan tingkat pengetahuan responden kurang baik (28,6%). Ada banayak faktor yang mempengaruhi penegetahuan responden, misalnya pengalaman serta sarana informasi. Pengetahuan bukan hanya didapat secara formal melainkan juga pengalaman. Pengetahuan juga didapat melalui sarana informasi yang tersedia EQ5D5L 30 28 19 21 221 20 10 8 11 13 7 4 10 5 01 10011 00000 0 Level 1 Level 2 Level 3 Level 4 Level 5 Mobilitas Perawatan Diri Kegiatan Yang Biasa Dilakukan Nyeri/Ketidaknyamanan Kecemasan/Depresi 2 Gambar 1 Persentase Respon Kualitas Hidup Berdasarkan EQ-5D-5L Pasien Hipertensi Gambar 1 Persentase Respon Kualitas Hidup Berdasarkan EQ-5D-5L Pasien Hipertens Berdasarkan gambar 1 diatas, dapat diketahui bahwa kualitas hidup responden berdasarkan EQ-5D-5L pada dimensi morbilitas level 1 sebanyak 19 responden (54,3%), level 2 sebanyak 11 responden (31,4%), level 3 sebanyak 4 responden (11,4%) level 4 sebanyak 1 responden (2,9%). Pada dimensi perawatan diri level 1 sebanyak 28 responden (80%) level 2 sebanyak 7 responden (20%). Pada dimensi kegiatan yang biasa dilakukan pada level 1 sebanyak 21 responden (60%) level 2 sebanyak 13 responden (37,1%) level 3 sebanyak 1 responden (2,9%). Pada dimensi Nyeri/ketidaknyamanan level 1 sebanyak 2 responden (5,7%) level 2 sebanyak 22 responden (62,9%) level 3 sebanyak 10 responden (28,6%) dan level 4 sebanyak 1 responden ( 2,9%). Pada dimensi kecemasan/depresi pada level 1 sebanyak 8responden (22,9%) level 2 sebanyak 21 responden (60%) level 3 sebanyak 5 responden (14,3%) dan level 4 sebanyak 2,9%). Semakin tinggi skor maka semakin baik kualitas hidup, skor berkisar antara 0 (kematian) hingga 1 (Kesehatan yang sempurna). Hasil penelitian ini serupa dengan yang dijelaskan oleh Fenwick dkk, beberapa respon emosi meliputi gejala depresi sebanyak 60% diikuti rasa nyeri yang sebesar 62,2%. Hasil penelitian novita (2019) menujukkan bahwa masalah lain yang dihadapi pasien adalah dimensi rasa nyeri/ketidaknyamanan sebesar 78%. Namun rasa nyeri/ketidaknyamanan ini tidak dapat dideskripsikan secara spesifik. PEMBAHASAN Rasa nyeri/ketidaknyamanan bisa terjadi karena adanya komorbid. Berdasarkan gambar 1 diatas, dapat diketahui bahwa kualitas hidup responden berdasarkan EQ-5D-5L pada dimensi morbilitas level 1 sebanyak 19 responden (54,3%), level 2 sebanyak 11 responden (31,4%), level 3 sebanyak 4 responden (11,4%) level 4 sebanyak 1 responden (2,9%). Pada dimensi perawatan diri level 1 sebanyak 28 responden (80%) level 2 sebanyak 7 responden (20%). Pada dimensi kegiatan yang biasa dilakukan pada level 1 sebanyak 21 responden (60%) level 2 sebanyak 13 responden (37,1%) level 3 sebanyak 1 responden (2,9%). Pada dimensi Nyeri/ketidaknyamanan level 1 sebanyak 2 responden (5,7%) level 2 sebanyak 22 responden (62,9%) level 3 sebanyak 10 responden (28,6%) dan level 4 sebanyak 1 responden ( 2,9%). Pada dimensi 133 Jurnal Buana Farma: Jurnal Ilmiah Farmasi, Vol. 3, No. 4, Desember 2023 dengan Mala (2022) Hasil analisis penelitian hubungan antara kepatuhan minum obat dengan kualitas hidup penderita hipertensi diperoleh hasil nilai p=0,000 < 0,05. Tabel 4. Hubungan kepatuhan minum obat berdasarkan MMAS-8 dengan Kualitas hidup EQ 5D-5L Tabel 4. Hubungan kepatuhan minum obat berdasarkan MMAS-8 dengan Kualitas hidup EQ 5D-5L Kepatuhan vs Kualitas Hidup (EQ5D5L) Kepatuhan Kualitas Hidup Kepatuhan Korelasi Koefisien 1,000 0,491 Hubungan Signifikan 0,003 Jumlah responden 35 35 Kualitas Hidup Korelasi Koefisien 0,491 1,000 Hubungan Signifikan 0,003 Jumlah responden 35 35 Tabel 5. Hubungan kepatuhan minum obat berdasarkan MMAS-8 dengan Kualitas hidup VAS Kepatuhan vs Kualitas Hidup (VAS) Kepatuhan Kualitas Hidup Kepatuhan Korelasi Koefisien 1,000 0,517 Hubungan Signifikan 0,001 Jumlah responden 35 35 Kualitas Hidup Korelasi Koefisien 0,517 1,000 Hubungan Signifikan 0,001 Jumlah responden 35 35 Berdasarkan tabel 4 diatas, menjelaskan hubungan kepatuhan berdasarkan kuesioner MMAS-8 dengan kualitas hidup menggunakan kuesioner EQ-5D-5L bahwa terdapat hubungan antara minum obat dengan kualitas hidup pasien. Berdasarkan korelasi ini p-value Berdasarkan tabel 5 diatas, menjelaskan hubungan kepatuhan berdasarkan kuesioner MMAS-8 dengan kualitas hidup menggunakan kuesioner VAS bahwa terdapat hubungan antara minum obat dengan kualitas hidup pasien. Berdasarkan korelasi ini p-value <0,05 maka terdapat hubungan yang signifikan antara kepatuhan dengan kualitas hidup VAS. Nilai koefisen korelasi dari hasil penelitian adalah 0,517. Pada uji korelasi spearman, nilai koefisien korelasi tersebut termasuk dalam rentang 0,51 – 0,75 yang menyatakan bahwa arah korelasi positif dan kekuatan hubungan kuat. Berdasarkan penelitian terdahulu (Hasibuan,2022) pada VAS dengan hasil uji statistik Spearman didapatkan p- value <0,05. Hasil ini menunjukkan bahwa ada hubungan yang signifikan antara kepatuhan minum obat dengan kualitas hidup pasien. Ketika kepatuhan terus meningkat, kualitas hidup meningkat. PEMBAHASAN Penelitian ini sejalan dengan Noviantika (2022) Hasil Hubungan antara tingkat kepatuhan pengobatan dengan kualitas <0,05 maka terdapat hubungan yang signifikan antara kepatuhan dengan kualitas hidup EQ- 5D-5L. Nilai koefisen korelasi dari hasil penelitian adalah 0,491. Pada uji korelasi spearman, nilai koefisien korelasi tersebut termasuk dalam rentang 0,26 – 0,50 yang menyatakan bahwa arah korelasi positif dan kekuatan hubungan cukup. Adapun korelasi spearman yang dimiliki menunjukkan hubungan yang searah, yaitu semakin tinggi tingkat kepatuhan semakin baik kualitas hidup pasien dengan kekuatan hubungan yang cukup. Hasil penelitian ini sama dengan hasil (Nufanesa et al,2020) menunjukkan hubungan yang signifikan antara kepatuhan minum obat dengan kualitas hidup. Pada analisis statistik, uji Spearman memberikan nilai p-value sebesar 0,001, dimana hasilnya adalah nilai yang signifikan antara observasi pengobatan dan kualitas hidup pasien. Penelitian Ini sejalan Jurnal Buana Farma: Jurnal Ilmiah Farmasi, Vol. 3, No. 4, Desember 2023 134 Jurnal Buana Farma: Jurnal Ilmiah Farmasi, Vol. 3, No. 4, Desember 2023 134 koefisien korelasi tersebut termasuk dalam rentang 0,26 – 0,50 yang menyatakan bahwa arah korelasi positif dan kekuatan hubungan cukup. Penelitian ini sejalan dengan penelitian Firda (2020) Hasil penelitian berdasarkan uji Spearman antara tingkat pengetahuan dengan kualitas hidup diperoleh p-value 0,000 dan korelasi koefisien nya adalah 0,449 dimana hasil tersebut menunjukkan adanya hubungan antar tingkat. pengetahuan dan kualitas hidup lansia dengan penyakit kronis dan terdapat hubungan positif yang cukup kuat. Kurniawati (2020) Hasil analisis data dilakukan berdasarkan data survey dengan menggunakan uji Spearman antara tingkat pengetahuan dengan kualitas hidup penderita hipertensi, diperoleh p value 0,000 dan correlation coefficient 0,449 imana hasil tersebut menunjukkan adanya hubungan antara tingkat pengetahuan dengan kualitas. hidup kualitas hidup pasien hipertensi di Poliklinik Tingkat III Baladhika Husada Jember dan memiliki hubungan positif dengan kekuatan hubungan cukup. hidup pasien hipertensi di uji dengan chi-square dan diperoleh p- value = 0,013 < α = 0,05 jadi Ha diterima, ada hubungan signifikan antara Kepatuhan Pengobatan dan Kualitas Hidup. Hubungan antara Kepatuhan Pengobatan dengan Kualitas Hidup, dapat disimpulkan bahwa tingkat kepatuhan pengobatan yang rendah berpengaruh terhadap kualitas hidup penderita hipertensi. Penderita lebih memilih meminum obat pada saat hanya sakit saja. Hal ini dikarenakan penderita malas meminum obat rutin atau lupa untuk meminum obat. Dengan meminum obat rutin maka kualitas hidup penderita akan lebih baik Tabel 6. PEMBAHASAN Hubungan Pengetahuan dengan Kualitas hidup EQ-5D-5L hidup EQ-5D-5L Pengetahuan vs Kualitas Hidup (EQ-5D-5L) Pengetahuan Kualitas Hidup Pengetahuan Korelasi Koefisien 1,000 0,481 Hubungan Signifikan 0,003 Jumlah Responden 35 35 Kualitas Hidup Korelasi Koefisien 0,481 1,000 Hubungan Signifikan 0,003 Jumlah Responden 35 35 Tabel 7. Hubungan Pengetahuan dengan Kualitas hidup VAS. Tabel 7. Hubungan Pengetahuan dengan Kualitas hidup VAS. Tabel 7. Hubungan Pengetahuan dengan Kualitas hidup VAS. Pengetahuan vs Kualitas Hidup (VAS) Pengetahuan Kualitas Hidup Pengetahuan Korelasi Koefisien 1,000 0,473 Hubungan Signifikan 0,004 Jumlah responden 35 35 Kualitas Hidup Korelasi Koefisien 0,473 1,000 Hubungan Signifikan 0,004 Jumlah responden 35 35 Blerdasarkan tabel 6 diatas, menjelaskan hubungan pengetahuan berdasarkan kuesioner dengan kualitas hidup menggunakan kuesioner EQ-5D-5L bahwa terdapat hubungan antara minum obat dengan kualitas hidup pasien. Berdasarkan korelasi ini p-value <0,05 maka terdapat hubungan yang signifikan antara pengetahuan dengan kualitas hidup EQ-5D-5L. Nilai koefisen korelasi dari hasil penelitian adalah 0,481. Pada uji korelasi spearman, nilai Berdasarkan tabel 7 diatas, menjelaskan hubungan pengetahuan berdasarkan kuesioner dengan kualitas hidup menggunakan kuesioner VAS bahwa terdapat hubungan antara minum 135 Jurnal Buana Farma: Jurnal Ilmiah Farmasi, Vol. 3, No. 4, Desember 2023 135 obat dengan kualitas hidup pasien. Berdasarkan korelasi ini p-value <0,05 maka terdapat hubungan yang signifikan antara pengetahuan dengan kualitas hidup VAS. Nilai koefisen korelasi dari hasil penelitian adalah 0,473. Pada uji korelasi spearman, nilai koefisien korelasi tersebut termasuk dalam rentang 0,26 – 0,50 yang menyatakan bahwa arah korelasi positif dan kekuatan hubungan cukup. Menurut Kurniawati (2020) hasil penelitian pada uji analisis data yang dilakukan berdasarkan penelitian menggunakan uji spearman antara tingkat pengetahuan dengan kualitas hidup didapatkan hasil p-value 0,004 dan korelasi koefisien nya adalah 0,449 dimana hasil ini menunjukkan adanya hubungan antara tingkat pengetahuan dengan kualitas hidup pada pasien hipertensi di Poli Klinik RS Tingkat III Baladhika Husada Jember dan mempunyai hubungan positif dengan kekuatan hubungan yang cukup. pengetahuan dengan kepatuhan. Nilai koefisen korelasi dari hasil penelitian adalah 0,688. Pada uji korelasi spearman, nilai koefisien korelasi tersebut termasuk dalam rentang 0,51 – 0,75 yang menyatakan bahwa arah korelasi positif dan kekuatan hubungan kuat. Penelitian ini diperkuat dengan adanya hasil penelitian terdahulu Hasbibuan (2022), hasil penelitian yang dilakukan menggunakan uji statistika pengetahuan penderita hipertensi dengan kepatuhan minum obat didapatkan p-value= 0,009. Secara keseluruhan terdapat hubungan antara pengetahuan dengan kepatuhan minum obat antihipertensi di wilayah kerja Puskesmas Batunadua. Tidak semua orang kepatuhan itu dipengaruhi oleh pengetahuan namun adapula faktor – faktor lain nya. Jurnal Buana Farma: Jurnal Ilmiah Farmasi, Vol. 3, No. 4, Desember 2023 Jurnal Buana Farma: Jurnal Ilmiah Farmasi, Vol. 3, No. 4, Desember 2023 136 KESIMPULAN Tabel 8. Hubungan Pengetahuan dengan Kepatuhan minum obat Berdasarkan penelitian yang telah dilakukan dengan judul “Analisis Kepatuhan Minum Obat dan Pengetahuan terhadap Kualitas Hidup Pasien Hipertensi di Puskesmas Karawang Kota” dapat disimpulkan bahwa : Kepatuhan minum obat Pengetahuan vs Kepatuhan Minum Obat (MMAS-8) Pengetahuan Kepatuhan Pengetahuan Korelasi Koefisien 1,000 0,688 Hubungan Signifikan 0,000 Jumlah responden 35 35 Kepatuhan Korelasi Koefisien 0,688 1,000 Hubungan Signifikan 0,000 Jumlah responden 35 35 1. Hasil dari penelitian menunjukkan bahwa terdapat hubungan antara tingkat kepatuhan minum obat dengan kualitas hidup (EQ-5D- 5L) pasien yang menunjukkan hubungan yang cukup berkorelasi. Berdasarkan tabel 8 diatas, menjelaskan hubungan pengetahuan berdasarkan kuesioner dengan kepatuhan menggunakan kuesioner MMAS-8 bahwa terdapat hubungan antara pengetahuan dengan kepatuhan minum obat. Berdasarkan korelasi ini p-value <0,05 maka terdapat hubungan yang signifikan antara 2. Hasil dari penelitian menunjukkan bahwa terdapat hubungan antara tingkat kepatuhan minum obat dengan kualitas hidup (VAS) pasien yang menunjukkan hubungan yang cukup berkorelasi. 3. Hasil dari penelitian menunjukkan bahwa 3. Hasil dari penelitian menunjukkan bahwa Jurnal Buana Farma: Jurnal Ilmiah Farmasi, Vol. 3, No. 4, Desember 2023 136 136 Translation and validation of the Malaysian version. Diabetes Research and Clinical Practice, 90(2), 216–221. terdapat hubungan antara pengetahuan dengan kualitas hidup (EQ-5D-5L) pasien menunjukkan hubungan yang cukup berkorelasi. terdapat hubungan antara pengetahuan dengan kualitas hidup (EQ-5D-5L) pasien menunjukkan hubungan yang cukup berkorelasi. Translation and validation of the Malaysian version. Diabetes Research and Clinical Practice, 90(2), 216–221. Alfian, R., & Putra, A. M. P. 2017. Uji validitas dan reliabilitas kuesioner medication adherence report scale (MARS) terhadap pasien Diabetes Mellitus. Jurnal Ilmiah Ibnu Sina, 2(2), 176–183. 4. Hasil dari penelitian menunjukkan bahwa terdapat hubungan antara pengetahuan dengan kualitas hidup (VAS) pasien menunjukkan hubungan yang cukup berkorelasi. Andayani T.M., 2013, Farmakoekonomi Prinsip dan Metodologi, Bursa ilmu,Yogyakarta. Arikunto, Suharsimi. 2012. Prosedur Penelitian Suatu Pendekatan Praktek. Jakarta: Rineka Cipta. 5. Hasil dari penelitian menunjukan bahwa terdapat hubungan antara pengetahuan dengan kepatuhan minum obat pasien menunjukkan hubungan yang kuat berkolerasi B G, C S, Ksv N, M M, Gunda RK, V S, et al. Cost-Effectiveness Analysis in the Management of Stroke. Asian J Pharm Clin Res. 2017;10(7):127. SARAN Basuni,H.L dan Saifurrahman.Analisis kualitas hidup pasien stroke berdasarkan respon time di ruang emergensi. Jurnal Kesehatan Primer. 2022. 7(1) 1-12. 1. 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Large Medical Books, Mc Graw, New York Jilao, Mareeya. 2017. Tingkat Kepatuhan Penggunaan Obat Antidiabetes Oral pada Pasien Diabetes Melitus di Puskesmas Koh-Libong Thailand. Malang: Fakultas Kedokteran dan Ilmu Ilmu Kesehatan Universitas Islam Negeri Maulana Malik Ibrahim. Fenwick EK, Pesudovs K, Khadka J, et al. The impact of diabetic retinopathy on quality of life: qualitative findings from an item bank development project. Qual Life Res. 2012;21(10):1771-1782. Firda. 2020. Hubungan antara Tingkat Pengetahuan dengan Kualitas Hidup Lansia Penderita Penyakit Kronis. Universitas Muhamadiyah Surakarta : Surakarta. JNC-8 Guideline Recommendations. Washington, Alabama: Pharmacy Assosiation. Kemenkes Republik Indonesia. 2014. Profil Kesehatan Indonesia 2014. Jakarta: Kemenkes Republik Indonesia. Gaili, A.A, Al-Ebrahem, S.Q, Metwali, Z.M. 2016. The Relationship Between Knowledge and Drug Adherence in Hypertensive Patients: A Cross Sectional Study in UAE. 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Innovation capability and organizational resources configuration

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©© Intangible Capital, 2009 – 5(3):301-320 – ISSN: 1697-9818 doi: 10.3926/ic.2009.v5n3.p301-320 Capacidad de innovación y configuración de recursos organizativos Edna R. Bravo-Ibarra Liliana Herrera Universitat Politècnica de Catalunya Universidad de León (Spain) (Spain) [email protected] [email protected] Received May, 2009 Accepted July, 2009 Resumen: El propósito de este artículo es contribuir a un mejor entendimiento de los recursos involucrados en el proceso de innovación continua en las empresas. En este trabajo se identifican un conjunto de buenas prácticas, las cuales integradas, forman actividades innovadoras que ayudan a las organizaciones a adquirir la capacidad para innovar continuamente por medio de los proyectos de desarrollo de nuevos productos. Además, en un esfuerzo por comprender como está conformada la capacidad de innovación, en esta investigación se presenta un modelo conceptual basado en la perspectiva de las capacidades dinámicas, el cual muestra esta capacidad como el resultado de cuatro procesos: creación de conocimiento, absorción de conocimiento, integración de conocimiento y reconfiguración de conocimiento. Estos procesos están soportados por cuatro tipos de recursos: capital humano, liderazgo, estructuras y sistemas y la cultura organizativa. Aplicando este modelo las organizaciones pueden identificar y estructurar las acciones organizativas más importantes en el proceso de innovación continua. Finalmente, el desarrollo del modelo, así como la identificación de las buenas prácticas, es realizada por medio de un estudio de casos exploratorio, el cual es aplicado a dos organizaciones de base tecnológica del sector audiovisual. Capacidad de innovación y configuración de recursos organizativos E.R. Bravo-Ibarra – L. Herrera 301 ©© Intangible Capital, 2009 – 5(3):301-320 – ISSN: 1697-9818 doi: 10.3926/ic.2009.v5n3.p301-320 Palabras clave: capacidades dinámicas, conocimiento, creación, integración, innovación. Código JEL: O32 Title: Innovation capability and organizational resources configuration. Abstract: The main purpose of this article is to contribute to a better understanding of the organizational sources pertaining to continuous innovation. This work identifies a set of best practices, which once integrated, create the innovative activities that help organizations to acquire a continuous innovation capability by means of the development of new products. Furthermore, in an effort to understand how the innovation capacity is created, based on the dynamic capability theory, a conceptual model is presented in this study. This model shows that the innovation capability is the result of four processes: knowledge creation, knowledge absorption, knowledge integration, and knowledge reconfiguration. These processes are leveraged by four kinds of resources: human capital, structures and systems, leadership, and company culture. Companies applying this model can identify and restructure the most important organizational actions in order to achieve a continuous innovation process. Finally, identification of best practices and model development are carried out by means of an exploratory case study, which is applied to two technological based organizations of the audiovisual sector. Keywords: dynamic capabilities, knowledge, creation, integration, innovation. JEL Code: O32 1. Introducción Se ha considerado que la competitividad y el desempeño económico de las empresas están influenciados por la capacidad de innovación. Por tal motivo, numerosas investigaciones han sido dedicadas al estudio y entendimiento del Capacidad de innovación y configuración de recursos organizativos E.R. Bravo-Ibarra – L. Herrera 302 ©© Intangible Capital, 2009 – 5(3):301-320 – ISSN: 1697-9818 doi: 10.3926/ic.2009.v5n3.p301-320 proceso de innovación y la capacidad innovadora de las empresas. Por la multidimensionalidad característica y complejidad estructural de esta capacidad, esta investigación utiliza la perspectiva de las capacidades dinámicas para identificar y analizar la combinación de los recursos que la componen. Esta teoría es reconocida como una herramienta útil en el estudio de fenómenos organizativos complejos (Brown & Eisenhardt, 1997). Además, varios autores han considerado la capacidad de innovación como un tipo de capacidad dinámica (Teece, Pisano & Shuen, 1997). En la literatura, es posible encontrar una amplia gama de definiciones de las capacidades dinámicas. No obstante, en general estas capacidades representan la habilidad de una organización para crear, ampliar o modificar deliberada y sistemáticamente las rutinas operacionales (Winter, 2003). Concretamente, la capacidad innovadora ha sido definida como la habilidad para transformar continuamente el conocimiento y las ideas en nuevos productos, procesos y sistemas que benefician a la organización y los stakeholders (Lawson & Samson, 2001). La asociación de las capacidades dinámicas con el cambio es una idea que se ajusta a la naturaleza epistemológica de la innovación y que es encontrada frecuentemente en estas definiciones. De acuerdo con algunos autores las capacidades dinámicas no muestran patrones característicos de las capacidades operacionales rutinarias, sino que ellas son similares a las buenas prácticas organizativas (Marsh & Stock, 2006). Por tanto, esta misma teoría puede ser aplicada para identificar las buenas prácticas asociadas a la implementación de la capacidad de innovación. Además, para identificar los recursos organizativos, que configurados efectivamente construyen la capacidad de innovación, esta investigación realiza una revisión profunda de las teorías de innovación y capacidades dinámicas, para construir un nuevo modelo de referencia conceptual de las componentes fundamentales que generan la capacidad de innovación. En este modelo, la capacidad de innovación se muestra como el resultado de cuatro procesos: creación de conocimiento, absorción de conocimiento, integración de conocimiento y reconfiguración de conocimiento. Capacidad de innovación y configuración de recursos organizativos E.R. Bravo-Ibarra – L. Herrera Estos procesos están 303 ©© Intangible Capital, 2009 – 5(3):301-320 – ISSN: 1697-9818 doi: 10.3926/ic.2009.v5n3.p301-320 soportados por cuatro tipos de recursos: capital humano, liderazgo, estructuras y sistemas y la cultura organizativa. Finalmente, este modelo conceptual es desarrollado por medio de un estudio de casos en dos empresas de base tecnológica del sector audiovisual. Uno de los aportes más importantes de este trabajo, es logrado cuando en el proceso de construcción del modelo conceptual de las componentes de la capacidad de innovación en las dos empresas de base tecnológica, se consigue identificar un conjunto de buenas prácticas que gestionadas permiten el desarrollo de dicha capacidad. Con este resultado se clarifican, no sólo las componentes clave que generan la capacidad de innovación, sino también las buenas prácticas que permiten su desarrollo. A diferencia de los estudios existentes en la literatura, que estudian de forma individual cada una de las partes que conforman la innovación, en esta investigación se estudian sus partes conjuntamente. Lo anterior, es un aporte valioso a la teoría de las capacidades dinámicas. 2. Marco teórico Capacidades que componen la capacidad de innovación Muchos de los estudios que han investigado los elementos clave que ayudan a las organizaciones a adquirir la capacidad de innovación han propuesto a los recursos y las competencias como elementos fundamentales para el entendimiento de la innovación (Verona & Ravasi, 2003). Para lograr la competitividad por medio de la innovación cada organización debe adaptar el proceso de innovación a sus propias posibilidades de desarrollo e integración de conocimiento, es decir a su propia capacidad de innovación. Esta capacidad proporciona el potencial para que el proceso de innovación sea efectivo e involucra distintas capacidades o procesos de la empresa. De acuerdo con la literatura que analiza la gestión del conocimiento, las capacidades dinámicas más comúnmente asociadas a la innovación son: la creación de conocimiento (Nonaka & Takeuchi, 1995), la absorción de conocimiento (Zahra & George, 2002), Capacidad de innovación y configuración de recursos organizativos E.R. Bravo-Ibarra – L. Herrera 304 ©© Intangible Capital, 2009 – 5(3):301-320 – ISSN: 1697-9818 doi: 10.3926/ic.2009.v5n3.p301-320 la integración de conocimiento (Grant, 1996) y la reconfiguración de conocimiento (Lavie, 2006). Creación de conocimiento Muchos términos han sido utilizados para describir los procesos de creación de conocimiento: adquisición, búsqueda, generación y colaboración. Todos estos términos tienen un tema en común: “la acumulación de conocimiento”. La creación de conocimiento organizativo requiere colaboración y diseminación de experiencias. Este tipo de colaboración tiene lugar en dos niveles dentro de la organización: entre individuos y entre la organización y sus redes de socios. La colaboración entre individuos es la base para la socialización de conocimiento (Nonaka & Takeuchi, 1995). La colaboración entre organizaciones es también una fuente potencial de conocimiento (Inkpen & Dinur, 1998). En resumen, podemos definir la creación de conocimiento como un proceso que incrementa y hace disponible el conocimiento creado por los individuos por medio del desarrollo de nuevos productos, creación de alianzas estratégicas, relaciones cercanas con el cliente etc. La absorción de conocimiento Varios estudios sobre la innovación consideran la capacidad de absorción como un elemento influyente en la capacidad para innovar. En este estudio se ha definido la capacidad de absorción como la habilidad y motivación de los empleados para obtener conocimiento externo y utilizarlo para el desarrollo de la capacidad de innovación (Cohen & Levinthal, 1990). Integración de conocimiento El concepto de integración ha sido definido como el conjunto de procedimientos Inter-organizativos y entre unidades funcionales orientados a la interacción y la colaboración. La interacción enfatiza la utilización e intercambio de información entre unidades funcionales. La colaboración se fundamenta en el trabajo colectivo entre departamentos o entre organizaciones. La capacidad de integración de una organización esta determinada por dos mecanismos críticos: la gestión del conocimiento y las rutinas organizativas. Capacidad de innovación y configuración de recursos organizativos E.R. Bravo-Ibarra – L. Herrera 305 ©© Intangible Capital, 2009 – 5(3):301-320 – ISSN: 1697-9818 doi: 10.3926/ic.2009.v5n3.p301-320 La gestión mejora la comunicación entre el personal por la codificación de conocimiento tácito en reglas explicitas. Las rutinas organizativas pueden reducir la necesidad de comunicar conocimiento explicito (Grant, 1996). Las capacidades internas existentes y su interacción con fuentes de conocimiento externo afectan positivamente el nivel de innovación de las compañías (Cohen & Levinthal, 1990). Reconfiguración de conocimiento Se ha definido la reconfiguración de conocimiento como el proceso de generación de nuevas alternativas de configuración de capacidades, actividades organizativas y formas de creación de valor (Lavie, 2006). En contextos de cambio continuo, es claro el valor que tiene la habilidad para flexibilizar la estructura de la organización y realizar la transformación necesaria (Amit & Schoemaker, 1993). Que se efectúen los ajustes necesarios depende de la habilidad para explorar y evaluar el contexto competitivo y efectuar rápidamente el proceso de reconfiguración. La descentralización y la autonomía facilitan este proceso (Teece, Pisano & Shuen, 1997). Capacidad de innovación y recursos organizativos Winter (2003), considera que la configuración de los recursos organizativos, especialmente, los orientados al incremento y transformación del conocimiento puede inhibir o promover el desarrollo de la capacidad de innovación. Eisenhardt y Martin (2000), han concluido que los recursos por si mismos no explican el desempeño; ellos requieren de procesos como el desarrollo de nuevos productos para que las habilidades y el conocimiento sean transformados en innovaciones. Los recursos organizativos más comúnmente relacionados con la capacidad de innovación en la literatura organizativa son: el capital humano (Leonard & Sensiper, 1998), el liderazgo (Oke, Munshi, & Walumbwa, 2009), la cultura (Hurley, 1995) y las estructuras y sistemas (Argote, McEvily & Reagans, 2003). Capital humano Investigaciones, como la de Leonard y Sensiper (1998), han subrayado la importancia del capital humano en el desarrollo de la capacidad de innovación. El concepto de capital humano se refiere al conocimiento y habilidades de los individuos que permiten los cambios y el crecimiento económico. Capacidad de innovación y configuración de recursos organizativos E.R. Bravo-Ibarra – L. Herrera 306 ©© Intangible Capital, 2009 – 5(3):301-320 – ISSN: 1697-9818 doi: 10.3926/ic.2009.v5n3.p301-320 El capital humano puede ser desarrollado por la formación profesional o por programas de formación orientados a la actualización y renovación de las capacidades. Los estudios que han relacionado el capital humano con la capacidad de innovación han concluido que el desarrollo del capital humano facilita la absorción del conocimiento y la creación de nuevas capacidades necesarias para la innovación (Subramaniam & Youndt, 2005). Liderazgo Estudios recientes han considerado el liderazgo como factor determinante de la capacidad de innovación (Gumusluoglu & Ilsev, 2009). En la literatura el término liderazgo ha adoptado distintos significados. Por tanto, una definición única de liderazgo no existe (Yukl, 2009). Sin embargo, la mayoría de definiciones del liderazgo reflejan algunos elementos básicos en común; entre estos se encuentran “grupo”, “influencia”, y “meta”. Algunos autores, han identificado dos tipos de liderazgo que afectan al proceso de innovación: el transformacional (Oke, Munshi, & Walumbwa, 2009) y el transaccional (Bass, 1990). Los líderes transformacionales tienen la capacidad de convencer a los demás para que abandonen sus propios intereses en beneficio de los intereses del grupo. El liderazgo transaccional defiende la existencia de una transacción entre el líder y los miembros del grupo, donde estos aceptan la influencia del líder siempre que este les proporcione recursos valiosos (Hull & Hage, 1982). Los estudios que han relacionado la capacidad de innovación con el liderazgo han concluido que las características del líder, sus habilidades de liderazgo, su filosofía de gestión orientada al cambio y capacidad de motivación para incrementar la transferencia de conocimiento, son elementos clave que influencian positivamente la capacidad innovadora de las organizaciones (Cooper & Kleinschmidt, 1996). Cultura Una cultura que fomenta la interacción entre individuos es esencial en el proceso de innovación; especialmente para la creación de nuevas ideas. Este tipo de interacción es importante cuando se intenta transmitir conocimiento tácito (Nonaka & Takeuchi, 1995). Además, los empleados deberían tener la habilidad de organizar sus propias redes y prácticas para facilitar la generación de soluciones y la producción de conocimiento (O'Dell & Grayson, 1998). En esta investigación se Capacidad de innovación y configuración de recursos organizativos E.R. Bravo-Ibarra – L. Herrera 307 ©© Intangible Capital, 2009 – 5(3):301-320 – ISSN: 1697-9818 doi: 10.3926/ic.2009.v5n3.p301-320 ha definido la cultura como: el conjunto de valores, normas y comportamientos de los miembros de una organización. Las investigaciones que relacionan la cultura con la capacidad de innovación han concluido que elementos de la cultura vinculados a la creación de normas para mejorar la creatividad, están asociados a un alto grado de innovación (OReilly, Chatman & Caldwell, 1991). Estructuras y sistemas Las características de los elementos estructurales pueden inhibir o facilitar la transferencia de conocimiento (Argote, McEvily & Reagans, 2003). Esta transferencia es fundamental para el desarrollo de la capacidad de innovación. Por tanto, para desarrollar esta capacidad es importante que la estructura organizativa sea flexible para facilitar los procesos de transferencia de conocimiento (Gold, Malhotra, & Segars, 2001). En esta investigación se ha definido la estructura y sistemas organizativos como la configuración formal de los componentes de la cadena de valor de la organización en términos de flujo de trabajo, canales de comunicación y jerarquía. Las investigaciones que han relacionado las estructuras y sistemas con la capacidad de innovación han concluido que las organizaciones que implementan políticas formales e informales, procedimientos, prácticas e incentivos específicamente orientados a la innovación consiguen un desempeño organizativo positivo (Christensen & Suarez, 1999). 3. Metodología de investigación Con el objeto de entender mejor el proceso de innovación, esta investigación utiliza el método de un múltiple estudio de casos exploratorio conducido en dos Empresas Innovadoras de Base Tecnológica del sector audiovisual (EIBT). Las empresas seleccionadas para la investigación fueron Activa Multimedia Digital (AMD) y la Corporación Catalana de Radio y Televisión Interactiva (CCRTVI). AMD es una empresa que proporciona soluciones y servicios para el sector audiovisual. La empresa cuenta con un equipo de más de 50 profesionales de distintas áreas: ingenieros, periodistas, meteorólogos, diseñadores gráficos, etc. También dispone de un laboratorio de desarrollo de aplicaciones interactivas para televisión digital. Dentro de este laboratorio se creó a “SAM” el hombre del tiempo virtual automático y multiplataforma, concebido como el primer presentador virtual automático. Capacidad de innovación y configuración de recursos organizativos E.R. Bravo-Ibarra – L. Herrera 308 ©© Intangible Capital, 2009 – 5(3):301-320 – ISSN: 1697-9818 doi: 10.3926/ic.2009.v5n3.p301-320 Este proyecto fue desarrollado por AMD en colaboración con el centro de innovación Barcelona Media, el Grupo de Tecnologías Interactivas de la Universidad Pompeu Fabra y la Universidad Ramon Llull. Algunas de las características que hacen que SAM sea un producto innovador son la evolución permanente de nuevas aplicaciones, máxima automatización del proceso y dificultad de imitabilidad por parte de la competencia. AMD participa desde hace años en distintos proyectos nacionales y europeos que le han permitido tener un alto nivel tecnológico para suministrar contenidos multimedia y soluciones para la televisión. La CCRTVI es una empresa que crea contenidos y los difunde a través de los nuevos medios de comunicación interactivos como Internet, Teletexto, telefonía móvil, agendas electrónicas, etc. La CCRTVI cuenta con más de 100 trabajadores de distintos perfiles: periodistas, informáticos, ingenieros, licenciados en gestión de empresas, etc. Dentro de los proyectos más importantes desarrollados por la CCRTVI están las páginas Web: 3alacarta.cat, icatfm.cat, 3xl.cat, super3.cat, elsesports.cat, ritmes.cat, tvcatalunya.com. En las dos EIBT se realizaron 27 entrevistas grupales semi-estructuradas a directivos, miembros de equipos de I+D y jefes de proyectos. En la selección de estos informantes se tuvo en cuenta la participación de diferentes áreas profesionales con diferentes niveles de responsabilidad, lo que permitió la integración de una variedad de perspectivas al estudio. Antes de la recogida de datos primarios a partir de las entrevistas, se desarrolló un protocolo enfocado a conseguir la respuesta a la pregunta de investigación ¿Cómo se construye la capacidad de innovación que permite a las empresas desarrollar nuevos productos?. Encauzando las entrevistas a través de preguntas como: ¿Cuáles son las fases de desarrollo del proceso de innovación?, Se consiguió guiar a los entrevistados al tratamiento de la unidad de análisis de esta investigación que son los proyectos de desarrollo de nuevos productos. Las entrevistas también incluyeron preguntas sobre la formación de los empleados, el rol desempeñado por el entrevistado en la organización, las características de los equipos de proyectos, las motivaciones para la participación en los proyectos, el proceso de toma de decisiones, las decisiones sobre el presupuesto que debe ser asignado a cada actividad de I+D, etc. Cada entrevista tuvo una duración de 90 Capacidad de innovación y configuración de recursos organizativos E.R. Bravo-Ibarra – L. Herrera 309 ©© Intangible Capital, 2009 – 5(3):301-320 – ISSN: 1697-9818 doi: 10.3926/ic.2009.v5n3.p301-320 minutos aproximadamente. En ellas, los investigadores tomaron nota de las ideas principales relacionadas con el proceso de innovación para resumir, la información en informes que fueron entregados personalmente a los entrevistados con posterioridad y, de este modo, verificar la recepción correcta de la información. Cuando se finalizó con el primer bloque de entrevistas realizadas entre los meses de abril y agosto de 2005 se hicieron revisiones y análisis que dieron como resultado un conjunto de ideas clave directamente relacionadas al proceso de innovación. Posteriormente a esta primera fase se inició el proceso de categorización de constructos por medio de la construcción de matrices. En la segunda fase, se realizaron y estudiaron las entrevistas efectuadas de septiembre a octubre de 2005. El objetivo principal fue la identificación de buenas prácticas relacionadas con los constructos identificados y categorizados en las matrices construidas en las fases previas de recogida de datos primarios. Simultáneamente con las entrevistas se recogieron datos secundarios como: estados financieros, memorias anuales, informes internos, publicaciones del sector audiovisual, y otros materiales elaborados por las empresas que facilitaron el proceso de triangulación entre los datos primarios, secundarios y la teoría. 4. Resultados del estudio de casos Proceso de creación y absorción de conocimiento La creación de productos para el sector audiovisual enmarca las actividades de las EIBT en cuatro áreas: TV Digital Interactiva, Producción y Gestión de Vídeo, Software de Gestión y Contenidos y Servicios. Para proporcionar estos productos innovadores las EIBT están compuestas por un equipo de trabajo con una base de conocimiento y habilidades esenciales entre las que destaca una capacidad fundamental relacionada a la naturaleza de los productos creados en las EIBT. Como la directora técnica de la CCRTVI remarcó: “Nuestro equipo técnico multidisciplinar tiene la habilidad de seleccionar la tecnología adecuada para las nuevas aplicaciones de nuestros productos”. Además del conocimiento tácito de cada uno de los empleados por su experiencia obtenida en el desarrollo de productos anteriores. Toda la actividad innovadora de Capacidad de innovación y configuración de recursos organizativos E.R. Bravo-Ibarra – L. Herrera 310 ©© Intangible Capital, 2009 – 5(3):301-320 – ISSN: 1697-9818 doi: 10.3926/ic.2009.v5n3.p301-320 las EIBT no sería posible sin las alianzas de conocimiento hechas en colaboración con los centros de investigación universitarios y empresas de la comunicación. El proyecto SAM es el internas con resultado de la combinación de recursos y capacidades la utilización de redes externas de conocimiento sobre aplicaciones potenciales de nuevas tecnologías. Estas relaciones han surgido porque las EIBT se han encargado de crear un capital social que se fundamenta en la confianza que han generado en el sector por su amplia aportación de conocimientos derivada de sus investigaciones pioneras en el campo digital. La evidencia de la investigación subraya la importancia del compromiso de las EIBT con el desarrollo de ciencia básica, que hace posible la creación, absorción y adquisición de conocimientos especializados, esta característica se puede observar en la afirmación hecha por la directora técnica de la CCRTVI: “Muchas veces, realizamos proyectos de experimentación con nuestros proveedores de tecnología. Por ejemplo, cuando surge una necesidad especifica de software, que nuestro proveedor todavía no ha creado se propone la realización de un proyecto de I+D para desarrollarla”. Además del compromiso de desarrollo de conocimientos básicos por medio de proyectos es importante destacar que el fin último de estos muchas veces no es la generación de rentabilidad por el lanzamiento de los productos al mercado, sino la obtención de conocimientos que no se poseen, o no están suficientemente desarrollados para generar una aplicación innovadora. Cómo el director de tecnología de AMD comentó: “Los proyectos se han concebido para sentar las bases de unas estructuras básicas de conocimiento que permitan a mediano plazo el desarrollo de nuevos productos”. En esta afirmación se refleja una tendencia hacia el desarrollo de actividades de exploración de conocimiento que es otra de las características que soporta el proceso de absorción de conocimiento. Para un mayor detalle de todas las características que componen este proceso en las EIBT ver el conjunto de buenas prácticas de la tabla 1. Capacidad de innovación y configuración de recursos organizativos E.R. Bravo-Ibarra – L. Herrera 311 ©© Intangible Capital, 2009 – 5(3):301-320 – ISSN: 1697-9818 doi: 10.3926/ic.2009.v5n3.p301-320 Proceso de integración de conocimiento Algunos de los proyectos introducidos por las EIBT en el sector audiovisual fueron fundamentados en innovaciones incrementales desarrolladas sobre iniciativas espontáneas de los ingenieros de la organización. Estas iniciativas fueron transmitidas por medio del diseño de prototipos que facilitaron el entendimiento de la idea de innovación. La estructura orgánica que tienen las EIBT incrementó la velocidad y eficiencia en la transferencia de ideas alrededor de la organización. Como el jefe de desarrollo de proyectos de la CCRTVI remarcó: “Los prototipos son el medio esencial para la transmisión de la información, la integración de conocimientos y la colaboración entre los diferentes equipos de I+D”. La facilidad de transferencia de ideas que se integran en los diferentes proyectos de I+D es facilitada por las reuniones fomentadas por la EIBT a las cuales asiste un representante de cada proyecto que describe las capacidades construidas en proyectos anteriores que pueden ser aplicadas a las nuevas actividades de I+D. Como la directora de tecnología de la CCRTVI señaló: “Se crean sinergias entre los equipos de I+D para conseguir propuestas creativas. Por tanto, podemos arriesgarnos a hacer productos nuevos porque la unión de nuestras capacidades técnicas no lo permite”. Esta sinergia de capacidades distintivas de las EIBT y la complejidad de los productos dificultan la imitabilidad de estos por parte de la competencia. Además, la mezcla de capacidades entre equipos multifuncionales y el hecho que los empleados se sienten responsables por el proyecto de forma global no únicamente por la aplicación de sus habilidades especificas permitió identificar algunas características relacionadas con la cultura organizativa: Abierta a la creatividad, ausencia de identificación departamental y dispuesta a la participación y al diálogo. En la capacidad de integración existe otra actividad importante de las EIBT que consiste en crear retroalimentación con los clientes, un ejemplo de este tipo de procesos consistió en la creación de comunidades virtuales en las que se discutían las nuevas aplicaciones que podrían ser implementadas de acuerdo a las necesidades comunicadas por los clientes en un entorno dinámico como Internet y con una utilización mínima de recursos. Las otras actividades identificadas que Capacidad de innovación y configuración de recursos organizativos E.R. Bravo-Ibarra – L. Herrera 312 ©© Intangible Capital, 2009 – 5(3):301-320 – ISSN: 1697-9818 doi: 10.3926/ic.2009.v5n3.p301-320 apoyan la capacidad de integración de conocimiento en las EIBT son descritas en la tabla 1. Proceso de reconfiguración de conocimiento El dinamismo del mercado en el sector audiovisual hace que las EIBT actualicen día a día las aplicaciones de sus productos y sus capacidades para asumir los cambios del entorno. En este proceso de adaptabilidad al cambio juega un papel clave la realización de proyectos orientados a explorar y explotar nuevos entornos tecnológicos que creen nuevos conocimientos que faciliten el mantenimiento de la renovación de los productos y en consecuencia la ventaja competitiva de la organización. Tener una estructura flexible facilita que las EIBT puedan adoptar los cambios. Estos también se apoyan en la decisión de las EIBT de invertir en recursos tecnológicos que les permitan desarrollar las capacidades técnicas de sus equipos de investigación. Precisamente, la formación de su equipo profesional y una estructura organizativa sin jerarquías rígidas que inhiban el desarrollo del conocimiento aportan características que aumentan la capacidad de reconfiguración del conocimiento, esta flexibilidad en las EIBT se refleja en su continua introducción de nuevos productos resultado de los proyectos de I+D que surgen de retroalimentaciones con los proveedores, clientes, otras empresas del sector y de una alta capacidad para asumir los cambios derivada de las características creativas de su cultura organizativa y el compromiso de los altos directivos con la innovación. Como el jefe de Procesos de AMD afirmo: “Nuestro director de tecnología es un motor que motiva la participación en proyectos europeos y la búsqueda de Know How complementario”. En resumen, la investigación sugiere que la capacidad de la EIBT para recombinar continuamente el conocimiento integrado a cada uno de sus productos y actividades se apoya en una estructura fundamentada en la ausencia de rigidez jerárquica y la existencia de múltiples modelos relacionales que se apoyan en una cultura organizativa abierta (tabla 1). Modelo conceptual de la capacidad de innovación Capacidad de innovación y configuración de recursos organizativos E.R. Bravo-Ibarra – L. Herrera 313 ©© Intangible Capital, 2009 – 5(3):301-320 – ISSN: 1697-9818 doi: 10.3926/ic.2009.v5n3.p301-320 En resumen, el modelo conceptual construido de la revisión de la literatura sobre las teorías de innovación y capacidades dinámicas muestra que la capacidad de innovación está compuesta por la presencia simultanea de cuatro procesos organizativos: creación de conocimiento, absorción de conocimiento, integración de conocimiento y reconfiguración de conocimiento. Además, estos cuatro procesos están soportados por cuatro tipos de recursos: capital humano, liderazgo, estructuras y sistemas y la cultura organizativa. Este modelo esta ilustrado en la (Figura 1). Figura 1. “Modelo conceptual de la capacidad de innovación”. Fuente: Elaboración propia Capacidad de innovación y configuración de recursos organizativos E.R. Bravo-Ibarra – L. Herrera 314 ©© Intangible Capital, 2009 – 5(3):301-320 – ISSN: 1697-9818 doi: 10.3926/ic.2009.v5n3.p301-320 Buenas prácticas asociadas a cada uno de los recursos organizativos Buenas prácticas-Capacidad de innovación Actores/capital humano Personal con diferente formación y experiencia X Empleados motivados y participativos X Liderazgo Directivos experimentados X Relaciones a largo plazo con redes de innovación X Libertad de exploración de temas relacionados con las competencias centrales Actitud abierta hacia la comunidad científica Cultura X X Predisposición a la creatividad X Constitución de equipos de diferentes áreas funcionales Aporte individualizado en los proyectos y responsabilidad global Grupos de I+D X X X Recolección y evaluación continua de propuestas Colaboración con expertos del ámbito académico Empleados capaces de trabajar en ambientes poco convencionales Buenas habilidades de gestión de los lideres Estructuras y Sistemas X X X X Estimulación de las actividades de investigación X Creación de procesos para evaluación de ideas X Ausencia de identificación departamental X Fomento del diálogo y la interacción X Amplia implicación en los procesos estratégicos X Comunicación frecuente, informal, directa, abierta X Sistemas de incentivos X Ausencia de barreras inter-departamentales X Participación global en el proceso de toma de decisiones Tiempo libre para la experimentación X X Utilización de la tecnología para la transferencia de conocimiento Codificación del conocimiento dentro de un sistema Aceptación al cambio Comunicación fluida entre los equipos de proyectos Desarrollo de los recursos humanos-programas de formación Aprendizaje continuo X X X X X Bases de datos de procedimientos Comunidades de intercambio de conocimiento online con clientes y empleados Participación del cliente en el proceso de innovación X X X Tabla 1. Buenas prácticas vs. Recursos organizativos. Fuente: propia Las buenas prácticas en esta investigación han sido definidas como: actividades derivadas de la efectiva combinación de los recursos que mejoran el desempeño organizativo. En la tabla 1 se pueden observar las buenas prácticas identificadas en Capacidad de innovación y configuración de recursos organizativos E.R. Bravo-Ibarra – L. Herrera 315 ©© Intangible Capital, 2009 – 5(3):301-320 – ISSN: 1697-9818 doi: 10.3926/ic.2009.v5n3.p301-320 las dos organizaciones estudiadas. Cada una de estas prácticas ha sido categorizada para cada uno de los recursos organizativos que soportan la construcción de la capacidad de innovación. 5. Conclusiones El análisis de las EIBT indica que para sostener el proceso de desarrollo de nuevos productos, las organizaciones deberían construir capacidades dinámicas de la combinación de sus principales recursos organizativos, que permitan la creación, absorción, integración y reconfiguración simultánea y continua de conocimiento. En las EIBT la creación de conocimiento esta apoyada en una amplia experiencia en el desarrollo de proyectos de I+D en el ámbito interno para la renovación de sus productos y en el ámbito externo para la evolución del “Know How” por medio de alianzas estratégicas con empresas del sector audiovisual Europeo. La habilidad para adquirir conocimiento esta directamente asociada a la presencia de conocimiento previo relacionado, lo que refleja la importancia de las inversiones hechas por las EIBT en capacidades técnicas que les han permitido absorber conocimiento externo. Además, la investigación ha encontrado la necesidad de nutrir las capacidades por medio de la identificación de los recursos y la combinación de estos. Los cuales deben adaptarse continuamente a la evolución de mercado. Este proceso de transformación de las capacidades esta apoyado en las EIBT por la continua distribución de roles y objetivos organizativos que ocurre cuando un empleado es asignado a un nuevo grupo de I+D que le exigirá desarrollar nuevas habilidades resultado de la combinación de su conocimiento y la adaptación de este para la generación de respuestas creativas. En las empresas estudiadas se ha observado que el liderazgo y sus buenas prácticas asociadas son un pilar fundamental en la construcción de la capacidad de innovación. Sin embargo, al igual que para los otros tres tipos de recursos esto puede cambiar dependiendo del sector en que se realice la investigación. De lo anterior se ha concluido que para construir la capacidad de innovación en determinadas industrias se deben potenciar específicamente alguno de los cuatro tipos de recursos que soportan el proceso de innovación. Capacidad de innovación y configuración de recursos organizativos E.R. Bravo-Ibarra – L. Herrera 316 ©© Intangible Capital, 2009 – 5(3):301-320 – ISSN: 1697-9818 doi: 10.3926/ic.2009.v5n3.p301-320 El análisis de las EIBT ha mostrado que cada uno de los tres procesos de conocimiento esta soportado en cuatro tipos de recursos que son: los actores/capital humano, el liderazgo, las estructuras y sistemas, y la cultura organizativa. La combinación de estos recursos con un carácter distintivo en cada organización crea una fórmula única de la capacidad de innovación y genera un conjunto de buenas prácticas que puede ser usado por los directivos para la toma de decisiones orientadas a la consecución de la innovación. 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https://openalex.org/W4286447745

https://bmcmededuc.biomedcentral.com/counter/pdf/10.1186/s12909-022-03615-0

English

Effectiveness of tutor shadowing on faculty development in problem-based learning

BMC medical education

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Abstract Background:  To enhance tutors’ teaching skills, tutor shadowing for novice tutors of problem-based learning (PBL) in addition to conventional faculty development (FD) was applied. This study aimed to develop a tutoring-skill scale (TS-scale) and evaluate the effect of shadowing on PBL tutors. Methods:  This study employed a before-and-after study design with three phases. In phase 1, a TS-scale was elabo‑ rated. A validity examination was performed in phase 2. Phase 3 was a study of the effectiveness using a TS-scale sur‑ vey of novice PBL tutors before and after the FD course. The FD course for novice PBL tutors included an FD workshop and PBL shadowing activities. Results:  A TS-scale with a 32-item questionnaire of self-rated confidence for PBL tutors was identified in phase 1. In phase 2, 7 experienced specialists in medical education were invited to evaluate the content validity of the scale. The item content validity index (I-CVI) ranged from 0.86 to 1, and the scale-CVI (S-CVI) was 0.95. A total of 85 novice PBL tutors completed the TS-scale before the FD course, yielding a Cronbach’s alpha of 0.98. An exploratory factor analysis with varimax rotation was performed. The twenty-four items with significant loadings greater than 0.5 were incor‑ porated into a new TS-scale and were grouped into three factors: student contact, medical expertise, and teaching expertise. In phase 3, 76 novice PBL tutors completed the 24-item TS-scale before (pretest) and after (posttest) the FD course. Their self-rated confidence improved significantly across the three factors after the FD course. The pretest and posttest scores did not differ according to the tutors’ gender, the grades they taught, or their specialty background. Conclusions:  Novice PBL tutors benefit from FD that incorporates tutor shadowing in the 3 key domains of tutoring competencies. The TS-scale developed in this study can be applied in future research on FD design. Conclusions:  Novice PBL tutors benefit from FD that incorporates tutor shadowing in the 3 key domains of tutoring competencies. The TS-scale developed in this study can be applied in future research on FD design. Keywords:  Tutor shadowing, Faculty development, Problem-based learning solution with students instead of imparting knowledge [1]. For medical students, PBL curricula are composed of clinical scenarios targeting learning goals. Medical students learn through a collaborative problem-solving process [2]. PBL tutors are expected to promote student- centred learning and drive the discussion toward the les- son’s objectives. © The Author(s) 2022. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://​creat​iveco​mmons.​org/​licen​ses/​by/4.​0/. The Creative Commons Public Domain Dedication waiver (http://​creat​iveco​ mmons.​org/​publi​cdoma​in/​zero/1.​0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Tsai et al. BMC Medical Education (2022) 22:564 https://doi.org/10.1186/s12909-022-03615-0 Open Access Abstract Using basic scientific techniques, PBL tutors help guide students and bridge the gaps in clini- cal subjects. Ultimately, PBL tutors must assess learning outcomes and provide feedback. To lead a successful PBL session, tutors must have strong facilitation skills, which Effectiveness of tutor shadowing on faculty development in problem‑based learning Chiao‑Ling Tsai1,2, Yen‑Lin Chiu3, Chia‑Ter Chao2,4, Mong‑Wei Lin2,5, Chao‑Chi Ho2,6, Huey‑Ling Chen3,7, Bor‑Ching Sheu2,8, Chiun Hsu2,9 and Chih‑Wei Yang3,10,11* Chiao‑Ling Tsai1,2, Yen‑Lin Chiu3, Chia‑Ter Chao2,4, Mong‑Wei Lin2,5, Chao‑Chi Ho2,6, Huey‑Ling Chen3,7, Bor‑Ching Sheu2,8, Chiun Hsu2,9 and Chih‑Wei Yang3,10,11* Backgroundh The recruitment and training of tutors are necessary when developing a problem-based learning (PBL) cur- riculum. In PBL, students usually solve an open-ended problem with peers. The tutors drive toward a possible *Correspondence: [email protected] p , Full list of author information is available at the end of the article *Correspondence: [email protected] 3 Graduate Institute of Medical Education and Bioethics, National Taiwan University College of Medicine, No. 1, Sec. 1, Ren’ai Rd., Zhongzheng Dist., 100 Taipei, Taiwan Full list of author information is available at the end of the article Tsai et al. BMC Medical Education (2022) 22:564 Page 2 of 9 Page 2 of 9 the discussion scenarios for M2 students include profes- sional norms, medical ethics, medical insurance, laws and regulations, stigmatization, vulnerable people, etc. The PBL cases comprise clear objectives, and paragraphs outlining the real clinical scenarios with problems and references. Before each session, the student self-studies the relevant literature according to the assignment and formulates his/her own knowledge required for problem- solving. The students might share the preparatory work before PBL class, present it on-site with slides, or write it down on the whiteboard directly. Some groups have course leaders during their PBL sessions, and some do not. Every student needs to express his/her own view- points on the learning objectives. The students would address their observations, raise questions, brainstorm, and propose some solutions for the issues to develop teamwork and problem-solving skills [10]. During class, the facilitator guides the seven steps in PBL [11]. The tutors promote the proper path for PBL discussion by aiding students to define and analyze the problem by ask- ing open-ended questions. The students are steered to formulate learning objectives, collect additional informa- tion and synthesize and test the newly acquired informa- tion [12]. Over the years, students’ learning strategies for PBL have changed from offline searches to online sur- veys. Among the students, the preparative discussion is held on social media rather than as face-to-face conver- sations as in previous years. The majority of PBL classes remain on campus courses that involve tutors. are difficult to improve through conventional faculty development [3].hf The skills needed for tutoring in PBL curricula differ from those needed for teaching in a lecture-based pro- gram. There is a consensus on the importance of faculty development (FD) for PBL tutors to assist in their respon- sibilities and roles [4]. The tutors can become proficient facilitators and activators through PBL training programs [1]. However, a lecture-based FD is unable to fill the gap between facilitating theory and actual practice. Most of the effective PBL FD for tutors includes novel designs, such as direct observation, video clips, interactive film, scene scenarios, and role-plays [5, 6]. Situated learning, including tutor shadowing during teaching, is mandatory for PBL tutors. Tutor shadowing, also known as peer observation, is an activity that involves inspecting colleagues’ teaching practices. The teachers can learn from each other and achieve professional growth. Tutor shadowing is gradu- ally becoming a feature of higher education practice and can be categorized into three basic models: evaluation, development, and peer review [7]. The goal of shadow- ing is to exchange and reflect on personal methods of teaching. Colleagues can work together to perfect their teaching approach and identify areas where they need to improve. For academic units, peer observation improves the quality of both education and instruction [8]. Tutors have to actively participate in the students’ self-directed learning processes while following the PBL curriculum. To improve facilitating abilities in PBL tutoring, the liter- ature indicates that the direct observation of PBL courses led by experienced tutors may be helpful [9]. To reinforce the FD of PBL, the National Taiwan University College of Medicine (NTUCM) adopted tutor shadowing for novice tutors in addition to lecture-based workshops.i Recruitment of tutors In the NTUCM, the qualified PBL tutors are attending physicians from the health care system of the National Taiwan University Hospital (NTUH) who volunteer to participate in tutoring. PBL tutors from different spe- cialty backgrounds join a coordinated FD program and learn how to become facilitators. The classic FD course at NTUCM consists of workshops or seminars aimed at teaching and learning strategies for novice PBL tutors. From 2018, NTUCM incorporated the tutor shadowing of PBL classes into the FD of novice tutors. In this study, the elaboration of a modified tutoring- skill scale (TS-scale) for measuring the facilitation skills of PBL tutors was addressed. The TS-scale was validated to assess the effects between conventional FD activity and tutor shadowing on the skills of novice tutors. Phase 3: study of effectivenessi Phase 3: study of effectiveness Qualified novice PBL tutors who participated in the training and completed the validated TS-scale (24 items) with self-rated confidence before (pretest) and after (posttest) the FD course were included for analysis. Phase 1: TS‑scale items elaboration A literature search of the PubMed database (from August 1, 1990, through July 31, 2016) with the following medi- cal subject heading terms and/or words in the main text: “problem-based learning,” “novice,” “facilitators,” and “questionnaire.” A comprehensive assessment scale for PBL tutors by Slattery and Douglas was identified [19]. The scale contains 32 items with a 5-point Likert scale to assess the self-rated confidence of tutors. The highest score (5) indicated the highest level of agreement on the item measured, and the lowest score (1) represented the lowest level of agreement on the item rated. The items can be categorized by four competencies, namely, (i) facilitation skills, (ii) programme/curriculum knowledge, (iii) personal qualities, and (iv) subject-matter expertise (Table 1). FD course descriptionh In NTUCM, PBL was first incorporated into the medical curriculum in 1993. The topics of the PBL class empha- size “humanity and society” for second-year medical (M2) students, “anatomy and physiology” for M3 stu- dents, and “pathology and pharmacology” for M4 stu- dents. In each PBL class, there are 8 to 11 students and a tutor to conduct the class, which lasts 2 h per week. The discussion topics are composed of several clinical scenar- ios with a preset schedule provided by the course admin- istrator at the beginning of each semester. For example, The FD course for PBL tutors consisted of an FD work- shop and tutor-shadowing activities. The FD workshop was composed of a one-day agenda with two panels. In the first panel, there were several lecture-based sessions about educational theory, advances in medical educa- tion, and professional development for educators. The second panel were group discussions divided according to which grade the tutors taught. The subjects included pedagogical and content training, PBL facilitation skills, Tsai et al. BMC Medical Education (2022) 22:564 Page 3 of 9 and evaluation. During tutor-shadowing activities, each of the novice tutors joined 2 PBL discussions, which were randomly selected from among the medical PBL courses. The novice tutors joined the PBL classes only as observ- ers. Before the shadowing activity, the observed tutor would introduce the steps of their group discussion to the observer. The observing tutor engaged in the obser- vation exercise silently by investigating the student-cen- tred learning process and the role of facilitating tutors. At the end of the shadowing activity, the observer and the observed had the chance to reflect and give feedback. They might exchange the practice of how their tutorial groups functioned. Afterward, tutors were encouraged to share their impressions from the shadowing activity by email or on tutor forums. consistency was tested by Cronbach’s alpha. A Cron- bach’s alpha value above 0.8 was considered acceptable [17]. y Content validity by professional commentaryh The commentary questionnaire of the TS-scale was sub- mitted to experts in the field of medical education to pro- vide content validity. Experts checked for statements and correspondence between the expression and conception of the items. Items from the shadowing scale for experts were scored on a Likert 4-point scale ranging from 1 to 4, with 4 as very appropriate, 3 as appropriate, 2 as inap- propriate, and 1 as very inappropriate. The recommen- dations of the experts were accordingly adopted when revising the questionnaire. The content validity indices (CVI) are reported as item-level (I-CVI) or scale-level (S-CVI) [15, 16]. An I-CVI of 0.78 or higher for three or more experts is considered to indicate agreeable con- tent validity. An S-CVI of 0.80 or higher is considered reasonable. Study procedure Th d l This study employed a before-and-after study design after a scale development process to explore the effectiveness of FD courses incorporating tutor-shadowing activities [13]. This research included three phases: Phase 1 was TS-scale item elaboration, phase 2 was an examination of validity, and phase 3 was a study of effectiveness. Construct validity EFA Concerning validity analysis based on the theoretical construct, an exploratory factor analysis (EFA) was per- formed. A new formation of the TS-scale was validated and tailored according to the EFA results. Statistical analysis A descriptive analysis for participant characteristics was conducted. The Cronbach’s alpha coefficient was calcu- lated for the internal consistency of the scales in our sam- ple. We obtained the Kaiser–Meyer–Olkin (KMO) index and conducted the Bartlett sphericity test to explore the sampling and data adequacy. The EFA using the maxi- mum likelihood method and varimax rotation follow- ing the recommended standards was performed [18]. Items with loading values greater than 0.5 were retained. Changes in self-rated confidence before and after tutor shadowing with a paired t test were compared. A p value of less than 0.05 was considered statistically significant. All statistical analyses were executed using SPSS (Statisti- cal Package for the Social Sciences) 20.0. Phase 1: literature review and item elaborationh The TS-scale elaboration process followed the recom- mendations of Hinkin [14]. A literature review on PBL and collected pre-existing scales were performed. Items were generated and modified according to the data from extensive investigations. Internal consistency A group of novice PBL tutors was invited to complete the 32-item TS-scale based on self-rated confidence before the FD activity. The reliability of the TS-scale on internal Tsai et al. BMC Medical Education (2022) 22:564 Page 4 of 9 Table 1  Questionnaire items and mean ratings of the self-rated confidence of tutors before tutor-shadowing (TS) a Items were adopted from the questionnaire proposed by Slattery and Douglas (2014) [17] Questionnaire ­Itema Mean SD Facilitation skills    Q1. Ensure group runs well 3.94 0.62   Q2. Monitor participation 4.01 0.66   Q3. Support group identification of learning needs 3.92 0.68   Q7. Feedback group process 4.09 0.61   Q8. Monitor group cohesion 3.75 0.75   Q16. Observe group not participate 4.07 0.65   Q18. Stimulate prior knowledge 3.73 0.82   Q21. Cultivate respect for group opinions 3.93 0.72   Q22. Monitor group agreement 3.91 0.61   Q23. Evaluate group progress 3.91 0.68   Q24. Model knowledge can change 4.01 0.68   Q25. Support use range of resources 3.92 0.71   Q27. Seek range of alternative solutions 3.93 0.69   Q29. Activate students prior experiences 3.74 0.73   Q30. Seek clarification of ideas 3.87 0.69   Q31. Clarify inconsistencies in problem solving 3.85 0.73   Q32. Encourage consideration of range of issues 3.96 0.76 Programme/curriculum knowledge   Q5. Assess students 3.69 0.71   Q6. Liaise between curriculum team and students 3.76 0.78   Q20. Check learning outcomes are achieved 3.92 0.73 Personal qualities   Q11. Role model 3.98 0.67   Q12. Mentor 3.88 0.73   Q13. Work as colleague in group 3.81 0.73   Q14. Share professional experiences 4.31 0.58   Q17. Evaluate own performance 3.74 0.74 Subject-matter expertise   Q4. Explain misunderstandings in knowledge 4.04 0.61   Q9. Give extra information 3.87 0.65   Q10. Offer content knowledge 4.01 0.59   Q15. Identify learning needs 3.82 0.68   Q19. Identify when problem solving is correct 3.82 0.68   Q26. Provide theory if not identified 3.88 0.73   Q28. Direct problem solving 3.99 0.66 from 0.86 to 1, and the S-CVI was 0.95, indicating rea- sonable content validity. Construct validity of the TS‑scaleh y The appropriateness of the dataset for EFA was exam- ined by estimating the KMO and conducting Bartlett’s test of sphericity. The KMO measure of the sampling adequacy was 0.91. Bartlett’s test was statistically signifi- cant at p < 0.001. The results suggested adequacy sam- pling for conducting the factor analysis [20]. The cut-off value of 0.5 was commonly used to determine an accept- able factor loading [21, 22]. Thus, 24 items with loadings greater than 0.5 were selected for the TS-scale. Accord- ing to the Kaiser rule, the factors with eigenvalues greater than 1 were considered significant [20]. Finally, the 24 items could be grouped into three factors: student con- tact, medical expertise, and teaching expertise. The total variance explained by the three factors reached 69.06% (Table 2). A total of 76 novice PBL tutors from the academic years 2018 to 2020 from 17 specialty backgrounds were recruited. The majority of tutors were men (77.6%). Twenty-two (28.9%) tutors, 24 (31.6%) tutors, and 30 (39.5%) tutors observed the PBL class for the M2 stu- dents, M3 students, and M4 students, respectively. The backgrounds and characteristics of the tutors are sum- marized in Table  3. Participants completed the ques- tionnaire based on their self-rated confidence before the tutor shadowing activities (Table 1). Phase 2: validity examination of the TS‑scale Internal consistency of the TS‑scalei A total of 85 qualified PBL tutors completed the TS-scale before the FD course. To evaluate the reliability of the scale, Cronbach’s alpha coefficients were calculated. The alpha coefficient for the total scale was.98. The alpha val- ues for the four key tutoring facilitation competencies, (i) facilitation skills, (ii) programme/curriculum knowledge, (iii) personal qualities, and (iv) subject-matter expertise, were 0.96, 0.84, 0.86, and 0.91, respectively. The Cron- bach’s alpha coefficients achieved reliable internal con- sistency estimates for the scale. Content validity of the TS‑scaleh Content validity of the TS‑scaleh The 76 novice PBL tutors completed the training course. The responses before (pretest) and after (posttest) the training course were used to evaluate the effectiveness of the FD course. A newly formed TS-scale was created according to the EFA results and was used after valida- tion. The Cronbach’s α coefficients for the three factors of student contact, teaching expertise and medical expertise The content validity of the questionnaire was assessed by seven experienced specialists in medical education. According to the experts, all 32 original items were rel- evant to the study design. The mean score of appropri- ation from the original 32 items ranged from 3.2 to 4 (mean 3.7). The I-CVI of the original 32 items ranged Tsai et al. BMC Medical Education (2022) 22:564 Page 5 of 9 Table 2  The results of exploratory factor analysis Item Factor1 Factor2 Factor3 Student contact   Q18 Stimulate prior knowledge 0.78   Q20 Check learning outcomes are achieved 0.77   Q29 Activate students prior experiences 0.77   Q19 Identify when problem solving is correct 0.75   Q6 Liaise between curriculum team and students 0.71   Q5 Assess students 0.69   Q23 Evaluate group progress 0.66   Q8 Monitor group cohesion 0.66   Q17 Evaluate own performance 0.63   Q22 Monitor group agreement 0.60   Q21 Cultivate respect for group opinions 0.56 Teaching expertise   Q11 Role model 0.79   Q12 Mentor 0.76   Q13 Work as colleague in group 0.72   Q16 Observe group not participate 0.64   Q2 Monitor participation 0.63 Medical expertise   Q10 Offer content knowledge 0.86   Q4 Explain misunderstandings in knowledge 0.81   Q14 Share professional experiences 0.63   Q32 Encourage consideration of range of issues 0.58   Q30 Seek clarification of ideas 0.57   Q9 Give extra information 0.57   Q31 Clarify inconsistencies in problem solving 0.54   Q24 Model knowledge can change 0.49 KMO = 0.913 Bartlett’s Test of Sphericity = 1799.914 Eigenvalues 7.04 4.93 4.60 Variance explanation (%) = 69.056 58.10 6.33 4.62 who were equally distributed according to the grades they taught were enrolled. These novice PBL tutors were experienced teachers in their medical fields but may be unfamiliar with how to efficiently facilitate a small-group PBL class. A training program incorporating workshops and course observation for novice tutors was imple- mented with tutor shadowing. With the aid of the vali- dated TS-scale, the advantage of tutor shadowing for novice PBL tutors in terms of 3 key facilitation compe- tency categories was clarified. were 0.95, 0.89, and 0.91, respectively. Content validity of the TS‑scaleh The self-rated con- fidence improved significantly after the FD across the same three factors, with pretest and posttest values of 3.8 ± 0.6 and 4.2 ± 0.5 with p < 0.001 for student contact, 3.9 ± 0.6 and 4.2 ± 0.5 with p < 0.001 for medical exper- tise, and 4.0 ± 0.5 and 4.3 ± 0.5 with p < 0.001 for teach- ing expertise (Fig. 1). The pretest and posttest scores did not differ according to the tutors’ gender, the grade they taught, or their specialty background (Fig. S1). were 0.95, 0.89, and 0.91, respectively. The self-rated con- fidence improved significantly after the FD across the same three factors, with pretest and posttest values of 3.8 ± 0.6 and 4.2 ± 0.5 with p < 0.001 for student contact, 3.9 ± 0.6 and 4.2 ± 0.5 with p < 0.001 for medical exper- tise, and 4.0 ± 0.5 and 4.3 ± 0.5 with p < 0.001 for teach- ing expertise (Fig. 1). The pretest and posttest scores did not differ according to the tutors’ gender, the grade they taught, or their specialty background (Fig. S1). Discussion i PBL has been widely used as a pedagogical strategy in medical education worldwide for decades. There is little guidance available on how to become a successful PBL tutor. Teaching and mentoring are essential components of PBL tutoring and need to be developed through a sys- temic process [23]. Over the years, the FD of PBL has been reformed, resulting in substantial modifications. Applying a robust process, a new TS-scale was success- fully developed and validated in our study. The validated TS-scale was composed of 24 items that were grouped into three categories. The scale items were reliable and had adequate internal consistency. In this study, highly representative participants with a variety of specialties Page 6 of 9 Page 6 of 9 Tsai et al. BMC Medical Education (2022) 22:564 Table 3  Background and characteristics of tutors Features N = 76 No (%) Gender   Men 59 (77.6%)   Women 17 (22.4%) Category by school years   2nd year (humanity/society) 22 (28.9%)   3rd year (anatomy/physiology) 24 (31.6%)   4th year (pathology/pharmacology) 30 (39.5%) Specialty   Internal Medicine 22 (28.9%)   Surgery 10 (13.2%)   Oncology 9 (11.8%)   Orthopedic Surgery 7 (9.2%)   Neurology 5 (6.6%)   Radiology 4 (5.3%)   Anesthesiology 3 (3.9%)   Nuclear Medicine 3 (3.9%)   Geriatrics 2 (2.6%)   Obsterics and Gynecology 2 (2.6%)   Ophthalmology 2 (2.6%)   Pediatrics 2 (2.6%)   Physical Medicine and Rehabilitation 2 (2.6%)   Family Medicine 1 (1.3%)   Otolaryngology 1 (1.3%)   Pathology 1 (1.3%)   Urology 1 (1.3%) measure of change is necessary [27]. Multiple methods and outcome measures would support the development of future FD [24]. The current evaluation instrument for tutor shadowing focuses on peer feedback and strategies for instructional practice [28]. A convenient instrument that could be easily implemented to value the effective- ness of shadowing activity is noteworthy. Quantification is a prerequisite for agreeing upon setting benchmarks [29]. In an attempt to determine the impact of shadow- ing on PBL with scale, the FD questionnaire for novice PBL tutors proposed by Slattery et  al. was adopted in our study [19]. The 3-phase study design was utilized to reformulate and validate the scale with 24 items. From our study, the new TS-scale was proved to be usable and applicable to FD of tutoring shadowing for PBL tutors. The new TS-scale is easy to use and efficient. h yfi The expert observation of teaching is a key element of FD in higher education and provides teaching practice and boosts confidence [30]. Discussion Similarly, the majority of medical doctors learned to be educators by observing the teaching practices of a senior faculty member in a clini- cal scenario. The incorporation of peer observation into tutor training in PBL curriculum is practicable. The peer observation of teaching with group feedback supports the development of teaching competencies [31]. Garcia et al. used video-recorded PBL sessions for self-observation and peer feedback as an FD approach for PBL tutors [28]. Self-observation strengthens awareness and cultivates student learning. Peer coaching helps tutors facilitate processes in PBL sessions. Informal teacher communities also enhance the professional development of PBL tutors [32]. To support tutoring practice, NTUCM introduced tutor shadowing in 2018. Most tutors agreed in the quali- tative interviews that the tutor shadowing made a posi- tive contribution to their development as tutors. From the commentary collected directly from tutors, emails, and tutor forums, tutors acknowledged the merit to learn from peers. Through shadowing, they had the chance to gain new insights into employing cognitive strategies, how to facilitate group dynamics, and provide structured feedback during PBL discussion. To objectively evaluate the effect of shadowing on tutoring, a quantitative scale was developed in this study. FD activities include reflection, educational projects to enhance teaching effectiveness, and formal, structured workshops designed as experiential learning [24]. Nev- ertheless, tutoring skills in facilitating the process of PBL could be further optimally cultivated via observa- tional learning [25]. Tutor shadowing is one activity that interests medical professionals, fosters peer learning and encourages teamwork. It is a highly personalized, learner- centred method to impel the socialization and profes- sional development of faculty members [26]. Through observing the PBL curriculum with an experienced tutor, the observer tutor has the chance to acquire the facilita- tion skill for PBL directly. In the 1990s, FD for PBL included general skill, devel- opmental, comprehensive, and course-based models [25]. With the integration of adult learning theory into FD for PBL tutors, new models of tutor training con- tinued to be created [33]. Program evaluation and out- come-based studies have also been developed to ensure that PBL curriculum is successful. Harden and Crosby identified twelve roles for the medical teacher [34]. The 12 roles are categorized into teaching expertise, medi- cal expertise, student contact, and students at a distance. A recent review found that most FD programs pro- vide excellent satisfaction and result in positive changes in teaching [24]. Discussion These evaluations of the FD programs depended on ascertained, self-assessments instead of observing and judging teaching practice [23]. How- ever, the majority of the assessments were not validated. A tailored questionnaire is essential for the quality improvement of individual FD programs. To evaluate the behaviour change in teaching skills, a performance-based Tsai et al. BMC Medical Education (2022) 22:564 Page 7 of 9 Fig. 1  The effectiveness of tutor shadowing for novice PBL tutors. ****: p < 0.001; PBL: problem-based learning; EFA: exploratory factor analysis Fig. 1  The effectiveness of tutor shadowing for novice PBL tutors. ****: p < 0.001; PBL: problem-based learning; EFA: explor Fig. 1  The effectiveness of tutor shadowing for novice PBL tutors. ****: p < 0.001; PBL: Conclusions According to Harden and Crosby, we arranged our new 24-item TS-scale into student contact, medical expertise, and teaching expertise (Table 3). These aspects are essen- tial components in the PBL class. The tutors’ perception of these three aspects helps us evaluate the effect of FD activities on facilitation skills. Tutoring skills are essential in PBL activities. Tutors need to learn to listen, ask inquiry-based questions, and moni- tor the PBL process. This curriculum mode abandons the traditional role of a teacher as a content expert and knowledge dispenser. Thus, facilitating the development of new tutoring skills while also transforming faculty beliefs about teaching and learning are the dual chal- lenges of PBL FD [25]. Tutor shadowing improves the success of FD for PBL tutors. Based on the results of our study, we found that tutor shadowing comprehensively enhanced three aspects of novice PBL tutors’ work: stu- dent contact, medical expertise, and teaching expertise. We were able to use the TS-scale that divided 24 items into 3 dimensions to measure the level of self-rated con- fidence of novice PBL tutors after tutor shadowing ses- sions. We believe that the TS-scale from this study can contribute to the evaluation of future FD activity. Availability of data and materials The datasets used and analyzed during the current study are available from the corresponding author on reasonable request. 12. Schmidt HG. Problem-based learning: rationale and description. Med Educ. 1983;17(1):11–6. 13. Sedgwick P. Before and after study designs. BMJ. 2014;349:g5074. Acknowledgements 5. Bosse HM, Huwendiek S, Skelin S, Kirschfink M, Nikendei C. Interactive film scenes for tutor training in problem-based learning (PBL): dealing with difficult situations. BMC Med Educ. 2010;10:52. g We are grateful for the assistance of Mrs. Yin-Li Tsao from the Center of Faculty Development and Curriculum Integration, National Taiwan University College of Medicine. We thank the participants for generously providing feedback. 6. Kukkamalla A, Lakshminarayana SK. Developing a facilitation skills training programme for problem-based learning tutors. Med Educ. 2011;45(11):1152–3. Consent for publication Not applicable. 18. Lloret S, Ferreres A, Hernández A, Tomás I. The exploratory factor analysis of items: guided analysis based on empirical data and software. Anales de Psicología. 2017;33(2):417–32. Funding 10. Shieh J-Y, Yao C-A, Hsu C, Chang S-C, Tseng F-Y. Group Report Contest Enhances Problem-Based Learning. J Med Educ. 2018;22(1):9–16. This work was supported by the National Taiwan University Hospital under Grant NTUH 111-S0244 and NTUH 111-S0312. 11. Bate E, Hommes J, Duvivier R, Taylor DC. Problem-based learning (PBL): getting the most out of your students - their roles and responsibilities: AMEE Guide No. 84. Med Teach. 2014;36(1):1–12. Limitation.h Limitation.h This study has some limitations. First, our tutors were experts in their medical fields and had experience in teaching techniques. The novice tutors had diverse spe- cialty backgrounds. However, we failed to define the individualized benefit of tutor shadowing according to gender or background. The TS-scale has to be examined with more tutors to confirm the conclusions drawn in this study. Second, the results of the TS-scale in our study were based solely on tutors’ self-perceptions. Self-recog- nized changes in PBL tutoring might not be reflected real behavioural changes. Further studies are needed to con- firm the effect of shadowing on tutoring behaviour. Page 8 of 9 Tsai et al. BMC Medical Education (2022) 22:564 Tsai et al. BMC Medical Education (2022) 22:564 Tsai et al. BMC Medical Education Additional file 1. 4. Papinczak T, Tunny T, Young L. Conducting the symphony: a qualita‑ tive study of facilitation in problem-based learning tutorials. Med Educ. 2009;43(4):377–83. The authors declare that they have no competing interests. 19. Slattery J, Douglas J. An exploration of novice and experienced problem- based learning facilitators’ perceptions of their roles in a speech-language pathology programme. Does experience matter? Clin Linguist Phonet. 2014;28(1–2):24–35. Declarations g y g ; g 14. Hinkin TR. A Brief Tutorial on the Development of Measures for Use in Survey Questionnaires Organ Res Methods 1998;1(1):104–21 14. Hinkin TR. A Brief Tutorial on the Development of Measures for Use in Survey Questionnaires. Organ Res Methods. 1998;1(1):104–21. Survey Questionnaires. Organ Res Methods. 1998;1(1):104–21. Received: 5 February 2022 Accepted: 6 July 2022 Received: 5 February 2022 Accepted: 6 July 2022 Authors’ contributions CLT drafted the manuscript and with CTC, MWL, and CWY. CCH, HLC, BCS and CH designed the study. CLT, YLC, and CWY were involved in data processing. CLT and YLC performed statistical analysis. CWY was the director responsible for general organization and instruction. All authors read and approved the final version of the manuscript. 7. Bennett S, Barp D. Peer observation – a case for doing it online. Teach Higher Educ. 2008;13(5):559–70. 8. Fletcher JA. Peer observation of teaching: A practical tool in higher edu‑ cation. J Faculty Develop. 2018;32(1):51–64. 8. Fletcher JA. Peer observation of teaching: A cation. J Faculty Develop. 2018;32(1):51–64. 9. Nayer M. Faculty development for problem-based learning programs. Teach Learn Med. 1995;7(3):138–48. Supplementary Information 1. Wood DF. Problem based learning. BMJ. 2003;326(7384):328–30. 1. Wood DF. Problem based learning. BMJ. 2003;326(7384):328–30. The online version contains supplementary material available at https://​doi.​ org/​10.​1186/​s12909-​022-​03615-0. The online version contains supplementary material available at https://​doi.​ org/​10.​1186/​s12909-​022-​03615-0. 2. Hmelo-Silver CE. Problem-Based Learning: What and How Do Students Learn? Educ Psychol Rev. 2004;16(3):235–66. 2. Hmelo-Silver CE. Problem-Based Learning: What and How Do Students Learn? Educ Psychol Rev. 2004;16(3):235–66. 3. Azer SA. Introducing a problem-based learning program: 12 tips for suc‑ cess. Med Teacher. 2011;33(10):808–13. Abbreviations Hospital, Taipei, Taiwan. 11 Department of Emergency Medicine, National Taiwan University Hospital, Taipei, Taiwan. CVI: content validity indexes; EFA: Exploratory Factor Analysis; FD: faculty development; I-CVI: item-level content validity indexes; KMO: Kaiser-Meyer- Olkin; TS: tutoring-skill; PBL: problem-based learning; NTUCM: National Taiwan University College of Medicine; NTUH: National Taiwan University Hospital; M2: second-year medical students; M3: third-year medical students; M4: fourth- year medical students; S-CVI: scale-level content validity indexes. Received: 5 February 2022 Accepted: 6 July 2022 Ethics approval and consent to participate 15. Polit DF, Beck CT. The content validity index: Are you sure you know what’s being reported? critique and recommendations. Res Nurs Health. 2006;29(5):489–97. Approval was obtained from the Institutional Review Board (IRB) of National Taiwan University Hospital, Taipei, Taiwan, for the collection and publication of data (NTUH-REC No.:202103035WB). Consent was not obtained from the participants because the study was observational and the participants cannot be identified from the data in the paper. The need for consent was waived by the IRB. The project plan was reviewed in the university’s educational development unit. All methods were performed in accordance with relevant guidelines and regulations. 16. Polit DF, Beck CT, Owen SV. Is the CVI an acceptable indicator of content validity? Appraisal and recommendations. Res Nurs Health. 2007;30(4):459–67. 17. Konings KD, de Jong N, Lohrmann C, Sumskas L, Smith T, O’Connor SJ, Spanjers IAE, Van Merrienboer JJG, Czabanowska K. Is blended learning and problem-based learning course design suited to develop future public health leaders? An explorative European study. Public Health Rev. 2018;39:13. Competing interests The authors declare that they have no competing interests. Author details 1 Ahmed SA, Hegazy NN, Kumar AP, Abouzeid E, Wasfy NF, Atta K, Wael D, Hamdy H. A guide to best practice in faculty development for health professions schools: a qualitative analysis. BMC Med Educ. 2022;22(1):150. 29. Ahmed SA, Hegazy NN, Kumar AP, Abouzeid E, Wasfy NF, Atta K, Wael D, Hamdy H. A guide to best practice in faculty development for health professions schools: a qualitative analysis. BMC Med Educ. 2022;22(1):150. 30. Bell A, Mladenovic R. The benefits of peer observation of tutor development. Higher Educ. 2007;55(6):735–52. 30. Bell A, Mladenovic R. The benefits of peer observation of teaching tutor development. Higher Educ. 2007;55(6):735–52. 31. Pattison AT, Sherwood M, Lumsden CJ, Gale A, Markides M. Foundation observation of teaching project – A developmental model of peer obser‑ vation of teaching. Med Teach. 2012;34(2):e136–42. 32. van Lankveld T, Schoonenboom J, Kusurkar R, Beishuizen J, Croiset G, Volman M. Informal teacher communities enhancing the professional development of medical teachers: a qualitative study. BMC Med Educ. 2016;16(1):109. 33. Farmer EA. Faculty development for problem-based learning. Eur J Dental Educ. 2004;8(2):59–66. 34. Crosby RMHJ. AMEE Guide No 20: The good teacher is more than a lecturer - the twelve roles of the teacher. Medical Teacher. 2000;22(4):334–347. 34. Crosby RMHJ. AMEE Guide No 20: The good teacher is more Author details 1 1 Division of Radiation Oncology, Department of Oncology, National Taiwan University Hospital, Taipei, Taiwan. 2 Center of Faculty Development and Cur‑ riculum Integration, National Taiwan University College of Medicine, Taipei, Taiwan. 3 Graduate Institute of Medical Education and Bioethics, National Taiwan University College of Medicine, No. 1, Sec. 1, Ren’ai Rd., Zhongzheng Dist., 100 Taipei, Taiwan. 4 Nephrology Division, Department of Internal Medicine, National Taiwan University Hospital, Taipei, Taiwan. 5 Department of Surgery, National Taiwan University Hospital, Taipei, Taiwan. 6 Chest Medicine Division, Department of Internal Medicine, National Taiwan University Hospital, Taipei, Taiwan. 7 Department of Pediatrics, National Taiwan University Hospital, Taipei, Taiwan. 8 Department of Obstetrics and Gynecology, National Taiwan University Hospital, Taipei, Taiwan. 9 Department of Education and Research, Department of Medical Oncology, National Taiwan University Cancer Center, Taipei, Taiwan. 10 Department of Medical Education, National Taiwan University 20. Hair JF, Babin BJ. Multivariate Data Analysis: Cengage; 2018. 21. Almutary H, Douglas C, Bonner A. Multidimensional symptom clusters: an exploratory factor analysis in advanced chronic kidney disease. J Adv Nurs. 2016;72(10):2389–400. 22. Tavakoli Ghouchani H, Niknami S, Aminshokravi F, Hojjat SK. Factors Related to Addiction Treatment Motivations; Validity and Reliability of an Instrument. J Res Health Sci. 2016;16(3):147–52. 23. McLean M, Cilliers F, Van Wyk JM. Faculty development: yesterday, today and tomorrow. Med Teach. 2008;30(6):555–84. 24. Steinert Y, Mann K, Anderson B, Barnett BM, Centeno A, Naism 24. Steinert Y, Mann K, Anderson B, Barnett BM, Centeno A, Naismith L, Prideaux D, Spencer J, Tullo E, Viggiano T, et al. A systematic review of fac‑ ulty development initiatives designed to enhance teaching effectiveness: A 10-year update: BEME Guide No. 40. Med Teach. 2016;38(8):769–86. Page 9 of 9 Tsai et al. BMC Medical Education (2022) 22:564 Tsai et al. BMC Medical Education (2022) 22:564 25. Irby DM. Models of faculty development for problem-based learning. Adv Health Sci Educ Theory Pract. 1996;1(1):69–81. 26. Steinert Y. Faculty development: From rubies to oak. Med Teach. 2019;42(4):429–35. 27. Stone S, Mazor K, Devaney-O’Neil S, Starr S, Ferguson W, Wellman S, Jacobson E, Hatem DS, Quirk M. Development and implementation of an objective structured teaching exercise (OSTE) to evaluate improvement in feedback skills following a faculty development workshop. Teach Learn Med. 2003;15(1):7–13. 28. Garcia I, James RW, Bischof P, Baroffio A. Self-Observation and Peer Feed‑ back as a Faculty Development Approach for Problem-Based Learning Tutors: A Program Evaluation. Teach Learn Med. 2017;29(3):313–25. 29. Publisher’s Note S Springer Nature remains neutral with regard to jurisdictional claims in pub‑ lished maps and institutional affiliations. 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https://figshare.com/articles/journal_contribution/Species_richness_and_identity_both_determine_the_biomass_of_global_reef_fish_communities/23008058/1/files/40759226.pdf

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Species richness and identity both determine the biomass of global reef fish communities

Nature communications

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1 Tennenbaum Marine Observatories Network and MarineGEO program, Smithsonian Environmental Research Center, Edgewater, MD 21037, USA. 2 Institute for Marine and Antarctic Studies, University of Tasmania, Hobart, TAS 7001, Australia. 3 Department of Ocean Sciences, Memorial University of Newfoundland, St. John’s, NF A1C 5S7, Canada. 4 Department of Biology, University of Victoria, Victoria, BC V8P 5C, Canada. 5 Landscape Ecology, Institute of Terrestrial Ecosystems, ETH Zürich, CH-8092 Zürich, Switzerland. 6 Aquatic Ecology and Evolution, Institute of Ecology and Evolution, University of Bern, Bern, Switzerland. 7 Department of Marine Science, The University of Texas at Austin, Marine Science Institute, Port Aransas, TX 78373, USA. 8 School of Marine Studies, The University of South Pacific, Laucala Bay Road, Suva, Fiji Islands. 9 Bigelow Laboratory for Ocean Sciences, East Boothbay, ME 04544, USA. 10 Department of Ecology & Evolutionary Biology, University of Toronto, Toronto, ON M5S 3B2, Canada. ✉email: [email protected] ARTICLE NATURE COMMUNICATIONS | (2021) 12:6875 | https://doi.org/10.1038/s41467-021-27212-9 | www.nature.com/naturecommunications Species richness and identity both determine the biomass of global reef fish communities https://doi.org/10.1038/s41467 021 27212 9 OPEN Jonathan S. Lefcheck 1✉, Graham J. Edgar 2, Rick D. Stuart-Smith2, Amanda E. Bates3,4, Conor Waldock 5,6, Simon J. Brandl 7, Stuart Kininmonth8, Scott D. Ling2, J. Emmett Duffy 1, Douglas B. Rasher9 & Aneil F. Agrawal10 Jonathan S. Lefcheck 1✉, Graham J. Edgar 2, Rick D. Stuart-Smith2, Amanda E. Bates3,4, Conor Waldock 5,6, Simon J. Brandl 7, Stuart Kininmonth8, Scott D. Ling2, J. Emmett Duffy 1, Douglas B. Rasher9 & Aneil F. Agrawal10 Changing biodiversity alters ecosystem functioning in nature, but the degree to which this relationship depends on the taxonomic identities rather than the number of species remains untested at broad scales. Here, we partition the effects of declining species richness and changing community composition on fish community biomass across >3000 coral and rocky reef sites globally. We find that high biodiversity is 5.7x more important in maximizing biomass than the remaining influence of other ecological and environmental factors. Differ- ences in fish community biomass across space are equally driven by both reductions in the total number of species and the disproportionate loss of larger-than-average species, which is exacerbated at sites impacted by humans. Our results confirm that sustaining biomass and associated ecosystem functions requires protecting diversity, most importantly of multiple large-bodied species in areas subject to strong human influences. 1 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-27212-9 addition of the previous four terms (RICH-L + COMP-L + RICH- G + COMP-G) summarizes the total difference in community biomass between the two sites due to both losses and gains in the number (richness) and identities (composition) of species, which we call the “total diversity effect” or DIV. E E xtinctions in nature are often biased towards species that are rare and large-bodied1,2, yet experiments designed to pre- dict the ecosystem consequences of such extinctions have typically removed species at random3. The few experiments that have employed more realistic extinction scenarios by removing rare and functionally-unique species have revealed greater changes in ecosystem functioning than from random losses4–6. Observational datasets provide a unique opportunity to examine how the nonrandom loss of species, particularly those that are too large or too rare to feasibly manipulate in experiments, affects ecosystem processes under natural circumstances. Such datasets reflect the cumulative processes—including natural environ- mental forcing, local losses and gains of species, and anthro- pogenic impacts—that generate gradients of biodiversity observed in the real world7. Species richness and identity both determine the biomass of global reef fish communities https://doi.org/10.1038/s41467 021 27212 9 OPEN p Here, we applied this decomposition to quantitatively assess the relative contributions of species richness, composition, and context-dependent effects to reef fish community biomass, a common proxy for secondary production, derived from a fisheries-independent global dataset of underwater visual cen- suses collected by the Reef Life Survey program (www.reeflifesurvey.com). We selected reefs with the highest biomass to serve as the reference sites, as high fish biomass is a desirable state and often the goal of conservation and restoration efforts. We then compared these high-biomass sites to all other sites within a 100-km radius (representing a potential upper limit on direct dispersal17) to determine whether variation in elements of biodiversity contributes to other nearby sites not reaching their biomass potential. We conducted 173 comparisons of reference sites (i.e., those with the highest biomasses) against a total of 2867 comparison sites, with each reference site having on average 16.6 (range = 5–136) comparison sites (Fig. 2). Sites were split near equally between tropical realms (mostly coral-dominated; n = 1603) and temperate realms (mostly rocky reefs; n = 1437). We go on to show that loss of biomass at the comparison site is most associated with biodiversity loss, which can be further attributed to both losses in the total number of species and the absence of uniquely large contributors to biomass. These effects were most pronounced in areas with high human populations, implicating humans as a primary driver of changes in fish bio- mass across the seascape. We must first describe the background of our decomposition (Fig. 1). Species that are absent or “lost” in the comparison community relative to the reference community contribute to the “richness loss” and the “compositional loss” components of the biomass difference, hereafter RICH-L and COMP-L. To quantify these components, it is necessary to choose a frame of reference for the “expected” biomass loss per absent species, under the null hypothesis that there is nothing unusual about the species that are absent. Here, we use the average biomass of species present in both communities (“shared species”) as this reference value (for more details, see Methods and Supplementary Materials). RICH-L is the amount of biomass lost based on the number of species absent from the comparison community weighted by this “expected” biomass per species. Species richness and identity both determine the biomass of global reef fish communities https://doi.org/10.1038/s41467 021 27212 9 OPEN The fifth and final term aims to estimate all additional factors contributing to the difference in biomass between sites that is not due to diversity, broadly speaking. These include, for example, differences in the number of individuals and their sizes, predator–prey interactions, resource availability, habitat quality and complexity, and/or underlying environmental drivers, col- lectively called the “context-dependent effect” (or CDE). The CDE term considers only differences in biomass among species shared by both communities rather than species, which are lost or gained between communities. We note that the CDE term cannot mechanistically distinguish among these various influences, as has been done using other experimental16 and observational datasets7, but its relative magnitude can be assessed against other terms to quantify the importance of losses or gains of whole populations (i.e., RICH and COMP) versus changes in individual biomass or abundance13. By extension, environmental factors can also drive changes in species richness and composition, so CDE should not be interpreted as exclusively indicative of environ- mental forcing. Rather, it can be thought of as the cumulative influence of all extrinsic factors on fish community properties that is independent of their realized effects on the observed dif- ferences in richness and composition. Even as observational evidence supports strong links between species richness and ecosystem functioning, specifically biomass production7, it remains unclear how much of this relationship stems from reducing the number of species (at random) versus the specific traits proxied by the identities of species. This dis- tinction is important if extinctions are biased towards individual species that contribute disproportionately to functioning, such as large-bodied fishes8. Selective removal of such species through overexploitation and sensitivity to human disturbance might be expected to have a larger-than-anticipated influence on com- munity productivity compared to random losses9. Separating the effects of changing species richness and com- munity composition on ecosystem properties in observational data has proved historically challenging10. One useful approach is to mathematically partition the total change in an aggregate property, such as community biomass, into component factors11–15. Inspired by these earlier approaches, we present a partitioning of the difference in standing stock biomass between pairs of communities—a reference site vs. a comparison site—into five additive components representing the effects of both gains and losses in species richness and composition, and the effects of any remaining influences such as environmental and ecological factors that are not already captured in the elements of com- munity diversity. Species richness and identity both determine the biomass of global reef fish communities https://doi.org/10.1038/s41467 021 27212 9 OPEN The actual loss in biomass at a community level can differ due to deviations in the biomass of the species that are absent compared to the species that are shared, which is captured by the COMP-L term. This term reflects the change in biomass between communities that is attributable to the kinds of species that are absent rather than simply the number of absent species. NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-27212-9 The RICH-G and COMP-G terms are analogous to RICH-L and COMP-L but arise from species that are present in the comparison site and absent from the reference (as illustrated in Contrasts III and IV). The final term, the “context-dependent effect” or CDE, considers only the species shared among both the reference and comparison sites. Differences in the contributions of shared species among the two sites reflect processes other than changes in richness and composition, such as changes in per species biomass, community size structure, resources, or the abiotic environment (e.g., temperature). In Contrasts I-IV, the shared species did not differ in their average per species contribution between the two sites (i.e., zuF = zcB) so there was no CDE. In Contrast V, the shared species differ in their average per species contribution to biomass, resulting in a nonzero CDE. In a real comparison, all five components can occur simultaneously. The five components sum to the observed difference in biomass between the reference and comparison communities. reference sites are not mathematically constrained to have more species, but apparently do in nature. The majority (69%) of comparisons to the reference site exhibited a net loss rather than a net gain of species (−11.4 ± 0.7% average (± SE) reduction in the number of species across all comparisons). Thus, our analysis indicates that differences in biodiversity, primarily those that reflect the absences of species from site to site, are the primary determinants of fish community biomass across the world’s coral and rocky reefs over the scale of 10 s of kilometers. large-bodied fishes) and/or those with extreme abundances (e.g., schooling fishes). Thus, we demonstrate that species’ identities and their traits (e.g., body size, gregariousness) are key factors in predicting the ecosystem consequences of species’ losses. We note that the strong effect of species identity does not negate the contributions of richness: indeed, reductions in the number of species necessarily change composition, and thus both operate simultaneously to influence community biomass. Our finding that biodiversity loss is the primary determinant of fish community biomass among reefs within a region is robust to the spatial extent over which the local comparisons are conducted (15-–100 km, Supplementary Fig. 1), with slightly weaker effects of composition observed at reduced scales (mean COMP- L = −0.38, −0.38, −0.40, and −0.43 for 15-, 25-, 50- and 100- km radii, respectively). NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-27212-9 In Contrasts I-IV, the shared species did not differ in their average per species contribution between the two sites (i.e., zuF = zcB) so there was no CDE. In Contrast V, the shared species differ in their average per species contribution to biomass, resulting in a nonzero CDE. In a real comparison, all five components can occur simultaneously. The five components sum to the observed difference in biomass between the reference and comparison communities tion of our decomposition quantifying the difference in biomass between two sites: a reference or reference site (B) Fig. 1 A conceptual representation of our decomposition quantifying the difference in biomass between two sites: a reference or reference site (B) and a comparison site (F). Notation: Sc, SuB, and SuF refers to the number of species: in common, unique to B and unique to F, respectively; and ZuB, ZcB, ZcF, and ZuF to average ecological function (in this example, biomass) of species: unique to B, common to both sites when present at B, common to both sites when present at F, and unique to F, respectively. The first term, RICH-L, reflects loss of species from the reference site that are most like the average of the species that are retained. In Contrast I, the comparison site lacks some species present at the reference site (left column), so the species richness is lower at the comparison site (second column). In this case, the species in common between the two sites have the same average contribution to biomass (per species) as species unique to the reference (i.e., zcB = zuB, third column). Thus, the difference in biomass between the two sites in Contrast I is entirely captured by the RICH-L effect (last column). The second term, COMP-L, reflects the loss of biomass beyond what is expected given the number of species lost and average per species contribution of the reference species, i.e., the shared species. Unlike in Contrast I, in the case shown for Contrast II the species unique to the reference site are larger and therefore greater contributors (per species) to total biomass than the species that are shared between sites (
zuB

zcB = δB > 0). The difference in total biomass between communities in Contrast II would be captured by the COMP-L term (last column) in addition to the RICH-L term (as shown for Contrast I). NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-27212-9 Fig. 1 A conceptual representation of our decomposition quantifying the difference in biomass between two sites: a reference or reference site (B) and a comparison site (F). Notation: Sc, SuB, and SuF refers to the number of species: in common, unique to B and unique to F, respectively; and ZuB, ZcB, ZcF, and ZuF to average ecological function (in this example, biomass) of species: unique to B, common to both sites when present at B, common to both sites when present at F, and unique to F, respectively. The first term, RICH-L, reflects loss of species from the reference site that are most like the average of the species that are retained. In Contrast I, the comparison site lacks some species present at the reference site (left column), so the species richness is lower at the comparison site (second column). In this case, the species in common between the two sites have the same average contribution to biomass (per species) as species unique to the reference (i.e., zcB = zuB, third column). Thus, the difference in biomass between the two sites in Contrast I is entirely captured by the RICH-L effect (last column). The second term, COMP-L, reflects the loss of biomass beyond what is expected given the number of species lost and average per species contribution of the reference species, i.e., the shared species. Unlike in Contrast I, in the case shown for Contrast II the species unique to the reference site are larger and therefore greater contributors (per species) to total biomass than the species that are shared between sites (
zuB

zcB = δB > 0). The difference in total biomass between communities in Contrast II would be captured by the COMP-L term (last column) in addition to the RICH-L term (as shown for Contrast I). The RICH-G and COMP-G terms are analogous to RICH-L and COMP-L but arise from species that are present in the comparison site and absent from the reference (as illustrated in Contrasts III and IV). The final term, the “context-dependent effect” or CDE, considers only the species shared among both the reference and comparison sites. Differences in the contributions of shared species among the two sites reflect processes other than changes in richness and composition, such as changes in per species biomass, community size structure, resources, or the abiotic environment (e.g., temperature). Results and discussion h l d h d When we applied the decomposition (Fig. 1) to our data, we found that low reef fish biomass between reference and com- parison sites was associated predominantly with changes in richness and composition rather than environmental and other drivers (Fig. 3). The cumulative biodiversity effect DIV was 5.7x stronger (95% confidence intervals: [5.1, 6.5]) than the CDE effect (Fig. 3). Furthermore, the biodiversity effect on community bio- mass was driven almost entirely by the loss (L) rather than the gain (G) of species (Fig. 3). We note that this finding is not an artifact of the partitioning equation: indeed, high-biomass p The third and fourth terms reflect changes in biomass from species present at the comparison site but absent from the reference site (“gained” species): RICH-G is the expected gain in biomass given the number of gained species (again based on the average biomass of species shared among both sites), and COMP- G reflects any additional biomass change from the gained species being different in average biomass from shared species. The NATURE COMMUNICATIONS | (2021) 12:6875 | https://doi.org/10.1038/s41467-021-27212-9 | www.nature.com/naturecommunications 2 ARTICLE NATURE COMMUNICATIONS | (2021) 12:6875 | https://doi.org/10.1038/s41467-021-27212-9 | www.nature.com/naturecommunications NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-27212-9 The proportion of species shared among any two sites, however, remained remarkably consistent from scales of 15–100 km (49.1–51.5% of the total community), and The strongest effect of biodiversity loss on community biomass was due to changes in species composition. Loss of biomass attributable to changing species identities (COMP-L) was 3.8x greater (95% CIs: [3.5, 4.1]) than that based on the “expected” or average outcome of removing species at random (RICH-L, Fig. 3). In approximately three-quarters of cases, the COMP-L term was negative, indicating that compositional effects were primarily due to the loss of greater-than-average contributors to biomass (i.e., 3 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-27212-9 Fig. 2 A map of study sites included in the analysis. Fig. 3 Declines in fish biomass between sites are driven primarily by loss of species (RICH-L) and compositional losses (COMP-L). Panels show the frequency distributions of each component based on differences between sites with the highest biomass and other nearby sites. Values have been standardized to the interval (−1, 1). Black points represent the mean of all observations ±95% confidence intervals (which are too small to be observed). RICH richness, COMP composition, L loss, G gain, CDE context-dependent effect, DIV total diversity effect (= RICH-L + COMP-L + RICH-G + COMP-G). Fig. 2 A map of study sites included in the analysis. Fig. 2 A map of study sites included in the analysis. Fig. 3 Declines in fish biomass between sites are driven primarily by loss of species (RICH-L) and compositional losses (COMP-L). Panels show the frequency distributions of each component based on differences between sites with the highest biomass and other nearby sites. Values have been standardized to the interval (−1, 1). Black points represent the mean of all observations ±95% confidence intervals (which are too small to be observed). RICH richness, COMP composition, L loss, G gain, CDE context-dependent effect, DIV total diversity effect (= RICH-L + COMP-L + RICH-G + COMP-G). effects of numerous factors such as reduction in suitable habitat and food availability9,19. In contrast, the context-dependent effect (CDE) was most influenced by environmental variables, specifi- cally salinity, nutrients (dissolved phosphorus), and temperature variation (Supplementary Fig. 3). NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-27212-9 This result would be expected when environmental variation affects the weight and abundance of individuals but does not exclude species entirely, clarifying our interpretation of the CDE term as capturing underlying gradients that might alter the capacity of species to acquire resources and produce biomass. Environmental factors also affected the diver- sity terms (e.g., phosphorus for RICH-L, Supplementary Fig. 3), albeit to a lesser degree, reflecting that environment can also be responsible for driving local species absences or latently indicate a degraded state which certain species find unsuitable. thus our results do not appear to be biased by an increased overlap in composition between communities at smaller scales or by more rare or unique species at larger scales. Additionally, our results are largely consistent across temperate and tropical realms (Supplementary Fig. 2), with two notable exceptions. First, we observed slightly more extreme declines in biomass due to rich- ness losses in tropical areas, presumably because tropical sites have, on average, 2.8x more species than temperate ones and therefore possess more scope for change. Second, we observed similarly larger declines in biomass due to compositional losses at temperate sites, reflecting that individuals there are, on average, 1.5x larger than at tropical sites and therefore more likely to contribute biomass in excess of the “expected” or average species. Nevertheless, these two differences cancel each other out such that the total diversity effect (DIV) does not differ between the two realms (Supplementary Fig. 2). The effects of species identity on community biomass recov- ered by our analysis are most likely related to selective removal of large species, as found in prior studies of fisheries20,21, rather than changes in the number of individuals. To test this hypothesis, we partitioned the contributions of species unique to reference and comparison sites as a function of size class. As expected, the unique contributors at the most productive reference sites were concentrated in the largest size class (>200 cm observed total length) (Fig. 5). Of species unique to the reference sites, 56.4% of the biomass was contributed by fishes >200 cm in length, whereas this size class contributed only 19.0% of the total biomass at the comparison sites. NATURE COMMUNICATIONS | (2021) 12:6875 | https://doi.org/10.1038/s41467-021-27212-9 | www.nature.com/naturecommunications NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-27212-9 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-27212-9 Fig. 4 Human population size predicts aspects of fish community structure. Proximity to high densities of humans is associated with: A reduced total fish community biomass; B smaller observed median size classes; and C fewer species. Lines are predicted trends from generalized linear models. Fig. 4 Human population size predicts aspects of fish community structure. Proximity to high densities of humans is associated with: A reduced total fish community biomass; B smaller observed median size classes; and C fewer species. Lines are predicted trends from generalized linear models. Fig. 5 Contributions of fish size to total fish biomass at comparison and reference sites. A A much larger proportion of the biomass of species unique to reference sites was attributable to large fishes (>200 cm), as compared to species that were only found at comparison sites. B Among those species that were shared between both the reference and comparison sites, small fish (<50 cm) made up a much larger proportion of the total biomass, especially at the comparison site. Points are medians ±95% quantiles, and the gray shaded area shows the underlying distribution of raw data. The total number of individuals represented in the plot is n = 41,267. Fig. 5 Contributions of fish size to total fish biomass at comparison and reference sites. A A much larger proportion of the biomass of species unique to reference sites was attributable to large fishes (>200 cm), as compared to species that were only found at comparison sites. B Among those species that were shared between both the reference and comparison sites, small fish (<50 cm) made up a much larger proportion of the total biomass, especially at the comparison site. Points are medians ±95% quantiles, and the gray shaded area shows the underlying distribution of raw data. The total number of individuals represented in the plot is n = 41,267. captured in the minority of positive values of COMP-L (Fig. 3). The lack of large species at comparison sites could have resulted from the inclusion of species that are more mobile and have larger home range sizes, and just so happen to have occurred at a particular reference site. To test whether the contributions of such species might have unduly influenced our results, we re-ran our analysis after removing all 156 pelagic/non-site attached species from our dataset (~5% of all species, classifications from22). NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-27212-9 These observations also explain the bimodal distribution of the COMP-L term: the majority of highly negative values reflect losses of larger-than-average species from reference sites, as opposed to smaller-than-average fishes which are To understand the potential drivers of our results, we used random forest analysis to identify the environmental and anthropogenic variables most associated with each component of our decomposition18. The top-ranked predictor for biomass loss associated with richness and compositional loss was consistently human population density, particularly for the COMP-L and DIV terms (Supplementary Fig. 3). Sites near even a minimal human population exhibited lower total fish biomass, smaller-bodied fishes, and fewer fish species (P < 0.001 in all cases, from gen- eralized linear mixed-effects models regressing fish community characteristics against human density) (Fig. 4). Our index pro- vides a proxy for a range of human impacts, including direct removal of species and biomass through fishing and the indirect 4 NATURE COMMUNICATIONS | (2021) 12:6875 | https://doi.org/10.1038/s41467-021-27212-9 | www.nature.com/naturecommunications NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-27212-9 The species present in the reference community can be classified into two types: species that are unique to the reference community (i.e., not present in the comparison We begin by assuming that the ecological function of the community, such as biomass, is a simple additive function of the contributions of its constituent species. We go on to compare two communities, one of which we consider the “reference” community and the other we refer to as the “comparison” community. The species present in the reference community can be classified into two types: species that are unique to the reference community (i.e., not present in the comparison community) and those that are in common with the comparison community. Let suB be the number of unique species in the reference community, and sc be the number in common between the two communities. Let
zuB be the average ecological function contributed per unique species to the reference community, and
zcB be the average ecological function contributed per shared species in the reference community. The total ecological function TB of the reference community can thus be decomposed as: reference community. The total ecological function TB of the reference community can thus be decomposed as: TB ¼ suB
zuB þ sc
zcB ð1Þ ð1Þ where the first term represents the ecological function contributed by species that are unique to the reference community (i.e., not present in the comparison community) and the latter term represents the contribution from species that are also found in the comparison community. Analogously, in the comparison community, the total ecological function can be decomposed as: TF ¼ suF
zuF þ sc
zcF ð2Þ ð2Þ with a similar interpretation to Eq. (1). Though there are sc species in common between the two communities, the average per species contribution need not be the same in the two communities (i.e.,
zcB may differ from
zcF). Our results strengthen previous findings that human actions have large impacts on fish biomass: many studies have demon- strated that humans are selectively removing large-bodied fishes from the ocean, largely on the basis of fisheries catch data9,18,20,21,27,28, resulting in a loss of functional diversity and disruption of ecosystem trophic structure29. Our study confirms a concerning ecosystem-scale consequence of this phenomenon: reductions in large-bodied species leads to greater divergence between high- and low-biomass sites. NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-27212-9 Our data, therefore, provide compelling new evidence that selective removal of species with particular traits and reduction of local biodiversity across the seascape through collective human impacts profoundly alter the structure and function of whole reef communities worldwide. This result has important implications for the continued man- agement of reef ecosystems: conservation practices that are tied to the restoration of high-performing species30 and those that aim to preserve the diversity of whole communities (e.g., marine pro- tected areas)31,32 are both required to maximize the provision of a critical ecosystem function in an increasingly human-dominated world33. The species in common between the two communities can serve as a reference point for comparison between communities. It is useful to define δB ¼
zuB

zcB and δF ¼
zuF

zcF as the difference in average ecological function per species of unique species versus shared species in reference and comparison communities, respectively. From this perspective, we consider the average ecological function of a species unique to the reference community as being equal to the average ecological function of shared species (as measured in the same community) plus the deviation from this value
zuB ¼
zcB þ δB. Using this equality and the analogous one for
zuF, along with Eqs. (1) and (2), the difference in the ecological function between communities can be decomposed as ΔT ¼ TF
TB ¼
suB
zcB
suBδB þ suF
zcF þ suFδF þ sc
zcF

zcB
 ð ð3Þ The first two terms represent the loss in ecological function in the comparison community due to the loss of species that are unique to the reference community. Specifically, the first term represents the loss in ecological function due to the absence of unique species if these species had the same average value of functioning as each of the shared species. In other words, it is the amount by which biomass is expected to decline if species were interchangeable. Therefore, we interpret this term as the “richness loss” or the loss in functioning due strictly to the loss of species: RICH-L (¼
suB
zcB). It will always be negative, assuming there is at least one species unique to the reference population. In cases where
zcB>
zuB, it is possible for RICH-L to exceed the total functioning observed at the reference site, which complicates interpretation of the raw values. Methods R f lif et ods Reef life survey. Reef fish communities were censused by a combination of experienced marine scientists and trained recreational SCUBA divers using globally standardized Reef Life Survey methods. All surveys were undertaken on 50 m long transects laid along a contour (at consistent depth) on predominantly hard sub- strate (usually rocky or coral reef) in shallow waters (depth range of transects 1 to 20 m, average ~7.2 m). Full details of fish census methods, data quality, and training of divers are provided in refs. 22,34,35 and in an online methods manual (www.reeflifesurvey.com). Fish abundance counts and size estimates per 500 m2 transect area (2 ×250 m2 blocks) were converted to biomass using length–weight relationships for each species obtained from Fishbase (www.fishbase.org). In cases where length–weight relationships were provided in Fishbase using standard length or fork length, rather than total length as estimated by divers, length–length relationships provided in Fishbase allowed conversion to the total length. For improved accuracy in biomass assessments, observed sizes were also adjusted to account for the bias in divers’ perception of fish size underwater using an empirical calibration36. Length–weight coefficients from similar-shaped close relatives were used for those species where length–weight relationships were not available in Fishbase. All transects were collapsed into a single average value of biomass for each species at a location to account for any differences in the total number of transect surveys performed. The second term accounts for the fact that the true loss in ecological function due to these lost species will often differ from the “richness expectation” because the lost species differ in value from the average value of shared species. In other words, this term reflects the deviation in the actual contributions of lost species from the average of shared species, which implies that not all species contribute equally (and that the identities of the species are important in determining differences in biomass between the two communities). We, therefore, interpret this term as indicating “compositional loss,” or the degree to which loss in biomass is due to loss of particular species: COMP-L (¼
suBδB). If the average lost species provide a higher contribution to the reference community than the average shared species (
zuB>
zcB), the COMP-L term will be negative. On the other hand, if the average lost species represent lower contributions, the COMP-L term will be positive (
zuB<
zcB). NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-27212-9 NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-27212-9 findings reflect the inherent organization of natural systems: high-biomass sites support large-bodied species that are not often found elsewhere23. Decomposition of difference in ecosystem functioning. Our equation was inspired by previous decompositions, principally the Price equation originally derived in the field of evolutionary biology as a means of separating genetic and environmental influences on phenotypic change over time37. Fox38 and later Fox and Kerr12 modified the Price equation to describe how the difference in the ecological function between two communities can be decomposed into components with different ecological interpretations. We follow a similar approach but use a different decomposition where the resulting components are similar to, but not the same as, the components proposed by Fox and Kerr12. Hundreds of experimental manipulations show that biodi- versity loss is among the pre-eminent factors contributing to variation in community biomass in a wide range of ecosystem types and taxa driven by both changes in richness and composition16,24. Our study extends theoretical inferences from this work to highly diverse, natural ecosystems across a range of contexts. A synthesis of >200 terrestrial plant manipulative experiments revealed roughly equivalent contributions by species richness and species identity10, a result which matches our findings for global reef fishes. Here, we also provide a new mathematical approach to address this question. It is important to note that, like other recent ecological decompositions, ours is not a mechanistic model12. Instead, it integrates across all potential mechanisms driving the aggregate loss and gain of species, including their response to abiotic drivers, and how they affect ecosystem functioning. Therefore, our application permits disentangling richness vs. compositional effects operating in these communities25, but how these effects mechanistically arise (e.g., through dominance, complementary resource use, indirect interactions such as facilitation, etc.) requires further investigation26. Similarly, assessing processes such as production (for which we substitute standing stock biomass as a proxy) using this new partitioning is necessary if the goal is to link diversity to more robust measures of ecosystem functioning. We begin by assuming that the ecological function of the community, such as biomass, is a simple additive function of the contributions of its constituent species. We go on to compare two communities, one of which we consider the “reference” community and the other we refer to as the “comparison” community. NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-27212-9 In this case, it is useful to consider only the relative quantities (each component is scaled by the sum of the absolute values of all components). We note that this situation arises only 41 times out of 2867 comparisons in our analysis, and removing these cases has no effect on our findings. We advise future applications be aware of this potential issue and test for its influence. ARTICLE ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-27212-9 This subsequent analysis yielded nearly identical results to those from the full dataset (Supplementary Fig. 4). captured in the minority of positive values of COMP-L (Fig. 3). The lack of large species at comparison sites could have resulted from the inclusion of species that are more mobile and have larger home range sizes, and just so happen to have occurred at a particular reference site. To test whether the contributions of such species might have unduly influenced our results, we re-ran our analysis after removing all 156 pelagic/non-site attached species from our dataset (~5% of all species, classifications from22). This subsequent analysis yielded nearly identical results to those from the full dataset (Supplementary Fig. 4). due primarily to cumulative changes in diversity, broadly defined (DIV), or losses within species (CDE). However, this choice of reference may also bias towards a compositional effect, since larger species that contribute most to biomass are more likely to be found at high-biomass sites. To test for this bias, we repeated our analysis on 1000 simulated versions of our original dataset that randomized composition while holding species numbers and per capita contributions constant. The value of COMP-L in the observed data was always more negative than those returned by our simulations (P < 0.001 based on a one-tailed comparison of means; Supplementary Fig. 5). Thus, our results do not appear to be an artifact of how the reference community was defined (see also Supplementary Materials for outcomes when alternatively establishing the reference as the most speciose sites). Instead, our We set the reference site as the highest biomass site to simplify the interpretation of biomass variation across communities because this makes the net change always negative13. Analyzing the data in this way allows us to ask whether losses in biomass are 5 NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-27212-9 NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-27212-9 shared species: RICH-G (¼ þsuF
zcF). It is always positive, assuming the comparison community has at least one unique species. The fourth term, COMP-G (¼ þsuFδF), reflects the difference in composition (with respect to average value) of gained versus shared species. This term can be positive or negative, being positive if the gained species have a higher per species value than the shared species. shared species: RICH-G (¼ þsuF
zcF). It is always positive, assuming the comparison community has at least one unique species. The fourth term, COMP-G (¼ þsuFδF), reflects the difference in composition (with respect to average value) of gained versus shared species. This term can be positive or negative, being positive if the gained species have a higher per species value than the shared species. In this case, Q = (COMP-L + RICH-L)/RICH-L, which reduces to the average value of unique species relative to the average value of shared species at the reference site. This quantity reflects the magnitude to which species unique to the reference site contribute to biomass relative to the “expected” contribution per species. To avoid biases associated with averaging ratios, we report the geometric mean of both quantities. Bootstrapped 95% confidence intervals were derived by randomly resampling DIV/CDE and Q for a total of 5000 times. For DIV/CDE, some values were negative, so we excluded them in both the original data and bootstrap samples. As an alternative approach that focused on the magnitude of effect, we examined the absolute value of |DIV | / | CDE | . In this case, the ratio was 6.9x with bootstrap 95% CIs of [6.2, 7.7]. The final term focuses on the changes in biomass considering only the species that are present in both communities. This can be thought of as holding richness and composition constant and considering changes in the community biomass that are controlled extrinsically, i.e., by underlying gradients in resource availability and other environmental factors. Historically, this term has been referred to as the “context-dependent effect,” or CDE, and is the number of shared species (sc), multiplied by the difference in biomasses among shared species at both sites (¼ scð
zcF

zcBÞ). It can be of either sign: positive if shared species have a higher value in the comparison community than in the reference, negative if they have a higher value in the reference community. NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-27212-9 The number of shared species has the potential to bias away from the CDE term if it is very low. However, we note that, on average, 49.1 ± 0.003% of species are shared for each comparison at the 100-km scale, and this value is remarkably consistent regardless of spatial scale (51.3–50.0% for 15–50 km). To explore the drivers of the components of our decomposition, we applied random forest analysis to account for potential collinearity and interactions among the suite of predictors previously selected in ref. 39. Depth was recorded on the surveys while the following predictors were obtained from the combination of remote sensed and in situ measurements compiled in the Bio-ORACLE database: mean, minimum, maximum, and range of sea surface temperature; mean, minimum and maximum for surface chlorophyll-a; mean salinity; mean PAR; mean dissolved oxygen; mean nitrate concentration; mean phosphate concentration40. Finally, an index of human population density was calculated by fitting a smoothly tapered surface to each settlement point on the year 2010 world- population density grid using a quadratic kernel function described previously41. Random forests were fit using the default settings in the randomForest package42 in R version 4.1.143. Variable importance was determined using the percent increase in the mean-square error after randomly permuting the predictor of interest for each tree in the random forest, averaging the error of the models, and then computing the difference relative to the accuracy of the original model. Our decomposition is similar to, but not the same as, that of Fox and Kerr12, though both are mathematically sound. Only the CDE term is mathematically identical across the two decompositions and, thus, shares the same interpretation. By extension, the sum across the loss and gain terms (the total diversity effect, or DIV) must also be identical, because both equations partition the same total quantity. Thus, it is important to note that using either decomposition yields the same inference with respect to comparisons of DIV and CDE. Our decomposition differs from Fox and Kerr’s because the two approaches use different reference points. We take the perspective that the shared species form the basis for comparison between two communities, so we then evaluate the average value of a unique species with respect to its deviation from an average value of a shared species. NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-27212-9 Beginning with the highest biomass site of all sites as the first reference site, we identified all other sites within a certain spatial radius (15-, 25-, 50-, or 100-km) to serve as the comparison sites. Setting the reference to be the site with the highest community biomass constrains the sum of the terms to be negative. This choice simplifies the language used to discuss the output13 and allows us to speak directly to the consequences of real-world activities like overharvesting (and their implications). Our simulation procedure focused on the site-by-species biomass matrix from each set of comparisons used in the main 100-km analysis. We divided this matrix by the corresponding site-by-species abundance matrix to yield the observed per capita contribution of each species in each community. We then averaged the per capita contributions of each species across all communities where the species was present to yield a single vector representing mean per capita contributions for all S species within that set of comparisons. We first ordered all sites by decreasing total biomass. Beginning with the highest biomass site of all sites as the first reference site, we identified all other sites within a certain spatial radius (15-, 25-, 50-, or 100-km) to serve as the comparison sites. Setting the reference to be the site with the highest community biomass constrains the sum of the terms to be negative. This choice simplifies the language used to discuss the output13 and allows us to speak directly to the consequences of real-world activities like overharvesting (and their implications). We initially constructed each simulated community by populating it with every species in the region (“maximum richness”). To determine the biomass of each species in each community we applied the following procedure. First, we identified the minimum and maximum observed abundance of each species across all communities where it is present. For a single community, we sampled an integer value between the minimum and maximum abundance for each species to yield a single vector of random abundance values of length S, and then multiplied this vector by the vector of average per capita contributions. This procedure yielded a new vector representing a new total contribution to biomass by every species. We repeated this for all n communities in the original site-by-species matrix and bound these vectors together in a new “maximum richness” version of the site-by-species matrix. Methods R f lif The next two terms are analogous to the first two terms but instead represent the increase in ecological function in the comparison community due to the “gain” of unique species that are lacking from the reference community. The third term represents the expected increase in ecological function due to an increase in species richness assuming these gained species had the same per species contribution as the NATURE COMMUNICATIONS | (2021) 12:6875 | https://doi.org/10.1038/s41467-021-27212-9 | www.nature.com/naturecommunications 6 NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-27212-9 In contrast, Fox and Kerr effectively evaluate the average value of a unique species with respect to its deviation from the average value of any species in that community (averaging over both unique and shared species). In both decompositions, the “composition” components only exist if there is some difference in the average value of shared and unique species. We prefer our decomposition for this case because it works with that difference directly rather than indirectly via the difference between unique and all species (which is the average of unique and shared species). Moreover, our composition makes intuitive sense that the function of the “average” species is determined by the ones that are known to exist at both sites. A full comparison of the Fox and Kerr formulation and ours is provided in the Supplementary Materials. Null simulations. A key finding of our analysis is that compositional losses are considerably greater than losses due to other aspects of the reef fish community. We wanted to evaluate the possibility of whether such a result could be an artifact of applying our decomposition to a dataset in which we assign the site with the higher total biomass as the “reference” community and the site with lower total biomass as the “focal” community. To do so, we conducted simulations in which we created communities with species richness values matching the observed data, but for community compositions that were random. Following the same procedure we used with the real communities, we applied our decomposition to these simulated communities to generate null distributions for the average values of each of the five terms when community composition is random. Comparing our observed values to these null distributions tells us if the values of the compositional components (or indeed any component) we observed arose as an artifact of our procedure or, alternatively, because high-biomass sites actually contain more high- biomass species than expected under random community assembly. Statistical analysis. A general function to conduct our new decomposition from a site-by-species biomass matrix, and a second function to perform the simulations, can be found here: https://gist.github.com/jslefche/76c076c1c7c5d200e5cb87113cdb9fb4. Statistical analysis. A general function to conduct our new decomposition from a site-by-species biomass matrix, and a second function to perform the simulations, can be found here: https://gist.github.com/jslefche/76c076c1c7c5d200e5cb87113cdb9fb4. We first ordered all sites by decreasing total biomass. NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-27212-9 For the ith row (community) in the original dataset, we calculated the richness, si. We then randomly subsampled si species at random from the simulated “maximum richness” site-by-species matrix and set the biomass of any remaining species to zero. We repeated this for each community to yield a simulated “observed richness” site-by-species matrix with the same dimensions as the original matrix. This procedure ensures that richness is held at the observed levels and that the biomass contribution of each species are within the observed range. We initially constructed each simulated community by populating it with every species in the region (“maximum richness”). To determine the biomass of each species in each community we applied the following procedure. First, we identified We then computed the components for each set of comparisons. We standardized the output to the same scale (−1, 1) by first taking the sum of the absolute value of all components, and then dividing each component by this value. This relativization was done to account for the fact that raw biomass may differ substantially among sites and regions and to make our results comparable across the entire dataset. Once the scaled components were computed, the reference and comparison sites were removed from the ordered list from any further comparisons to prevent any bias that might arise from including the same site multiple times. We then moved onto the next most productive site in the list, identified the comparison sites within 100 km, computed the components, and so on, until all sites were analyzed. From these individual comparisons, we computed the means of all components while omitting any reference sites for which there were fewer than five comparison sites. We alternately averaged the components for all comparisons for each reference site and then took the grand mean of these averaged values, although this additional level of aggregation did not qualitatively change our results (Supplementary Fig. 6). We have chosen to present the raw values in the main text to demonstrate the full range of variability inherent in the individual comparisons, which might otherwise be condensed by showing only the means for each reference site. We repeated the analysis over multiple spatial radii to assess whether the spatial extent and therefore the size and composition of the species pool, might influence our results. species to zero. NATURE COMMUNICATIONS | (2021) 12:6875 | https://doi.org/10.1038/s41467-021-27212-9 | www.nature.com/naturecommunications References 34. Edgar, G. J. & Stuart-Smith, R. D. Systematic global assessment of reef fish communities by the Reef Life Survey program. Sci. Data 1, 1–8 (2014). 1. Pimm, S. L., Jones, H. L. & Diamond, J. On the risk of extinction. Am. Nat. 132, 757–785 (1988). 35. Edgar, G. J. & Stuart-Smith, R. D. Ecological effects of marine protected areas on rocky reef communities-A continental-Scale analysis. Mar. Ecol. Prog. Ser. 388, 51–62 (2009). 2. Payne, J. L., Bush, A. M., Heim, N. A., Knope, M. L. & McCauley, D. J. 2. Payne, J. L., Bush, A. M., Heim, N. A., Knope, M. L. & McCauley, D. J. Ecological selectivity of the emerging mass extinction in the oceans. Science 353, 1284–1286 (2016). 36. Edgar, G. J., Barrett, N. S. & Morton, A. J. Biases associated with the use of underwater visual census techniques to quantify the density and size-structure of fish populations. J. Exp. Mar. Bio. Ecol. 308, 269–290 (2004). 3. Srivastava, D. S. & Vellend, M. Biodiversity-ecosystem function research: is it relevant to conservation? Annu. Rev. Ecol. Evol. Syst. 36, 267–294 (2005). 37. Price, G. R., others. Selection and covariance. Nature 227, 520–521 (1970). 4. Solan, M. Extinction and ecosystem function in the marine benthos. Science 306, 1177–1180 (2004). 38. Fox, J. W. Using the Price Equation to partition the effects of biodiversity loss on ecosystem function. Ecology 87, 2687–2696 (2006). 5. Zavaleta, E. S. & Hulvey, K. B. Realistic species losses disproportionately reduce grassland resistance to biological invaders. Science 306, 1175–1177 (2004). y gy 39. Breiman, L. Random forests. Mach. Learn 45, 5–32 (2001). 6. Bracken, M. E. S. & Low, N. H. N. Realistic losses of rare species disproportionately impact higher trophic levels. Ecol. Lett. 15, 461–467 (2012). 40. Tyberghein, L. et al. Bio-ORACLE: a global environmental dataset for marine species distribution modelling. Glob. Ecol. Biogeogr. 21, 272–281 (2012). p disproportionately impact higher trophic levels. Ecol. Lett. 15, 461–467 (2012). ff d d l d ff h ld 41. Silverman, B. W. Density Estimation for Statistics and Data Analysis (Chapman & Hall, 1986). 7. Duffy, J. E., Godwin, C. M. & Cardinale, B. J. Biodiversity effects in the wild are common and as strong as key drivers of productivity. Nature 549, 261–264 (2017). 42. Liaw, A. & Wiener, M. Classification and regression by randomForest. R. N. 2, 18–22 (2002). 8. Estes, J. A., Heithaus, M., McCauley, D. Acknowledgements We acknowledge the many Reef Life Survey (RLS) divers who participated in data collection; Sue Baker, Hugh Sweatman, Beth Strain, and Russell Thomson for productive discussions; Jeremy Fox for constructive comments on an earlier draft; and Katie May Laumann and Berlioz the cat for moral support. J.S.L. was supported by the Michael E. Tennenbaum Secretarial Scholar gift to the Smithsonian Institution. G.J.E. and R.D.S.-S. were supported by the Australian Research Council, Institute for Marine and Antarctic Studies, and the Marine Biodiversity Hub, a collaborative partnership supported through the Australian Government’s National Environmental Science Program. J.E.D. was supported by the Tennenbaum Marine Observatories Network. D.B.R. was supported by the Maxwell Hanrahan Foundation. A.E.B. was supported by the Canada Research Chairs program. This is contribution #92 from the Tennenbaum Marine Observatories Network and MarineGEO program. We acknowledge the many Reef Life Survey (RLS) divers who participated in data collection; Sue Baker, Hugh Sweatman, Beth Strain, and Russell Thomson for productive discussions; Jeremy Fox for constructive comments on an earlier draft; and Katie May Laumann and Berlioz the cat for moral support. J.S.L. was supported by the Michael E. Tennenbaum Secretarial Scholar gift to the Smithsonian Institution. G.J.E. and R.D.S.-S. were supported by the Australian Research Council, Institute for Marine and Antarctic Studies, and the Marine Biodiversity Hub, a collaborative partnership supported through the Australian Government’s National Environmental Science Program. J.E.D. was supported by the Tennenbaum Marine Observatories Network. D.B.R. was supported by the Maxwell Hanrahan Foundation. A.E.B. was supported by the Canada Research Chairs program. This is contribution #92 from the Tennenbaum Marine Observatories Network and MarineGEO program. 10. Cardinale, B. J. et al. The functional role of producer diversity in ecosystems. Am. J. Bot. 98, 572–592 (2011). 11. Fox, J. W. Using the Price Equation to partition the effects of biodiversity loss on ecosystem function. Ecology 87, 2687–2696 (2006). 12. Fox, J. W. & Kerr, B. Analyzing the effects of species gain and loss on ecosystem function using the extended Price equation partition. Oikos 121, 290–298 (2012). 13. Winfree, R., Fox, J. W., Williams, N. M., Reilly, J. R. & Cariveau, D. P. Abundance of common species, not species richness, drives delivery of a real- world ecosystem service. Ecol. Lett. 18, 626–635 (2015). 14. Genung, M. A. et al. The relative importance of pollinator abundance and species richness for the temporal variance of pollination services. Ecology 98, 1807–1816 (2017). 15. NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-27212-9 NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-27212-9 Reporting Summary. Further information on research design is available in the Nature Research Reporting Summary linked to this article. 25. Loreau, M. & Hector, A. Partitioning selection and complementarity in biodiversity experiments. Nature 412, 72–76 (2001). biodiversity experiments. Nature 412, 72–76 (2001). 26. Clark, A. T. et al. How to estimate complementarity and selection effects from an incomplete sample of species. Methods Ecol. Evol. 10, 2141–2152 (2019). Data availability 27. McCauley, D. J. et al. Marine defaunation: animal loss in the global ocean. Science 347, 1255641 (2015). y The data generated in this study have been deposited in the figshare database at: https:// doi.org/10.25573/serc.16847029. 28. Mora, C. et al. Global human footprint on the linkage between biodiversity and ecosystem functioning in reef fishes. PLoS Biol. 9, e1000606 (2011). References J., Rasher, D. B. & Worm, B. Megafaunal impacts on structure and function of ocean ecosystems. Ann. Rev. Environ. Resour. 41, 83–116 (2016). 43. R: a language and environment for statistical computing (R Core Team, 2021). 9. Mellin, C. et al. Humans and seasonal climate variability threaten large-bodied coral reef fish with small ranges. Nat. Commun. 7, 10491 (2016). Author contributions G.J.E., R.D.S.-S. led the data collection; J.S.L., A.F.A., G.J.E., R.D.S.-S., A.E.B., C.W., S.J.B., S.K., S.D.L., J.E.D. and D.B.R. developed the concepts; J.S.L. and A.F.A. ran the analyses; J.S.L. and A.F.A. wrote the paper with input from all authors. G.J.E., R.D.S.-S. led the data collection; J.S.L., A.F.A., G.J.E., R.D.S.-S., A.E.B., C.W., S.J.B., S.K., S.D.L., J.E.D. and D.B.R. developed the concepts; J.S.L. and A.F.A. ran the analyses; J.S.L. and A.F.A. wrote the paper with input from all authors. 16. Hooper, D. U. et al. A global synthesis reveals biodiversity loss as a major driver of ecosystem change. Nature 486, 105–108 (2012). y g 17. Kinlan, B. P. & Gaines, S. D. Propagule dispersal in marine and terrestrial environments: a community perspective. Ecology 84, 2007–2020 (2003). p g The authors declare no competing interests. 18. Duffy, J. E., Lefcheck, J. S., Stuart-Smith, R. D., Navarrete, S. A. & Edgar, G. J. Biodiversity enhances reef fish biomass and resistance to climate change. Proc. Natl Acad. Sci. USA 113, 6230–6235 (2016). Acknowledgements Genung, M. A., Fox, J. & Winfree, R. Species loss drives ecosystem function in experiments, but in nature the importance of species loss depends on dominance. Glob. Ecol. Biogeogr. 29, 1–11 (2020). Received: 17 July 2020; Accepted: 8 November 2021; ecosystem services. Front. Ecol. Environ. 7, 204–211 (2008). 32. Edgar, G. J. et al. Global conservation outcomes depend on marine protected areas with five key features. Nature 506, 216–220 (2014). 33. Topor, Z. M., Rasher, D. B., Duffy, J. E. & Brandl, S. J. Marine protected areas enhance coral reef functioning by promoting fish biodiversity. Conserv. Lett. 12, 1–9 (2019). ARTICLE ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-27212-9 We repeated this for each community to yield a simulated “observed richness” site-by-species matrix with the same dimensions as the original matrix. This procedure ensures that richness is held at the observed levels and that the biomass contribution of each species are within the observed range. g g although this additional level of aggregation did not qualitatively change our results (Supplementary Fig. 6). We have chosen to present the raw values in the main text to demonstrate the full range of variability inherent in the individual comparisons, which might otherwise be condensed by showing only the means for each reference site. We repeated the analysis over multiple spatial radii to assess whether the spatial extent and therefore the size and composition of the species pool, might influence our results. These communities were intentionally constructed randomly with respect to composition as our goal was to test whether the observed compositional effects in the real data are significantly different than under this null hypothesis with respect to composition. Thus, using the simulated “observed richness” site-by-species matrix, we computed the (scaled) components as we had with the real data and took their means across all communities. We repeated the randomization procedure 1000 times to yield 1000 total average values of each component. We compared the observed mean to the distribution of expected means using a one- tailed t-test to determine whether the observed components were more or less extreme than would be expected by chance. We calculated the relative strength of the total diversity effect vs. the context- dependent effect for each comparison as the ratio of DIV/CDE, and of compositional vs. richness losses as: Q ¼ ð
suBδB
suB
zcBÞ
suB
zcB ¼
zuB
zcB ð4Þ 7 MMUNICATIONS | (2021) 12:6875 | https://doi.org/10.1038/s41467-021-27212-9 | www.nature.com/naturecommunications Code availability 29. Graham, N. A. J. et al. Human disruption of coral reef trophic structure. Curr. Biol. 27, 231–236 (2017). The code to reproduce all analyses has been deposited in the figshare database at: https:// doi.org/10.25573/serc.16847029. 30. Cinner, J. E. et al. Bright spots among the world’s coral reefs. Nature 535, 416–419 (2016). 31. Palumbi, S. R. et al. Managing for ocean biodiversity to sustain marine ecosystem services. Front. Ecol. Environ. 7, 204–211 (2008). 31. Palumbi, S. R. et al. Managing for ocean biodiversity to sustain marine ecosystem services Front Ecol Environ 7 204 211 (2008) Received: 17 July 2020; Accepted: 8 November 2021; Received: 17 July 2020; Accepted: 8 November 2021; Received: 17 July 2020; Accepted: 8 November 2021; Additional information 19. Graham, N. A. J. et al. Dynamic fragility of oceanic coral reef ecosystems. Proc. Natl Acad. Sci. USA 103, 8425–8429 (2006). Supplementary information The online version contains supplementary material available at https://doi.org/10.1038/s41467-021-27212-9. Supplementary information The online version contains supplementary material available at https://doi.org/10.1038/s41467-021-27212-9. Supplementary information The online version contains supplementary material available at https://doi.org/10.1038/s41467-021-27212-9. 20. Jackson, J. B. C. et al. Historical overfishing and the recent collapse of coastal ecosystems. Science 293, 629–637 (2001). Correspondence and requests for materials should be addressed to Jonathan S. Lefcheck. Correspondence and requests for materials should be addressed to Jonathan S. Lefcheck. y 21. Zgliczynski, B. J. & Sandin, S. A. Size-structural shifts reveal intensity of exploitation in coral reef fisheries. Ecol. Indic. 73, 411–421 (2017). Peer review information Nature Communications thanks Hudson Pinheiro and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available. Peer review information Nature Communications thanks Hudson Pinheiro and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available. 22. Stuart-Smith, R. D. et al. Integrating abundance and functional traits reveals new global hotspots of fish diversity. Nature 501, 539–542 (2013). 23. Stevenson, C. et al. High apex predator biomass on remote Pacific islands. Coral Reefs 26, 47–51 (2007). Reprints and permission information is available at http://www.nature.com/reprints Reprints and permission information is available at http://www.nature.com/reprints f 24. Cardinale, B. J. et al. Biodiversity loss and its impact on humanity. Nature 489, 326–326 (2012). 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. 8 NATURE COMMUNICATIONS | (2021) 12:6875 | https://doi.org/10.1038/s41467-021-27212-9 | www.nature.com/naturecommunications ARTICLE © The Author(s) 2021, corrected publication 2021 NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-27212-9 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/ licenses/by/4.0/. NATURE COMMUNICATIONS | (2021) 12:6875 | https://doi.org/10.1038/s41467-021-27212-9 | www.nature.com/naturecommunications

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Exemples de recadrage du discours direct en français. Pour une approche historique du discours théâtral

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SHS Web of Conferences 8 (2014) DOI 10.1051/shsconf/20140801033 © aux auteurs, publié par EDP Sciences, 2014 Congrès Mondial de Linguistique Française – CMLF 2014 SHS Web of Conferences Exemples de recadrage du discours direct en français Pour une approche historique du discours théâtral Cigada, Sara Università Cattolica del Sacro Cuore (Milan, Italie) [email protected] 1 Introduction Pour cet article, nous avons voulu reprendre une suggestion proposée par Bernard Combettes lors du CMLF de 2012. Il avait souligné que les structures linguistiques changent de format et de fonction dans le temps. Ce phénomène est facilement appréciable, par exemple, pour ce qui concerne le lexique: un mot change de forme et de sens, sous l'influence du changement phonétique, de phénomènes systémiques déclenchés par l'interférence d’autres langues (emprunts qui « occupent » p. ex. un espace sémantique en modifiant par conséquent la valeur des mots qui font partie de cet espace), de l'usage et de besoins communicatifs liés au contexte historique, politique etc. Étudier l'histoire d'un lexème suppose donc la considération de chacun de ces niveaux, et d'autres, selon les cas. Combettes soulignait que ce même procédé devrait être appliqué à d'autres structures linguistiques, textuelles et, ajoutons-nous, discursives. Il parlait par exemple de la structure et de la fonction de la description : si nous considérons la description des vêtements d’Énide habillée par Guenièvre dans le roman de Chrétien de Troyes, nous remarquons que le temps de la narration se prolonge d'une manière qui, aujourd'hui, apparaît peu naturelle, excessive. Si ces passages sont linguistiquement précieux, comme source documentaire sur les habits de l’époque, ils produisent aussi sur nous un effet d'extranéité presque physique : le texte s’avère aujourd'hui peu « proportionné ». On pourrait dire la même chose à propos des dialogues de la Princesse de Clèves, dans lesquels les personnages dissertent de leurs sentiments réciproques (Cigada 2005 : 212-213). Pour s’emparer des présupposés communicatifs permettant d'interpréter ces longues répliques, il ne suffit pas de rentrer dans le monde du récit : celui-ci est en effet clair, tout comme les sentiments et les relations qu’il inclut. Du point de vue du contenu nous percevons, il est vrai, une distance, par exemple entre la fidélité inconditionnelle de la jeune princesse de Clèves à son mari, vivant puis défunt, contre son sentiment même, et la pratique actuelle de la fidélité dans le mariage. Mais nous comblons cette distance – de manière un peu hâtive et superficielle peut-être – par des clichés historico-culturels. Ce qui nous demeure vraiment étranger, par contre, est la manière dont les personnages parlent, en termes de quantité, à cause de la longueur des répliques rapportées et des nombreux détails qu’elles contiennent. L'effet de distance relève autant (ou plus) de ce niveau, que de celui du contenu. À ce propos, deux implications de la suggestion de Combettes nous ont frappée. 1. D'abord, son idée d'étendre une méthodologie de recherche bien connue, en l’appliquant à des niveaux de la construction du texte (ou du discours) moins étudiés sous cette perspective. Parcourir systématiquement ce chemin pourrait donner comme résultat quelque chose de semblable à la compétence qu'un linguiste exploite pour l'analyse d'un lexème : il a une multitude de remarques à faire – et de questions à poser – devant n'importe quel mot, car il a une connaissance systématique des phénomènes divers qui peuvent l’affecter dans son évolution historique. Il domine en effet une méthodologie (ou une pluralité de méthodologies) d'analyse lexicale, permettant de questionner un mot de manière pertinente. La perspective de développer une compétence analogue concernant d'autres niveaux de la construction du discours a sans aucun doute de l'attrait. 2. Le deuxième aspect concerne la langue française, l’objectif étant d’étudier l'identité « française » des phénomènes linguistiques analysés. En effet, la démarche préconisée par Combettes épouse cette perspective, car la comparaison entre les différents « états de langue » permettrait de mieux connaître la langue française en soi, mais elle permettrait aussi d’y discerner des états linguistiquement et Article en accès libre placé sous licence Creative Commons Attribution 4.0 (http://creativecommons.org/licenses/by/4.0) Article available at http://www.shs-conferences.org or http://dx.doi.org/10.1051/shsconf/20140801033 1937 Congrès Mondial de Linguistique Française – CMLF 2014 SHS Web of Conferences SHS Web of Conferences 8 (2014) DOI 10.1051/shsconf/20140801033 © aux auteurs, publié par EDP Sciences, 2014 culturellement différents, tout en demeurant dans les limites de la même langue-culture française. Nous nous proposons en effet d'analyser le discours direct en tant que structure discursive se manifestant dans des « cultures » éloignées, en diachronie plutôt qu'en synchronie. Nous dressons donc l'hypothèse qu’il est possible de reconnaître des « états de culture » correspondant à des « états de langue ». 2 Objet linguistique de la recherche Comme objet de cette recherche, nous avons choisi le discours rapporté (dorénavant DR) direct dans un contexte particulier, celui du discours de l'action dramatique dans le théâtre français. Attention : nous ne songeons pas du tout au théâtre comme « texte en discours direct ». Notre objet concerne le fait qu'un personnage rapporte la parole d'un autre, en le citant en discours direct (dorénavant DD). Un cas exemplaire : dans Phèdre, la mort d'Hippolyte ne se déroule pas sur scène ; elle est racontée à son père, Thésée, par Théramène (§ 4.5 de cet article), qui insère dans son récit les derniers mots prononcés par le jeune prince mourant, en DD : après avoir rapporté les derniers mots d’Hippolyte, Théramène reprend la parole pour terminer son récit. Racine utilise donc le DD comme l’un des éléments de ce récit. Nous avons choisi le théâtre à cause de la pertinence, qualitative et quantitative, de ce genre dans l'histoire de la culture française. Il est relativement facile d'établir un (ou plusieurs) corpus, par auteur, par époque, par typologie... et donc d'utiliser ces textes de manière méthodologiquement correcte pour une recherche, même un peu « quantitative-mécanique », au départ du moins. Nous nous référons, par exemple, à la possibilité de chercher de manière automatique les guillemets qui ouvrent les passages en DD en utilisant des éditions numériques des ouvrages, même si cela n’est pas toujours suffisant, comme nous le verrons plus loin. En outre, l'interdiscours du théâtre est extrêmement riche : il suffit de penser aux textes des auteurs qui commentent ou expliquent leurs pièces, à la critique, à l'histoire des mises en scène, aux déclarations d’acteurs fameux qui ont joué ces rôles..., mais aussi aux rapports explicites entre théâtre et éducation, théâtre et idéologie, théâtre et pouvoir… 2.1 Le DD dans le discours théâtral Du point de vue de sa nature discursive, le DD enchâssé dans le discours théâtral exalte la nature dramatique que Bakhtine attribue même « à l’énoncé le plus simple » (Todorov 1981 : 75-76). Si le discours théâtral est conçu – dès sa formulation – pour réaliser une action (drame), la mise en scène du DD dans le drame multiplie les effets discursifs. En outre, la récitation pose à l’acteur le problème de mettre en scène la voix qu’il cite. Cet aspect est même inscrit dans ce type de DD et il serait très intéressant de réaliser un travail d’analyse du discours pour comparer les différentes performances de grands acteurs dont nous possédons les enregistrements, pour étudier les manières qu’ils ont inventées pour réaliser ce type d’action ainsi que les discours concernant ce sujet. En même temps, l’étude de la voix au sens physique du mot nous rapprocherait de manière très pertinente de ce que Bakhtine évoque en parlant de l’intonation (Todorov 1981 : 194 ss ; 301 ss et passim). Nous ne voulons pas, pour autant, borner notre regard à l’organisation polyphonique de ces discours, au sens bakhtinien de « discours littéraire » (cf. Nowakowska 2005 : 23-24 et passim), mais surtout prendre en compte leur nature dialogique. Étudier la dialogisation dans le contexte du discours théâtral pose un problème, parce que (selon la caractérisation fournie par Bres 2005 : 55) le discours-pièce manifeste une nature monologale (en tant qu’il est créé par un seul auteur). Nous en considérons toutefois le dialogisme (en tant que discours s’adressant à un auditoire dont la « culture » – y compris la langue – est commune à l’auteur et prise en charge par celui-ci). La question est justement là : si la manière d’organiser le discours (comme par exemple la manière d’insérer les DD ou les autres paramètres que nous allons étudier) change dans le temps, il faut pouvoir saisir et « mesurer » ce changement. Ce changement concerne le système langueculture, qui appartient en commun aux interlocuteurs (dans notre cas, à l’auteur de la pièce et à son auditoire). Or, la « manière d’organiser le dialogisme » constitue un phénomène beaucoup plus vaste que l’insertion d’un DD dans la réplique d’une pièce. Toutefois, justement du fait que le DR peut être 1938 Article en accès libre placé sous licence Creative Commons Attribution 4.0 (http://creativecommons.org/licenses/by/4.0) SHS Web of Conferences 8 (2014) DOI 10.1051/shsconf/20140801033 © aux auteurs, publié par EDP Sciences, 2014 Congrès Mondial de Linguistique Française – CMLF 2014 SHS Web of Conferences considéré comme « la partie émergée de l’iceberg dialogisme » (Bres 2005 : 59), nous avons expérimenté cette voie pour sonder notre hypothèse. Du point de vue linguistique, en effet, l'objet DD est « plus facile » à saisir, car il a une forme linguistique (dont la manifestation est morphosyntaxique, mais aussi typographique) assez déterminée – encore plus que d'autres types de discours rapportés donc – ce qui donne un ancrage solide à la recherche pour ce qui concerne l’individuation des données pertinentes. En même temps, ce type de DD présente un rapport immédiat soit avec le dialogisme, soit avec la polyphonie, éléments centraux de l'organisation du discours. En effet, la stratégie discursive en DD que nous souhaitons étudier se trouve au carrefour entre polyphonie et dialogisme dans les sens identifiés par Rosier (2008 : 38-40) : il y a, dans ces occurrences du DD, du dialogisme, à savoir « la capacité de l’énoncé à faire entendre, outre la voix de l’énonciateur, une (ou plusieurs) autre(s) voix qui se hiérarchise(nt) du point de vue énonciatif », mais aussi de la polyphonie car l’effet de « mise en scène des différents points de vue et voix dans le discours » est, si l’on peut dire, réalisé au pied de la lettre. Sur dialogisme vs polyphonie nous renvoyons aussi à Bres – Mellet (2009 : 6-9) : dans le contexte du discours que nous étudions, la « conception théâtrale de l’énonciation » n’est donc pas prise au sens métaphorique. Cependant, il nous semble évident qu’il ne serait pas trop raisonnable de nous proposer d’ores et déjà un résultat théorique, du fait qu’il faut tout d’abord vérifier le côté empirique : nous exploitons donc les catégories issues des travaux cités, de manière cohérente, pour vérifier si les données linguistiques subsistent. Pour ce qui concerne le « cadre énonciatif », nous considérons que, grâce au DD, les répliques des énonciateurs-personnages (ces répliques constituant le discours-pièce) enchâssent dans l’énonciation principale d’autres énonciateurs (Rosier 2008 : 40 et passim) et leurs énoncés. On remarque que, par conséquent, l’organisation énonciative change selon les rapports qui relient les énonciateurs entre eux, aux différents niveaux d’enchâssement. Les ‘recadrages’ énonciatifs dépendent soit de la structure de l’enchâssement, soit de la nature énonciative du DR (modalité et temps du verbe citant). L’intérêt de cette analyse dépend tout d’abord de l’organisation de l’enchâssement, parce que les énonciateurs cités en DD peuvent coïncider ou non avec les personnages. Pour ce qui concerne l’auteur et l’auditoire/lecteur (c’est-à-dire le producteur et le destinataire physiques du discours théâtral), nous en considérons l’existence comme un présupposé, ainsi que le caractère monogal de la pièce, la nature fictive des dialogues, du ‘recadrage’ et du caractère dialogal du discourspièce (cf. § 2.1) : les énonciateurs et les énoncés « enchâssés » ne représentent en effet que des « feuilletages » de l’auteur et du discours-pièce1. Finalement, nous avons adopté une terminologie qui permet de repérer le plus immédiatement possible le cadre énonciatif : énonciateur-personnage et allocutaire-personnage ; énoncé ou discours enchâssant ; énonciateur et allocutaire du discours enchâssé, etc. Tous ces phénomènes ne relèvent que du dialogisme. 2.2 Repères bibliographiques À propos du DD nous soulignons tout d’abord sa nature « intermédiaire » – en tant que linguistique – entre structure et fonction : Rigotti – Rocci (2006), ce qui justifie sa présence dans des discours très variés, un certain dégré de souplesse de sa structure et sa versatilité par rapport aux fonctions qu’il peut remplir (cf. Cigada 2012a). Sur la structure linguistique du DD en tant que forme particulière du DR, cf. Rosier (2008). À propos de l'autonymie comme caractéristique du DD, cf. la bibliographie citée dans Cigada (2012a). La distinction entre « autonymie de langue » et « autonymie de parole » se montre intéressante par rapport au corpus étudié dans cet article, surtout pour comparer quelques uns des DD, plus évidemment « autonymiques », avec d’autres qui le paraissent moins. Nous n’allons toutefois pas parcourir ce chemin. Il faut dire aussi que le phénomène spécifique que nous avons abordé ne semble pas avoir été traité de manière systématique auparavant : cf Siouffi (2013 :141) sur le Cid, ou Dufiet (2005) qui parle de « prouesse discursive » à propos de Molière, ou encore Petitjean (2005) sur Marivaux. Dans le joli volume Le discours rapporté dans tous ses états édité par Lopez Muños, Marnette et Rosier (2004), ce type de DD n'est pas cité. La reprise en écho revient dans Granier (2003 : 217-231) et Salvan (2005) : par Article en accès libre placé sous licence Creative Commons Attribution 4.0 (http://creativecommons.org/licenses/by/4.0) 1939 Congrès Mondial de Linguistique Française – CMLF 2014 SHS Web of Conferences SHS Web of Conferences 8 (2014) DOI 10.1051/shsconf/20140801033 © aux auteurs, publié par EDP Sciences, 2014 ailleurs, Sorin Stati affrontait déjà le problème (1991 : 56-58), à propos de la « fonction écho », avec un exemple tiré de Cocteau. Authier-Revuz (2003 : 86-87) considère la scène du Misanthrope qui se base sur l'autocitation. Pour ce qui concerne les fonctions de ce type de DD, Adam (1992 : 188-192) ne considère pas l'insertion d'un DD dans le récit de Théramène, quand il analyse la mort d'Hippolyte. Il considère par contre ‘Le récit dans la conversation’ (Adam 1992 : 172-175). Adam considère aussi le dialogue entre Agnès et le jeune homme rapporté par Agnès à Arnolphe en DD (Adam 1992 : 172-175). L’auteur n’étudie toutefois que l’organisation interne du récit, et plus particulièrement la fonction des questions et des commentaires d’Arnolphe, qui orientent la construction de la narration d’Agnès : de fait, Adam réduit le DD à un élément quelconque du récit d’Agnès. Comme l'affirme Calaseru en clôture de son livre Testuali parole (2004 : 211), « Il discorso riportato ci appare come un punto di osservazione strategico anche per il discorso in genere» (cependant, Calaresu ne considère pas le type d'objet dont nous nous occupons ici)2. 3 But, méthode et plan de l’article Ce que nous nous proposons donc ici est d’émettre une première hypothèse pour vérifier si la suggestion de Combettes donne un résultat intéressant et, d'autre part, de mettre à nouveau en cause la structure linguistique du DD et les fonctions qu'elle peut remplir, dans le contexte d'un discours précis, celui de l'action théâtrale. Le but de cet article est partant de sonder le terrain, pour donner un aperçu général de la faisabilité d’une telle recherche. Nous avons commencé ce sondage avec beaucoup de liberté par l’analyse de sept pièces, sans tenir compte du genre auquel elles appartiennent. En voici la liste et la date de leur création : 1637 le Cid (Pierre Corneille) ; 1666 le Misanthrope (Molière) ; 1677 Phèdre (Jean Racine) ; 1725 l’Île des esclaves (Marivaux) ; 1897 Cyrano de Bergerac (Edmond Rostand) ; 1928 Topaze (Marcel Pagnol) ; 1953 En attendant Godot (Samuel Beckett). Mais, après une longue réflexion, nous avons résolu de présenter les exemples suivant l’ordre diachronique renversé, du plus récent au plus ancien. La démarche est discutable, mais méditée : cette disposition permet de percevoir plus aisément, à notre avis, les différences entre les dialogismes. Dans chacune de ces pièces, nous avons pris en compte la totalité des DD. Si quelques exemples n’ont pas été analysés, nous l’avons signalé. La recherche automatique n’est en fait pas possible, car le DD, tout en étant reconnaissable grâce à sa structure syntaxique et discursive, n’est pas toujours marqué du point de vue typographique par des guillemets. Le discours rapporté en DD peut être oral ou bien écrit. Pour l’analyse, nous avons considéré la fréquence et la distribution des DD dans la pièce, les aspects morphologiques, le cadre énonciatif et ses variations/ recadrages et les aspects pragmatique-fonctionnels, selon une grille en quatre points que nous proposons ci-dessous. 1. Fréquence des DD et leur distribution. Les DD se trouvent distribués de manière inégale tout au long des pièces et d’une pièce à l’autre : ces données, concernant la fréquence des DD dans chaque pièce et leur collocation préférentielle, pourraient être pertinentes pour évaluer le changement de l’organisation discursive théâtrale au cours du temps. Nous avons partant mis en relief cet aspect, de manière descriptive. 2. Aspects morphosyntaxiques (concernant le cadre énonciatif et l’énoncé en DD). Modes et temps du verbe citant3 . À l’intérieur de l’énoncé en DD : termes d’adresse ; formes de politesse et usage de tutoiement-vouvoiement ; exclamations/interjections ; phrases interrogatives, impératives, exclamatives. 3. Cadre énonciatif. Autour du DD, les répliques des énonciateurs-personnages (fictifs) enchâssent dans l’énonciation principale d’autres ‘énonciateurs’ (fictifs) et leurs énoncés. Nous avons étudié les relations entre les énonciateurs aux différents niveaux d’enchâssement (identité ou échange de rôle énonciatif entre personnages, énoncés exogènes). Le dialogisme en est élaboré de manière différente dans les différentes pièces. 1940 Article en accès libre placé sous licence Creative Commons Attribution 4.0 (http://creativecommons.org/licenses/by/4.0) SHS Web of Conferences 8 (2014) DOI 10.1051/shsconf/20140801033 © aux auteurs, publié par EDP Sciences, 2014 Congrès Mondial de Linguistique Française – CMLF 2014 SHS Web of Conferences 4. Aspects pragmatique-fonctionnels. Réinterprétation pragmatique du DD rapporté et amalgames pragmatiques (selon l’expression de Kerbrat-Orecchioni) ; exploitation du cadre pour des effets de sens ; éthopée ; narrativisation ; argumentation ; implication émotive. Les remarques sur la manipulation rhétorique que l’on trouvera au cours de l’analyse ont affaire à ce niveau de la description du discours : en effet, la rupture morphosyntaxique et le recadrage énonciatif entrainent des effets de sens spécifiques. Un énoncé en DD peut servir par exemple d’argument, mais il peut remplir d’autres fonctions (faire poursuivre la narration, montrer le caractère d’un personnage, etc.). 4 Analyse des exemples 4.1 Godot Dans En attendant Godot, pièce en deux actes, le DD n’apparaît que dans le deuxième acte, où il est utilisé deux fois. Deux autres usages, toujours dans le deuxième acte, ont un statut ambigu, comme dans l’exemple qui suit : au début du deuxième acte, Vladimir chantonne à plusieurs reprises cette chansonnette. 4.1.1 Exemple – Un chien vint dans l’office (Acte II, pp. 79-804) Vladimir : Un chien vint dans l’office Et prit une andouillette. Alors à coups de louche Le chef le mit en miettes. Les autres chiens ce voyant Vite vite l’ensevelirent Au pied d’une croix en bois blanc Où le passant pouvait lire : Un chien vint dans l’office… Il n’y a pas de verbe citant. L’énonciateur-personnage est seul sur scène, il n’y a pas d’allocutaire. L’énonciateur-cité a un statut indéfini, que l’on ne peut reconstruire que par la référence à plusieurs éléments de nature culturelle et contextuelle (voir plus bas). L’énoncé enchâssé ne s’adresse à personne. Le sujet de l’énoncé cité n’a aucun rapport avec les énonciateurs, à aucun niveau du discours. L’énoncé cité comporte un enchâssement secondaire : des énonciateurs (les autres chiens) et un allocutaire (le passant). L’énoncé cité est constitué par le texte écrit sur la croix en bois blanc, qui répète l’énoncé enchâssant. Virtuellement, cette structure se répète à l’infini. On pourrait considérer la mélodie comme marquant le statut discursif différent. Du point de vue fonctionnel, ce discours commence comme un récit d’évènements, mais se poursuit comme un récit de paroles (écrites en épitaphe sur le tombeau du chien) : ce DR, à l’oral, se répète à l’infini grâce à sa structure de cité-et-citant ou de mise en abyme. La mélodie de la chanson, que Beckett aurait traduite de l’allemand au français (et ensuite en anglais), est reprise du Carnaval de Venise straussien5 (cf. Sebellin 2003 : 41-58). La superposition de plusieurs éléments, dont quelques-uns oxymoriques (mélodie fameuse, écrite en souvenir du violoniste feu Heinrich W. Ernst, ami de Strauss ; mélodie connue sous le titre de ‘Carnaval’ de Venise ; mélodie fantaisiste et légère, bâtie sur la variation sur un thème très simple et employée pour une chanson enfantine ; contenu violent de l’histoire racontée et référence à la mort ; construction répétitive circulaire à l’infini ; traduction des paroles de l’allemand en français), fait peut-être de ce DD (s’il l’est) la clef de voûte de la pièce, du point de vue de l’exploitation discursive du dialogisme et de la polyphonie. Nous proposons en effet de considérer cette chanson comme du DR en DD, même si ce statut n’est pas indiscutable à cause de la nature autonymique du discours cité. Vladimir répète un discours dont on ne connaît pas le premier énonciateur, un discours d’enfant, de tous et de personne6. Article en accès libre placé sous licence Creative Commons Attribution 4.0 (http://creativecommons.org/licenses/by/4.0) 1941 Congrès Mondial de Linguistique Française – CMLF 2014 SHS Web of Conferences 4.1.2 SHS Web of Conferences 8 (2014) DOI 10.1051/shsconf/20140801033 © aux auteurs, publié par EDP Sciences, 2014 Exemple – Dis, Pense, cochon ! (Acte II, p. 103) Vladimir : Dis-moi de penser. Estragon : Comment ? V. : Dis, Pense, cochon ! E. : Pense, cochon ! V. : Je ne peux pas ! Ici l’acte de parole est ordonné en DD et ensuite exécuté. Le discours citant contient en effet un impératif (dis) qui exprime un ordre. Marque typographique : Pense, avec majuscule. Terme d’adresse : cochon. Du point de vue de l’organisation énonciative, l’énonciateur-personnage (Vladimir) s’adresse à l’allocutairepersonnage (Estragon) en lui demandant d’exécuter un énoncé en DD dont l’allocutaire (Estragon) sera l’énonciateur (Estragon), et l’énonciateur (Vladimir) sera l’allocutaire (Vladimir. Estragon dira donc a Vladimir « Pense, cochon !). Un enchâssement de la sorte présente une structure assez inusitée, sauf, peut-être, dans les jeux d’enfants : la reprise du DD précède-t-elle le DR même ? Le dialogisme en est-il déformé ? Remarquons par ailleurs que le DD ordonné exprime lui aussi un ordre, manifesté à l’impératif (Pense !). Toute cette scène pourrait être considérée du point de vue discursif comme la reprise de la rencontre avec Pozzo et Lucky du premier acte, du fait qu’elle est introduite par la proposition de Vladimir « On pourrait jouer à Pozzo et Lucky » (p. 102). 4.1.3 Exemple – Memoria praeteritorum bonorum (Acte II, p. 121) Vladimir : […] laisse-le tranquille. Ne vois-tu pas qu’il est en train de se rappeler son bonheur. (Un temps.) Memoria praeteritorum bonorum – ça doit être pénible. Il n’y a pas de verbe introducteur, mais la citation est marquée soit typographiquement, par l’italique, soit prosodiquement, par la pause indiquée dans la glose. Toutefois, comme pour 4.1.1, le statut de cette citation est ambigu. L’énonciateur reprend en variation (renversement sémantique) le dicton de Cicéron « etiam iucunda est memoria praeteritorum malorum » (« le souvenir des maux passés est agréable », Cic. de Finibus bonorum et malorum, II, 105, texte retrouvé sur Perseus, qui devient donc « le souvenir des biens passés est pénible »). Cicéron citerait lui-même un proverbe grec issu d’un vers d’Euripide. Même si l’énoncé n’est pas attribué, il est donc possible d’en reconstruire le premier énonciateur ; en tant que doxa, toutefois, l’énoncé n’appartient à personne et s’adresse à tous. La fonction de cet énoncé est finalement argumentative : il sert à justifier l’ordre que Vladimir donne à Estragon de ne pas déranger Pozzo. 4.1.4 Exemple – Un autre me regarde, en se disant, Il dort (Acte II, p. 128) Le DD n’est pas marqué typographiquement, mais la majuscule ainsi que la structure syntaxique l’indiquent de manière claire, comme dans 4.1.2. Le verbe introducteur est en disant, marqué par la réflexivité (se). Vladimir : […] Moi aussi, un autre me regarde, en se disant, Il dort, il ne sait pas, qu’il dorme. (Un temps.) L’énonciateur (un autre) n’est pas précisé ; il s’adresse à lui-même. Dans cet exemple, l’effet discursif est curieux parce que l’énonciateur-personnage qui rapporte le DD (Vladimir) est censé dormir, ce qui impliquerait qu’il n’a pas pu entendre le discours de « l’autre ». En fait il ne dort pas mais parle. On pourrait dire que ce dialogisme est fonctionnel à l’absurde, parce que l’absurde relève de l’emploi insensé du cadre énonciatif et non pas du contenu du DD7. La rupture du dialogisme rompt en effet la distinction entre la réalité et le rêve. 1942 Article en accès libre placé sous licence Creative Commons Attribution 4.0 (http://creativecommons.org/licenses/by/4.0) SHS Web of Conferences 8 (2014) DOI 10.1051/shsconf/20140801033 © aux auteurs, publié par EDP Sciences, 2014 4.2 Congrès Mondial de Linguistique Française – CMLF 2014 SHS Web of Conferences Topaze Dans la comédie Topaze, Marcel Pagnol insère plusieurs DR directs et aussi des textes écrits qui sont lus sur la scène par Topaze (une lettre anonyme ; un article de journal et le document qui accompagne les Palmes). Les DD se trouvent en quantité majoritaire dans le troisième et le quatrième acte8. Dans les exemples 4.2.1, 4.2.2, 4.2.3 Topaze, hanté par les abus qu’il est en train de commettre, voit partout des accusateurs. 4.2.1 Exemple – Tripoteur ! Tripoteur ! Tripoteur ! (Acte III, scène 2, p. 167) Topaze : […] le bruit de leurs moteurs m’arrivait encore, et savez-vous ce qu’ils disaient, ces moteurs ? Ils disaient : « Tripoteur ! Tripoteur ! Tripoteur ! » Et les brosses obliques, en frôlant les pavés, chuchotaient : « Topaze escroc ! Topaze escroc ! ». Les verbes introducteurs à l’imparfait signalent une action répétée (disaient, chuchotaient). Topaze s’adresse à Suzy. Les énonciateurs dont le discours est rapporté sont les moteurs et les brosses des balayeuses, objets dont la pièce s’occupe largement mais qui n’ont jamais le statut d’énonciateurs en tant que personnages, si ce n’est dans ce DD, enchâssé dans une réplique de Topaze. Les énoncés en DD concernent Topaze lui-même, qui se considère comme l’objet et la cible de ce discours (qui n’existe pas). Le DD sert donc, en fonction d’éthopée, pour animer ces énonciateurs fictifs, ce qui montre l’obsession de Topaze9. 4.2.2 Exemple – Cachât ! (Acte III, scène 2, p. 167) En fonction typiquement autonymique, nous remarquons la reprise de la correction de la forme verbale dans l’exemple suivant, et plusieurs autres reprises en écho que nous ne considérons pas : Suzy : Mon cher, si vous ne l’aviez pas compris tout de suite, vous méritiez qu’on vous le cache. Topaze : Cachât ! Suzy : Comment, cachât ? Topaze : Qu’on vous le cachât. Ainsi, vous avouez !... La reprise en écho ne prévoit pas dans ce cas-là de verbe introducteur. La phrase interrogative (Comment, cachât ?) pose cependant une question relative à l’énoncé (Cachât !) de Topaze, telle « Quel est le sens du propos que vous venez d’énoncer ? ». 4.2.3 Exemple – le sympathique idiot (Acte III, scène 2, p. 167) Topaze : […] j’entendis une conversation effroyable. Suzy : Effroyable ? Topaze : Hideuse. Mais pleine de sens pour moi. M. Castel-Bénac disait : « Chérie, pourquoi as-tu invité le sympathique idiot ? » et vous avez répondu : « Le sympathique idiot est très utile et il faut un peu l’amadouer ». Le sympathique idiot, c’était moi. Quant au mot « chérie », il m’a suffisamment renseigné sur la nature de vos relations avec cet homme. L’imparfait disait du verbe citant constitue l’arrière-plan de l’action discursive, ponctuelle, véritablement visée, vous avez répondu. À remarquer la valeur, différente, de l’imparfait habituel (p. ex. Et je disais à Mme Muche : « S’il veut partir, je le laisserai libre ! », p. 198 ; Elles me répondaient toujours : « Monsieur le directeur n’est pas là », p. 234) qui peut correspondre au présent habituel (on me répond : « Monsieur est chez son tailleur », p. 215). Encore différent, l’imparfait de Rostand (Le Duc : […] Et quelqu’un me disait, hier, au jeu, chez la Reine :/ « Ce Cyrano pourrait mourir d’un accident », Acte V, scène 2, p. 292) qui, uni au pronom quelqu’un, opacifie la source (l’identité de l’énonciateur). Le DD reproduit le tutoiement entre Castel-Bénac et Suzy, tandis que Topaze continue de vouvoyer Suzy. Article en accès libre placé sous licence Creative Commons Attribution 4.0 (http://creativecommons.org/licenses/by/4.0) 1943 Congrès Mondial de Linguistique Française – CMLF 2014 SHS Web of Conferences SHS Web of Conferences 8 (2014) DOI 10.1051/shsconf/20140801033 © aux auteurs, publié par EDP Sciences, 2014 Le dialogue rapporté en DD n’a jamais été joué sur scène : son contenu et ses conséquences sont appris par le spectateur au moment même où Topaze accuse Suzy d’avoir trompé sa confiance. Le cadre énonciatif comporte en effet Topaze et Suzy comme personnages, tandis que les deux répliques rapportées en DD par Topaze ont été respectivement prononcées par son patron (Castel-Bénac) et par sa maîtresse (Suzy). Les deux énonciateurs des énoncés enchâssés sont donc des personnages, doués de parole dans la pièce. Dans cet exemple, toutefois, le dialogue rapporté s’est déroulé entre deux énonciateurs (Suzy et Castel-Bénac) dont aucun ne coïncide avec l’énonciateur actuel (Topaze) : celui-ci l’a entendu de manière directe, mais hasardeuse, parce qu’il ne participait pas du cadre énonciatif du dialogue entre Suzy et Castel-Bénac. L’énonciateur-Topaze reconstruit donc à posteriori le cadre énonciatif de départ constitué par deux énonciateurs à plein statut plus un allocutaire à statut mineur (non intentionnel ni par les énonciateurs ni par lui-même). Le contexte exige une interprétation de littéralité presque totale du DR. Il y a en outre la reprise en écho de sympathique idiot dans la conversation entre Castel-Bénac et Suzy et ensuite autonymique dans le discours de Topaze ; chérie est une reprise autonymique du discours de Castel-Bénac insérée dans celui de Topaze. L’ensemble des informations reconstruites à partir de cette construction discursive a une fonction narrative, du fait qu’il permet de faire évoluer les rapports entre les personnages en constituant une nouvelle étape de la pièce. 4.2.4 Exemple – ce que je dirais à mes élèves (Acte IV, scène 4, p. 239) Topaze : Sais-tu ce que je dirais à mes élèves ? (Il s’adresse soudain à sa classe du premier acte.) « Mes enfants, … » La forme je dirais introduit un DD fictif, qui pourrait se dérouler dans le présent ou l’avenir. Bres – Mellet (2009 : 6 et passim) ont recours au verbe feuilleter, pour décrire le rapport multiple entre les énonciations. Si ce phénomène caractérise le discours théâtral en tant que « dialogue montré masquant un dialogisme interne, qui fait s’exprimer des voix hiérarchisées » (Salvan 2004 : 276), ce « feuilletage » est démasqué lors de l’enchâssement d’un énoncé en DD, surtout si l’énonciateur en est le même, comme dans cet exemple. Le verbe citant conditionnel (je dirais) met en relation le monde réel du discours-pièce (ce que Topaze a donc effectivement dit à ses élèves et qu’il est censé professer encore) avec le monde conditionnel du DD (ce qu’il dirait aujourd’hui s’il se trouvait à nouveau en classe et qu’il juge être le plus correct), les deux mondes étant reliés par le biais de l’énonciateur (Topaze). L’allocutaire Tamise, son ancien collègue à l’école, est témoin du changement du discours tenu par Topaze et donc du changement de l’énonciateur. Du point de vue du récit, en effet, ce DD constitue la charnière entre l’ancien et le nouveau Topaze. 4.3 Cyrano Cette pièce est très riche en DD, ce qui est assez compréhensible si l’on songe que l’action tourne autour de la parole, authentique et/ou fictive, de Cyrano et Christian. Nous n’avons choisi que quelques exemples en en laissant beaucoup d’autres de côté10. La parole « prêtée » par Cyrano à Christian soustend en effet toute la pièce (Christian : Il me faudrait de l’éloquence ! Cyrano : Je t’en prête !/ Toi, du charme physique et vainqueur, prête-m’ en/ Et faisons à nous deux un héros de roman !/ Christian : Quoi ? Cyrano : Te sentirais-tu de répéter les choses/ Que chaque jour je t’apprendrais ?... Acte II, scène 10, p. 155). Le dialogisme joue un rôle décisif dans la scène du balcon, dont l’effet émotif découle de l’identification retrouvée entre l’énonciateur (Cyrano) et l’auteur de la parole (Cyrano) : c’est en effet Cyrano qui, en se cachant dans la nuit et feignant d’être Christian, parle directement à Roxane sans qu’elle le reconnaisse (Acte III, scène 7-9, pp. 187-199). De plus, Cyrano récite souvent les lettres qu’il est en train d’écrire à Roxane ; Roxane déclame à Cyrano ces lettres dont Cyrano est le véritable auteur, en les croyant de Christian (Acte III, scène 1, p. 166-7). Finalement, l’anagnorèse est caractérisée par un dialogisme très fort : Cyrano lit à haute voix la dernière lettre qu’il a écrite à Roxane de la part de 1944 Article en accès libre placé sous licence Creative Commons Attribution 4.0 (http://creativecommons.org/licenses/by/4.0) SHS Web of Conferences 8 (2014) DOI 10.1051/shsconf/20140801033 © aux auteurs, publié par EDP Sciences, 2014 Congrès Mondial de Linguistique Française – CMLF 2014 SHS Web of Conferences Christian, dont Roxane pense toujours qu’elle a été écrite par Christian ; Roxane se rend compte en l’écoutant qu’il en connaît déjà les mots et, finalement, elle comprend que la voix de l’écrivain décédé quatorze ans auparavant, la voix écrite et lue, la voix du lecteur, sont la même : Cyrano se réapproprie ce qu’il est en train de lire et Roxane comprend qu’il l’aime et qu’il est maintenant en train de mourir (Cyrano : Et je crie : « Adieu !... ». Acte V, p. 305). Le feuilletage discursif est donc au cœur de la pièce. 4.3.1 Exemple – On pouvait dire […] vous m’auriez dit (Acte I, scène 4, pp. 72-74) Cyrano : […] On pouvait dire… Oh ! Dieu !... bien des choses en somme/ En variant le ton, – par exemple, tenez :/ Agressif : « Moi, monsieur, si j’avais un tel nez/ Il faudrait sur-le-champ que je me l’amputasse ! »/ Amical : « Mais il doit tremper dans votre tasse:/ Pour boire, faites-vous fabriquer un hanap ! »…11 Ce long passage, où le discours citant alterne avec le discours cité en en caractérisant le « ton », se conclut par un tiret qui correspond à celui qui ouvre la liste des exemples (cf. extrait ci-dessus). Après quoi Cyrano garde la parole pour terminer sa tirade : Cyrano : […] – Voilà ce qu’à peu près, mon cher, vous m’auriez dit/ Si vous aviez un peu de lettres et d’esprit/… Rostand entoure ce DD par un discours citant qui le précède et un discours citant qui le suit, les deux discours citants ayant la même fonction. Toutefois, le passage de la forme impersonnelle (on pouvait dire) à la formule de politesse à la deuxième personne du pluriel (vous m’auriez dit) joue un rôle important car il permet de viser la cible des insultes qui vont suivre. Comme dans l’exemple 4.2.4, ces verbes introducteurs modalisent le rapport entre le discours-pièce et le DD, en reliant cette fois-ci une condition présente irréelle (si vous aviez…) à une action qui n’a pas eu lieu (vous m’auriez dit). Dans le discours qu’il suggère à son allocutaire (le vicomte), Cyrano se fait vouvoyer et traiter de monsieur. Dans cet exemple le vicomte, c’est-à-dire l’énonciateur du DD qui est enchâssé dans la réplique de Cyrano, coïncide avec l’allocutaire de la réplique (le vicomte), tandis que l’allocutaire de ce DD enchâssé (Cyrano) est l’énonciateur du discours enchâssant (Cyrano). Tous les deux sont personnages de la pièce, ayant donc le statut d’énonciateurs : le DD ne fait que renverser la direction du discours (dans la réplique, Cyrano s’adresse au vicomte ; par le DD enchâssé dans la réplique, le vicomte s’adresse à Cyrano). 4.3.2 Exemple – Qu’est-ce que c’est encore que cette égratignure ? (Acte II, scène 6, p. 120) L’énonciateur (Roxane) rappelle à l’allocutaire (Cyrano) la question qu’elle lui posait autrefois lorsqu’ils étaient enfants. En ce faisant, elle découvre que le contexte réactualise la question (Cyrano a à présent la main blessée) : Roxane : […] Alors, jouant à la maman,/ Je disais d’une voix qui tâchait d’être dure :/ (Elle lui prend la main) « Qu’est-ce que c’est encore que cette égratignure ? »/ (Elle s’arrête stupéfaite) Oh ! C’est trop fort ! Et celle-ci ! Le verbe citant (je disais) comporte l’auto-citation d’un discours tenu habituellement dans un passé très éloigné. Comme dans l’exemple 4.2.4, un des résultats en est le feuilletage discursif. Toutefois, ce feuilletage se conclut ici par la découverte que la situation de référence du DD se reproduit au présent de manière identique : le feuilletage en est réduit à une seule couche, ramenant le passé au présent discursif. Énonciateur-personnage (Roxane) et allocutaire-personnage (Cyrano) coïncident tous deux avec l’énonciateur (Roxane) et avec l’allocutaire (Cyrano) enchâssés. À propos du verbe citant, remarquons aussi un des peu d’exemples comportant une note caractérisant la voix du point de vue phonétique (d’une voix qui tâchait d’être dure). Dans cet échange, la fonction de l’énoncé rapporté, au départ narrative et référée au passé, est réinterprétée comme actuellement performative : l’interrogation rapportée change de statut et le récit de parole se transforme en une question véritable. Article en accès libre placé sous licence Creative Commons Attribution 4.0 (http://creativecommons.org/licenses/by/4.0) 1945 Congrès Mondial de Linguistique Française – CMLF 2014 SHS Web of Conferences 4.3.3 SHS Web of Conferences 8 (2014) DOI 10.1051/shsconf/20140801033 © aux auteurs, publié par EDP Sciences, 2014 Exemple – Passez, señorita ! (Acte IV, scène 4, p. 244) Le DD permet de citer la parole de l’interlocuteur étranger dans sa langue, comme les Espagnols que Roxane a rencontrés sur son chemin vers le camp français : Roxane : […] Alors je répondais : « Je vais voir mon amant. »/ – Aussitôt l’Espagnol à l’air le plus féroce/ […] S’inclinait en disant : « Passez, señorita ! »/ […] J’ai dit : mon amant, oui… pardonne !/ Tu comprends, si j’avais dit : mon mari, personne/ Ne m’eût laissé passer… Comme dans 4.3.2, le verbe introducteur à l’imparfait (je répondais) signale une action discursive réitérée dans le passé. De même pour le verbe citant la réponse, où, toutefois, l’imparfait se déplace sur le verbe décrivant le geste qui accompagne le discours (s’inclinait), tandis que le verbe introducteur se trouve au gérondif (en disant). Le cadre énonciatif du DD comporte un échange dialogal entre Roxane, qui est aussi l’énonciateur de la réplique enchâssante, et l’Espagnol (plusieurs Espagnols en fait, qui lui ont posé la question au fur et à mesure qu’elle avançait dans son voyage). L’Espagnol n’a pas le statut de personnage dans la pièce, sa parole n’y entre donc que par le discours de Roxane. L’usage du terme d’adresse en espagnol (señorita), ainsi que la longue description de l’attitude du « hidalgo », suggère une interprétation ironique. Puis Roxane reprend un élément de son propre discours pour se justifier devant son époux de l’avoir traité d’amant : ce mot, repris de manière autonymique (et donc littérale), est introduit par le verbe j’ai dit et deux points, ce qui suggère plutôt une auto-citation. Ensuite, elle explique son choix en s’imaginant par l’absurde avoir dit la vérité. Ici, le verbe citant est (si) j’avais dit, exprimant une condition irréelle, suivi lui aussi des deux points et du DD mon mari. Le statut de cette occurrence est plus ambigu du fait que l’expression mon mari n’a jamais été prononcée avant, il ne s’agit donc pas d’une reprise autonymique. 4.3.4 Exemple – C’était une Fâcheuse (Acte V, scène 5, p 298) Dans ce dialogue figé, Cyrano raconte s’être adressé à la mort qui venait le chercher et ce faisant, il la désigne comme une Fâcheuse. Roxane ne comprend en effet pas de qui il parle. Cyrano : Cousine, c’était une/ Fâcheuse. Roxane : Vous l’avez renvoyée ? Cyrano : Oui, j’ai dit :/ Excusez-moi, mais c’est aujourd’hui samedi,/ Jour où je dois me rendre en certaine demeure ;/ Rien ne m’y fait manquer : repassez dans une heure ! Le verbe citant (j’ai dit) renvoie à une action discursive ponctuelle dans un passé qui est proche (cf. repassez dans une heure). Le feuilletage de l’énonciateur comporte Cyrano-personnage et Cyranoénonciateur de l’énoncé enchâssé en DD. L’allocutaire par contre change : dans la réplique Cyrano s’adresse à Roxane tandis que, dans le DR, il s’adresse à la Fâcheuse. Dans son DR, Cyrano parle, de manière indirecte, de Roxane. Comme l’Espagnol, la Fâcheuse n’a pas le statut de personnage dans la pièce ; mais, à la différence de l’Espagnol, elle ne parle pas, même comme énonciateur d’un DR. Toutefois, le fait d’être représentée comme allocutaire du DD présuppose son statut d’interlocuteur au même niveau que les autres. La construction du cadre énonciatif sert donc ici à conférer à la Mort le statut de personnage. Sous cette forme mitigée, Cyrano s’excuse d’être arrivé en retard en expliquant sans se faire comprendre par Roxane ce qui s’est passé. Le spectateur par contre, renseigné par les répliques précédentes, se rend compte du vrai sens. L’effet d’éthopée est bâti sur l’emploi de la personnification, mais encore plus sur la participation de la Fâcheuse à l’énonciation. Même si, dans le contexte de cette scène, Rostand exploite l’équivoque pour mitiger l’effet sur Roxane, dans la scène suivante – quand la vérité est désormais connue de tout le monde – la personnification continue de jouer un rôle pertinent, car c’est en se battant que Cyrano décède. L’auteur met en scène la métaphore du dernier combat entre le protagoniste et la Mort en le représentant comme un duel effectif, pendant lequel Cyrano ne cesse de s’adresser (à la deuxième personne) à son ennemie et à ses copains : le Mensonge, les Préjugés etc. Cet exemple exploite 1946 Article en accès libre placé sous licence Creative Commons Attribution 4.0 (http://creativecommons.org/licenses/by/4.0) SHS Web of Conferences 8 (2014) DOI 10.1051/shsconf/20140801033 © aux auteurs, publié par EDP Sciences, 2014 Congrès Mondial de Linguistique Française – CMLF 2014 SHS Web of Conferences la distance entre le discours monologal constitué par l’énoncé-pièce, échangé entre l’auteur et le spectateur, et le discours dialogal mis en scène, comme dans l’exemple 4.2.3. 4.4 L’Île des esclaves Dans l’Île des esclaves, comédie en un acte, les DD n’apparaissent que dans la scène 3, lors de la description du caractère d’Euphrosine, faite en sa présence, par Cléanthis, sa servante, interrogée par Trivelin. Le DD apparaît plusieurs fois. D’abord Cléanthis l’utilise pour ridiculiser son ancienne patronne. Basculant entre différents types de discours indirects, Cléanthis emploie aussi le DD ([…] la journée sera glorieuse : Qu’on m’habille ! Madame verra du monde…). 4.4.1 Exemple – Comment vous portez-vous, Madame ? (scène 3, pp. 48) Si Euphrosine a le visage fatigué parce qu’elle a mal dormi, elle utilise, selon Cléanthis, son discours pour se justifier devant ses amies. Cléanthis : […] il y a remède à tout : vous allez voir. Comment vous portez-vous, Madame ? Très mal, Madame : j’ai perdu le sommeil ; il a huit jours que je ne ferme l’œil ; je n’ose pas me montrer, je fais peur. Et cela veut dire : Messieurs, figurez-vous que ce n’est point moi, au moins ; ne me regardez pas ; remettez à me voir ; ne me jugez pas aujourd’hui ; attendez que j’aie dormi. J’entendais tout cela, moi… Il n’y a pas de verbe citant, le dialogue est mis en scène grâce à l’expression « vous allez voir », par une métaphore visuelle. L’énonciateur de l’énoncé enchâssant, Cléanthis, ne participe pas de l’énonciation enchâssée, rapporté en DD, si ce n’est comme allocutaire aléatoire (J’entendais tout cela, moi). Elle s’adresse à Trivelin, qui jugera de la culpabilité d’Euphrosine. Cette dernière écoute l’accusation de sa servante. Les énonciateurs des DR en DD sont Euphrosine, l’un des personnages, et ses amies, qui par contre ne sont présentes dans la pièce qu’à travers ce cadre énonciatif (comme l’Espagnol de l’exemple 4.3.3). L’une et les autres se vouvoient et s’appellent Madame, avec un effet de politesse maniérée. La conversation citée en DD est suivie d’une paraphrase, qui la traduit en son vrai sens. Cette paraphrase est introduite par l’expression et cela veut dire, deux points et le terme d’adresse Messieurs. La paraphrase est construite en effet comme un autre DD, adressé d’Euphrosine à ses allocutaires à la deuxième personne du pluriel. La comparaison entre le sens affiché et la réalité montre la fausseté du premier. La comparaison entre le sens affiché et le sens sous-entendu montre un désir démesuré de n’être jugée que favorablement, possédant donc une fonction d’éthopée. Le DD apparaît ensuite dans une scène galante entre Euphrosine et un cavalier : Cléanthis y montre la coquetterie d’Euphrosine, sa manière frivole de s’attacher les hommes. 4.4.2 Exemple – Continuez, folâtre, continuez (scène 3, pp. 49) Cléanthis : […] vous parliez d’une femme qu’il voyait souvent. Cette femme-là est aimable, disiez-vous ; elle a les yeux petits, mais très doux ; et là-dessus vous ouvriez les vôtres […] Vous réussîtes pourtant, le cavalier s’y prit ; il vous offrit son cœur. À moi ? lui dîtes-vous. Oui, Madame, à vous-même, à tout ce qu’il y a de plus aimable au monde. Continuez, folâtre, continuez, dîtes-vous, en ôtant vos gants sous prétexte de m’en demander d’autres […] Dans sa tirade, constituée par une série de répliques, Cléanthis s’adresse tantôt à Trivelin tantôt à Euphrosine. Dans la première réplique en DD, elle parle directement à sa patronne en la vouvoyant par la forme disiez-vous, qui constitue le verbe introducteur du DD. L’imparfait indique une action d’une certaine durée située dans le passé. Le DD n’est pas marqué du point de vue typographique, si ce n’est par la ponctuation. Ce premier DR est adressé d’Euphrosine au cavalier, tandis que Cléanthis, à nouveau, a Article en accès libre placé sous licence Creative Commons Attribution 4.0 (http://creativecommons.org/licenses/by/4.0) 1947 Congrès Mondial de Linguistique Française – CMLF 2014 SHS Web of Conferences SHS Web of Conferences 8 (2014) DOI 10.1051/shsconf/20140801033 © aux auteurs, publié par EDP Sciences, 2014 assisté à la scène en tant que servante. Le cavalier n’est pas un personnage de la pièce, il n’existe discursivement qu’en tant qu’allocutaire (puis énonciateur) d’un discours cité. Le deuxième DD présente le même cadre énonciatif, sauf pour le verbe introducteur (lui dîtes-vous) qui désigne une action ponctuelle. Le troisième DD manifeste la réponse du cavalier, sans verbe citant. Ce DD contient le terme d’adresse Madame. Le quatrième est introduit à nouveau par la forme dîtes-vous, accompagnée par la description du geste accompagnant la parole (en ôtant vos gants…). Ce dernier DD, comportant l’introducteur dîtes-vous, contient le terme d’adresse folâtre, qui, tout comme le précédent, n’est possible qu’en DR direct. Les paroles du cavalier, dont l’apparence est sincère dans le discours de Cléanthis, mettent encore davantage en évidence le caractère rusé d’Euphrosine, qui joue à ne pas comprendre qu’il est sérieux quand elle le traite de folâtre. Le DD permet ici à Trivelin de juger par lui-même de l’attitude d’Euproshine, en comparant ses contenances à ses mots, ses contenances et ses mots à ceux du cavalier. La parole d’Euphrosine, comparée à ses actions, en démontre la frivolité et l’insincérité : ces séquences en DD revêtent donc une fonction d’éthopée mais aussi, en amalgame pragmatique, narrative (elles permettent au récit de se développer) et argumentative (les paroles d’Euphrosine prouvent son délit). 4.4.3 Exemple – Un jour qu’elle pouvait m’entendre (scène 3, pp. 49-50) Ensuite, Cléanthis révèle avoir utilisé un discours pour duper sa patronne : Cléanthis : […] Un jour qu’elle pouvait m’entendre, et qu’elle croyait que je ne m’en doutais pas, je parlais d’elle, et je dis : Oh ! pour cela, il faut l’avouer, Madame est une des plus belles femmes du monde. Que de bontés, pendant huit jours, ce petit mot-là ne me valut-il pas !... Le DD est introduit par le verbe je dis, définissant l’action comme ponctuelle et passée. L’énonciateurpersonnage, Cléanthis, s’adresse à Trivelin. Euphrosine assiste mais n’intervient pas. Cléanthis explique le cadre énonciatif du DD qu’elle va rapporter : elle-même en est l’énonciateur, tandis que la fonction allocutaire du discours enchâssé comporte un dédoublement intentionnel : il y a celui avec qui Cléanthis est en train de parler (qui n’est pas indiqué) et puis Euphrosine, qui est le véritable allocutaire visé, mais qui ne doit pas s’en apercevoir. L’incise il faut l’avouer insérée dans le DD souligne la sincérité de l’énonciateur en augmentant la crédibilité du propos. Remarquons dans cette autocitation la présence de l’interjection Oh !, uniquement possible en DD (cf. Cigada 2012a : 464 et 471). Le récit prouve encore une fois la vanité d’Euphrosine. La littéralité qu’on imagine est fonction du statut d’épreuve, presque judiciaire, de ces DR. 4.4.4 Exemple – Regardez mes grâces (scène 3, pp. 50) Finalement, Cléanthis dénonce la mise faussement négligée de sa patronne, visant en réalité à souligner la beauté sans façon de la dame et, en même temps, la simplicité de sa tenue. Cléanthis : […] on y montre sa bonne façon naturelle ; on y dit aux gens : Regardez mes grâces, elles sont à moi, celles-là ; et d’un autre côté on veut leur dire aussi : Voyez comme je m’habille, quelle simplicité, il n’y a point de coquetterie dans mon fait. L’esclave s’adresse toujours à Trivelin, en présence d’Euphrosine. L’expression on y dit sert pour introduire le premier DD : c’est la dame qui parle en DD (cf. l’adjectif possessif dans mes grâces), en annonçant aux gens le message véhiculé par ses habits. Ce DD « traduit » en énonciation l’intention communicative des habits, intention que Cléanthis rend par un impératif (Regardez). L’allocutaire, les gens, est presque universel. Ce discours s’adresse en effet à tous ceux qui voient Euphrosine. 1948 Article en accès libre placé sous licence Creative Commons Attribution 4.0 (http://creativecommons.org/licenses/by/4.0) SHS Web of Conferences 8 (2014) DOI 10.1051/shsconf/20140801033 © aux auteurs, publié par EDP Sciences, 2014 Congrès Mondial de Linguistique Française – CMLF 2014 SHS Web of Conferences Le deuxième DD comporte les mêmes énonciateurs, mais il est introduit par la locution vouloir dire, qui récupère ici sa nature compositionnelle en exprimant l’intention de tromper par une fausse évidence (Voyez). 4.5 Phèdre Il n’y a qu’un DD dans Phèdre, les derniers mots d’Hippolyte : Théramène raconte à Thésée la fin du prince en rapportant ses paroles. Le récit commence au vers 1498, en réponse à la question de Thésée, qui s’inquiète de son fils, et se termine au vers 1570, au cours de la scène 6, soit l’avant-dernière de la pièce. 4.5.1 Exemple – À ces mots, ce héros expiré (Acte V, scène 6, vv. 1561-1567) Théramène : […] J’arrive, je l’appelle, et me tendant la main,/ Il ouvre un œil mourant qu’il referme soudain :/ « Le ciel, dit-il, m’arrache une innocente vie./ Prends soin après ma mort de la triste Aricie./ Cher ami, si mon père un jour désabusé/ Plaint le malheur d’un fils faussement accusé,/ Pour apaiser mon sang et mon ombre plaintive,/ Dis-lui qu’avec douceur il traite sa captive,/ Qu’il lui rende… » À ces mots, ce héros expiré,/ N’a laissé dans mes bras… Le verbe du discours citant est dit-il, cohérent au temps présent employé par Théramène dans son récit. À remarquer que, d’après le récit de Théramène, Hippolyte parle les yeux fermés, après les avoir rouverts à son approche. Cette notation concernant le geste qui accompagne le discours est intéressante aussi : en se raccordant à la description de l’agonie, elle souligne surtout que l’action jouée sur scène à ce moment n’est faite que des paroles d’Hippolyte, le contact entre les interlocuteurs étant représenté – de manière beaucoup plus forte que par le regard – par la main tendue à l’ami12. C’est pourquoi on imagine ce discours comme rapporté de manière plutôt littérale, dans le cadre fictionnel du discours-pièce. Pour ce qui concerne le cadre énonciatif : l’énonciateur-personnage Théramène s’adresse à l’allocutairepersonnage Thésée. Le DR a été prononcé par un énonciateur, Hippolyte, qui coïncide avec un autre personnage, et adressé à Théramène. Le DD comporte par ailleurs un effet de mise en abyme parce qu’il contient la prière, adressée du fils à son père, de concéder la liberté à Aricie. Cette prière est formulée en DR indirect (dis-lui qu’avec douceur il traite sa captive, qu’il lui rende…). Le DR d’Hippolyte demeure inachevé. C’est à travers le récit de Théramène qu’il trépasse sur scène, sa dernière « action » étant son discours : Théramène le rapporte, puis, après la pause qui marque le trépas d’Hippolyte, récupère son rôle d’énonciateur pour poursuivre la narration (À ces mots, ce héros expiré…)13. Du point de vue fonctionnel, ce DD contient le dernier témoignage de l’innocence d’Hippolyte et sa consigne à Théramène de prendre soin de son aimée. En racontant ce qu’Hippolyte a dit, Théramène exécute aussi la volonté de ce dernier de demander à Thésée sa clémence envers Aricie. Théramène est bien plus qu’un rapporteur, car il n’attend pas que Thésée soit un jour désabusé pour lui dévoiler l’innocence de son fils : le DD qu’il rapporte a justement la fonction de prouver ce qu’il a déjà affirmé en arrivant auprès de Thésée (J’ai vu des mortels mourir le plus aimable,/ Et j’ose dire encore, Seigneur, le moins coupable, vv. 1493-4). Son récit, et son récit de parole en particulier, remplit en effet une fonction argumentative, en amalgame pragmatique (récit et argumentation). Finalement, ce DD remplit la fonction d’éthopée en nous montrant jusqu’aux dernières conséquences le caractère entier d’Hippolyte, pieux envers les dieux et envers son père dont il ne veut pas dénoncer la honte, confiant dans l’amitié de Théramène, amoureux d’Aricie et inquiet de son sort, étranger à la vengeance. Par ses mots, il suggère au père une action réparatrice de tout le mal qui lui a été fait. 4.6 Le Misanthrope Dans cette pièce (comme dans Cyrano), le rôle de la parole est central : en tant que franche et immuable, la parole d’Alceste distingue cet homme des autres, qui changent constamment d’avis selon les intérêts du moment. Les DD se trouvent au début de la pièce et dans l’Acte IV : dans le dernier acte c’est plutôt la Article en accès libre placé sous licence Creative Commons Attribution 4.0 (http://creativecommons.org/licenses/by/4.0) 1949 Congrès Mondial de Linguistique Française – CMLF 2014 SHS Web of Conferences SHS Web of Conferences 8 (2014) DOI 10.1051/shsconf/20140801033 © aux auteurs, publié par EDP Sciences, 2014 parole écrite (la lettre de Célimène, Acte V, scène 4, insérée en prose après le vers 1690) qui est citée, en prenant une place importante dans le récit. 4.6.1 Exemple – Quel besoin si pressant avez-vous de rimer ? (Acte I, scène 2, 362-372) Avant de répondre de manière directe à Oronte, qui s’inquiète de son jugement à propos de ses vers, Alceste essaie de lui faire comprendre son opinion en feignant de lui rapporter ce qu’il avait dit à un autre poète dans une occasion semblable. Oronte : Est-ce que j’écris mal ? […] Alceste : Je ne dis pas cela ; mais enfin lui disois-je :/ Quel besoin si pressant avezvous de rimer ?/ Et qui diantre vous pousse à vous faire imprimer ?/ Si l’on peut pardonner l’essor d’un mauvais livre,/ Ce n’est qu’aux malheureux qui composent pour vivre./ Croyez-moi, résistez à vos tentations,/ Dérobez au public ces occupations ;/ Et n’allez point quitter, de quoi que l’on vous somme,/ Le nom que dans la cour vous avez d’honnête homme,/ Pour prendre, de la main d’un avide imprimeur,/ Celui de ridicule et misérable auteur./ C’est ce que je tâchais de lui faire comprendre. L’énoncé en DD est introduit par le verbe lui disois-je, renvoyant à une situation passée d’une certaine durée (une conversation). L’usage du DD permet la présence de structures syntaxiques qui ne pourraient pas apparaître en discours indirect, comme les deux énoncés interrogatifs en fonction de question (balançant entre question et question rhétorique). La deuxième question comporte aussi l’exclamation (diantre), typique du DD. Les impératifs en fonction d’incise (croyez-moi) et d’ordre (résistez ; dérobez ; n’allez point quitter) requièrent eux aussi le DD. Alceste est l’énonciateur-personnage et Oronte l’allocutaire-personnage. Pour ce qui concerne l’énoncé en DD inséré dans la réplique d’Alceste, Alceste en est l’énonciateur et « quelqu’un, dont je tairai le nom » (v. 343) l’allocutaire. Celui-ci n’a jamais la parole, sa présence demeurant bornée à celle d’allocutaire de ce discours d’Alceste. Le DR est ici le stratagème discursif utilisé par Alceste pour permettre à son interlocuteur (Oronte) de saisir à travers le dialogisme que ce discours qui a été adressé à un autre est, maintenant, adressé à lui. Cette interprétation délie le DR du contrat de littéralité. À un niveau plus stratégique, c’est Molière qui met en scène la sottise d’Oronte, incapable de comprendre une fonction discursive si évidente. À la différence de, par exemple, 4.3.4 (C’était une Fâcheuse), ce dialogisme n’a aucune pertinence pour l’éthopée de l’allocutaire. 4.6.2 Exemple – Si le Roi m’avoit donné/ Paris, sa grand’ville/… (Acte I, scène 2, 393400 et 405-412) Alceste cite la « vieille chanson » en la comparant au sonnet d’Oronte, qu’Oronte a déclamé peu de vers auparavant et dont Alceste vient de reprendre quelques expressions pour les commenter. Alceste répète la chanson deux fois14 : Alceste : […] une vieille chanson que je m’en vais vous dire : Si le Roi m’avoit donné/ Paris, sa grand’ville,/ Et qu’il me fallût quitter/ L’amour de ma mie,/ Je dirois au roi Henri :/ « Reprenez votre Paris :/ j’aime mieux ma mie, au gué !/ J’aime mieux ma mie. »… La chanson est introduite par la forme je m’en vais vous dire : le futur proche annonce la réalisation immédiate de l’acte, mais le texte rapporté relève d’un passé éloigné, comme l’expression une vieille chanson l’indique. L’énonciateur-personnage Alceste s’adresse à l’allocutaire-personnage Oronte ; la chanson en tant que DR n’est attribuée à aucun énonciateur en particulier, mais le chanteur y parle, à la première personne du singulier, sans s’adresser à aucun allocutaire en particulier. Le statut de cet exemple est ambigu, de manière analogue à 4.1.1 (Un chien vint dans l’office), même s’il y a ici la présence du verbe introducteur. Mais par contre la notion de littéralité n’a pas beaucoup de sens par rapport à ce type de citation. 1950 Article en accès libre placé sous licence Creative Commons Attribution 4.0 (http://creativecommons.org/licenses/by/4.0) SHS Web of Conferences 8 (2014) DOI 10.1051/shsconf/20140801033 © aux auteurs, publié par EDP Sciences, 2014 Congrès Mondial de Linguistique Française – CMLF 2014 SHS Web of Conferences À remarquer qu’il y a un autre DD adressé par le chanteur au roi Henri, enchâssé dans la chanson avec un effet de mise en abyme. Ce DD, introduit par le conditionnel je diroit, comporte une énonciation irréelle, en fonction de paradoxe. L’énonciateur-chanteur se feuillette dans le passé. Alceste introduit la deuxième occurrence de la chanson en invitant Oronte à percevoir la pureté de la passion qui inspire ce texte, en dépit de la pauvreté de son style. Il l’invite à l’écouter en proposant un déchiffrage lié au contexte : c’est justement la pauvreté du style (l’absence de colifichets) qui rend la passion parlante : Alceste : La rime n’est pas riche, et le style en est vieux :/ Mais ne voyez-vous pas que cela vaut bien mieux/ Que ces colifichets, dont le bon sens murmure,/ Et que la passion parle là toute pure ?/ Si le Roi… La répétition de la citation sert donc à préciser l’écoute : la voix, qui a été entendue (et probablement reconnue) lors de la première citation, doit ensuite être écoutée et observée selon des critères précis15. Le cadre énonciatif ne change pas, à part pour l’absence de verbe citant substitué par l’invitation à regarder l’objet-discours (ne voyez-vous pas… ?). 4.6.3 Exemple – je ne me dédis point (Acte IV, scène 1, 1139-1154 et 1158-1160) Philinte raconte à Éliante le procès entre Alceste et Oronte. La scène n’a pas été jouée sur scène, c’est à travers ce récit qu’Éliante et le spectateur en connaissent l’issue : Philinte : […] « Non, Messieurs, disoit-il, je ne me dédis point,/ Et tomberais d’accord de tout, hors de ce point./ De quoi s’offense-t-il ? et que veut-il me dire ?/ Y va-t-il de sa gloire à ne pas bien écrire ?/ Que lui fait mon avis, qu’il a pris de travers ?/ […] lorsque d’en mieux faire on n’a pas le bonheur,/ On ne doit de rimer avoir aucune envie,/ Qu’on n’y soit condamné sur peine de la vie. »/ Enfin toute la grâce et l’accommodement/ où c’est, avec effort, plié son sentiment,/ C’est de dire, croyant adoucir bien son style :/ « Monsieur, je suis fâché d’être si difficile,/ Et pour l’amour de vous, je voudrois, de bon cœur,/ Avoir trouvé tantôt votre sonnet meilleur. »… Le premier verbe citant (disoit-il) à l’imparfait signale une action d’une certaine durée ou répétée, c’est-adire qu’il reproduit – sans prétension d’exactitude – l’attitude d’Alceste au procès. L’énonciateur de cet énoncé enchâssé est un personnage, Philinte, qui s’adresse à un autre personnage, Éliante, en rapportant le discours d’Alceste (personnage) qui parle aux juges (Messieurs). Ceux-ci ne sont pas par contre des personnages et n’ont jamais la parole à aucun niveau du discours-pièce. Oronte est présent, mais non pas en tant qu’allocutaire visé par ce premier DD : Alceste parle en effet de lui à la troisième personne (De quoi s’offense-t-il ?...). Les termes d’adresse (Messieurs puis Monsieur dans le deuxième DD) ne sont possibles qu’en DR direct, tout comme les quatre énoncés interrogatifs. Le deuxième DD prévoit le verbe introducteur …c’est de dire, accompagné par une annotation qui caractérise l’attitude d’Alceste (croyant adoucir bien son style) et manifeste celle de l’énonciateur enchâssant, Éliante. L’énonciateur en est toujours Alceste, qui s’adresse maintenant à Oronte en le vouvoyant, tandis que les juges écoutent sans prendre la parole. Si, par le premier DD, Alceste confirme son jugement négatif des vers d’Oronte, par le deuxième, il s’excuse : tous deux collaborent à l’éthopée, en montrant la bizarrerie du tempérament excessif d’Alceste. Le maintien de la parole prononcée est le trait qui distingue Alceste de tous les autres et son refus de la changer en est une conséquence16. Ceci est perceptible en comparant ces DR aux discours mis en scène dans les répliques d’Alceste : les paroles d’Alceste rapportées ici en DD reprennent ce qu’il a déjà déclaré plusieurs fois pendant la pièce. Ces DR reprennent donc un discours antérieur, prononcé directement par le protagoniste, en constituant un dialogisme que le spectateur est censé reconnaitre. Article en accès libre placé sous licence Creative Commons Attribution 4.0 (http://creativecommons.org/licenses/by/4.0) 1951 Congrès Mondial de Linguistique Française – CMLF 2014 SHS Web of Conferences 4.7 SHS Web of Conferences 8 (2014) DOI 10.1051/shsconf/20140801033 © aux auteurs, publié par EDP Sciences, 2014 Le Cid Dans le Cid nous avons repéré quatre cas de DD, dont l’un se trouve dans la première scène du premier Acte, les autres dans le cinquième Acte, scène 1, 6 et 7 (la dernière). 4.7.1 Exemple – Un si charmant discours ne se peut… (Acte I, scène 1, vv. 25-38) La gouvernante Elvire rapporte à Chimène le discours de don Gomès son père à propos de ses deux prétendants, la pièce s’ouvrant sur le récit de ce discours, qu’Elvire a déjà rapporté, mais que Chimène souhaite écouter à nouveau. Elvire : […] Et puisqu’il vous en faut encor faire un récit,/ Voici d’eux et de vous ce qu’en hâte il m’a dit :/ « Elle est dans le devoir, tous deux sont dignes d’elle,/ Tous deux formés d’un sang noble, vaillant, fidèle,/ Jeunes, mais qui font lire aisément dans leurs yeux/ L’éclatante vertu de leurs braves aïeux./ Don Rodrigue surtout n’a trait en son visage/ Qui d’un homme de cœur ne soit la haute image,/ Et sort d’une maison si féconde en guerriers/ Qu’ils y prennent naissance au milieu des lauriers./ La valeur de son père en son temps sans pareille,/ Tant qu’a duré sa force, a passé pour merveille ;/ Ses rides sur son front ont gravé ses exploits,/ Et nous disent encor ce qu’il fut autrefois./ Je me promets du fils ce que j’ai vu du père ;/ Et ma fille, en un mot, peut l’aimer et me plaire »./ Il allait au conseil… Le DD est introduit par un verbe citant au passé proche, il m’a dit, qui indique une action ponctuelle, passée mais encore prochaine. Le discours précédent signale explicitement qu’il s’agit d’un récit et que, à l’intérieur du discours fictif de la pièce, Elvire l’a déjà répété plusieurs fois. Par ailleurs, l’énoncé cité a été prononcé en hâte. L’énonciateur et l’allocutaire de la réplique enchâssante sont respectivement Elvire et Chimène ; l’énonciateur et l’allocutaire de l’énoncé enchâssé sont don Gomès père de Chimène, personnage de la pièce ayant la parole en propre, et Elvire elle-même. Le DR porte sur Chimène et ses prétendants. En accord avec les attentes de l’allocutaire (Chimène) et avec la bonne volonté de l’énonciateur (Elvire), ce DD est censé reproduire de manière très fidèle le discours qu’il rapporte. L’énoncé en DD manifeste une fonction narrative en constituant un élément important du récit-pièce (le spectateur apprend les relations entre plusieurs personnages principaux ; Chimène apprend que son père approuve l’amour qui la lie à Rodrigue). Il manifeste aussi une fonction argumentative parce qu’il établit pour la première fois dans le discours-pièce la hiérarchie de valeurs qui en définissent le monde. De plus, pour la même raison, il est fonctionnel à l’éthopée de Rodrigue, mais aussi à celles de Chimène et de don Gomès. 4.7.2 Exemple – Il adorait Chimène… » (Acte V, scène 1, vv. 1533-1542) Don Rodrigue : […] On dira seulement : « Il adorait Chimène ;/ Il n’a pas voulu vivre et mériter sa haine ;/[…] Pour venger son honneur il perdit son amour,/ Pour venger sa maîtresse il a quitté le jour,/ Préférant (quelque espoir qu’eût son âme asservie)/ Son honneur à Chimène, et Chimène à sa vie ». La locution on dira introduit un DD placé dans l’avenir et destiné à être répété plusieurs fois : cette dernière valeur est exprimée par l’impersonnel on. L’indicatif signale que cette action discursive est certaine et l’adverbe seulement accompagnant le verbe exclut la possibilité d’autres discours concernant la mort de Rodrigue. L’énonciateur-personnage est Rodrigue même, qui s’adresse à Chimène, allocutairepersonnage. L’énonciateur et l’allocutaire de l’énoncé enchâssé n’ont pas d’identité précise, ce discours étant plutôt destiné au consensus universel. Le DD concerne les deux énonciateurs qui se l’échangent. Nonobstant sa collocation dans l’avenir, l’énonciateur garantit à son discours un haut degré de « littéralité »… Ce DD contient l’argument fondamental que Rodrigue émet pour expliquer à Chimène qu’il se laissera tuer par Sanche, sans pourtant sacrifier son honneur et l’honneur de Chimène. L’honneur a en effet dans cette pièce une dimension exquisément sociale : c’est devant les autres qu’il doit être préservé. L’effet 1952 Article en accès libre placé sous licence Creative Commons Attribution 4.0 (http://creativecommons.org/licenses/by/4.0) SHS Web of Conferences 8 (2014) DOI 10.1051/shsconf/20140801033 © aux auteurs, publié par EDP Sciences, 2014 Congrès Mondial de Linguistique Française – CMLF 2014 SHS Web of Conferences que Rodrigue cherche est de reproduire les discours que sa mort causera, ce que l’on en dira, pour vérifier et montrer à Chimène que ces discours rehausseront – au lieu d’amoindrir – l’honneur de chacun d’eux. La démonstration force Chimène, dans la réplique suivante, à changer d’attitude : elle demande en effet à Rodrigue de vaincre, pour l’affranchir de Sanche. 4.7.3 Exemple – Ne crains rien (Acte V, scène 6, vv. 1747-1753) Dans le récit du duel, don Sanche rapporte en DD les mots que Rodrigue lui a adressés après l’avoir désarmé. Don Sanche : […] Ce généreux guerrier, dont son cœur est charmé,/ « Ne crains rien, » m’a-t-il dit, quand il m’a désarmé :/ « Je laisserais plutôt la victoire incertaine,/ Que de répandre un sang hasardé pour Chimène ;/ Mais puisque mon devoir m’appelle auprès du roi,/ Va de notre combat l’entretenir pour moi,/ De la part du vainqueur lui porter ton épée »./ Sire,… Le verbe qui introduit l’énoncé enchâssé est dire (m’a-t-il dit), relatant en évènement ponctuel, passé mais récent. L’énonciateur-personnage don Sanche parle devant la cour, en présence de Chimène, en s’adressant directement au roi qu’il appelle Sire. Il rapporte dans l’énoncé en DD le discours tenu par ce généreux guerrier (Rodrigue, énonciateur-personnage) à lui-même. Rodrigue n’est pas présent et le DR n’a pas été joué sur scène. Ce DD couronne l’éthopée de Rodrigue : il est encore une fois fidèle à la hiérarchie de ses devoirs, il obéit à Chimène en vainquant Sanche et son amour justifie qu’il l’épargne, mais l’amour ne l’empêche pas de courir avant tout chez le roi. 4.7.4 Exemple – S’il ne m’avait aimée, il ne serait pas mort (Acte V, scène 7, v. 1800) Le dernier DD de la pièce contient les mots que Rodrigue demande à Chimène de répéter en son souvenir : Don Rodrigue : […] Et dites quelquefois, en déplorant mon sort :/ « S’il ne m’avait aimée, il ne serait pas mort ». La requête est formulée par le verbe citant à l’impératif dites : elle concerne un avenir hypothétique. L’énonciateur et l’allocutaire-personnage en sont respectivement Rodrigue et Chimène, tandis que, pour l’énoncé enchâssé, l’énonciateur en est Chimène s’adressant à soi-même. Le cadre énonciatif rappelle donc 4.1.2 (Pense, cochon !) mais n’est pas identique. L’avenir dont on parle dépendra du sentiment de Chimène, de son choix entre l’amour de Rodrigue ou la mort de celui-ci (car en tout cas il ne veut plus survivre sans elle). Et si elle décrétait sa mort, il suggère à Chimène de se répéter souvent que seul l’amour a causé sa fin. Cette suggestion fortement émotive adressée à Chimène déclenche à nouveau la réponse de son cœur : elle accepte l’amour de Rodrigue. 5 Résultats et conclusions préliminaires Nous allons résumer les résultats en suivant les premiers trois niveaux de la grille proposée au début (§ 3). Nous ne considérerons pas le quatrième niveau : il suffit, à cette étape de notre sondage, d’avoir vérifié que les DD peuvent remplir les fonctions, très variées, résultant de l’analyse des exemples. 1.Fréquence des DD et leur distribution 4.1 Godot 4.2 Topaze 4.3 Cyrano Structure de la pièce 2 actes 4 actes 5 actes Distribution des DD Acte II (4) un peu partout ; surtout aux Actes III (3) et IV (1) Nombreux et distribués partout Article en accès libre placé sous licence Creative Commons Attribution 4.0 (http://creativecommons.org/licenses/by/4.0) 1953 SHS Web of Conferences 8 (2014) DOI 10.1051/shsconf/20140801033 © aux auteurs, publié par EDP Sciences, 2014 Congrès Mondial de Linguistique Française – CMLF 2014 SHS Web of Conferences 4.4 4.5 4.6 4.7 l’Île des esclaves Phèdre le Misanthrope le Cid 11 scènes 5 actes 5 actes 5 actes Que dans la scène 3, une dizaine Acte V, scène 6 (avant-dernière scène), un seul Acte I et IV (+Acte V, la lettre) Acte I scène 1 ; Acte V scène 1, 6, 7 La distribution n’est vraisemblablement pas fortuite : les DD sont distribués de manière cohérente à des moments décisifs pour le déroulement du récit. 2.Aspects morphosyntaxiques 4.1 Godot 4.2 Topaze 4.3 Cyrano 4.4 l’Île des esclaves 4.5 Phèdre 4.6 le Misanthrope 4.7 le Cid Verbes citants 4.1.1 – 4.1.2 dis-moi 4.1.3 – 4.1.4 en disant 4.2.1 ils disaient ; chuchotaient 4.2.2 – 4.2.3 (il) disait ; vous avez répondu 4.2.4 je dirais 4.3.1 on pouvait dire ; vous m’auriez dit 4.3.2 je disais 4.3.3 je répondais ; en disant ; j’ai dit ; si j’avais dit 4.3.4 j’ai dit 4.4.1 (vous allez voir) ; et cela veut dire 4.4.2 disiez-vous; lui dîtes-vous; – ; dîtes-vous 4.4.3 je dis (passé) 4.4.4 on y dit ; on veut leur dire 4.5.1 dit-il (prés.) 4.6.1 lui disois-je 4.6.2 je m’en vais vous dire; (ne voyez-vous pas ?) 4.6.3 disoit-il ; c’est de dire 4.7.1 il m’a dit 4.7.2 on dira 4.7.3 m’a-t-il dit 4.7.4 dites (imp.) Dire G * * * V * * * * * ° ** * ° ° * ° Nous avons indiqué dans la colonne Dire l’occurrence de verbes différents de dire, qui se réduisent à deux occurrences de répondre (4.2.3 Topaze et 4.3.3 Cyrano), à chuchoter (4.2.1, dans Topaze) et à deux occurrences de voir (4.4.1 et 4.6.2). L’absence de verbe citant coïncide avec les cas d’autonymie sauf pour 4.4.2 (troisième DD au cours du dialogue entre Euphrosine et le cavalier). Dans la colonne G nous avons indiqué la présence de marques concernant l’action ou les gestes qui accompagnent le dire : parmi les gestes, nous avons noté aussi 4.7.1 (en hâte) ; 4.6.3 et 4.7.4 signalent plutôt une attitude psychologique attribuée à l’énonciateur (croyant adoucir bien son style et en déplorant mon sort). Les gestes accompagnent donc assez régulièrement la parole, ce qui semble plutôt naturel si l’on considère que le récit comporte l’action verbale mêlée dans l’action. À remarquer qu’aucun cas comportant un geste, sauf pour 4.3.2, ne corresponde à une autocitation : l’énonciateur-personnage décrit donc « l’action et le discours » d’un autre. La présence de marques qui décrivent la voix ou la manière de parler se trouve dans la colonne V : il n’y a que dans 4.3.2 que le verbe citant est connoté du point de vue métaphonologique (je disais d’une voix qui cherchait d’être dure). Ces données sont probablement à imputer à la situation discursive : d’un côté au besoin de baisser le niveau de difficulté d’interprétation. De l’autre, le texte étant écrit pour être dit, les effets prosodiques n’ont pas besoin d’être décrits : l’auteur peut laisser à l’acteur le soin de donner au DD la voix la plus adaptée. Ce résultat est significatif par rapport au statut de la voix physique (l’acteur) qui joue le 1954 Article en accès libre placé sous licence Creative Commons Attribution 4.0 (http://creativecommons.org/licenses/by/4.0) SHS Web of Conferences 8 (2014) DOI 10.1051/shsconf/20140801033 © aux auteurs, publié par EDP Sciences, 2014 Congrès Mondial de Linguistique Française – CMLF 2014 SHS Web of Conferences personnage : on ne peut pas effacer tout court cette médiation car elle a apparemment une influence sur l’organisation du cadrage énonciatif. À propos des termes d’adresse, on remarque une évolution du registre, du plus formel au moins formel ; les interjections (en italique) n’apparaissent que dans les pièces les plus anciennes ; les énoncés interrogatifs, impératifs et exclamatifs semblent être distribués de manière plutôt uniforme, comme le tableau ci-dessous le montre, mais il serait sans aucun doute convenable de les étudier davantage. Godot Topaze Cyrano l’Île des esclaves Phèdre le Misanthrope le Cid Termes d’adresse interjections 4.1.1 4.1.2 cochon 4.1.3 4.1.4 4.2.1 4.2.2 4.2.3 chérie 4.2.4 mes enfants 4.3.1 monsieur 4.3.2 4.3.3 señorita 4.3.4 4.4.1 Madame, Messieurs 4.4.2 Madame, folâtre 4.4.3 Oh ! 4.4.4 4.5.1 cher ami 4.6.1 4.6.2 au gué ! 4.6.3 Messieurs, Monsieur 4.7.1 4.7.2 4.7.3 4.7.4 Syntaxe de l’énoncé en DD déclaratif (un chien vint) impératif (Pense !) déclaratif tronqué déclaratif (il dort…) ; subjonctif (qu’il dorme) exclamation sans verbe (tripoteur ! Topaze escroc !) exclamatif (cachât !) interrogatif (pourquoi… ?) ; déclaratif (il est très utile) déclaratif (les proverbes correspondaient…) exclamatif (…) interrogatif (qu’est-ce que c’est… ?) déclaratif (je vais) ; impératif (passez) impératif (excusez-moi ; repassez !) interrogatif ; déclaratif ; impératif déclaratif; interrogatif (à moi ?) ; exclamatif (à vous !..) déclaratif impératif (regardez mes grâces ! voyez… !) déclaratif ; impératif (prends soin ; dis-lui que) interrogatif rhét.; déclaratif ; exclamatif (croyez-moi...) déclaratif conditionnel interrogatif ; déclaratif ; déclaratif déclaratif (elle est dans le devoir) déclaratif (il adorait Chimène) exclamatif (ne crains rien) ; déclaratif ; exclamatif déclaratif conditionnel (…il ne serait pas mort) 3.Ces signaux permettent d’étudier le cadre énonciatif. L’analyse des verbes introducteurs rend possible en effet de distinguer les cas d’autocitation (colonne AC) des autres cas, que nous avons classés en énoncés attribués à l’allocutaire du discours citant (colonne All) ; énoncés attribués à un autre personnage (colonne AutreP) ; énoncés attribués à un énonciateur qui n’est pas un personnage (colonne autre). On constate que la distribution des phénomènes observés change d’un ouvrage à l’autre. L’autocitation ne concerne pas Godot, Phèdre et le Cid. La citation d’autres énonciateurs externes au discours concerne plutôt les pièces plus récentes. Il faudra étudier plus de données, parce que la distribution paraît être significative. Par rapport au feuilletage des énonciateurs, on remarque la possibilité d’un « auto-feuilletage », qui peut être conditionnel (comme dans 4.2.4, je dirais), passé ponctuel mais habituel (4.3.2 je disais ; 4.3.3 je répondais), passé ponctuel (4.3.3 j’ai dit ; 4.3.4 j’ai dit ; 4.4.3 je dis), irréel (4.3.3 si j’avais dit), passé prolongé (4.6.1 lui disais-je), futur proche (4.6.2 je m’en vais vous dire). D’autre part, le feuilletage peut concerner aussi l’allocutaire-énonciateur (colonne All), dont le discours peut être convoqué par l’énonciateur à travers un impératif qui concerne l’avenir (4.1.2 dis-moi ; 4.7.4 dites), ou par une déclarative qui concerne le passé (4.2.3 vous avez répondu ; 4.4.2 disiez-vous, lui dîtes-vous et dîtes-vous) Article en accès libre placé sous licence Creative Commons Attribution 4.0 (http://creativecommons.org/licenses/by/4.0) 1955 Congrès Mondial de Linguistique Française – CMLF 2014 SHS Web of Conferences SHS Web of Conferences 8 (2014) DOI 10.1051/shsconf/20140801033 © aux auteurs, publié par EDP Sciences, 2014 mais aussi le présent (4.4.4 on y dit et on veut leur dire), comme une possibilité irréelle (4.3.1 on pouvait dire et vous m’auriez dit). Plusieurs cas de figure apparaissent donc dans notre exemplier : aux remarques de Rosier sur le « ‘sujet’ se feuilletant en des ‘mois’ passés » (Rosier 2008 : 126-132), on peut ajouter que, outre la possibilité de « re-convoquer son propre dit » (127), le feuilletage peut convoquer aussi les ‘mois’ que l’on vient de considérer. Par ailleurs, les discours des autres personnages (AutreP) se situent toujours dans le passé. En revanche, les discours attribués à des énonciateurs externes à la pièce (autre) peuvent se situer dans le passé (4.1.1 un passant pouvait lire; 4.1.3 Cicéron ; 4.2.1 les balayeuses disaient et chuchotaient ; 4.3.3 l’Espagnol s’inclinait en disant), mais aussi dans le présent (4.1.4 un autre me regarde en se disant) et dans l’avenir (4.7.2 on dira). Parmi les formes impersonnelles (on) qu’on retrouve en 4.3.1, 4.4.4 (deux fois) et 4.7.2, les trois premières renvoient en réalité à l’allocutaire correspondant en effet respectivement à vous pouviez dire et vous y dites. Le troisième représente par contre un véritable impersonnel, comme on l’a dit au cours de l’analyse. Verbes citants AC All AutreP autre 4.1 Godot 4.1.1 – * 4.1.2 dis-moi * 4.1.3 – * 4.1.4 en se disant * ** 4.2 Topaze 4.2.1 ils disaient ; chuchotaient ° 4.2.2 – * * 4.2.3 (il) disait ; vous avez répondu * 4.2.4 je dirais 4.3 Cyrano 4.3.1 on pouvait dire ; vous m’auriez dit ** 4.3.2 je disais * 4.3.3 je répondais ; en disant ; j’ai dit ; si j’avais dit *** * 4.3.4 j’ai dit * ? 4.4 l’Île 4.4.1 (vous allez voir) ; et cela veut dire *** des esclaves 4.4.2 disiez-vous; lui dîtes-vous; – ; dîtes-vous (passé) * 4.4.3 je dis (passé) ** 4.4.4 on y dit ; on veut leur dire 4.5 Phèdre 4.5.1 dit-il (prés.) * 4.6 4.6.1 lui disois-je * Misanthrope 4.6.2 je m’en vais vous dire; (ne voyez-vous pas ?) * 4.6.3 disoit-il ; c’est de dire ** * 4.7 le Cid 4.7.1 il m’a dit * 4.7.2 on dira * 4.7.3 m’a-t-il dit * 4.7.4 dites (imp.) Pour ce qui que concerne le cadre énonciatif, il ne nous reste à considérer que la position de l’allocutaire du DD, au sein de la pièce. Les cas de figure sont nombreux, parce que le DR peut s’adresser :  à l’énonciateur, qui rapporte le discours d’un autre (4.3.3 en [me] disant ; 4.4.1. et 4.4.2 présentent des situations de double allocutaire du fait que Cléanthis à assisté aux conversations entre la dame et d’autres personnes ; 4.5.1 [me] dit-il ; 4.7.1 il m’a dit ; 4.7.3 m’a-t-il dit) ;  à l’énonciateur, en échangeant le rôle des deux (comme dans Godot 4.1.2 dis-moi ; Topaze 4.2.2 cachât et Cyrano 4.3.1 on pouvait dire et vous m’auriez dit) ;  à l’allocutaire du discours citant (Topaze 4.2.3 il [vous] disait ; Île des esclaves 4.4.2 le DD du cavalier, sans verbe introducteur) ; en cas d’autocitation, le DD reproduit alors le cadre citant (Cyrano 4.3.2 je [vous] disais ; Misanthrope 4.6.2 je m’en vais vous dire) ;  à un autre des personnages (Topaze 4.2.3 vous [lui] avez répondu) ; 1956 Article en accès libre placé sous licence Creative Commons Attribution 4.0 (http://creativecommons.org/licenses/by/4.0) SHS Web of Conferences 8 (2014) DOI 10.1051/shsconf/20140801033 © aux auteurs, publié par EDP Sciences, 2014  Congrès Mondial de Linguistique Française – CMLF 2014 SHS Web of Conferences à un allocutaire externe au discours-pièce, particulier (4.1.4 un autre disant à soi-même ; 4.3.3 je [à l’Espagnol] répondais ; j’ai dit ; si j’avais dit ; 4.3.4 j’ai dit [à la Fâcheuse] ; 4.6.1 lui disois-je [à quelqu’un dont je tairai le nom] ; 4.6.3 disoit-il [aux juges]) ou universel (4.1.1 ; 4.1.3 ; 4.2.1 ; 4.7.4 où l’allocutaire pourrait aussi être à vous-même). Quand les énonciateurs rapportés n'apparaissent pas dans la pièce comme personnages, le DD fait rentrer leurs discours dans le discours dit en scène, de manière totalement exogène. À remarquer que, parfois, ces « énonciateurs » ne participent du cadre énonciatif qu'en tant qu’allocutaires du DR, sans jamais prendre la parole. En récapitulant, on observe que les cadres énonciatifs constitués par les répliques d’une pièce de théâtre peuvent enchâsser des DD selon plusieurs types de cadres énonciatifs subordonnés. Nous soulignons en particulier la possibilité d’inclure dans le discours-pièce, par le biais du DR, des énoncés qui mettent en rapport le discours-pièce et ses énonciateurs avec des discours prononcés par ou adressés à d’autres énonciateurs/allocutaires qui ne font pas partie du cadre citant. *** Ces premières conclusions confirment l’intérêt de la démarche proposée, tout en indiquant la nécessité absolue d’élargir systématiquement le corpus, avant qu’on ne puisse préciser le projet de recherche. En effet, si ce sondage ne nous permet pas de confirmer l’hypothèse émise à propos d’une évolution historique du cadre énonciatif du DD dans le discours théâtral, il permet toutefois d’apprécier des variations très fortes de celui-ci en suggérant d’approfondir la recherche, surtout en ce qui concerne 1.l’exploitation du cadre énonciatif, cf. les pièces de Rostand, Pagnol et Beckett (énonciateurs qui ne sont pas des personnages et recadrages énonciatifs conséquents) ; 2.les ouvrages où la parole est au cœur du message (cf. Molière et Rostand) ; 3.la quantité de DD utilisé et sa collocation dans la pièce (cf. Corneille, Racine et Marivaux). Références bibliographiques Adam, J.-M. (1992). Les Textes : types et prototypes. Paris : Nathan. Authier-Revuz, J. (2003). Le Fait autonymique : langage, langue, discours Quelques repères. In : Parler des mots. Le fait autonymique en discours, Authier-Revuz, J., Doury, M., Reboul-Touré, S. (éds.). Paris : Presses Sorbonne Nouvelle, 67-96. Bres, J. (2005). Savoir de quoi on parle : dialogue, dialogal, dialogique ; dialogisme, polyphonie… In : Dialogisme et polyphonie, Bres, J. et al. (éds.). Bruxelles : de boeck.duculot, 47-61. Bres, J. & Mellet, S. (2009). Une Approche dialogique des faits grammaticaux. Langue française, 163, 3-20. Calaseru, E. (2004). Testuali parole. Milano : F. Angeli. Cigada, S. (2005). L’Expression des émotions dans les dialogues de ‘La Princesse de Clèves’. Phénomènes d’exploitation de la coordination. In : Dialogue in Literature and the Media, Betten, A. & Dannerer, M. (éds.). Tübingen : Niemeyer, vol. 1 Literature, 209-217. Cigada, S. (2012a). Sur la plurifonctionnalité du discours direct. In : 3e Congrès Mondial de Linguistique Française, Neveu, F. et al. (éds.). SHS Web of Conferences, vol. 1, 459-476. Cigada, S. (2012b). Remarques sur la fonction du discours direct dans la narration. In : Modernitate şi interdisciplinaritate în cercetarea lingvistică. Omagiu doamnei profesor Liliana Ionescu-Ruxăndoiu [Modernité et interdisciplinarité dans la recherche linguistique. Hommage au professeur Liliana Ionescu-Ruxăndoiu], Constantinescu, M.N., Stoica, G., Bărbulescu, O.U. (éds.). Bucarest : Editura Universităţii din Bucureşti, 143160. Combettes, B. (2012). Linguistique textuelle et diachronie. In : 3e Congrès Mondial de Linguistique Française, SHS Web of Conferences, 1, 13-10. Ducrot, O. et al. (1980). Les Mots du discours. Paris : Éditions de minuit. Article en accès libre placé sous licence Creative Commons Attribution 4.0 (http://creativecommons.org/licenses/by/4.0) 1957 Congrès Mondial de Linguistique Française – CMLF 2014 SHS Web of Conferences SHS Web of Conferences 8 (2014) DOI 10.1051/shsconf/20140801033 © aux auteurs, publié par EDP Sciences, 2014 Dufiet, J.-P. (2005). Le Discours rapporté direct dans la comédie classique (Le cas de Molière). In : Dans la jungle des discours : genres de discours et discours rapportés, Lopez, J.M., Marnette, S., Rosier, L. (éds.). Cádiz : Servicio de publicaciones de la Universidad de Cádiz, 183-192. Dufiet, J.-P. & Petitjean, A. (2013). Approches linguistiques des textes dramatiques. Paris : Classiques Garnier. Granier, J.-M. (2003). Faire référence à la parole de l'autre : quelques questions sur l'enchaînement « sur le mot » chez Marivaux. In : Parler des mots. Le fait autonymique en discours, Authier-Revuz, J., Doury, M., ReboulTouré, S. (éds.). Paris : Presses Sorbonne Nouvelle, 217-231. Lopez Muños, J.M., Marnette, S., Rosier, L. (2004). Le Discours rapporté dans tous ses états. Paris : Harmattan. Nowakowska, A. (2005). Dialogisme, polyphonie : des textes russes de M. Bakhtine à la linguistique contemporaine. In : Dialogisme et polyphonie, Bres, J. et al. (éds.). Bruxelles : de boeck.duculot, 19-32. Petitjean, A. (2005). Textualité dramatique et discours rapporté : l’exemple de Marivaux. In : Dans la jungle des discours : genres de discours et discours rapportés, Lopez, J.M., Marnette, S., Rosier, L. (éds.). Cádiz : Servicio de publicaciones de la Universidad de Cádiz, 193-202. Rigotti, E. & Rocci, A. (2006). Le Signe linguistique comme structure intermédiaire. In : Nouveaux Regards sur Saussure. Mélanges offerts à René Amacker, Saussure, L. (éd.). Genève : Droz, 219-247. Rosier, L. (2008). Le Discours rapporté en français. Paris : Ophrys. Salvan, G. (2005). Dites-vous ou le dialogique à l’épreuve du dialogal (et vice-versa). In : Dialogisme et polyphonie, Bres, J. et al. (éds.). Bruxelles : de boeck.duculot, 265-279. Sebellin, R. (2003). Quattro cani in cucina : La filastrocca in Waiting for Godot di Samuel Beckett, Linguae & 2003/2, 41-58. Siouffi, R. (2013). Mensonges héroïques : autour de la fonction défensive de la parole chez Corneille. In : Héros ou personnages ?. Dufour-Maître, M. (éd.). Mont-Saint-Aignan : PURH, 129-145. Stati, S. (1991). Le Transphrastique. Paris : PUF. Todorov, Tz. (1981). Mikhaïl Bakhtine. Le principe dialogique. Paris : Éditions du Sueil. Sources des exemples (éditions utilisées) Beckett, S. (1952). En attendant Godot. Paris : Les Éditions de minuit (pour les exemples : 4.1.1-4). Corneille, P (2012). Le Cid. Milan : Feltrinelli. Lunari, L. éd. (pour les exemples : 4.7.1-4). Marivaux (1999). L’Île des esclaves. Paris : Le livre de poche (pour les exemples : 4.4.1-4). Molière (1982). Le Misanthrope. Milan : BUR. Lunari, L. éd. (pour les exemples : 4.6.1-3). Pagnol, M (2004). Topaze. Paris : Éditions de Fallois (pour les exemples : 4.2.1-4). Racine, J (2011). Phèdre. Paris : Le livre de poche (pour l’exemple : 4.5.1). Rostand, E. (1983). Cyrano de Bergerac. Paris : Gallimard (pour les exemples : 4.3.1-4). 1 Le statut de l’acteur est par ailleurs un peu plus complexe car, si la pièce est mise en scène (et non pas lue), il prête physiquement sa voix au personnage, mais aussi aux énonciateurs que le personnage cite en DD : dans ce cas-là, l’acteur joue donc « à la frontière » entre l’énoncé enchâssé et l’énoncé enchâssant. 2 L’ouvrage de Dufiet – Petitjean (2013) qui vient de paraître sera sans doute décisif pour ce sujet. La table des matières ne semble en tout cas pas indiquer une approche historique du sujet. 3 Temps et modes du discours citant et, par conséquent, rapports entre le temps et la modalité du DD et le temps et la modalité de l’acte « originaire » (Rosier 2008 : 118 parle d’anticipation du discours d’autrui par pseudo-discours rapportés introduits par un verbe au futur). 4 Pour renvoyer aux textes, nous avons utilisé les éditions citées en bibliographie, en indiquant les pages ou les vers, selon les cas. 1958 Article en accès libre placé sous licence Creative Commons Attribution 4.0 (http://creativecommons.org/licenses/by/4.0) SHS Web of Conferences 8 (2014) DOI 10.1051/shsconf/20140801033 © aux auteurs, publié par EDP Sciences, 2014 Congrès Mondial de Linguistique Française – CMLF 2014 SHS Web of Conferences 5 Cf. Sebellin 2003 qui renvoie à J. Strauss I, Erinnerung an Ernst oder Der Carnaval in Venedig, Fantasie Op. 126. Mélodie de la comptine allemande Ein Hund kam in die Küche (mais aussi Mein Hut der hat drei Ecken). La chansonnette de Vladimir contient un discours direct écrit rapporté à l’oral qui se répète à l’infini. Cf. http://www.youtube.com/watch?v=HowoftxyPWs. 6 Effet semblable mais beaucoup moins fort quand Vladimir chante « Do do do do… » pour faire dormir Estragon (Acte II, p. 99). 7 Phénomène partiellement analogue dans ce discours, cité en DD mais jamais prononcé : Roxane : […] J’ai songé : s’il voulait, lui que tous ils craindront… (Acte II, scène 6, p. 126). A propos de ce type de DD, cf. Cigada (2012b : 155, corpus Bosco Gutierrez). 8 Les deux occurrences du DD dans la première partie de l’ouvrage sont utilisées par le directeur M. Muche, l’une (2004 : 21) pour citer le règlement de la pension Muche (acte I, scène 3), l’autre (2004 : 28) pour communiquer à Topaze qu’il n’a pas reçu les Palmes académiques (acte I, scène 3), avec un effet ironique qui sert pour souligner sa niaiserie : Muche : Il m’a dit « M. Topaze mérite dix fois les Palmes ! » / Topaze : Dix fois ! / M. : « Mérite dix fois les Palmes, et j’ai eu presque honte quand j’ai appris qu’il ne les avait pas encore. » / T. (il rougit de joie) : Oh ! je suis confus, monsieur le directeur ! / M. : « D’autant plus, a-t-il ajouté, que je ne puis pas les lui donner cette année ! » / T. (consterné) : Ah ! Il ne peut pas ! / M. : Hé, non. Il a dû distribuer tous les rubans dont il disposait à des maîtres plus anciens que vous… Tenez, reprenez votre dossier. Ses dernières paroles ont été : « Dites bien à M. Topaze que pour cette année je lui décerne les Palmes moralement. » / T. Moralement ? / M. Moralement. C’est peutêtre encore plus beau. 9 La version cinématographique de 1950, réalisée par Pagnol avec Fernandel, ne conserve malheureusement pas cette réplique de Topaze. Elle est par contre gardée dans la première version, de 1936 (Pagnol, avec Arnaudy dans le rôle de Topaze). Arnaudy y exprime de manière marquée l’effet onomatopéique des deux DD reproduisant le bruit des balayeuses. 10 Nous ne considérons pas, par exemple, les scènes 1 à 4 du premier acte qui se passent au théâtre et dans lesquelles se déroule le début d’une représentation précieuse, avec un effet de mise en abyme. Nous ne considérons pas non plus la ballade qui accompagne le duel (dans cette même scène 4) et que l’on pourrait classer comme DD en tant qu’autocitation ou auto-récitation (Cyrano joue son personnage de spadassin et poète, en l’affichant avec ostentation). Quelques vers de la ballade seront repris par Ragueneau (Acte II, scène 3, p. 107) qui les cite à Cyrano pour le complimenter. Un autre type encore (d’autocitation en DD) concerne l’écriture de lettres sur scène (Acte II, scène 3, p. 109), ou la déclamation de la « recette en vers » de Ragueneau (Acte II, scène 4, p. 113-4). Fonctions de mise en abyme dans la fiction de la « gazette » de Cyrano, p. 302. 11 La longue liste de choses que l’on aurait pu dire contient aussi deux fonctions autonymiques introduites par le verbe appeler, dont la première est la fausse citation d’Aristophane (l’animal qu’Aristophane appelle Hippocampelephantocamélos), reprise des Lettres de Le Bret : appeler a ici la fonction de récupérer parmi les objets connus celui qui correspond au nom utilisé. La deuxième fois, appeler est utilisé pour introduire l’expression avoir pignon sur rue dans Cyrano : […] Respectueux : « Souffrez, monsieur, qu’on vous salue/ C’est là ce qui s’appelle avoir pignon sur rue ! » L’usage autonymique de l’expression figée n’est pas souligné du point de vue typographique, mais seulement par la présence du discours citant. Le contexte déclenche la réinterprétation de l’expression figée. Finalement Rostand introduit la dernière variation par une mise en abyme qui renvoie à Théophile de Viau, auteur de Pyrame et Thisbé : […] Enfin parodiant Pyrame en un sanglot :/ « Le voilà donc ce nez… » (Rostand 1983 : 352, note 29) : ce DD a donc la fonction de ‘pasticher’ le texte de De Viau. 12 Il faudrait peut-être étudier systématiquement le rapport entre regard et parole dans les pièces de théâtre en utilisant les travaux de Lorenza Mondada. 13 Nous avons retrouvé sur internet un enregistrement de la Société des Comédiens Français, réalisation de J. Reynier, radiodiffusé le 16/07/1953 http://www.youtube.com/watch?v=b1zvkJwdOxY (13’05’’-30’’) : la récitation du DD, plutôt rapide, souligne la souffrance du mourant. À comparer à la mise en scène de P. Chéreau, Odéon-Théâtre de l’Europe, Paris 2003, http://www.youtube.com/watch?v=W_lpICaJkG8 (2h8’20’’-9’12’’) : la récitation du DD est beaucoup plus lente et inexpressive du point de vue prosodique, ce qui souligne la littéralité du DR (de plus, l’acteur ferme ses yeux, comme s’il était en train de se rappeler exactement les mots). Article en accès libre placé sous licence Creative Commons Attribution 4.0 (http://creativecommons.org/licenses/by/4.0) 1959 Congrès Mondial de Linguistique Française – CMLF 2014 SHS Web of Conferences SHS Web of Conferences 8 (2014) DOI 10.1051/shsconf/20140801033 © aux auteurs, publié par EDP Sciences, 2014 14 Selon la tradition interprétative, cette chanson serait chantée la première fois et dite la deuxième (Molière 1982 : 91, note 6). En fait nous avons pu consulter deux versions enregistrées : la chanson du Roi Henri est dite deux fois dans la version jouée en 2000 à la Comédie Française (mise en scène de J.-P. Miquel, Alceste joué par D. Podalydès, http://www.youtube.com/watch?v=ImlUzaTaM7Q, minute 28 :15’), mais chantée deux fois dans la mise en scène amateur de 2011 par l’Atelier théâtre de l’École alsacienne (http://www.youtube.com/watch?v=Z57hVx624J4, minute 20 :45’). 15 La répétition peut revêtir une autre fonction, de reprise en écho, quand par exemple la prude Arsinoé rend visite à Célimène pour lui rapporter les médisances qui la concernent, afin qu’elle se corrige. Célimène la remercie en lui rendant le même service. En conclusion de sa tirade, Célimène reprend exactement les derniers mots de la tirade d’Arsinoé, en la parodiant (Molière, Le Misanthrope, Acte III, scène 4, 909-912 et 957-960) : Arsinoé : […] Madame, je vous crois l’âme trop raisonnable,/ Pour ne pas prendre bien cet avis profitable,/ Et pour l’attribuer qu’aux mouvements secrets/ D’un zèle qui m’attache à tous vos intérêts. Célimène : […] Madame, je vous crois aussi trop raisonnable,/ Pour ne pas prendre bien cet avis profitable,/ Et pour l’attribuer qu’aux mouvements secrets/ D’un zèle qui m’attache à tous vos intérêts. 16 Nous ne considérons pas ici le billet dont il est question dans l’Acte IV, scène 3, dont l’interprétation serait susceptible de changer totalement selon qu’il s’adresse à un homme ou à une femme (mais dont le texte n’est pas cité). Nous ne considérons pas non plus toute la scène 4 du cinquième Acte, qui tourne autour de la lecture des billets de Célimène à Clitandre et Acaste, en présence de ses amoureux, et dans lesquels elle se plaint de chacun d’eux en en décrivant les défauts. Le DD manifeste également ici le dialogisme par l’usage de la prose, insérée parmi les vers de la pièce. 1960 Article en accès libre placé sous licence Creative Commons Attribution 4.0 (http://creativecommons.org/licenses/by/4.0) 

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English

Dedicated networks for IoT: PHY / MAC state of the art and challenges

EAI endorsed transactions on internet of things

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To cite this version: Claire Goursaud, Jean-Marie Gorce. Dedicated networks for IoT : PHY / MAC state of the art and challenges. EAI endorsed transactions on Internet of Things, 2015, ￿10.4108/eai.26-10-2015.150597￿. ￿hal-01231221￿ 1. Introduction infrastructure and featuring a very long life time with very small battery or energy needs. Internet of Things (IoT) is going to take a major place in the telecommunications market as announced in technical and public medias. The paradigm of IoT relies on the deployment of billions of objects having the capability of transmitting information about their context and environment and to create a real-time, secured and efficient interaction between the real and the virtual worlds, pushing them to evolve from the state of cousins to the state of Siamese twins. IoT revealed to be a key technology for solving societal issues such as digital cities, intelligent transportation, green environment monitoring or medical care and elderly person monitoring. In this global picture, we may consider different technical issues. M2M has been first defined to connect MTDs in their vicinity. The proposed solutions extensively rely on the research results produced over the last 20 years for ad-hoc and wireless sensor networks. Starting 20 years ago from theoretical concepts, this very active research area went up to the definition of full standards (802.15.4, 802.15.6, Zigbee, Bluetooth) which already found a market. More recently, the IoT paradigm has been extended to the problem of connecting all these MTDs to the Internet, and through Internet to anyone or anything. The massive connection of objects spread over the world is a challenge that has some similarities with the paradigm of cellular networks which aimed at connecting people. This similarity attracted the interest of mobile network providers, to exploit such attractive potential market and IoT has been identified as a target for the future 5G [1], while several proposals already exist to adapt the 4G technology to IoT [2]. Nevertheless the IoT paradigm may present some very specific features that cannot be easily integrated with the constraints of cellular networks. In many applications, the individual targeted throughput is very low and the capacity is not a relevant criterion. On the opposite, the latency, the energy efficiency or the reliability are more IoT has strong connections with machine-to-machine (M2M), and sometimes in literature, both refer to the same idea. From our point of view, IoT covers a broader scope including as well the technology and the applications. On the opposite, M2M refers to the technologies that allow machines or objects to communicate. EAI Endorsed Transactions on the Internet of Things Research Article Abstract This paper focuses on the the emerging transmission technologies dedicated to IoT networks. We first analyze the classical cellular network technologies when taking into account the IoT requirements, and point out the need of dedicated technologies for IoT. Then, we present the PHY and MAC layers of the technologies that are already deployed, or likely to be deployed: UNB by SigFox, CSS by LoRaT M , Weighless, and RPMA by Ingenu. We then compare their performances to highlight their pros and cons. Finally, we discuss on the open research challenges that still need to be addressed. Received on 22 September 2015; accepted on 21 October 2015; published on 26 October 2015 Keywords: Internet of Things, M2M, PHY and MAC layer, Long Range transmissions. Copyright © 2015 C. Goursaud, J.M. Gorce, licensed to EAI. This is an open access article distributed under the terms of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/), which permits unlimited use, distribution and reproduction in any medium so long as the original work is properly cited. doi:10.4108/eai.26-10-2015.150597 ∗Corresponding author. Email: [email protected] Dedicated networks for IoT : PHY / MAC state of the art and challenges C. Goursaud1,∗, J.M. Gorce1 C. Goursaud1,∗, J.M. Gorce1 1Univ Lyon, INSA Lyon, Inria, CITI, F-69621 Villeurbanne, France HAL Id: hal-01231221 https://hal.science/hal-01231221v1 Submitted on 19 Nov 2015 L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. 3. UNB In 2004, Walker [5, 6] first proposed the use of VMSK (Very Minimum Shift Keying) to compress data transmission in the smallest possible band. However, in practice, this modulation technique did a step forward but didn’t reach the claimed ultra narrow frequency occupancy [7]. Nonetheless, the french company SigFox has successfully attempted to develop, patent, and deploy a new technology for UNB [8]. UNB standardization is now ongoing. 1. Introduction Whatever, from the technical point of view, the main challenge of this new paradigm is to let a very huge number of machine type devices (MTDs) be connected to the Internet at a low cost, with a limited EAI Endorsed Transactions on Internet of Things 10 - 2015 | Volume 1 | Issue 1| e3 EAI Endorsed Transactions on Internet of Things 10 - 2015 | Volume 1 | Issue 1| e3 1 C. Goursaud, J.M. Gorce critical. Except for cars or few other mobile objects, IoT may rely mostly on static nodes. But the dynamic of the problem comes from the fact that these nodes may transmit a packet with a very low probability (e.g. once a week, once a month, or even once a year !!!). Keeping these nodes continuously connected would be not efficient and an important issue is to allow a fast and reliable bursty connection. In section 2 we first summarize the recent results attempting to use 4G networks for serving IoT nodes at a large scale and we also discuss the challenges for 5G. We then enumerate the features that may claim for the design of new Physical (PHY) layers. Section 3 and section 4 detail the Ultra-narrow band (UNB) and the Chirp Spread Spectrum (CSS) technologies. Section 5 briefly describes two additional technologies. We compare the performances of the four technologies in section 6. Furthermore, we discuss and provide perspectives in section 7. critical. Except for cars or few other mobile objects, IoT may rely mostly on static nodes. But the dynamic of the problem comes from the fact that these nodes may transmit a packet with a very low probability (e.g. once a week, once a month, or even once a year !!!). Keeping these nodes continuously connected would be not efficient and an important issue is to allow a fast and reliable bursty connection. In section 2 we first summarize the recent results attempting to use 4G networks for serving IoT nodes at a large scale and we also discuss the challenges for 5G. We then enumerate the features that may claim for the design of new Physical (PHY) layers. Section 3 and section 4 detail the Ultra-narrow band (UNB) and the Chirp Spread Spectrum (CSS) technologies. Section 5 briefly describes two additional technologies. We compare the performances of the four technologies in section 6. 1. Introduction Furthermore, we discuss and provide perspectives in section 7. objectives, the separation seems inevitable but the right question is to find the best level at which the separation should occur. Therefore, using a separate network for MTDs is appealing even if additional deployment costs have to be supported. This drawback is balanced by the efficiency gain a dedicated PHY layer may bring to MTDs. More precisely the challenge is to design jointly a PHY/MAC protocol being able to manage a high density of nodes with bursty transmissions, with a reduced signaling overhead, a long range capability and very low energy requirements. Different technologies have been already investigated to address in this way this IoT paradigm. The following sections investigate these recent technologies and present a comparison of their main features. 2. PHY/MAC design for massive IoT services 2. PHY/MAC design for massive IoT services Because the massive connection of MTDs is commonly recognized as an important challenge [1], different technologies and strategies are currently under inves- tigation. The most straightforward approach relies on exploiting the current cellular networks to absorb this new traffic as summarized in [2]. The first idea relies on exploiting the second generation (2G) technology (e.g. GSM) which is progressively freed, the voice traffic being progressively offloaded toward 3G and 4G net- works. If 2G networks present interesting features in a mid-term perspective [3], some technical limitations cannot be alleviated: the granularity offered by the native GSM cannot serve a million of MTDs, the system is not energetically efficient and the signaling is over- sized for such small packets. Further, if the provider would keep the 2G system active only for MTDs, it may be anticipated that using these radio resources with a more appropriate technology would offer a more efficient perspective. 3.1. UNB PHY Principle 3.1. UNB PHY Principle The UNB PHY layer used in SigFox ’s network is very simple. Binary data are broadcast with a BPSK modulation at a very low rate Rb = 100 bps. The transmitted signal thus occupies a band of about b = 100 Hz. The novelty comes from the fact that the multiple transmissions are performed at a carrier frequency chosen in a much larger band B (typically 192 kHz in the 868 MHz (resp 915 MHz) ISM band in Europe (resp in the America)). As narrow band signals may suffer from flat fading, Frequency Hopping (FH) inside B is supported to introduce diversity and improve the reliability. While UMTS is assumed less efficient for MTDs for energy and coverage range reasons, LTE appeared more appealing [2]. However several issues have to be risen. Above some classical considerations such as energy consumption or coverage limitation, the most challenging issues are related to the medium access protocols which are inappropriate for massive access. More specifically, the RACH (Random Access Channel) channel used for contention access for mobile users would be overloaded by massive MTDs request. Although different subtleties have been suggested to share optimally the RACH channel [4], the most efficient way relies on providing MTDs and UEs (User Equipment) with separate access channels. Due to the different nature of the data flows and their QoS A key parameter of such system is the oscillator precision. which induces an offset between the targeted frequency and the actual one. Indeed, the very low signal bandwidth used in UNB implies a high sensitivity to the oscillators precision. Objectively, the system is referred as UNB when the frequency uncertainty is higher than the signal bandwidth. In this case, contrarily to NB systems, it is not possible to obtain non-overlapping frequency channels with reasonable guard intervals [11]. This leads to a new paradigm for the multiple access scheme, described in the next section. EAI Endorsed Transactions on Internet of Things 10 - 2015 | Volume 1 | Issue 1| e3 2 Dedicated networks for IoT : PHY / MAC state of the art and challenges Figure 1. Example of temporal & spectral repartitions of nodes. (Software Design Radio) algorithms designed to analyze the total band, to detect transmitted signals and to retrieve sent data. 3.1. UNB PHY Principle This is done with a FFT block applied to the received signal followed by an adaptive detector which aims at identifying the spectral signatures of the transmitted UNB signals. So any uncontrolled frequency shift at any transmitter is not problematic if this shift is fix during the short message duration. For each detected transmission, the appropriate frequency band is filtered and demodulated with a standard BPSK demodulator. To improve reliability, each message can be sent up to 3 times on different frequencies. The base station then responds exactly on the same frequency which allows to keep the node reception algorithm simpler as the band analysis is not necessary. g p y y Finally, the transmitted packet is made of : Figure 1. Example of temporal & spectral repartitions of nodes. Figure 1. Example of temporal & spectral repartitions of nodes. • a preamble of 4 bytes, • a frame synchronization part of 2 bytes, 3.2. UNB associated MAC 3.2. UNB associated MAC • a device identifier of 4 bytes, The inherent MAC associated to UNB is RFTDMA (Random Frequency and Time Division Multiple Access). Nodes access to the wireless medium randomly both in time and frequency domain, and without any contention-based protocol. This corresponds to an Aloha-based protocol without preliminary sensing of the channel occupancy. Nonetheless, contrary to classical Aloha transmissions, the carrier frequencies are chosen in B inside a continuous interval, instead of a predefined discrete set. To illustrate the system behavior, an example is drawn in Fig.1, representing the signal generated by 100 nodes and observed by a receiver in the time-frequency plan. • a payload of up to 12 bytes, • a Hash code to authenticate the packet in SigFox network (variable length), • Cyclic Redundancy Check (CRC) syndroms of 2 bytes for security and error detection. • Cyclic Redundancy Check (CRC) syndroms of 2 bytes for security and error detection. 3.3. UNB Analysis As explained in section 1, three main criteria are of major importance in IoT networks which are now used to evaluate the UNB-based technology. Communication range. The range usually depends on the channel characteristics, interference and the receiver noise level. At a first glance, we consider a Line of Sight (LoS) transmission and interference free conditions. Thus, the range is first constrained by the noise level given by: The benefits of RFTDMA are: • no energy-consumption for medium sensing. Indeed, due to the very large size of the cells, sensing the channel at the node location would not ensure collision-free signals at the base station. NdBm = −174 + NF + 10 · log10(B), (1) (1) • no need for time-synchronization over the net- work. Thus beacon packets can be eliminated. with NF the noise figure of the receiver. with NF the noise figure of the receiver. g In the reference UNB case described above, the noise floor is thus −154 + NF dBm. • no constraint on the oscillator precision. As any frequency can be chosen in the whole band, even the cheapest oscillators can be used without performance degradation. Therefore, in free-space conditions and assuming the antenna gains balance roughly the noise factor loss, the signal to noise ratio is : Nevertheless, the uncontrolled medium access leads to interference or packet collisions between the active users. This will be discussed in section 3.3. SNR = Pr −N = Tx + 132 −20 log  r λ  , (2) (2) where r is the range and λ the wavelength. From the receiver’s point of view, as shown in Fig.1, the monitored bandwidth B is filled by a combination of narrow-banded signals randomly located in time and frequency. Their demodulation relies on efficient SDR Now considering a SNR threshold of 8 dB and a link margin of 4 dB, the received power is required to be Pr ≥−142 dBm. EAI Endorsed Transactions on Internet of Things 10 - 2015 | Volume 1 | Issue 1| e3 3 C. Goursaud, J.M. Gorce C. Goursaud, J.M. Gorce Figure 2. Behavior of the rejection coefficient in linear and logarithmic scale vs frequency difference δf . Thus, an emission power of 14 dBm (maximum radiation power allowed by ETS 300-220 regulation) may ensure thousands kilometers range in free-space. In practice, about a 63 kms range is observed for terrestrial communications. 4. CSS The rejection coefficient relies on the product of the pulse-shapping filter 2 and the matching filter 1: In this section, we present the general properties of CSS modulation with a deeper focus on a specific technique. The use of CSS for IoT networks was initially developed and patented by Cycléo, a french compagny acquired by Semtech in 2012. The later includes this modulation in LoRaT Mdevices, along with FSK modulation. Standardization of Low Power Wide Area Network (LPWAN) using such technology is targeted by the LoRaT MAlliance [12]. In this section, we present the general properties of CSS modulation with a deeper focus on a specific technique. The use of CSS for IoT networks was initially developed and patented by Cycléo, a french compagny acquired by Semtech in 2012. The later includes this modulation in LoRaT Mdevices, along with FSK modulation. Standardization of Low Power Wide Area Network (LPWAN) using such technology is targeted by the LoRaT MAlliance [12]. Pi(t, f1, f2) =| G(f1, t) · G(f2, t) | . (6) (6) More precisely, the rejection coefficient β(δf , t) is a function of the frequency shift between the 2 active users δf = |f1 −f2|, and is normalized by the power of the desired signal: β(δf , t) = Pi(t, f1, f2) Pi(t, f1, f1) (7) CSS is a rather classical technique in radar systems but was proposed for the first time for communication systems by Winkler in 1962 [13] and barely used since. For readers unfamiliar with this technique, the following paragraph provides its main features. (7) In Fig.2, the rejection coefficient is plotted as a function of the frequency difference (6), for a given point in time. We can observe 2 main areas, with transitions around ±100 Hz. In the central area, i.e. for low δf in the range [−100, 100] Hz, the interference level is important (up to 0 dB when δf = 0). Contrarily, in the outer area, i.e. for high δf , the interference level 3.3. UNB Analysis Thus, UNB is suitable for long distance transmissions (up to the horizon). Interference Sensitivity. Interference occurs when some packets are transmitted simultaneously, which is inherent to RFTDMA. The few interference situations observed in Fig.1 are characterized by the peaks above the average. The characteristics of this interference evaluated in [10] are summarized below. Consider a multiple access channel with N = 2 active transmitters. The received signal can be expressed as: r(t) = 2 X i=1 si(t) · g(fi, t) ⊗hi(t) + n(t) (3) (3) Figure 2. Behavior of the rejection coefficient in linear and logarithmic scale vs frequency difference δf . where si(t), ∀i ∈{1, 2} are the BPSK symbols sent by the active user i, g(fi, t) the impulse response of the emission FIR filter (centered at fi); hi(t) is the path-loss of the corresponding link, and n(t) is an additive white Gaussian noise with zero mean, and variance σ2. is low, and mainly distributed around −90 dB. As, the considered band is much larger than 200 Hz (at least 12 kHz), the interferers are positioned with a highest probability in the second area, thus not impacting the desired user detection. For the sake of simplicity, consider the case where hi(t) = δ(t), ∀i ∈[1, . . . , k + 1]. This corresponds to the worst case where both users are at the same distance from the base station and experience the same flat channel. Without loss of generality, we consider that the desired user is #1. The signal used for data recovery is thus: Energy Consumption. Typical emission consumption varies from 20mA to 70mA, while it drops to almost 0 when inactive. The actual energy consumption depends on the message size as well as the emission power. For the considered frequencies, the nodes are allowed to transmit up to 14 dBm in Europe, and up to 21.7 dBm in America. Thus, the energy consumption can be kept very low to comply with the battery-powered IoT nodes. r ′(t) = r(t) ⊗g(f1, t) (4) = k+1 X i=1 si(t) · g(fi, t) ⊗g(f1, t) + n(t) ⊗g(f1, t). (5) (4) (5) 4.1. CSS Principle 4.1. CSS Principle In contrast to UNB, a CSS transmission occupies a bandwidth much larger than what is actually needed EAI Endorsed Transactions on Internet of Things 10 - 2015 | Volume 1 | Issue 1| e3 EAI Endorsed Transactions on Internet of Things 10 - 2015 | Volume 1 | Issue 1| e3 4 Dedicated networks for IoT : PHY / MAC state of the art and challenges −60 −40 −20 0 20 40 60 −500 0 500 (a) −60 −40 −20 0 20 40 60 0 50 100 150 (b) −60 −40 −20 0 20 40 60 −1 0 1 (c) −60 −40 −20 0 20 40 60 1 1 1 1 (d) 0 20 40 60 80 100 120 −400 −200 0 200 (e) −60 −40 −20 0 20 40 60 −500 0 500 (a) −60 −40 −20 0 20 40 60 0 50 100 150 (b) −60 −40 −20 0 20 40 60 −1 0 1 (c) −60 −40 −20 0 20 40 60 1 1 1 1 (d) 0 20 40 60 80 100 120 −400 −200 0 200 (e) Figure 3. Up raw chirp : (a) frequency evolution during the chirp, (b) the corresponding instantaneous phase, (c) varying period of the phase and quadrature components, (d) the output of the matched filter and (e) its FFT. for the considered data rate. It is thus a subcategory of Direct-Sequence Spread Spectrum (DSSS), which takes advantage of the controlled frequency diversity to recover data from weak signals (even under the noise level). Thus, compared to narrow band transmissions, DSSS permits to alleviate the constraint on the receiver’s sensitivity and increase the communication range, but at the cost of a reduced data rate. Thus, DSSS is compliant with IoT networks needs. Usually, in DSSS, data are spread with a sequence. Each symbol duration is divided into F small chips (with F the spreading factor), whose level is in a finite set. The sequence of chips used by the transmitter is known by the receiver, which searches such pattern in the signal. However, in CSS modulation, the spreading effect is obtained through a continuously varying carrier frequency [14]. In CSS case, chips do not correspond to physical realizations anymore, but are considered by analogy with coded DSSS. Nevertheless, the spreading factor still characterizes the increase in band occupation. 4.1. CSS Principle LoRaT M defines the spreading factor SF as follows [15]: 2SF = B Rs = B · T (8) (8) where B is the spread bandwidth, Rs the symbol rate, and T = 1 Rs the chirp duration. s So, the original basic element of CSS modulation is the chirp. Its waveform is written as follow : c(t) =  exp (jφ(t)) if −T 2 ≤t ≤T 2 0 otherwise (9) (9) Figure 3. Up raw chirp : (a) frequency evolution during the chirp, (b) the corresponding instantaneous phase, (c) varying period of the phase and quadrature components, (d) the output of the matched filter and (e) its FFT. with φ(t) the chirp phase. φ( ) p p The instantaneous frequency is given by : φ( ) p p The instantaneous frequency is given by : f (t) = 1 2π dφ(t) dt (10) (10) an up-chirp (resp down-chirp), as instantaneous frequencies are added, For linear chirps, such as the ones used by LoRaT M, f (t) is defined by For linear chirps, such as the ones used by LoRaT M, f (t) is defined by flc(t) = fc + µ · B T · t (11) (11) • the multiplication of an up-chirp with the corre- sponding down-chirp (i.e. the same instantaneous frequency expression with opposite µ, also called conjugate chirp) leads to a narrow peak at twice the carrier frequency. with fc the central carrier frequency. If µ = 1, an up-chirp is obtained, while µ = −1 corresponds to a down-chirp. One may note that for CSS modulation, B corresponds to the spectral occupancy, as well as the difference between the maximum and minimum instantaneous frequency during the chirp [16]. Fig.3 shows an example of an up-chirp. We can verify the linearity of the frequency (a), the quadratic evolution of the phase (b), and the varying period of the in-phase and quadrature components (c). Thus, the matched receiver for a linear chirp is performed by multiplication with the conjugate chirp, and leads, as shown in Fig.3(d) to a constant. The output signal is then analyzed to identify the presence or absence of the narrow interference peak (which is at 0 in Fig.3(e)). This basic CSS modulation permits to send one bit per chirp. 4.1. CSS Principle Consequently, before the transmission of coded chirps, a preamble made of raw chirps has to be sent, to estimate this offset and determine the reference frequency. One may note that Nanoscale has evaluated that an uncertainty up to 40 ppm is supported [17], which enables the use of cheap devices. Once the syn- chronization is done, the decoder evaluates the offset of the coded symbols with respect to the reference frequency. For sake of simplicity, from now on, we suppose that the reference is known, and neglect fc + fd, to get the baseband signal. • all previous results are obtained when consider- ing an ideal time and frequency synchronization between the transmitter and the receiver. How- ever, any imprecision in time or frequency will be perceived as an additive offset compared to the nominal frequency. Consequently, before the transmission of coded chirps, a preamble made of raw chirps has to be sent, to estimate this offset and determine the reference frequency. One may note that Nanoscale has evaluated that an uncertainty up to 40 ppm is supported [17], which enables the use of cheap devices. Once the syn- chronization is done, the decoder evaluates the offset of the coded symbols with respect to the reference frequency. For sake of simplicity, from now on, we suppose that the reference is known, and neglect fc + fd, to get the baseband signal. Figure 4. Coded chirp : SF = 27, raw chirp shifted by 10 4.1. CSS Principle At the receiver, the following properties are used: • the multiplication of an up-chirp with an up- chirp (resp down-chirp with down-chirp) leads to • the multiplication of an up-chirp with an up- chirp (resp down-chirp with down-chirp) leads to EAI Endorsed Transactions on Internet of Things 10 - 2015 | Volume 1 | Issue 1| e3 5 C. Goursaud, J.M. Gorce −60 −40 −20 0 20 40 60 −500 0 500 (a) −60 −40 −20 0 20 40 60 −350 −300 −250 −200 (b) −60 −40 −20 0 20 40 60 −1 0 1 (c) −60 −40 −20 0 20 40 60 −1 0 1 (d) 0 20 40 60 80 100 120 −400 −200 0 200 (e) Figure 4. Coded chirp : SF = 27, raw chirp shifted by 10 At the receiver, the multiplication of the received signal with the raw down-chirp (supposedly perfectly synchronized) modulated at a carrier frequency fd, the instantaneous frequency becomes: fp(t) =  fc + fd −µ · k T + B if −T 2 ≤t ≤k B fc + fd −µ · k T if k B ≤t ≤T 2 . (13) (13) The product signal is thus made of two periods, each having a constant frequency (Fig.4(d)). The transition between them occurs at the time index corresponding to the value of the coded chirp. In both periods, the frequency linearly depends on the offset and the difference between the 2 levels is B Hz . Therefore sampling the signal at the chip rate, i.e., at B Hz, the instantaneous frequency becomes continuous over the whole chirp and, after subtracting the carrier fc + fd, is proportional to the shift k. Thus, the FFT of the sampled signal provides a flat response with the peak shifted by the chip shifting value (Fig.4(e)). To do so, two constraints must be verified : • the phase of the transmitted signal must be continuous, especially at the transition. Besides, the instantaneous phase must be identical at the beginning and at the end of the symbol. This ensures an exploitable FFT result. This feature is controlled by the transmitter. • all previous results are obtained when consider- ing an ideal time and frequency synchronization between the transmitter and the receiver. How- ever, any imprecision in time or frequency will be perceived as an additive offset compared to the nominal frequency. 4.4. CSS Performance Analysis Communication range. As in the UNB section, we first evaluate the noise floor. For CSS spread over a frequency band with width B, the noise level is NdBm = −174 + NF + 10 · log10(B) (15) (15) with 125kHz ≤B ≤500kHz for the 900 MHz band. Thus, in free-space, the link budget is : with 125kHz ≤B ≤500kHz for the 900 MHz band. Thus, in free-space, the link budget is : Thus, in free-space, the link budget is : SNR = Tx + 132 −20 log  r λ  + CG (16) (16) Data transmission can be bi-directional, even if the uplink is expected to be dominant. The specification thus defines 3 categories of end-devices : with CG the coding gain due to the spreading. This coding gain is estimated by CG = 2, 5 · SF [18]. • Class A : the communication is initiated by the end-device. The uplink transmission triggers two short downlink receive windows. The uplink transmission slot is scheduled when needed by the end-device on a random time basis (ALOHA based protocol). This Class A is more suitable for energy constrained nodes. The theoretical maximal range is about hundreds kilometers but was evaluated to be 22 km in practice. Thus, long ranges are achieved. Interference Sensitivity. There are two sources of inter- ference: from non-LoRaT Msignals, and LoRaT Msignals. For the first category, it was estimated [18] that a single tone pulse is not a problem if it is less than 5 dB (resp 19, 5 dB) above the desired signal for SF = 7 (resp SF = 12) with an error correcting scheme of 4/6. Interference Sensitivity. There are two sources of inter- ference: from non-LoRaT Msignals, and LoRaT Msignals. For the first category, it was estimated [18] that a single tone pulse is not a problem if it is less than 5 dB (resp 19, 5 dB) above the desired signal for SF = 7 (resp SF = 12) with an error correcting scheme of 4/6. • Class B: this class adds scheduled receive win- dows to the Class A random ones. The required time synchronization between the gateway and the end-device is obtained thanks to a time syn- chronized Beacon sent by the gateway. For the second category, one may note that two devices can not use the same SF, on the same frequency at the same time. 4.3. MAC for CSS networks • a payload of 2up to 255 bytes, • a payload of 2up to 255 bytes, The LoRaT MAlliance is currently defining the LoRaWAN protocol which precises the MAC protocol envisioned for LoRaT M. An end device is driven by the LoRaT MMAC master if its Adaptive Data Rate (ADR) mode is enabled. In this case, the LoRaT MMAC has the ability to control the spreading factor, the bandwidth occupation and the RF output power of each node in order to both maximize the node battery life and the network overall capacity. This also permits to transmit with the highest possible rate, thus reducing the time occupancy. Consequently, closer nodes to the gateway benefit from a higher data rate than the nodes at the cell edge. • the CRC calculated for the payload (2 bytes). • the CRC calculated for the payload (2 bytes). 4.2. CSS Adaptation for LoRaT M The CSS modulation used in LoRaT Mnetworks is more evolved and constrained to fit with IoT requirements [17]. First of all, a single LoRaT Mchirp may code up to SF = 12 bits. To do so, during one chirp period, a specific frequency trajectory is defined for each of the 2SF symbols. This is done by shifting the frequency ramp based on the symbol value, as illustrated in Fig.4. Thus each coded chirp is obtained by a cyclic shift of the reference chirp. This introduces a sharp edge in the instantaneous frequency trajectory, occurring in the example at the chip number 10. In short, the whole modulation process defined by LoRaT Mfollows. First, a preamble is sent for the intrinsic offset estimation. Bits are divided into words of SF bits. There are 2SF code words, mapped to the 2SF possible offsets. Gray indexing is used to reduce the bit error rate, when a symbol is erroneous. The new expression for the instantaneous frequency of the coded chirp is : fcc(t) =  fc + µ · B T ·  t −k B  + B if −T 2 ≤t ≤k B fc + µ · B T ·  t −k B  if k B ≤t ≤T 2 , (12) fcc(t) =  fc + µ · B T ·  t −k B  + B if −T 2 ≤t ≤k B fc + µ · B T ·  t −k B  if k B ≤t ≤T 2 , (12) The typical bandwidth values are 125, 250 and 500 kHz in the HF ISM 900 MHz band, while they are 7.8, 10.4, 15.6, 20.8, 31.2, 41.7 and 62.5 kHz in the LF 160 and 480 MHz band. Besides, the spreading factor SF with k the number of shifted chips. with k the number of shifted chips. with k the number of shifted chips. Thus, as seen in Fig.4(a-c), the raw chirp pattern is cyclic shifted of 10 chips toward the right. EAI Endorsed Transactions on Internet of Things 10 - 2015 | Volume 1 | Issue 1| e3 EAI Endorsed Transactions on Internet of Things 10 - 2015 | Volume 1 | Issue 1| e3 6 Dedicated networks for IoT : PHY / MAC state of the art and challenges can vary from 7 to 12. 4.2. CSS Adaptation for LoRaT M The chip rate remains unchanged, and equals to B, so the chirp duration is impacted when modifying the spreading factor. A high spreading factor corresponds to a long chirp. Therefore, the raw data rate can be calculated as follows : In class A and class B, nodes initiate the uplink transmission. In this case, whenever it wants to transmit data, a node pseudo-randomly chooses a channel from the list of available channels, and performs a Listen Before Talk (LBT) before actually emitting its data. Transmission is performed with the fixed default SF and B values. However, if a node needs to check its connection to the network, a short frame (17 bytes) is sent first at SF = 8, then SF = 10 and finally SF = 12. In this case, the end-device gets a feedback on the link margin of the received test frame. Rb = SF · BW 2SF . (14) (14) It thus varies from 22 bit/s (B = 7.8 kHz and SF = 12) to 27 kbit/s (B = 500 kHz and SF = 7). These degrees of freedom permit to adapt the rate and frequency occupancy to the transmission conditions. In particular, devices at the edge of the gateway range are likely to be assigned with a high SF, while closer devices use lower SFs. To improve reliability, a retransmission scheme is available. The number of retransmissions is defined by the end-device, but the LoRaTMMAC master can also modify it. Finally, the transmitted PHY packet is composed by : Finally, the transmitted PHY packet is composed by : Finally, for a high amount of data to transmit, FH is usedThe frequency pattern is predefined, and known by the receiver. • a preamble of at least 12 raw chirps • a header (optional) of 4 to 8 symbols 4.3. MAC for CSS networks 4.3. MAC for CSS networks 4.4. CSS Performance Analysis A FH algorithm is used to counteract interference and fading. This standard is intended for devices that need one-way communications at a very low cost. Table 1. Cochannel rejection (dB) for all combinations of spreading factor for the desired and interferer user Weightless-P. This second standard improves the first one by allowing two-way communications. This permits to enhance the reliability by using acknowledgment protocols. A multiple access is performed with FDMA+TDMA in 12.5kHz narrow band channels. Thus the BSs are time-synchronized to schedule the transmissions in the slots. However, the range is slightly reduced to 2 km in urban environment. successful if one signal is received at least 6 dB above the other (Table 1). Finally, we have computed and reported in Table 1, the co-channel rejection when considering all couples of SF. We can observe that two devices using different spreading factors can transmit their data simultaneously, as long as none is received with a power significantly higher. We can also note that the rejection coefficient increases with the spreading factors. Thus, the high SF usually assigned to distant nodes for the noise sensitivity also permits to overcome the impact of closer devices which are likely to be received with a higher power level. Weightless-W. The last standard permits to take advantage of the available white-space in the spectrum use. Use of TV white spaces is the primary idea of the founding members of Weightless. The modulation modes vary from 16-QAM to DBPSK, in the 470 −790 MHz TV band. Besides, spreading is also enabled (with a spreading factor up to 1024) to dynamically adapt the rate and the range to the actual needs. One may note that the BS transmission power is 20 dB higher than the end-device’s one. To balance the link budget, the end-device uses a channel with a bandwidth 64 smaller than the BS one. Time Division Duplexing is used to provide uplink and downlink pairing, as spectrum is not guaranteed in the TV white space. The indoor (resp. outdoor) range is up to 5 km (resp. 10 km). Energy Consumption. As for SigFox, the emission power may vary up to 14 dBm. Besides, the circuit consumption depends on the device state. During idle or sleep mode, the supply current is negligible (around 1 µA), while reception (resp. transmission) consumes up to 11 mA (resp. 125 mA). 4.4. CSS Performance Analysis A complete simulator, which takes into account all specificities of the device behavior is available at [19]. Complementary information on these standards is available only for members of the Weightless SIG. 5.2. RPMA In this section, we present two others emerging communications systems dedicated to IoT networks. Contrarily to UNB and CSS, they are based on more usual technologies, which are adapted to IoT needs. RPMA (Random Phase Multiple Access) was developed by On-Ramp Wireless. This American company was founded in 2008, to provide connectivity to oil and gas actors. In september 2015, it was renamed Ingenu, and targets to extend its technology to the IoT and M2M market [23]. 4.4. CSS Performance Analysis Indeed, the detection is a linear process. Thus, with two devices transmitting, the FFT output would provide the summation of each FFT, leading to 2 indiscernible peaks. The receiver would not be able to identify which offset to take into account. Nevertheless, one transmission over the two can be • Class C: the end-device is always available for reception, except when transmitting. A low latency is granted, at the cost of a higher energy consumption. EAI Endorsed Transactions on Internet of Things 10 - 2015 | Volume 1 | Issue 1| e3 EAI Endorsed Transactions on Internet of Things 10 - 2015 | Volume 1 | Issue 1| e3 7 C. Goursaud, J.M. Gorce interferer 7 8 9 10 11 12 desired 7 -6 16 18 19 19 20 8 24 -6 20 22 22 22 9 27 27 -6 23 25 25 10 30 30 30 -6 26 28 11 33 33 33 33 -6 29 12 36 36 36 36 36 -6 Table 1. Cochannel rejection (dB) for all combinations of spreading factor for the desired and interferer user interferer 7 8 9 10 11 12 desired 7 -6 16 18 19 19 20 8 24 -6 20 22 22 22 9 27 27 -6 23 25 25 10 30 30 30 -6 26 28 11 33 33 33 33 -6 29 12 36 36 36 36 36 -6 Table 1. Cochannel rejection (dB) for all combinations of spreading factor for the desired and interferer user Weightless-N. This standard is based on narrow band technology, with a differential binary phase shift keying (DBPSK) digital modulation scheme, and is mainly based on Nwave technology [22]. The announced data rates are 30 −100 kbps. Transmissions are performed in the sub-GHz ISM bands 868 MHz. This leads to a range of 5 kms even in challenging urban environments. A FH algorithm is used to counteract interference and fading. This standard is intended for devices that need one-way communications at a very low cost. Weightless-N. This standard is based on narrow band technology, with a differential binary phase shift keying (DBPSK) digital modulation scheme, and is mainly based on Nwave technology [22]. The announced data rates are 30 −100 kbps. Transmissions are performed in the sub-GHz ISM bands 868 MHz. This leads to a range of 5 kms even in challenging urban environments. 5. Additional technologies 5.2. RPMA 5.1. Weightless For a fair comparison, we compare the raw data rate, i.e., the actual bit rate transmitted on the medium independently of the nature of the traffic (preamble, data, code syndrome, ...). Numerical values were obtained from [25] for Ingenu , and from [20] for Weightless . Uplink and downlink are performed in an half- duplex way, with a downlink period of 2s followed by an uplink period of 2s. This permits to dynamically adapt the spreading factor to the channel conditions depending on the received power. One may note that a smaller SF can be used in downlink compared to uplink, as the BS is not energy-constrained and can transmit at a higher power level. For Sigfox, (6) corresponds to the maximum number of users guarantying that there is no collision on a given user for at least 90% transmissions. In addition, for LoRaT M, this value is constrained by the number of available codes which is 6. Finally, for RPMA, Ingenu claims 1000 simultaneous users [25]. Note that such value mainly corresponds to high SF codes. Unfortunately, for Weightless, such information is neither estimated on public documents, nor computable with the available data. Random multiple access is performed by delaying the signal to transmit at each end-device as illustrated in Fig.5. The slot is first divided into Ns subslots such that Ns = 8192 2k , with 2k the used spreading factor. For k ≤13, the transmitter selects one (or several to increase the effective data rate) subslot called access slot. Within the subslot (gray area in Fig.5), transmission is delayed by a random number d ∈ h 0; 2k −1 i . Ingenu estimates that up to 1000 uplink users can be served in each slot. (7) was evaluated by normalizing the overall data rate for all active users, with the spectrum occupancy. We can note that in spite of the wide range of raw data rate (1:300), the spectral efficiency is almost the same (1:2). Furthermore, cell size is also reported, constraint for which SigFox and LoRaT Mare the most efficient ones. Besides, we can observe that all technologies allow two-ways communications, and that Weightless permits to select either one-way or two-way transmissions. Communication range. 5.1. Weightless RPMA is based on DSSS. Transmissions are made in the 2.4 GHz ISM band. Data are first encoded (1/2 rate) and interleaved. The resulting stream is then D-BPSK modulated, before being spread by a Gold Code. The signal is then randomly delayed before transmission. One may note that additional blocks ensure time and frequency synchronization between the BS and the end- devices [24], but are not detailed in this paper. k The third communication technology is proposed by Weightless. Weightless SIG [20] is an organization which aims at providing Weightless standards for IoT networks. These standards are based on the technology initially developed by the British company Neul [21], recently acquired by Huawei in fall 2014. At the time of this article publication, 3 different standards are proposed : Weightless-N, Weightless-P and Weightless- W. Each standard targets different use-cases, but each of them complies with the low-cost, large range and low power consumption as required for IoT. The spreading factor of the Gold Codes is 2k with 2 ≤k ≤13. Each time the spreading factor is doubled, the processing gain increases of 3 dB. This permits to EAI Endorsed Transactions on Internet of Things 10 - 2015 | Volume 1 | Issue 1| e3 EAI Endorsed Transactions on Internet of Things 10 - 2015 | Volume 1 | Issue 1| e3 8 Dedicated networks for IoT : PHY / MAC state of the art and challenges Figure 5. RPMA Multiple Access Principle On the contrary, there is no consensus on the spectrum use. Indeed, one half of the technologies aim at minimizing the bandwidth (Narrow Band (NB) or Ultra NB) to reduce the probability of interference, while the second half spread the information on the available channel to take advantage of spectral diversity, and to benefit from adaptable data rate (with varying spreading factor). In addition, the channel width is also varying. One may note that for Sigfox, 192 kHz is the observed channel, while the signals width is 100 Hz. For the other technologies, the two values coincide. Figure 5. RPMA Multiple Access Principle adapt the data rate to the propagation conditions. For the uplink, or the downlink broadcast transmission, a unique Gold code is used. On the contrary, for unicast downlink transmission, the Gold code is built with the end-device ID, such that no other end-device is able to decode the data. 5.1. Weightless By using eq.15 and eq.16 with CG = 3 · k, we can evaluate that for B = 106, and k = 13, the minimal received power is −145 dBm, which corresponds to 200 kms in free space. In [26], the range was estimated to 10 kms with Okumura-Hata model. Last but not least, the receiver sensitivity to a frequency offset is discussed, as cheap (thus imprecise) oscillators are favored for dense deployment. Ingenu algorithm to compensate for this offset tolerates a deviation up to 10 ppm. In addition, the intrinsic behavior of LoRaT Mpermits to correctly transmit even with a 40ppm oscillator. Finally, SigFox has no limitation and can decode the signals as long as they are in the monitored band. Interference Sensitivity. The random delay permits to shift the time of arrival of the different signals. As Gold codes have low auto-correlation, each arrival can be decoded as long as at most one end-devices selected the considered delay. The multiple access is thus a slotted ALOHA protocol. 6. Technologies comparison IoT is a relatively new market which may lead to the deployment of billions of connected devices. The associated networking paradigm is quite different from those of cellular systems. Despite that in some specific applications (e.g. for transmitting videos) MTDs may need high rate communications, it may be anticipated that a majority of these MTDs will require only a We present in Table 2 some important features of the presented emerging technologies for IoT applications. We can first note that the sub-GHz bands are favored. Indeed, the pathloss is smaller for low frequencies. Thus, higher ranges are obtained with the same emission power. EAI Endorsed Transactions on Internet of Things 10 - 2015 | Volume 1 | Issue 1| e3 EAI Endorsed Transactions on Internet of Things 10 - 2015 | Volume 1 | Issue 1| e3 9 C. Goursaud, J.M. Gorce SigFox LoRaTM Weightless Ingenu N P W (1) Band 868/915 MHz 868/915 MHz Sub-GHz Sub-GHz 470 −790 MHz 2.4 GHz ISM Yes Yes Yes Yes No Yes (2) PHY UNB CSS NB NB DSSS RPMA (3) Spreading factor NA 27 −212 NA NA 1 −210 24 −213 (4) Typical channel bandwidth 192 kHz 500 −125 kHz NAD 12.5 kHz 6 −8 MHz 1 MHz (5) Raw rate (kbps) 0.1 27 −0.37 30 −100 0.2 −100 1 −10000 0.06 −30 (6) Simultaneous active users in (4) for OP = 10−1 100 6 NAD NAD NAD 1000 (7) Raw spectral efficiency (b/s.Hz) 0.05 0.12 NAD NAD NAD 0.1 (8) Range (km) 63 22 5 2 10 10 −2 (9) Downlink Yes Yes No Yes Yes Yes (10) Doppler sensitivity Unconstrained Up to 40 ppm NAD NAD NAD Up to 10 ppm Table 2. Technologies summary and comparison (NA= Non Applicable; NAD= Non Available Data) the architecture cost and to ensure low latency and high reliability. This optimization will rely on joint PHY/MAC strategies which may permit to use original distributed coding approaches as proposed for instance in [28]. In addition the energy consumption of MTDs will play an important role for ensuring long life-time and small battery requirements. bursty access, with very small packets but under high QoS constraints such as high reliability or low latency. Therefore, two strategies are competing to absorb this new traffic. The first strategy relies on broadening the capabilities of cellular technologies especially with the preparation of the future 5G at horizon 2020. References [16] Patrick Flandrin, Time-Frequency/Time-Scale Analysis, ISBN 0-12-259670-9, in Academic Press, San Diego, 1999 [1] F. Boccardi, R. Heath, A. Lozano, T. Marzetta, and P. Popovski, “Five disruptive technology directions for 5g,” Communications Magazine, IEEE, vol. 52, pp. 74–80, February 2014. [17] Nanoscale Labs, Communications system, Patent US8406275, submitted 2010-03-09, published 2013-03- 26 [2] A. Biral, M. Centenaro, A. Zanella, L. Vangelista, and M. Zorzi, “The challenges of m2m massive access in wireless cellular networks,” Digital Communications and Networks, vol. 1, no. 1, pp. 1–19, 2015. [18] Semtech Corporation, SX1272/73 - 860 MHz to 1020 MHz Low Power Long Range Transceiver Datasheet, March 2015, rev3, available www.semtech.com/images/datasheet/sx1272.pdf accessed 2015-09-14 [3] P. Jain, P. Hedman, and H. Zisimopoulos, “Machine type communications in 3gpp systems,” Communications Magazine, IEEE, vol. 50, pp. 28–35, November 2012. [19] http://www.semtech.com/apps/filedown/down. php?file=SX1272LoRaCalculatorSetup1%271.zip, accessed 2015-09-10 [4] A. Laya, L. Alonso, and J. Alonso-Zarate, “Is the random access channel of lte and lte-a suitable for m2m communications? a survey of alternatives,” Communications Surveys & Tutorials, IEEE, vol. 16, no. 1, pp. 4–16, 2014. [20] www.weightless.org accessed 2015-09-14 [21] www.neul.com/neul/ accessed 2015-09-14 [22] www.nwave.io/ accessed 2015-09-14 [23] www.ingenu.com/ accessed 2015-09-14 [5] Walker, H.R. (2004) Ultra narrow band modulation in 2004 IEEE/SarnoffSymposium on Advances in Wired and Wireless Communication, pp.19-22, 26-27 Apr 2004 [24] On-Ramp Wireless, Inc., Random phase multiple access communication interface system and method, Patent WO 2009117284 A3, submitted 2009-03-11, published 2009- 12-17 [6] Shikai Zhang (2013) Spectrum aanlyses of UNB modu- lation formats in 2013 3rd International Conference on Consumer Electronics, Communications and Networks (CECNet), pp.594-597, 20-22 Nov. 2013 [25] OnRamp White Paper RPMA Technology for the Internet of Things, 010-0064-00 Rev. B, May 11, 2015 [26] Project IEEE P802.15 Working Group for Wireless Personal Area Networks (WPANs) On-Ramp Wireless Dynamic Direct Sequence Spread Spectrum (D-DSSS) Proposal for 802.15.4g, IEEE 802.15-09-0307-00-004g, May 2009 [7] Phil Karn (2013) The VMSK Delusion http://www.ka9q.net/vmsk/, accessed 2015-08-28 [8] www.sigfox.com accessed 2015-09-14 [9] www.sigfox.com/static/media/Files/ [9] www.sigfox.com/static/media/Files/ Documentation/SIGFOX_Whitepaper.pdf accessed 2015-09-14 y [27] H. S. Dhillon, H. Huang, H. Viswanathan, R. Valenzuela, et al., “Fundamentals of throughput maximization with random arrivals for m2m communications,” Communications, IEEE Transactions on, vol. 62, no. 11, pp. 4094–4109, 2014. [10] Minh-Tien Do, Claire Goursaud, and Jean-Marie Gorce, Interference Modelling and Analysis of Random FDMA scheme in Ultra Narrowband Networks, in AICT 2014 [28] M. Shirvanimoghaddam, Y. Li, M. Dohler, B. Vucetic, and S. 6. Technologies comparison In this case, 5G intends to jointly manage M2M and mobile H2H traffics with appropriate dynamic sharing rules. The advantage of this strategy is to foster the development of IoT by avoiding the development of a specific access network and minimize infrastructure costs. For the best of our knowledge the competition is still open between the actual technologies. To foster the analysis of these technologies and to evaluate the possible gains, a suitable general theoretical model is needed. Some works exist, such as the recent work proposed in [27] which exploits stochastic geometry and related models previously used for cellular networks. However, it is important to emphasize that the classical Shannon capacity established in asymptotic regime does not hold for short packets communications. If the underlying theoretical The second strategy relies on the deployment of a new network technology which may better comply with the specific features of IoT. This objective needs a cross- layer design of the PHY and MAC layers. The winning technology will be surely the one which will be able to maximize the manageable MTDs density, to minimize EAI Endorsed Transactions on Internet of Things 10 - 2015 | Volume 1 | Issue 1| e3 EAI Endorsed Transactions on Internet of Things 10 - 2015 | Volume 1 | Issue 1| e3 10 Dedicated networks for IoT : PHY / MAC state of the art and challenges model is quite clear for the uplink situation and relies on the Multiple Access Channel (MAC, see e.g. [29]), the specific bursty nature related to short packets imposes to develop a new framework allowing to establish the achievable region balancing reliability, latency, MTDs density and energy. [14] A. Springer, W. Gugler, M. Huemer, L. Reindl, C. C. W. Ruppel, and R. Weigel,, Spread spectrum communications using chirp signals, in IEEE Proc. Eurocomm, pp.166 - 170, 2000 [15] Semtech Corporation, AN1200.22 LoRaT M Modulation Basics, May 2015, Rev2, available at www.semtech.com/images/datasheet/an1200.22.pdf accessed 2015-09-14 References Feng, “Probabilistic rateless multiple access for machine-to-machine communication,” Wireless Commu- nications, IEEE Transactions on, 2015. [11] Minh-Tien Do, Claire Goursaud, and Jean-Marie Gorce, On the benefits of random FDMA schemes in ultra narrow band networks, in IEEE 12th International Symposium on Modeling and Optimization in Mobile, Ad Hoc, and Wireless Networks (WiOpt), pp. 672-677, 2014 [29] T.M. Cover, J.A. Thomas, “Elements of information theory.” John Wiley & Sons, 2012. [12] www.lora-alliance.org/ accessed 2015-09-14 [13] M. Winkler, Chirp signals for communications, in IEEE WESCON Convention Record, vol. Pt. 7, 1962 EAI Endorsed Transactions on Internet of Things 10 - 2015 | Volume 1 | Issue 1| e3 EAI Endorsed Transactions on Internet of Things 10 - 2015 | Volume 1 | Issue 1| e3 11

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DNA oxidation induced by fetal exposure to BPA agonists impairs female meiosis

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. CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted August 20, 2020. ; https://doi.org/10.1101/2020.08.17.253724 doi: bioRxiv preprint . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted August 20, 2020. ; https://doi.org/10.1101/2020.08.17.253724 doi: bioRxiv preprint . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted August 20, 2020. ; https://doi.org/10.1101/2020.08.17.253724 doi: bioRxiv preprint . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprin this version posted August 20, 2020. ; https://doi.org/10.1101/2020.08.17.253724 doi: bioRxiv preprint DNA oxidation induced by fetal exposure to BPA agonists impairs female meiosi DNA oxidation induced by fetal exposure to BPA agonists impairs female meiosis. Sonia Abdallah1, Delphine Moison1, Margaux Wieckowski1, Sébastien Messiaen1, Emmanuelle Martini1, Anna Campalans2, J. Pablo Radicella2, René Habert1, Gabriel Livera1*, Virginie Rouiller- Fabre1, Marie-Justine Guerquin1*. 1 2 3 4 Sonia Abdallah1, Delphine Moison1, Margaux Wieckowski1, Sébastien Messiaen1, Emmanuelle Martini1, Anna Campalans2, J. Pablo Radicella2, René Habert1, Gabriel Livera1*, Virginie Rouiller- Fabre1, Marie-Justine Guerquin1*. 2 3 4 1 Laboratory of Development of the Gonads, UMRE008 Genetic Stability Stem cells and Radiations, Université de Paris, Université Paris Saclay, CEA/DRF/IBFJ/IRCM, F-92265 Fontenay aux Roses, France. 5 6 7 1 Laboratory of Development of the Gonads, UMRE008 Genetic Stability Stem cells and Radiations, Université de Paris, Université Paris Saclay, CEA/DRF/IBFJ/IRCM, F-92265 Fontenay aux Roses, France. 5 6 7 2 Laboratory of Genetic Instability Research, UMRE008 Genetic Stability Stem cells and Radiations, Université de Paris, Université Paris Saclay, CEA/DRF/IBFJ/IRCM, F-92265 Fontenay aux Roses, France. 8 9 10 2 Laboratory of Genetic Instability Research, UMRE008 Genetic Stability Stem cells and Radiations, Université de Paris, Université Paris Saclay, CEA/DRF/IBFJ/IRCM, F-92265 Fontenay aux Roses, France. 8 9 10 * Correspondences: [email protected], [email protected] 11 Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead contact, [email protected]. 12 13 Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead contact, [email protected]. 12 13 1 1 . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted August 20, 2020. ; https://doi.org/10.1101/2020.08.17.253724 doi: bioRxiv preprint . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted August 20, 2020. ; https://doi.org/10.1101/2020.08.17.253724 doi: bioRxiv preprint Keywords: development, oogenesis, meiosis, DNA oxidation, bisphenols, endocrine disruptor. 33 Introduction 34 Moreover, fetal exposure to BPA alters the distribution of recombination events signaled by MLH1, a DNA mismatch repair protein required for 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 In females, aneuploidy (aberrant number of chromosomes) is an important cause of adverse reproductive outcomes such as miscarriages and congenital abnormalities. Aneuploid eggs can be induced by numerous lifestyle factors (age, obesity, environmental pollutants…) (Nagaoka et al., 2012) and can derive from alterations occurring during meiotic prophase I in fetal life. Indeed, proper chromosome segregation at adulthood requires organized reciprocal DNA exchanges between homologous chromosomes (crossover) occurring during prophase I as a result of the repair of meiotic double strand break (DSB) by homologous recombination (Patricia A. Hunt & Hassold, 2008; Ottolini et al., 2015; S. Wang et al., 2017). Crossover regulation depends on a correct implementation of the meiotic program in primordial germ cells (PGCs) initiated after their migration into the gonad. At this stage, pluripotent and proliferative PGCs acquire the competence to initiate meiosis through the expression of Deleted In Azoospermia-like (Dazl) (Nicholls et al., 2019). Depending on the somatic environment and under the control of meiotic orchestrators such as Stra8 that direct the switch from mitosis to meiosis, female PGCs initiate prophase I at 13.5 day post-conception (dpc) (Bailey et al., 2017; Hargan-Calvopina et al., 2016a; Ishiguro et al., 2020; Le Bouffant et al., 2010; Spiller & Bowles, 2019; Trautmann et al., 2008). Alterations that occur during the establishment of the meiotic program lead to meiotic defects in prophase I that can hamper future fertility (Bailey et al., 2017; Hargan-Calvopina et al., 2016a; Ishiguro et al., 2020; Nicholls et al., 2019). It is well known that implementation and progression of prophase I are extremely sensitive to environmental factors such as toxicants and endocrine disrupting chemicals. Among those, bisphenol A (BPA) is the first and the most studied environmental compound known to alter meiosis and folliculogenesis in females in numerous mammalian and non-mammalian organisms (Brieno-Enriquez et al., 2012; Brieño-Enríquez et al., 2011; P. A. Hunt et al., 2012; Lawson et al., 2011; Susiarjo et al., 2007; W. Wang et al., 2014; H.-Q. Zhang et al., 2012; T. Zhang et al., 2014). Summary 14 Many endocrine disruptors have been proven to impair the meiotic process that is mandatory to produce healthy gametes. Bisphenol A is emblematic as it impairs meiotic prophase I and causes oocyte aneuploidy following in utero exposure. However, the mechanisms underlying these deleterious effects remain poorly understood. Furthermore, the increasing uses of BPA analogs raise concerns for public health. Here, we investigated the effect on oogenesis in mouse of fetal exposure to two BPA analogs, Bisphenol A Diglycidyl Ether (BADGE) or Bisphenol AF (BPAF). These analogs delay meiosis initiation, increase MLH1 foci per cell and induce oocyte aneuploidy. We further demonstrate that these defects are accompanied by a deregulation of gene expression and aberrant mRNA splicing in fetal premeiotic germ cells. Interestingly, we observed an increase in DNA oxidation after exposure to BPA analogs. Specific induction of oxidative DNA damages during fetal germ cell differentiation causes similar defects during oogenesis, as observed in 8- Oxoguanine DNA Glycosylase (OGG1) deficient mice or after in utero exposure to potassium bromate (KBrO3), an inducer of oxidative DNA damages. Moreover, the supplementation of N- acetylcysteine (NAC) with BPA analogs counteracts the bisphenol-induced meiotic effect. Together our results position oxidative stress as a central event that negatively impacts the female meiosis with major consequences on oocyte quality. This could be a common mechanism of action for so called endocrine disruptors pollutants and it could lead to novel strategies for reprotoxic compounds. 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 2 2 . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted August 20, 2020. ; https://doi.org/10.1101/2020.08.17.253724 doi: bioRxiv preprint . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted August 20, 2020. ; https://doi.org/10.1101/2020.08.17.253724 doi: bioRxiv preprint Introduction 34 In females, aneuploidy (aberrant number of chromosomes) is an important cause of adverse reproductive outcomes such as miscarriages and congenital abnormalities. Aneuploid eggs can be induced by numerous lifestyle factors (age, obesity, environmental pollutants…) (Nagaoka et al., 2012) and can derive from alterations occurring during meiotic prophase I in fetal life. Indeed, proper chromosome segregation at adulthood requires organized reciprocal DNA exchanges between homologous chromosomes (crossover) occurring during prophase I as a result of the repair of meiotic double strand break (DSB) by homologous recombination (Patricia A. Hunt & Hassold, 2008; Ottolini et al., 2015; S. Wang et al., 2017). Crossover regulation depends on a correct implementation of the meiotic program in primordial germ cells (PGCs) initiated after their migration into the gonad. At this stage, pluripotent and proliferative PGCs acquire the competence to initiate meiosis through the expression of Deleted In Azoospermia-like (Dazl) (Nicholls et al., 2019). Depending on the somatic environment and under the control of meiotic orchestrators such as Stra8 that direct the switch from mitosis to meiosis, female PGCs initiate prophase I at 13.5 day post-conception (dpc) (Bailey et al., 2017; Hargan-Calvopina et al., 2016a; Ishiguro et al., 2020; Le Bouffant et al., 2010; Spiller & Bowles, 2019; Trautmann et al., 2008). Alterations that occur during the establishment of the meiotic program lead to meiotic defects in prophase I that can hamper future fertility (Bailey et al., 2017; Hargan-Calvopina et al., 2016a; Ishiguro et al., 2020; Nicholls et al., 2019). It is well known that implementation and progression of prophase I are extremely sensitive to environmental factors such as toxicants and endocrine disrupting chemicals. Among those, bisphenol A (BPA) is the first and the most studied environmental compound known to alter meiosis and folliculogenesis in females in numerous mammalian and non-mammalian organisms (Brieno-Enriquez et al., 2012; Brieño-Enríquez et al., 2011; P. A. Hunt et al., 2012; Lawson et al., 2011; Susiarjo et al., 2007; W. Wang et al., 2014; H.-Q. Zhang et al., 2012; T. Zhang et al., 2014). In female primates and rodents, fetal exposure to BPA induces alteration of the expression of meiotic genes at the time of meiosis onset and during prophase I (Brieno-Enriquez et al., 2012; Lawson et al., 2011; H.-Q. Zhang et al., 2012). Introduction 34 In female primates and rodents, fetal exposure to BPA induces alteration of the expression of meiotic genes at the time of meiosis onset and during prophase I (Brieno-Enriquez et al., 2012; Lawson et al., 2011; H.-Q. Zhang et al., 2012). Moreover, fetal exposure to BPA alters the 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 3 . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted August 20, 2020. ; https://doi.org/10.1101/2020.08.17.253724 doi: bioRxiv preprint . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted August 20, 2020. ; https://doi.org/10.1101/2020.08.17.253724 doi: bioRxiv preprint resolution of double Holliday junctions as crossovers (Ashley et al., 2001; Cheng et al., 2009; Patricia A. Hunt et al., 2003). This increase observed during pachytene stage is correlated with the occurrence of aneuploid oocytes at adulthood. The precise action of BPA on meiosis has been proposed to involve estrogen receptor signaling (Gibert, 2015; Susiarjo et al., 2007; M. Yu et al., 2018; H.-Q. Zhang et al., 2012). Moreover, several studies suggest that BPA exposure induces epigenetic alterations in germ cells such as DNA or histone methylation resulting in gene expression alterations (Chao et al., 2012; Chianese et al., 2017; Kim et al., 2014; T. Wang et al., 2016; H.-Q. Zhang et al., 2012). Lastly, some studies have shown that post-natal exposure to BPA increases the amount of reactive oxygen species (ROS) compromising oocyte maturation and chromosome segregation and inducing DNA damages such as 8-hydroxydeoxyguanosine (8OdG) (Ganesan & Keating, 2016; M. Zhang et al., 2017; T. Zhang et al., 2018). 62 63 64 65 66 67 68 69 70 71 72 BPA is a member of the bisphenol family like other structural analogs that are commonly used in the industry. Due to recent regulations and growing commercialization of BPA-free labeled products, BPA analogs are increasingly used in the manufacturing of consumer products. BADGE or BPAF fetal exposure reproduces BPA defects on folliculogenesis and recombination events. 89 90 In mammals, fetal exposure to BPA disrupts follicle formation causing multioocyte follicles and increases the incidence of missegregation after meiosis resumption in post-natal ovaries. To study the effects of analogs of BPA on oocyte and folliculogenesis at adulthood, we exposed pregnant mice to 10 µM of BADGE or BPAF in drinking water from 10.5 to 18.5 days post-conception. 91 92 93 94 Figure 1: BADGE or BPAF fetal exposures fetal exposure impairs folliculogenesis in post- natal mouse ovaries. (A) Haematoxylin and eosin staining of ovarian section from 3 months old mice exposed during fetal life to vehicle (ETOH), BADGE (BADGE) or BPAF (BPAF). Scale bar, 200µm or 10µm for the higher magnification (multi-oocyte follicle). (B) Quantification of total follicle count in bisphenol-treated or control adult ovaries. Each bar represents the percentage of primordial, primary, secondary and antral follicles. (C) Percentage of multioocytes follicle in bisphenols-treated or control post-natal (8 dpp) and adult (3 months) ovaries. Error bars indicate mean ± s.e.m. n=3-5 mice from 3 independent exposures, * p <0.05 (Mann-Whitney’s test). At 3 th ld th t t l b f f lli l d i t t d (BADGE BPAF) d t l A ETOH BADGE BPAF B ETOH BADGE BPAF primordial primary secondary antral 0 2000 4000 6000 8000 total follicle count per ovary * * C multi-oocyte follicle 0.0 multi-oocyte follicle (%) ETOH BADGE BPAF 1.0 2.0 3.0 4.0 5.0 ETOH BADGE BPAF * * * * 8 dpp 3 months 3 months 95 96 97 98 99 100 101 102 103 A ETOH BADGE BPAF B 8000 C multi-oocyte follicle 5 0 * Figure 1: BADGE or BPAF fetal exposures fetal exposure impairs folliculogenesis in post- A ETOH BADGE BPAF B ETOH BADGE BPAF primordial primary secondary antral 0 2000 4000 6000 8000 total follicle count per ovary * * C multi-oocyte follicle 0.0 multi-oocyte follicle (%) ETOH BADGE BPAF 1.0 2.0 3.0 4.0 5.0 ETOH BADGE BPAF * * * * 8 dpp 3 months 3 months 95 C 0.0 multi-oocyte follicle (%) ETOH BADGE BPAF 1.0 2.0 3.0 4.0 5.0 ETOH BADGE BPAF * * * * 8 dpp 3 months B ETOH BADGE BPAF primordial primary secondary antral 0 2000 4000 6000 8000 total follicle count per ovary * * 3 months C 5 B Figure 1: BADGE or BPAF fetal exposures fetal exposure impairs folliculogenesis in post- natal mouse ovaries. Introduction 34 (A) Haematoxylin and eosin staining of ovarian section from 3 months old mice exposed during fetal life to vehicle (ETOH), BADGE (BADGE) or BPAF (BPAF). Scale bar, 200µm or 10µm for the higher magnification (multi-oocyte follicle). (B) Quantification of total follicle count in bisphenol-treated or control adult ovaries. Each bar represents the percentage of primordial, primary, secondary and antral follicles. (C) Percentage of multioocytes follicle in A ETOH BADGE BPAF B ETOH BADGE BPAF primordial primary secondary antral 0 2000 4000 6000 8000 total follicle count per ovary * * C multi-oocyte follicle 0.0 multi-oocyte follicle (%) ETOH BADGE BPAF 1.0 2.0 3.0 4.0 5.0 ETOH BADGE BPAF * * * * 8 dpp 3 months 3 months 88 89 90 91 92 93 94 95 96 97 98 99 100 Introduction 34 Among those, we choose to focus our study on emerging bisphenols: Bisphenol A Diglycidyl Ether (BADGE) and Bisphenol AF (BPAF) because very little data exists on their effects on mammalian germ cells. However, recent studies have shown that, as BPA, BPAF induces the release of ROS in adult oocytes, delaying in vitro maturation (Ding et al., 2017; Jones et al., 2018). 73 74 75 76 77 78 79 The aim of this study was to explore the effects of prenatal exposure of murine oocytes to BADGE and BPAF and their consequences on fertility at adulthood. We show that exposure of pregnant mice to the BPA analogs, BADGE and BPAF causes oxidative DNA damage, alterations during mitosis to meiosis transition and an increase of crossovers number leading to aneuploidy and oocyte loss. Specific induction of oxidative DNA damages during fetal germ cell differentiation causes similar defects in prophase I as observed in 8-Oxoguanine DNA Glycosylase (OGG1) deficient mice and after fetal exposure to potassium bromate. Thus, we unveiled the central role of oxidative DNA damage in the meiotic response to bisphenols. 80 81 82 83 84 85 86 87 4 . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted August 20, 2020. ; https://doi.org/10.1101/2020.08.17.253724 doi: bioRxiv preprint . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted August 20, 2020. ; https://doi.org/10.1101/2020.08.17.253724 doi: bioRxiv preprint Results BADGE or BPAF fetal exposure reproduces BPA defects on folliculogenesis and recombination events. In mammals, fetal exposure to BPA disrupts follicle formation causing multioocyte follicles and increases the incidence of missegregation after meiosis resumption in post-natal ovaries. To study the effects of analogs of BPA on oocyte and folliculogenesis at adulthood, we exposed pregnant mice to 10 µM of BADGE or BPAF in drinking water from 10.5 to 18.5 days post-conception. Figure 1: BADGE or BPAF fetal exposures fetal exposure impairs folliculogenesis in post- natal mouse ovaries. BADGE or BPAF fetal exposure reproduces BPA defects on folliculogenesis and recombination events. 89 90 (A) Haematoxylin and eosin staining of ovarian section from 3 months old mice exposed during fetal life to vehicle (ETOH), BADGE (BADGE) or BPAF (BPAF). Scale bar, 200µm or 10µm for the higher magnification (multi-oocyte follicle). (B) Quantification of total follicle count in bisphenol-treated or control adult ovaries. Each bar represents the percentage of primordial, primary, secondary and antral follicles. (C) Percentage of multioocytes follicle in bisphenols-treated or control post-natal (8 dpp) and adult (3 months) ovaries. Error bars indicate mean ± s.e.m. n=3-5 mice from 3 independent exposures, * p <0.05 (Mann-Whitney’s test). 95 96 97 98 99 100 101 102 At 3 months old, the total number of follicles was assayed in treated (BADGE or BPAF) and control (ethanol [ETOH]) ovaries (Figure 1A-C). A significant decrease of follicular pool was detected in treated ovaries (Figure 1B). The distribution of follicle classes (primordial to antral follicle) was not affected by the treatment suggesting that the progression of folliculogenesis was not affected 103 104 105 106 5 . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted August 20, 2020. ; https://doi.org/10.1101/2020.08.17.253724 doi: bioRxiv preprint . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted August 20, 2020. ; https://doi.org/10.1101/2020.08.17.253724 doi: bioRxiv preprint (Figure 1B). However, we observed abnormal multioocyte follicles in BADGE- and BPAF- treated 3-months ovaries (0 follicle in ETOH group VS 34 ± 11 follicles and 58 ± 14 follicles in BADGE and BPAF groups respectively; Figure 1C). The incidence of multioocyte follicles was even more pronounced at the initiation of the folliculogenesis (ie 8 days postpartum) in the BADGE- and BPAF- treated ovaries (0 follicle in ETOH group VS 647 ± 262 follicles and 75 ± 11 follicles in BADGE and BPAF groups respectively; Figure 1C). 107 108 109 110 111 112 As observed with BPA, exposure to bisphenol A analogs induces chromosome missegragation during meiotic divisions. Ploidy in metaphase II oocytes from bisphenols-treated ovaries was assessed by the presence of isolated chromosomes. BADGE or BPAF fetal exposure reproduces BPA defects on folliculogenesis and recombination events. 89 90 (A) Aneuploid MII oocytes in 3 months old mice exposed during fetal life with vehicle (ETOH), BADGE or BPAF were quantified after CREST immunofluorescence staining. Left panel: photomicrographs of representative euploid (20 chromosomes, 40 centromeres) or aneuploid oocytes (≠20 chromosomes, ≠40 centromeres). Arrowhead indicates extra chromatids. Scale bar: 10 µm. Right panel: quantification of the percentage of aneuploid MII oocytes. n= 28 (ETOH), 40 (BADGE) and 42 (BPAF) oocytes from 5-12 independent exposures. Mean± s.e.m, * p < 0.05 (Mann-Whitney’s test). (B) The number of chiasmata per bivalent (tetrad) in MI oocytes from 3 months old mice exposed during fetal life with vehicle (ETOH), BADGE or BPAF were determined according to the shape of the bivalent after CREST immunostaining (green). Left panel: representative photomicrograph of MI oocyte that contains tetrads with one (1), two (2), three or more (3) chiasmata. Scale bar: 10 µm. Right panel: percentage of bivalent with 3 or more chiasmata. n= 33 (ETOH), 56 (BADGE) and 23 (BPAF) oocytes from 5 independent exposures. Mean (red bar), *** p < 0.001, **** p < 0.0001 (Mann-Whitney’s test). (C) Ovaries from fetuses exposed to vehicle (ETOH) or bisphenols (BADGE and BPAF) from 10.5 dpc to 18.5 dpc were used for the MLH1 quantification (long exposure). The number of crossovers per synaptonemal complexes (synapsis) in pachytene oocytes from 18.5 dpc fetuses was quantified using MLH1 immunostaining (green). The pachytene stage is determined on the basis of the SYCP3 staining (red). Left panel: Representative photomicrographs of pachytene cells from vehicle (ETOH) and BADGE treated ovaries. White arrow shows a synaptonemal complexe with 3 MLH1 foci. Right panel: Percentage of synaptonemal complexes with 3 or more MLH1 foci. n= 86 (ETOH), 78 (BADGE) and 82 (BPAF) oocytes from 12 independent exposures. Mean: red bar, ** p < 0.01; ***p < 0.001. c progression and initiation Figure 2: BADGE or BPAF fetal exposures increase the recombination events and missegregation during meiosis. (A) Aneuploid MII oocytes in 3 months old mice exposed during fetal life with vehicle (ETOH), BADGE or BPAF were quantified after CREST immunofluorescence staining. Left panel: photomicrographs of representative euploid (20 chromosomes, 40 centromeres) or aneuploid oocytes (≠20 chromosomes, ≠40 centromeres). Arrowhead indicates extra chromatids. Scale bar: 10 µm. Right panel: quantification of the percentage of aneuploid MII oocytes. n= 28 (ETOH), 40 (BADGE) and 42 (BPAF) oocytes from 5-12 independent exposures. Mean± s.e.m, * p < 0.05 (Mann-Whitney’s test). BADGE or BPAF fetal exposure reproduces BPA defects on folliculogenesis and recombination events. 89 90 BADGE or BPAF fetal exposure significantly increases the percentage of aneuploid metaphase II (MII) oocytes (Figure 2A). During prophase I, crossovers and chiasmata distribution in oocyte was also modified after BPAF or BADGE in utero exposure. We quantified MLH1 foci in pachytene cells at 18.5 days post-conception (dpc) in bisphenols-treated ovaries. Pachytene stage was identified on the basis of SYCP3 staining patterns and the number of MLH1 foci per bivalent (chromosome pair) was determined. The number of bivalent with three or more MLH1 foci was increased in bisphenols-treated oocytes (Figure 2C). The increase of reciprocal exchanges between homologs was confirmed by the quantification of chiasmata in metaphase I oocytes. In bisphenols-treated oocytes, we observed an increase in bivalent with three or more chiasmata identified according to the shape of the bivalent (Figure 2B). Thus BPAF and BADGE mimic the known hallmarks of BPA during oogenesis like impaired crossovers distribution and aneuploidy. 113 114 115 116 117 118 119 120 121 122 123 124 125 126 6 6 . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted August 20, 2020. ; https://doi.org/10.1101/2020.08.17.253724 doi: bioRxiv preprint . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted August 20, 2020. At 18.5 dpc, we observed a delay in meiotic progression in bisphenols-treated mice with a decrease in the proportion of diplotene stages in favor to the early and late pachytene stages (Figure 3A). 175 176 177 BADGE or BPAF fetal exposure reproduces BPA defects on folliculogenesis and recombination events. 89 90 ; https://doi.org/10.1101/2020.08.17.253724 doi: bioRxiv preprint BADGE or BPAF fetal exposures delay meiot At 18.5 dpc, we observed a delay in meiotic A euploid aneuploid aneuploid oocytes (%) 0 20 40 60 * * ETOH BADGE BPAF adult oocytes B metaphase I 0 10 20 30 40 50 > 3 chiasmata / bivalent (%) ETOH BADGE BPAF **** bivalents adult oocytes C 0 ETOH 5 10 15 20 > 3 MLH1 foci / bivalent (%) ETOH BADGE BPAF ETOH BADGE 18.5 dpc oocytes long term exposure 18.5 (dpc) 14.5 10.5 meiotic prophase I *** *** ** CREST / DAPI CREST / DAPI MLH1 / SYCP3 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150 151 152 153 154 155 156 157 158 159 160 161 162 163 164 165 166 167 168 169 170 171 172 173 174 175 CC made available under a (which was not certified by peer review) is the author/funder, who h t s ; ttps //do o g/ 0 0 / 0 0 08 53 do b o p ep t 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150 151 152 153 154 155 156 157 158 159 160 161 162 163 164 165 166 167 168 169 170 171 172 173 174 175 176 177 A euploid aneuploid aneuploid oocytes (%) 0 20 40 60 * * ETOH BADGE BPAF adult oocytes CREST / DAPI BA 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150 151 152 153 154 155 156 157 158 159 160 161 162 163 164 165 166 167 168 169 170 171 172 173 174 A A 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150 151 152 153 154 155 156 157 158 159 160 161 162 163 164 165 166 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150 151 Figure 2: BADGE or BPAF fetal exposures increase the recombination events and missegregation during meiosis. BADGE or BPAF fetal exposure reproduces BPA defects on folliculogenesis and recombination events. 89 90 (B) The number of chiasmata per bivalent (tetrad) in MI oocytes from 3 months old mice exposed during fetal life with vehicle (ETOH), BADGE or BPAF were determined according to the shape of the bivalent after CREST immunostaining (green). Left panel: representative photomicrograph of MI oocyte that contains tetrads with one (1), two (2), three or more (3) chiasmata. Scale bar: 10 µm. Right panel: percentage of bivalent with 3 or more chiasmata. n= 33 (ETOH), 56 (BADGE) and 23 (BPAF) oocytes from 5 independent exposures. Mean (red bar), *** p < 0.001, **** p < 0.0001 (Mann-Whitney’s test). (C) Ovaries from fetuses exposed to vehicle (ETOH) or bisphenols (BADGE and BPAF) from 10.5 dpc to 18.5 dpc were used for the MLH1 quantification (long exposure). The number of crossovers per synaptonemal complexes (synapsis) in pachytene oocytes from 18.5 dpc fetuses was quantified using MLH1 immunostaining (green). The pachytene stage is determined on the basis of the SYCP3 staining (red). Left panel: Representative photomicrographs of pachytene cells from vehicle (ETOH) and BADGE treated ovaries. White arrow shows a synaptonemal complexe with 3 MLH1 foci. Right panel: Percentage of synaptonemal complexes with 3 or more MLH1 foci. n= 86 (ETOH), 78 (BADGE) and 82 (BPAF) oocytes from 12 independent exposures. Mean: red bar, ** p < 0.01; ***p < 0.001. c progression and initiation euploid aneuploid B metaphase I 0 10 20 30 40 50 > 3 chiasmata / bivalent (%) ETOH BADGE BPAF **** bivalents adult oocytes *** CREST / DAPI B B metaphase I 148 149 150 151 152 153 154 155 156 157 158 159 160 161 162 163 164 165 166 167 168 169 170 171 172 173 174 bivalents C 0 ETOH 5 10 15 20 > 3 MLH1 foci / bivalent (%) ETOH BADGE BPAF ETOH BADGE 18.5 dpc oocytes long term exposure 18.5 (dpc) 14.5 10.5 meiotic prophase I *** ** MLH1 / SYCP3 C ETOH BADGE 18.5 dpc oocytes 18.5 dpc oocytes At 18.5 dpc, we observed a delay in meiotic progression in bisphenols-treated mice with a decrease in the proportion of diplotene stages in favor to the early and late pachytene stages (Figure 3A). 175 176 177 7 Figure 3: BADGE or BPAF fetal exposures delay the meiosis initiation and prophase I progression. (A) Distribution of meiosis prophase I late stages in 18.5 dpc treated (BADGE, BPAF) and control (ETOH) ovaries. BADGE or BPAF fetal exposure reproduces BPA defects on folliculogenesis and recombination events. 89 90 It is The copyright holder for this preprint this version posted August 20, 2020. ; https://doi.org/10.1101/2020.08.17.253724 doi: bioRxiv preprint Figure 3: BADGE or BPAF fetal exposures delay the meiosis initiation and prophase I progression. (A) Distribution of meiosis prophase I late stages in 18.5 dpc treated (BADGE, BPAF) and control (ETOH) ovaries. Error bars show mean ± s.e.m.; mice analysed n=3-6; * p < 0.05 (Mann-Whitney’s test). (B-C) prophase I initiation was assayed in 14.5 dpc ovaries after immunostaining of SYCP3 (meiosis), BrdU (mitosis and meiosis S phase) and TRA98 (germ cell marker). (B) Representative photomicrographs of SYCP3 (green), BrdU (red) and TRA98 (blue) immunostaining. Scale bar 5 µm. Pre PL = pre- leptotene; post PL= post-leptotene. Error bars show mean ± s.e.m. n=5 mice from independent exposures; * p ≤ 0.05; ** p ≤ 0.01 (Mann-Whitney’s test). (C) Graphs showing the percentage of preleptotene (BrdU and SYCP3 positive) and meiotic (BrdU negative, SYCP3 positive) oocytes. Error bars show mean ± s.e.m. n=5 mice from independent exposures; * p < 0.05; ** p < 0.01 (Mann-Whitney’s test). A ETOH BADGE BPAF 0 20 40 60 80 100 germ cells (%) early pachynema late pachynema diplonema * * * A B y p y p y p BRDU TRA98 SYCP3 ME R GE oogonia pre PL post PL B C C 0 5 10 15 100 95 90 85 0 BrdU+/ SYCP3+ (%) ETOH BADGE BPAF BrdU-/ SYCP3+ (%) preleptotene post-preleptotene * ** * * C 0 5 10 15 100 95 90 85 0 BrdU+/ SYCP3+ (%) ETOH BADGE BPAF BrdU-/ SYCP3+ (%) preleptotene post-preleptotene * ** * * This defect in meiosis progression could be the consequence of a delay in meiosis initiation. Immunostainings for BrdU, SYCP3 and a germ cells marker (TRA98), were used to identify three germinal populations 14.5 dpc ovaries: oogonia, the female PGCs (SYCP3-negative cells), premeiotic cells in S-phase also called pre-leptonema (BrdU-positive/SYCP3-positive) and oocytes (BrdU-negative/SYCP3-positive; Figure 3B-C). In untreated 14.5 dpc ovaries almost all germ cells had initiated meiosis (over 96% are SYCP3+) and very few oogonia and preleptotene cells were still present. In bisphenols-treated mice, we observed a significant increase in mitotic PGCs (Supplementary Figure 1A) and pre-leptonema while oocyte number was reduced (Figure 3C). 201 202 203 204 205 206 207 208 This defect in meiosis progression could be the consequence of a delay in meiosis initiation. BADGE or BPAF fetal exposure reproduces BPA defects on folliculogenesis and recombination events. 89 90 Error bars show mean ± s.e.m.; mice analysed n=3-6; * p < 0.05 (Mann-Whitney’s test). (B-C) prophase I initiation was assayed in 14.5 dpc ovaries after immunostaining of SYCP3 (meiosis), BrdU (mitosis and meiosis S phase) and TRA98 (germ cell marker). (B) Representative photomicrographs of SYCP3 (green), BrdU (red) and TRA98 (blue) immunostaining. Scale bar 5 µm. Pre PL = pre- leptotene; post PL= post-leptotene. Error bars show mean ± s.e.m. n=5 mice from independent exposures; * p ≤ 0.05; ** p ≤ 0.01 (Mann-Whitney’s test). (C) Graphs showing the percentage of preleptotene (BrdU and SYCP3 positive) and meiotic (BrdU negative, SYCP3 positive) oocytes. Error bars show mean ± s.e.m. n=5 mice from independent exposures; * p < 0.05; ** p < 0.01 (Mann-Whitney’s test). This defect in meiosis progression could be the consequence of a delay in meiosis initiation. Immunostainings for BrdU, SYCP3 and a germ cells marker (TRA98), were used to identify three i l l ti 14 5 d i i th f l PGC (SYCP3 ti ll ) A B C ETOH BADGE BPAF 0 20 40 60 80 100 germ cells (%) early pachynema late pachynema diplonema BRDU TRA98 SYCP3 ME R GE oogonia pre PL post PL 0 5 10 15 100 95 90 85 0 BrdU+/ SYCP3+ (%) ETOH BADGE BPAF BrdU-/ SYCP3+ (%) preleptotene post-preleptotene * ** * * * * * 178 179 180 181 182 183 184 185 186 187 188 189 190 191 192 193 194 195 196 197 198 199 200 201 202 203 . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted August 20, 2020. ; https://doi.org/10.1101/2020.08.17.253724 doi: bioRxiv preprint . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted August 20, 2020. ; https://doi.org/10.1101/2020.08.17.253724 doi: bioRxiv preprint . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. BADGE or BPAF fetal exposure reproduces BPA defects on folliculogenesis and recombination events. 89 90 Immunostainings for BrdU, SYCP3 and a germ cells marker (TRA98), were used to identify three germinal populations 14.5 dpc ovaries: oogonia, the female PGCs (SYCP3-negative cells), premeiotic cells in S-phase also called pre-leptonema (BrdU-positive/SYCP3-positive) and oocytes (BrdU-negative/SYCP3-positive; Figure 3B-C). In untreated 14.5 dpc ovaries almost all germ cells had initiated meiosis (over 96% are SYCP3+) and very few oogonia and preleptotene cells were still present. In bisphenols-treated mice, we observed a significant increase in mitotic PGCs (Supplementary Figure 1A) and pre-leptonema while oocyte number was reduced (Figure 3C). 201 202 203 204 205 206 207 208 This defect in meiosis progression could be the consequence of a delay 201 8 8 . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted August 20, 2020. ; https://doi.org/10.1101/2020.08.17.253724 doi: bioRxiv preprint . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted August 20, 2020. ; https://doi.org/10.1101/2020.08.17.253724 doi: bioRxiv preprint A ETOH BADGE BPAF SYCP3 negative (%) ns * 209 210 211 212 213 214 215 216 217 218 219 220 ETOH BADGE BPAF 0 20 40 60 STRA8 positive (%) B ns ns Supplementary Figure 1 : (A) Percentage of oogonia (SYCP3-/ TRA98 positive). Error bars show mean ± s.e.m. n=5 mice from independent exposures; * p <0.05 (Mann-Whitney’s test).(B) Percentage of germ cells expressing STRA8 in 14.5 dpc female gonads. n=5 mice from independent exposures; p= 0.08 (BADGE), p= 0.07(BPAF) (Mann- Whitney’s test). In addition, we observed an increasing trend for STRA8 positive germ cells in 14.5 dpc female ETOH BADGE BPAF 0 20 40 60 STRA8 positive (%) B A ETOH BADGE BPAF SYCP3 negative (%) ns * ns ns 209 210 211 212 213 214 215 216 217 218 219 220 221 Supplementary Figure 1 : (A) Percentage of oogonia (SYCP3-/ TRA98 positive). Error bars show mean ± s.e.m. n=5 mice from independent exposures; * p <0.05 (Mann-Whitney’s test).(B) Percentage of germ cells expressing STRA8 in 14.5 dpc female gonads. n=5 mice from independent exposures; p= 0.08 (BADGE), p= 0.07(BPAF) (Mann- Whitney’s test). BADGE or BPAF fetal exposure reproduces BPA defects on folliculogenesis and recombination events. 89 90 ETOH BADGE BPAF 0 20 40 60 STRA8 positive (%) B ns ns Supplementary Figure 1 : (A) Percentage of oogonia (SYCP3-/ TRA98 positive). Error bars show mean ± s.e.m. n=5 mice from independent exposures; * p <0.05 (Mann-Whitney’s test).(B) Percentage of germ cells expressing STRA8 in 14.5 dpc female gonads. n=5 mice from independent exposures; p= 0.08 (BADGE), p= 0.07(BPAF) (Mann- Whitney’s test). 60 B A B In addition, we observed an increasing trend for STRA8 positive germ cel 221 In addition, we observed an increasing trend for STRA8 positive germ cells in 14.5 dpc female gonads (20% ± 4 in ETOH group VS 36% ± 1 p=0.08 and 39% ± 6, p=0.07 in BADGE and BPAF groups respectively, Supplementary Figure 1B). The presence of STRA8 correlated with the initiation of the meiotic program and declines rapidly just after the initiation in prophase I. The observed increase confirmed the bisphenols-induced delay of meiotic initiation. A defect of crossover distribution could be the consequence of a delay and/or an alteration of meiosis initiation. To confirm this hypothesis, we performed exposure to bisphenols until meiosis initiation (short term exposure). 221 222 223 224 225 226 227 228 Supplementary Figure 2: Ovaries from fetuses exposed to vehicle (ETOH) or bisphenols (BADGE and BPAF) from 10.5 dpc to 14.5 dpc were used for the MLH1 quantification (short exposure). Left panel: Representative photomicrographs of pachytene cells from vehicle (ETOH) and BADGE treated ovaries. White arrow shows a synaptonemal complexe with 3 MLH1 foci. Right panel: Percentage of synaptonemal complexes with 3 or more MLH1 foci. n= 62 (ETOH), 55 (BADGE) and 55 (BPAF) oocytes from 3 independent exposures. Mean (red bar), * p < 0.05, ** p < 0.01 (Mann- Whitney’s test). Fetuses were exposed from 10.5 to 14.5 dpc (Supplementary Figure 2, upper panel) and the MLH1 foci was quantified at 18.5 dpc. As observed for bisphenols exposures during meiosis progression (long-term exposure), the number of MLH1 foci was significantly increased after bisphenols exposure (Supplementary Figure 2). BADGE or BPAF fetal exposure reproduces BPA defects on folliculogenesis and recombination events. 89 90 This suggests that changes in crossovers distribution were short term exposure 18.5 (dpc) 14.5 10.5 meiotic prophase I 0 10 20 30 40 50 > 3 MLH1 foci / bivalent (%) MLH1 / SYCP3 ETOH BADGE 18.5 dpc oocytes ETOH BADGE BPAF ** * 229 230 231 232 233 234 235 236 237 238 239 240 241 242 243 244 245 246 short term exposure 18.5 (dpc) 14.5 10.5 meiotic prophase I 0 10 20 30 40 50 > 3 MLH1 foci / bivalent (%) MLH1 / SYCP3 ETOH BADGE 18.5 dpc oocytes ETOH BADGE BPAF ** * 229 230 231 232 233 234 235 236 237 238 239 240 241 242 229 230 231 232 233 234 235 236 237 238 239 240 241 242 Supplementary Figure 2: Ovaries from fetuses exposed to vehicle (ETOH) or bisphenols (BADGE and BPAF) from 10.5 dpc to 14.5 dpc were used for the MLH1 quantification (short exposure). Left panel: Representative photomicrographs of pachytene cells from vehicle (ETOH) and BADGE treated ovaries. White arrow shows a synaptonemal complexe with 3 MLH1 foci. Right panel: Percentage of synaptonemal complexes with 3 or more MLH1 foci. n= 62 (ETOH), 55 (BADGE) and 55 (BPAF) oocytes from 3 independent exposures. Mean (red bar), * p < 0.05, ** p < 0.01 (Mann- Whitney’s test). Supplementary Figure 2: Ovaries from fetuses exposed to vehicle (ETOH) or bisphenols (BADGE and BPAF) from 10.5 dpc to 14.5 dpc were used for the MLH1 quantification (short exposure). Left panel: Representative photomicrographs of pachytene cells from vehicle (ETOH) and BADGE treated ovaries. White arrow shows a synaptonemal complexe with 3 MLH1 foci. Right panel: Percentage of synaptonemal complexes with 3 or more MLH1 foci. n= 62 (ETOH), 55 (BADGE) and 55 (BPAF) oocytes from 3 independent exposures. Mean (red bar), * p < 0.05, ** p < 0.01 (Mann- Whitney’s test). ETOH BADGE 18.5 dpc oocytes 18.5 dpc oocytes Fetuses were exposed from 10.5 to 14.5 dpc (Supplementary Figure 2, upper panel) and the MLH1 foci was quantified at 18.5 dpc. As observed for bisphenols exposures during meiosis progression (long-term exposure), the number of MLH1 foci was significantly increased after bisphenols exposure (Supplementary Figure 2). This suggests that changes in crossovers distribution were 243 244 245 246 9 . BADGE or BPAF fetal exposure reproduces BPA defects on folliculogenesis and recombination events. 89 90 CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted August 20, 2020. ; https://doi.org/10.1101/2020.08.17.253724 doi: bioRxiv preprint . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted August 20, 2020. ; https://doi.org/10.1101/2020.08.17.253724 doi: bioRxiv preprint probably due to alterations occurring before or during mitosis to meiosis transition in germ cells (GCs). 247 248 BADGE or BPAF fetal exposures alter gene expression and mRNA splicing in ge p.adjust (Log) Meiosis Stem Cell Differentiation ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● BADGE (Log2FC) BPAF (Log2FC) Gpm6a Msx1 Snai2 Jag1 Sox9 Lin28a Nrp1 Erbb4 Shh Hnf1b Pdgfra Runx2 Bmp4 Hes5 Alx1 Mir99a Mir130a Setd6 Lbh Rbm24 Fgf15 Hnrnpu Wnt7b Folr1 Pef1 Pax6 Cfl1 Frzb Gbx2 Gata4 Aldh1a2 Efnb1 Foxc2 Foxa1 Foxc1 Sox21 Kitl Sema6d Hoxd4 Fzd1 Htr2b Hoxa7 Cdk6 Sox18 Sema5b Pax2 Ptn Sema6a Gsk3b r=0.31 pvalue=0.005648 −1.0 −0.5 0.0 0.5 1.0 −1.0 −0.5 0.0 0.5 1.0 BPAF (Log2FC) Sfrp1 Tgfb2 Ednra Tcf7l1 Myocd Gpm6a Nrp2 Snai2 Jag1 Gdnf Epcam Sox9Sema3d Nrp1 Hnf1b Pdgfra Anxa6 Bmp4 Rest Hes1 Sox2 Sox11 Twist1 Tacstd2 Lbh Nanog Wnt7a Folr1 Frzb Gata4 Aldh1a2 Ednrb Prickle1 Kitl Sema3a Gata6 Pax2 Cited2 Ptn Sema6a BADGE (Log2FC) r=0.45 pvalue=5.455E-05 −1.0 −0.5 0.0 0.5 1.0 −1.0 −0.5 0.0 0.5 1.0 BADGE (Log2FC) BPAF (Log2FC) Zfp318 Sycp2 Mybl1 Stag3 Brca2 Tex15Sycp1 Sycp3 Xlr Ccdc36 Prdm9 Hormad2 Dmc1 Rnf212 Ythdc2 Spo11 Mei1 Hormad1 Syce1 Gm1140 Mcmdc2 Slc26a8 Dmrtc2 Spdya Spata22 Meiob Topaz1 Boll Tex11 Ccne2 Xlr5b Syce3 r=0.87 pvalue<2.2E-16 −1.0 −0.5 0.0 0.5 1.0 −1.0 −0.5 0.0 0.5 1.0 BADGE (Log2FC) BPAF (Log2FC) Smc1b Sycp3 Hormad2Mei4 Cpeb1 Brca2 Stag3 Mael Hormad1 Syce1 Tex15 Hfm1 Tex12 Fanca Mybl1 Stra8 Topaz1 Tex11 Sycp1 1700013H16Rik Xlr4a M1ap Lfng Fmn2 Cdc25b r=0.37 pvalue= 1.408E-09 −1.0 −0.5 0.0 0.5 1.0 −1.0 −0.5 0.0 0.5 1.0 C 11.5 dpc 13.5 dpc 272 273 274 275 276 277 278 279 280 281 282 Meiosis Stem Cell Differentiation ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● BADGE (Log2FC) BPAF (Log2FC) Gpm6a Msx1 Snai2 Jag1 Sox9 Lin28a Nrp1 Erbb4 Shh Hnf1b Pdgfra Runx2 Bmp4 Hes5 Alx1 Mir99a Mir130a Setd6 Lbh Rbm24 Fgf15 Hnrnpu Wnt7b Folr1 Pef1 Pax6 Cfl1 Frzb Gbx2 Gata4 Aldh1a2 Efnb1 Foxc2 Foxa1 Foxc1 Sox21 Kitl Sema6d Hoxd4 Fzd1 Htr2b Hoxa7 Cdk6 Sox18 Sema5b Pax2 Ptn Sema6a Gsk3b r=0.31 pvalue=0.005648 −1.0 −0.5 0.0 0.5 1.0 −1.0 −0.5 0.0 0.5 1.0 BPAF (Log2FC) Sfrp1 Tgfb2 Ednra Tcf7l1 Myocd Gpm6a Nrp2 Snai2 Jag1 Gdnf Epcam Sox9Sema3d Nrp1 Hnf1b Pdgfra Anxa6 Bmp4 Rest Hes1 Sox2 Sox11 Twist1 Tacstd2 Lbh Nanog Wnt7a Folr1 Frzb Gata4 Aldh1a2 Ednrb Prickle1 Kitl Sema3a Gata6 Pax2 Cited2 Ptn Sema6a BADGE (Log2FC) r=0.45 pvalue=5.455E-05 −1.0 −0.5 0.0 0.5 1.0 −1.0 −0.5 0.0 0.5 1.0 BADGE (Log2FC) BPAF (Log2FC) Zfp318 Sycp2 Mybl1 Stag3 Brca2 Tex15Sycp1 Sycp3 Xlr Ccdc36 Prdm9 Hormad2 Dmc1 Rnf212 Ythdc2 Spo11 Mei1 Hormad1 Syce1 Gm1140 Mcmdc2 Slc26a8 Dmrtc2 Spdya Spata22 Meiob Topaz1 Boll Tex11 Ccne2 Xlr5b Syce3 r=0.87 pvalue<2.2E-16 −1.0 −0.5 0.0 0.5 1.0 −1.0 −0.5 0.0 0.5 1.0 BADGE (Log2FC) BPAF (Log2FC) Smc1b Sycp3 Hormad2Mei4 Cpeb1 Brca2 Stag3 Mael Hormad1 Syce1 Tex15 Hfm1 Tex12 Fanca Mybl1 Stra8 Topaz1 Tex11 Sycp1 1700013H16Rik Xlr4a M1ap Lfng Fmn2 Cdc25b r=0.37 pvalue= 1.408E-09 −1.0 −0.5 0.0 0.5 1.0 −1.0 −0.5 0.0 0.5 1.0 C 11.5 dpc 13.5 dpc C Figure 4: BADGE or BPAF fetal exposures induce transcriptional alteration before meiosis initiation. BADGE or BPAF fetal exposures alter gene expression and mRNA splicing in ge r= Pearson’s correlation B p.adjust (Log) 11.5 dpc 13.5 dpc 0 25 50 75 100 transmembrane receptor protein serine/threonine kinase signaling pathway Notch signaling pathway negative regulation of Wnt signaling pathway retinoic acid receptor signaling pathway stem cell differentiation promoter DNA−binding tactivator activity, RNA polymerase II−specific DNA recombination meiotic cell cycle meiotic nuclear division synapsis BADGE BPAF 0 25 50 75 100 Meiosis Stem Cell Differentiation ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● BADGE (Log2FC) BPAF (Log2FC) Gpm6a Msx1 Snai2 Jag1 Sox9 Lin28a Nrp1 Erbb4 Shh Hnf1b Pdgfra Runx2 Bmp4 Hes5 Alx1 Mir99a Mir130a Setd6 Lbh Rbm24 Fgf15 Hnrnpu Wnt7b Folr1 Pef1 Pax6 Cfl1 Frzb Gbx2 Gata4 Aldh1a2 Efnb1 Foxc2 Foxa1 Foxc1 Sox21 Kitl Sema6d Hoxd4 Fzd1 Htr2b Hoxa7 Cdk6 Sox18 Sema5b Pax2 Ptn Sema6a Gsk3b r=0.31 pvalue=0.005648 −1.0 −0.5 0.0 0.5 1.0 −1.0 −0.5 0.0 0.5 1.0 BPAF (Log2FC) Sfrp1 Tgfb2 Ednra Tcf7l1 Myocd Gpm6a Nrp2 Snai2 Jag1 Gdnf Epcam Sox9Sema3d Nrp1 Hnf1b Pdgfra Anxa6 Bmp4 Rest Hes1 Sox2 Sox11 Twist1 Tacstd2 Lbh Nanog Wnt7a Folr1 Frzb Gata4 Aldh1a2 Ednrb Prickle1 Kitl Sema3a Gata6 Pax2 Cited2 Ptn Sema6a BADGE (Log2FC) r=0.45 pvalue=5.455E-05 −1.0 −0.5 0.0 0.5 1.0 −1.0 −0.5 0.0 0.5 1.0 BADGE (Log2FC) BPAF (Log2FC) Zfp318 Sycp2 Mybl1 Stag3 Brca2 Tex15Sycp1 Sycp3 Xlr Ccdc36 Prdm9 Hormad2 Dmc1 Rnf212 Ythdc2 Spo11 Mei1 Hormad1 Syce1 Gm1140 Mcmdc2 Slc26a8 Dmrtc2 Spdya Spata22 Meiob Topaz1 Boll Tex11 Ccne2 Xlr5b Syce3 r=0.87 pvalue<2.2E-16 −1.0 −0.5 0.0 0.5 1.0 −1.0 −0.5 0.0 0.5 1.0 BADGE (Log2FC) BPAF (Log2FC) Smc1b Sycp3 Hormad2Mei4 Cpeb1 Brca2 Stag3 Mael Hormad1 Syce1 Tex15 Hfm1 Tex12 Fanca Mybl1 Stra8 Topaz1 Tex11 Sycp1 1700013H16Rik Xlr4a M1ap Lfng Fmn2 Cdc25b r=0.37 pvalue= 1.408E-09 −1.0 −0.5 0.0 0.5 1.0 −1.0 −0.5 0.0 0.5 1.0 C 11.5 dpc 13.5 dpc B p.adjust (Log) 11.5 dpc 13.5 dpc 0 25 50 75 100 transmembrane receptor protein serine/threonine kinase signaling pathway Notch signaling pathway negative regulation of Wnt signaling pathway retinoic acid receptor signaling pathway stem cell differentiation promoter DNA−binding tactivator activity, RNA polymerase II−specific DNA recombination meiotic cell cycle meiotic nuclear division synapsis BADGE BPAF 0 25 50 75 100 B p.adjust (Log) 11.5 dpc 13.5 dpc 0 25 50 75 100 transmembrane receptor protein serine/threonine kinase signaling pathway Notch signaling pathway negative regulation of Wnt signaling pathway retinoic acid receptor signaling pathway stem cell differentiation promoter DNA−binding tactivator activity, RNA polymerase II−specific DNA recombination meiotic cell cycle meiotic nuclear division synapsis BADGE BPAF 0 25 50 75 100 B Figure 4: BADGE or BPAF fetal exposures induce transcriptional alteration before meiosis initiation. BADGE or BPAF fetal exposures alter gene expression and mRNA splicing in ge SSEA1 positive germ cells from 11.5 or 13.5 dpc ovaries treated by vehicle (ETOH) or bisphenols (BADGE and BPAF) were used for transcriptomic analyses. Differentially expressed genes (DEGs) between bisphenols and control condition (BPAF vs ETOH or BADGE vs ETOH) were filtered according to the log fold change (abs[Log2FC] ≥ 0.5) and the significativity (p < 0.05). Three pools of cells each of them from 10-15 fetuses were analyzed. (A) The table represents the number (and percentage) of DEGs after BADGE or BPAF exposure in 11.5 or 13.5 dpc germ cells. (B) Gene Ontology enrichment associated to meiosis and stem cell differentiation among DEGs. (C) Correlation analysis between BADGE and BPAF condition of the differential expression (Log2FC) of genes associated to meiosis or stem cell differentiation. r= Pearson’s correlation coefficient (Pearson’s test). BADGE or BPAF fetal exposures alter gene expression and mRNA splicing in ge ; https://doi.org/10.1101/2020.08.17.253724 doi: bioRxiv preprint A BADGE BPAF BADGE BPAF 1817 733 886 1376 11.5dpc 13.5dpc DEG (LFC>0.5, pvalue <0.05 ) 802 (44.2%) 243 (34.6%) 640 (72.2%) 846 (61.4%) upregulated downregulated 1015 (55.8 %) 490 (65.4%) 247 (27.8%) 528 (38.6%) A Figure 4: BADGE or BPAF fetal exposures induce transcriptional alteration before meiosis initiation. SSEA1 positive germ cells from 11.5 or 13.5 dpc ovaries treated by vehicle (ETOH) or bisphenols (BADGE and BPAF) were used for transcriptomic analyses. Differentially expressed genes (DEGs) between bisphenols and control condition (BPAF vs ETOH or BADGE vs ETOH) were filtered according to the log fold change (abs[Log2FC] ≥ 0.5) and the significativity (p < 0.05). Three pools of cells each of them from 10-15 fetuses were analyzed. (A) The table represents the number (and percentage) of DEGs after BADGE or BPAF exposure in 11.5 or 13.5 dpc germ cells. (B) Gene Ontology enrichment associated to meiosis and stem cell differentiation among DEGs. (C) Correlation analysis between BADGE and BPAF condition of the differential expression (Log2FC) of genes associated to meiosis or stem cell differentiation. BADGE or BPAF fetal exposures alter gene expression and mRNA splicing in ge BADGE or BPAF fetal exposures alter gene expression and mRNA splicing in germ cells In order to understand the bases of bisphenols-induced alterations in PGCs during acquisition of meiotic competence and initiation of the meiotic program, we performed transcripomic analyses on 11.5 dpc (during acquisition of the meiotic competence) and 13.5 dpc (during meiosis initiation) germ cells. We sorted PGCs by Magnetic Activated Cell Sorting (MACS) using the cell surface protein stage-specific embryonic antigen 1 (SSEA-1). Gene expression analysis was conducted using murine Affymetrix GeneChip Gene 2.0 TS (11.5 dpc) and ClariomTM D (13.5 dpc) microarrays. At 11.5 dpc, we identified 1817 and 733 differentially expressed genes (DEGs) in BADGE and BPAF treated germ cells respectively (compared to vehicle treated PGCs with a |Log2 Fold-Change| ≥ 0.5 and p < 0.05) (Figure 4). DEGs were mostly downregulated (56 to 65 % of DEGs; Figure 4A). At 13.5 dpc when the program of meiosis is initiated, we identified 886 and 1376 DEGs in BADGE and BPAF treated germ cells respectively (Figure 4A). Contrary to what it is observed at 11.5 dpc, two third of the DEGs were upregulated in 13.5 dpc bisphenols-treated germ cells (Figure 4A). Using EnrichGO function from Clusterprofiler package to identify enrichment of gene ontologies, we observed that DEGs at 11.5 dpc as well as 13.5 dpc were mostly related to meiosis, stem cell differentiation and regulation of stem cell signaling pathway such as Wnt pathway (Figure 4B and Supplementary Figure 3). Meiosis-associated DEGs such as Stag3, Sycp1&3, Hormad1&2, Spo11, Spata22, Meiob, Dmc1 or Brca2 were strongly enriched in genes associated to synapsis and DNA recombination processes (Figure 4A-C, Supplementary Table 1). Surprisingly, genes linked to meiosis and stem cell differentiation showed opposite transcriptional response at 11.5 dpc and 13.5 dpc (Supplementary Figure 3 and Figure 4C). Stemness genes tend to be up-regulated at 11.5 dpc and mostly down-regulated at 13.5 dpc and meiotic genes were preferentially down-regulated at 11.5 dpc and up-regulated at 13.5 dpc (Figure 4C). 249 250 251 252 253 254 255 256 257 258 259 260 261 262 263 264 265 266 267 268 269 270 271 10 . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted August 20, 2020. BADGE or BPAF fetal exposures alter gene expression and mRNA splicing in ge Enrichment of Gene Ontology (biological process, molecular function and cellular component) of DEGs after bisphenols exposure in 11.5 and 13.5 dpc. Ontologies associated to down-regulated genes is presented as negative values of pvalues. 11.5 dpc 13.5 dpc −50 0 50 100 −50 0 50 100 canonical Wnt signaling pathway cell junction assembly cell junction organization cell−cell junction assembly cell−cell junction organization cell−cell signaling by wnt cell−matrix adhesion chromosome organization involved in meiotic cell cycle chromosome segregation cofactor metabolic process collagen−containing extracellular matrix condensed chromosome condensed nuclear chromosome developmental cell growth developmental growth involved in morphogenesis DNA recombination drug catabolic process embryonic appendage morphogenesis embryonic organ development embryonic organ morphogenesis endocrine system development extracellular matrix female gamete generation gland development gland morphogenesis homologous chromosome segregation hormone metabolic process hormone secretion male meiotic nuclear division meiosis I meiosis I cell cycle process meiotic cell cycle meiotic cell cycle process meiotic chromosome segregation meiotic nuclear division mesenchymal cell development mesenchymal cell differentiation mesenchyme development mesonephric epithelium development mesonephric tubule development mesonephros development negative regulation of canonical Wnt signaling pathway negative regulation of cell development negative regulation of response to wounding negative regulation of Wnt signaling pathway negative regulation of wound healing Notch signaling pathway nuclear chromosome segregation nuclear division oogenesis organelle fission organic acid binding organic hydroxy compound metabolic process oxidoreductase activity, acting on CH−OH group of donors oxidoreductase activity, acting on peroxide as acceptor oxidoreductase activity, acting on the CH−OH group of donors, NAD or NADP as acceptor oxygen binding oxygen carrier activity positive regulation of epithelial cell proliferation positive regulation of ERK1 and ERK2 cascade positive regulation of reproductive process proximal promoter DNA−binding transcription activator activity, RNA polymerase II−specific regulation of developmental growth regulation of epithelial to mesenchymal transition regulation of stem cell differentiation regulation of transforming growth factor beta receptor signaling pathway regulation of transmembrane receptor protein serine/threonine kinase signaling pathway regulation of Wnt signaling pathway regulation of wound healing renal system process renal system vasculature development retinoic acid receptor signaling pathway sex differentiation SMAD protein signal transduction small molecule catabolic process smooth muscle cell proliferation stem cell development stem cell differentiation steroid biosynthetic process steroid metabolic process synapsis synaptonemal complex synaptonemal complex assembly synaptonemal complex organization synaptonemal structure transforming growth factor beta receptor signaling pathway transmembrane receptor protein kinase activity transmembrane receptor protein serine/threonine kinase signaling pathway Wnt signaling pathway wound healing logpvalue BADGE BPAF Supplementary Figure 3. BADGE or BPAF fetal exposures alter gene expression and mRNA splicing in ge SSEA1 positive germ cells from 11.5 or 13.5 dpc ovaries treated by vehicle (ETOH) or bisphenols (BADGE and BPAF) were used for transcriptomic analyses. Differentially expressed genes (DEGs) between bisphenols and control condition (BPAF vs ETOH or BADGE vs ETOH) were filtered according to the log fold change (abs[Log2FC] ≥ 0.5) and the significativity (p < 0.05). Three pools of cells each of them from 10-15 fetuses were analyzed. (A) The table represents the number (and percentage) of DEGs after BADGE or BPAF exposure in 11.5 or 13.5 dpc germ cells. (B) Gene Ontology enrichment associated to meiosis and stem cell differentiation among DEGs. (C) Correlation analysis between BADGE and BPAF condition of the differential expression (Log2FC) of genes associated to meiosis or stem cell differentiation. r= Pearson’s correlation coefficient (Pearson’s test). 272 273 274 275 276 277 278 279 280 281 282 11 . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted August 20, 2020. ; https://doi.org/10.1101/2020.08.17.253724 doi: bioRxiv preprint . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted August 20, 2020. ; https://doi.org/10.1101/2020.08.17.253724 doi: bioRxiv preprint Supplementary Figure 3. Enrichment of Gene Ontology (biological process, molecular function and cellular component) of DEGs after bisphenols exposure in 11.5 and 13.5 dpc. Ontologies associated to down-regulated genes is presented as negative values of pvalues. BADGE or BPAF fetal exposures alter gene expression and mRNA splicing in ge 11.5 dpc 13.5 dpc −50 0 50 100 −50 0 50 100 canonical Wnt signaling pathway cell junction assembly cell junction organization cell−cell junction assembly cell−cell junction organization cell−cell signaling by wnt cell−matrix adhesion chromosome organization involved in meiotic cell cycle chromosome segregation cofactor metabolic process collagen−containing extracellular matrix condensed chromosome condensed nuclear chromosome developmental cell growth developmental growth involved in morphogenesis DNA recombination drug catabolic process embryonic appendage morphogenesis embryonic organ development embryonic organ morphogenesis endocrine system development extracellular matrix female gamete generation gland development gland morphogenesis homologous chromosome segregation hormone metabolic process hormone secretion male meiotic nuclear division meiosis I meiosis I cell cycle process meiotic cell cycle meiotic cell cycle process meiotic chromosome segregation meiotic nuclear division mesenchymal cell development mesenchymal cell differentiation mesenchyme development mesonephric epithelium development mesonephric tubule development mesonephros development negative regulation of canonical Wnt signaling pathway negative regulation of cell development negative regulation of response to wounding negative regulation of Wnt signaling pathway negative regulation of wound healing Notch signaling pathway nuclear chromosome segregation nuclear division oogenesis organelle fission organic acid binding organic hydroxy compound metabolic process oxidoreductase activity, acting on CH−OH group of donors oxidoreductase activity, acting on peroxide as acceptor oxidoreductase activity, acting on the CH−OH group of donors, NAD or NADP as acceptor oxygen binding oxygen carrier activity positive regulation of epithelial cell proliferation positive regulation of ERK1 and ERK2 cascade positive regulation of reproductive process proximal promoter DNA−binding transcription activator activity, RNA polymerase II−specific regulation of developmental growth regulation of epithelial to mesenchymal transition regulation of stem cell differentiation regulation of transforming growth factor beta receptor signaling pathway regulation of transmembrane receptor protein serine/threonine kinase signaling pathway regulation of Wnt signaling pathway regulation of wound healing renal system process renal system vasculature development retinoic acid receptor signaling pathway sex differentiation SMAD protein signal transduction small molecule catabolic process smooth muscle cell proliferation stem cell development stem cell differentiation steroid biosynthetic process steroid metabolic process synapsis synaptonemal complex synaptonemal complex assembly synaptonemal complex organization synaptonemal structure transforming growth factor beta receptor signaling pathway transmembrane receptor protein kinase activity transmembrane receptor protein serine/threonine kinase signaling pathway Wnt signaling pathway wound healing logpvalue BADGE BPAF Supplementary Figure 3. Enrichment of Gene Ontology (biological process, molecular function and cellular component) of DEGs after bisphenols exposure in 11.5 and 13.5 dpc. Ontologies associated to down-regulated genes is presented as negative values of pvalues. BADGE or BPAF fetal exposures alter gene expression and mRNA splicing in ge 11.5 dpc 13.5 dpc −50 0 50 100 −50 0 50 100 canonical Wnt signaling pathway cell junction assembly cell junction organization cell−cell junction assembly cell−cell junction organization cell−cell signaling by wnt cell−matrix adhesion chromosome organization involved in meiotic cell cycle chromosome segregation cofactor metabolic process collagen−containing extracellular matrix condensed chromosome condensed nuclear chromosome developmental cell growth developmental growth involved in morphogenesis DNA recombination drug catabolic process embryonic appendage morphogenesis embryonic organ development embryonic organ morphogenesis endocrine system development extracellular matrix female gamete generation gland development gland morphogenesis homologous chromosome segregation hormone metabolic process hormone secretion male meiotic nuclear division meiosis I meiosis I cell cycle process meiotic cell cycle meiotic cell cycle process meiotic chromosome segregation meiotic nuclear division mesenchymal cell development mesenchymal cell differentiation mesenchyme development mesonephric epithelium development mesonephric tubule development mesonephros development negative regulation of canonical Wnt signaling pathway negative regulation of cell development negative regulation of response to wounding negative regulation of Wnt signaling pathway negative regulation of wound healing Notch signaling pathway nuclear chromosome segregation nuclear division oogenesis organelle fission organic acid binding organic hydroxy compound metabolic process oxidoreductase activity, acting on CH−OH group of donors oxidoreductase activity, acting on peroxide as acceptor oxidoreductase activity, acting on the CH−OH group of donors, NAD or NADP as acceptor oxygen binding oxygen carrier activity positive regulation of epithelial cell proliferation positive regulation of ERK1 and ERK2 cascade positive regulation of reproductive process proximal promoter DNA−binding transcription activator activity, RNA polymerase II−specific regulation of developmental growth regulation of epithelial to mesenchymal transition regulation of stem cell differentiation regulation of transforming growth factor beta receptor signaling pathway regulation of transmembrane receptor protein serine/threonine kinase signaling pathway regulation of Wnt signaling pathway regulation of wound healing renal system process renal system vasculature development retinoic acid receptor signaling pathway sex differentiation SMAD protein signal transduction small molecule catabolic process smooth muscle cell proliferation stem cell development stem cell differentiation steroid biosynthetic process steroid metabolic process synapsis synaptonemal complex synaptonemal complex assembly synaptonemal complex organization synaptonemal structure transforming growth factor beta receptor signaling pathway transmembrane receptor protein kinase activity transmembrane receptor protein serine/threonine kinase signaling pathway Wnt signaling pathway wound healing logpvalue BADGE BPAF Ontology (biological process, molecular function ols exposure in 11.5 and 13.5 dpc. Ontologies as negative values of pvalues. 11.5 dpc 13.5 dpc −50 0 50 100 −50 0 50 100 logpvalue Supplementary Figure 3. BADGE or BPAF fetal exposures alter gene expression and mRNA splicing in ge For this reason, we studied the impact of bisphenols exposure on RNA splicing events during meiosis initiation. We took advantage of the ClariomTM D that allows the detection of most RNA splicing events by covering independently exons and exons junctions of all transcripts. After bisphenols exposure, 3543 (BADGE) and 2678 (BPAF) coding or non-coding genes showed one or more alternative RNA splicing events compared to the control condition (Figure 5A-C). Differentially spliced genes were strongly associated with chromosome segregation, meiotic cell cycle and DNA repair and recombination (Figure 5A). Half of them were identical in BADGE and BPAF -treated germ cells including genes such as Smc4, Sun1, Mcmdc2, Dazl, Rad51, Mlh1, Dmc1, Sycp2 (Table1 and Supplementary Table 2). Different modes of alternative splicing including skipped exons, alternative 5′ splice sites, alternative 3′ splice sites, and retained intron are described in metazoans. TAC software classifies some of these events in function of the coverage of the probes sets of the array Bisphenols- 286 287 288 289 290 291 292 293 294 295 296 297 298 299 300 301 302 303 304 305 306 307 308 309 310 311 Interestingly, we observed a significant correlation between BPAF and BADGE of the differential expression of genes associated to meiosis after bisphenols exposure (r=0.37, pvalue < 0.0001, 11.5dpc and r=0.87, pvalue < 0.0001, 13.5 dpc) and stem cell (r=0.32, p<0.0001, 11.5 dpc and r=0.45, p < 0.0001, 13.5 dpc) at 11.5 dpc and 13.5 dpc (Figure 4C). These positive correlations demonstrate a common transcriptional signature between BPAF and BADGE exposure and suggest similar mechanisms of action for both bisphenols. Interestingly, as observed in the chromosomes 3, 4, 7, 10, 14, and 17, genome mapping of DEGs (up- and down-regulated) showed a concentration of genes on specific genome locations that were differentially expressed after BADGE and BPAF exposure at 11.5 dpc as well as 13.5 dpc (Supplementary Figure 5). 286 287 288 289 290 291 292 293 294 A regulated alternative splicing program has been reported during meiosis initiation in male and in female germ cells (Naro et al., 2017; J. Wang et al., 2019). The exact role of this program is unclear but is though to be required for stabilization of meiotic transcripts during prophase I of meiosis (Liu et al., 2017; J. Wang et al., 2019). BADGE or BPAF fetal exposures alter gene expression and mRNA splicing in ge Enrichment of Gene Ontology (biological process, molecular function and cellular component) of DEGs after bisphenols exposure in 11.5 and 13.5 dpc. Ontologies associated to down-regulated genes is presented as negative values of pvalues. 283 284 285 Supplementary Figure 3. Enrichment of Gene Ontology (biological process, molecular function and cellular component) of DEGs after bisphenols exposure in 11.5 and 13.5 dpc. Ontologies associated to down-regulated genes is presented as negative values of pvalues. 283 284 285 12 12 . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted August 20, 2020. ; https://doi.org/10.1101/2020.08.17.253724 doi: bioRxiv preprint . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted August 20, 2020. ; https://doi.org/10.1101/2020.08.17.253724 doi: bioRxiv preprint Interestingly, we observed a significant correlation between BPAF and BADGE of the differential expression of genes associated to meiosis after bisphenols exposure (r=0.37, pvalue < 0.0001, 11.5dpc and r=0.87, pvalue < 0.0001, 13.5 dpc) and stem cell (r=0.32, p<0.0001, 11.5 dpc and r=0.45, p < 0.0001, 13.5 dpc) at 11.5 dpc and 13.5 dpc (Figure 4C). These positive correlations demonstrate a common transcriptional signature between BPAF and BADGE exposure and suggest similar mechanisms of action for both bisphenols. Interestingly, as observed in the chromosomes 3, 4, 7, 10, 14, and 17, genome mapping of DEGs (up- and down-regulated) showed a concentration of genes on specific genome locations that were differentially expressed after BADGE and BPAF exposure at 11.5 dpc as well as 13.5 dpc (Supplementary Figure 5). A regulated alternative splicing program has been reported during meiosis initiation in male and in female germ cells (Naro et al., 2017; J. Wang et al., 2019). The exact role of this program is unclear but is though to be required for stabilization of meiotic transcripts during prophase I of meiosis (Liu et al., 2017; J. Wang et al., 2019). As observed in adult testis, modulation of this program results in spermatogenesis defects, highlighting the essential role of the RNA splicing during meiosis (Hannigan et al., 2017; Liu et al., 2017). BADGE or BPAF fetal exposures alter gene expression and mRNA splicing in ge Cassette Exon Intron Retention Unclassified event Gene ratio C Smc4 splicing site primers reference primers 1 reference primers 2 Smc4-201 relative quantification 0.0 0.4 0.8 1.2 BADGE BPAF * relative to: primers 1 primers 2 BADGE BPAF * * ETOH Smc4-202 Sycp2-201 Sycp2-202 reference primers 1 reference primers 2 Sycp2 alternative 3' acceptor site splicing site primers relative quantification 0.0 0.5 1.0 1.5 BADGE BPAF * relative to: primers 1 primers 2 BADGE BPAF * ETOH alternative 3' acceptor site 5' UTR 1 2 3 4 5 2 3 4 5 1 5' UTR 18 17 16 15 14 40 39 37 36 38 5' UTR D ratio Smc4 201/ total Smc4 ratio Sycp2-202/ total Sycp2 314 315 316 317 318 319 320 321 322 323 324 325 326 327 328 i Sycp2-201 Sycp2-202 reference primers 1 reference primers 2 Sycp2 alternative 3' acceptor site splicing site primers 18 17 16 15 14 40 39 37 36 38 D i Sycp2-201 Sycp2-202 reference primers 1 reference primers 2 Sycp2 alternative 3' acceptor site splicing site primers relative quantification 0.0 0.5 1.0 1.5 BADGE BPAF * relative to: primers 1 primers 2 BADGE BPAF * ETOH 18 17 16 15 14 40 39 37 36 38 D ratio Sycp2-202/ total Sycp2 C Smc4 splicing site primers reference primers 1 reference primers 2 Smc4-201 Smc4-202 alternative 3' acceptor site 5' UTR 1 2 3 4 5 2 3 4 5 1 5' UTR 5' UTR D C relative quantification 0.0 0.5 1.0 1.5 BADGE BPAF * relative to: primers 1 primers 2 BADGE BPAF * ETOH ratio Sycp2-202/ total Sycp2 relative quantification 0.0 0.4 0.8 1.2 BADGE BPAF * relative to: primers 1 primers 2 BADGE BPAF * * ETOH ratio Smc4 201/ total Smc4 primers 2 BADGE BPAF Figure 5: BADGE or BPAF fetal exposures induce RNA splicing alterations in premeiotic germ cells. Splicing events were determined in 13.5 dpc germ cells using the high resolutive microarray. Using TAC software, differentially spliced genes (BPAF vs ETOH and BADGE vs ETOH) were filtered according to the exon splicing index (abs[exon splicing index]≥1) and the exon pvalue (p<0.05).n=3 pools of cells from 10-15 fetuses each. (A) dot plot showing enrichment on ontology associated to meiosis of differentially spliced genes. (B) distribution of splicing events in all genes and specifically meiotic genes. BADGE or BPAF fetal exposures alter gene expression and mRNA splicing in ge Modes of alternative splicing including skipped exons, alternative 5′ splice sites, alternative 3′ splice sites, and retained intron. Unclassified events correspond to events that cannot be included in any of the standard categories. (C) and (D) Quantification of the relative incidence of Smc4 (C) and Sycp2 (D) splice variants (Ensembl variants:) in bisphenols-treated germ cells. n=3 independent exposures. Upper panel: Representation of splicing sites leading to different splice variants (Smc4-201, Smc4-202, Sycp2- 201 and Sycp2-202, Ensembl name). Lower pannel: relative quantification of expression of Smc4- 201 or Sycp2-202 to total Smc4 transcripts and Sycp2 transcripts respectively in bisphenols treated germ cells in comparison to ETOH-treated germ cells. * p <0.05 (Mann-Whitney’s test). BADGE or BPAF fetal exposures alter gene expression and mRNA splicing in ge Using TAC software, differentially spliced genes (BPAF vs ETOH and BADGE vs ETOH) were filtered according to the exon splicing index (abs[exon splicing index]≥1) and the exon pvalue (p<0.05).n=3 pools of cells from 10-15 fetuses each. (A) dot plot showing enrichment on ontology associated to meiosis of differentially spliced genes. (B) distribution of splicing events in all genes and specifically meiotic genes. Modes of alternative splicing including skipped exons, alternative 5′ splice sites, alternative 3′ splice sites, and retained intron. Unclassified events correspond to events that cannot be included in any of the standard categories. (C) and (D) Quantification of the relative incidence of Smc4 (C) and Sycp2 (D) splice variants (Ensembl variants:) in bisphenols-treated germ cells. n=3 independent exposures. Upper panel: homologous recombination chromosome organization involved in meiotis meiotic chromosome segregation meiosis I meiosis I cell cycle process meiotic nuclear division meiotic cell cycle process DNA recombination 0.000 0.025 GeneRatio 0.01 0.02 0.03 Alternative 3' Acceptor Site Alternative 5' Donor Site Cassette Exon Intron Retention Unclassified event BPAF (n=3879) splicing events Gene ratio reciprocal DNA recombination C Smc4 splicing site primers reference primers 1 reference primers 2 Smc4-201 relative quantification 0.0 0.4 0.8 1.2 BADGE BPAF * relative to: primers 1 primers 2 BADGE BPAF * * ETOH Smc4-202 Sycp2-201 Sycp2-202 reference primers 1 reference primers 2 Sycp2 alternative 3' acceptor site splicing site primers relative quantification 0.0 0.5 1.0 1.5 BADGE BPAF * relative to: primers 1 primers 2 BADGE BPAF * ETOH alternative 3' acceptor site 5' UTR 1 2 3 4 5 2 3 4 5 1 5' UTR 18 17 16 15 14 40 39 37 36 38 5' UTR D ratio Smc4 201/ total Smc4 ratio Sycp2-202/ total Sycp2 14 15 16 17 18 19 20 21 22 23 24 Figure 5: BADGE or BPAF fetal exposures induce RNA splicing alterations in premeiotic germ cells. Splicing events were determined in 13.5 dpc germ cells using the high resolutive microarray. Using TAC software, differentially spliced genes (BPAF vs ETOH and BADGE vs ETOH) were filtered according to the exon splicing index (abs[exon splicing index]≥1) and the exon pvalue (p<0.05).n=3 pools of cells from 10-15 fetuses each. (A) dot plot showing enrichment on ontology associated to meiosis of differentially spliced genes. (B) distribution of splicing events in all genes and specifically meiotic genes. BADGE or BPAF fetal exposures alter gene expression and mRNA splicing in ge As observed in adult testis, modulation of this program results in spermatogenesis defects, highlighting the essential role of the RNA splicing during meiosis (Hannigan et al., 2017; Liu et al., 2017). For this reason, we studied the impact of bisphenols exposure on RNA splicing events during meiosis initiation. We took advantage of the ClariomTM D that allows the detection of most RNA splicing events by covering independently exons and exons junctions of all transcripts. After bisphenols exposure, 3543 (BADGE) and 2678 (BPAF) coding or non-coding genes showed one or more alternative RNA splicing events compared to the control condition (Figure 5A-C). Differentially spliced genes were strongly associated with chromosome segregation, meiotic cell cycle and DNA repair and recombination (Figure 5A). Half of them were identical in BADGE and BPAF -treated germ cells including genes such as Smc4, Sun1, Mcmdc2, Dazl, Rad51, Mlh1, Dmc1, Sycp2 (Table1 and Supplementary Table 2). Different modes of alternative splicing including skipped exons, alternative 5′ splice sites, alternative 3′ splice sites, and retained intron are described in metazoans. TAC software classifies some of these events in function of the coverage of the probes sets of the array. Bisphenols- treated germ cells showed an enrichment of transcripts with skipped exon features in all genes including meiotic specific genes (Figure 5B, Table 2). 295 296 297 298 299 300 301 302 303 304 305 306 307 308 309 310 311 312 313 13 . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted August 20, 2020. ; https://doi.org/10.1101/2020.08.17.253724 doi: bioRxiv preprint . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted August 20, 2020. ; https://doi.org/10.1101/2020.08.17.253724 doi: bioRxiv preprint Figure 5: BADGE or BPAF fetal exposures induce RNA splicing alterations in premeiotic germ cells. Splicing events were determined in 13.5 dpc germ cells using the high resolutive microarray. Using TAC software, differentially spliced genes (BPAF vs ETOH and BADGE vs ETOH) were filtered according to the exon splicing index (abs[exon splicing index]≥1) and the exon pvalue (p<0.05).n=3 pools of cells from 10-15 fetuses each. BADGE or BPAF fetal exposures alter gene expression and mRNA splicing in ge (A) dot plot showing enrichment on ontology associated to meiosis of differentially spliced genes. (B) distribution of splicing events in all genes and specifically meiotic genes. Modes of alternative splicing including skipped exons, alternative 5′ splice sites, alternative 3′ splice sites, and retained intron. Unclassified events correspond to events that cannot be included in any of the standard categories. (C) and (D) Quantification of the relative incidence of Smc4 (C) and Sycp2 (D) splice variants (Ensembl BADGE BPAF homologous recombination chromosome organization involved in meiotis meiotic chromosome segregation meiosis I meiosis I cell cycle process meiotic nuclear division meiotic cell cycle process DNA recombination nuclear chromosome segregation meiotic cell cycle chromosome segregation regulation of chromosome organization DNA repair 0.000 0.025 0.050 0.075 0.100 p.adjust GeneRatio 0.01 0.02 0.03 Alternative 3' Acceptor Site Alternative 5' Donor Site Cassette Exon Intron Retention Unclassified event BADGE (n=5085) BPAF (n=3879) splicing events all transcripts meiotic transcripts Gene ratio A B reciprocal DNA recombination C Smc4 splicing site primers reference primers 1 reference primers 2 Smc4-201 relative quantification 0.0 0.4 0.8 1.2 BADGE BPAF * relative to: primers 1 primers 2 BADGE BPAF * * ETOH Smc4-202 Sycp2-201 Sycp2-202 reference primers 1 reference primers 2 Sycp2 alternative 3' acceptor site splicing site primers relative quantification 0.0 0.5 1.0 1.5 BADGE BPAF * relative to: primers 1 primers 2 BADGE BPAF * ETOH alternative 3' acceptor site 5' UTR 1 2 3 4 5 2 3 4 5 1 5' UTR 18 17 16 15 14 40 39 37 36 38 5' UTR D ratio Smc4 201/ total Smc4 ratio Sycp2-202/ total Sycp2 Alternative 3' Acceptor Site Alternative 5' Donor Site Cassette Exon Intron Retention Unclassified event BADGE (n=5085) BPAF (n=3879) splicing events all transcripts meiotic transcripts B BADGE BPAF homologous recombination chromosome organization involved in meiotis meiotic chromosome segregation meiosis I meiosis I cell cycle process meiotic nuclear division meiotic cell cycle process DNA recombination nuclear chromosome segregation meiotic cell cycle chromosome segregation regulation of chromosome organization DNA repair 0.000 0.025 0.050 0.075 0.100 p.adjust GeneRatio 0.01 0.02 0.03 Gene ratio A reciprocal DNA recombination B B A Figure 5: BADGE or BPAF fetal exposures induce RNA splicing alterations in premeiotic germ cells. Splicing events were determined in 13.5 dpc germ cells using the high resolutive microarray. BADGE or BPAF fetal exposures alter gene expression and mRNA splicing in ge Modes of alternative splicing including skipped exons, alternative 5′ splice sites, alternative 3′ splice sites, and retained intron. Unclassified events correspond to events that cannot be included in any of the standard categories. (C) and (D) Quantification of the relative incidence of Smc4 (C) and Sycp2 (D) splice variants (Ensembl variants:) in bisphenols-treated germ cells. n=3 independent exposures. Upper panel: Representation of splicing sites leading to different splice variants (Smc4-201, Smc4-202, Sycp2- 201 and Sycp2-202, Ensembl name). Lower pannel: relative quantification of expression of Smc4- 201 or Sycp2-202 to total Smc4 transcripts and Sycp2 transcripts respectively in bisphenols treated germ cells in comparison to ETOH-treated germ cells. * p <0.05 (Mann-Whitney’s test). 314 315 316 317 318 319 320 321 322 323 324 325 326 327 328 14 . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted August 20, 2020. ; https://doi.org/10.1101/2020.08.17.253724 doi: bioRxiv preprint . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted August 20, 2020. ; https://doi.org/10.1101/2020.08.17.253724 doi: bioRxiv preprint Table 1: List of common genes between BADGE and BPAF conditions showing an alternative spllcing relative to control germ cell. BADGE or BPAF fetal exposures alter gene expression and mRNA splicing in ge ; https://doi.org/10.1101/2020.08.17.253724 doi: bioRxiv preprint . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted August 20, 2020. ; https://doi.org/10.1101/2020.08.17.253724 doi: bioRxiv preprint Table 2: Splicing event estimation by TAC software in bisphenols treated germ cells. EEJ: one exon-exon junction was covered by probes, E: one exon was covered by probes. Only PSRs have assigned « Event Estimation Name ». Embedded text: common significative splicing events between BADGE and BPAF 331 332 333 334 Table 2: Splicing event estimation by TAC software in bisphenols treated germ cells. EEJ: one exon-exon junction was covered by probes, E: one exon was covered by probes. Only PSRs have assigned « Event Estimation Name ». Embedded text: common significative splicing events b BADGE d BPAF 1 2 3 4 have assigned « Event Estimation Name ». Embedded text: common significative splicing even between BADGE and BPAF. BADGE or BPAF fetal exposures alter gene expression and mRNA splicing in ge Go term Differentially Spliced genes count (adj pvalue) Common genes between bisphenols % common genes Chromosome Segregation (GO:0007059) 105 (BADGE ; pvalue 9 x10-8) 85 (BPAF; pvalue 2 x10-6) 46% (BADGE) 56% (BPAF) Smc4, Espl1, Mms19, Srpk1, Cit, Ccne1, Psrc1, Bub1b, Pttg1, Kif4, Mlh1, Dync1h1, Zw10, Zwint, Cep63, Hecw2, Spag5, Pogz, Pum1, Mcmdc2, Top2b, Bub1, Setdb2, Anapc1, Atm, Pibf1, Msto1, Mau2, Cdc20, Ndc1, Fancd2, Usp9x,Top3b , Tpr, Ago4, Ncapd3, Sun1, Cenph, Nipbl, Ddx11, Hira, Csnk2a1, Lrrk1, Smc1b, Ino80, Dmc1, Hfm1 DNA repair (GO:0006281) 151 (BADGE; pvalue: 5 x10-10) 108 (BPAF; pvalue: 7 x10-06) 44 % (BADGE) 61 % (BPAF) Nhej1, Nabp1, Pold2, Gins4, Wrn, Uchl5, Smc4, Hdac9, Npas2, Usp47, Mms19, Prkdc, Pms2, Fanci, Pif1, Upf1, Pttg1, Rad51, Mlh1, Cdc45, Kdm2a, Usp3, Otub1, Abl1, Dclre1a, Shprh, Parp9, Cep164, Neil1, Marf1, Rbm17, Mcmdc2, Npm1, Top2b, Taok3, Ticrr, Cdc5l, Supt16, Xpc, Atm, Rad52, Uimc1, Poli, Ube2t, Taok1, Rbbp8, Fancd2, Setx, Sprtn, Ercc6l2, Gtf2h1, Nipb, Atr, Alkbh1, Ddx11, Usp28, Eya1, Rtel1, Rif1, Rev1, Cdc7, Ino80, Dmc1, Huwe1, Helq Nuclear Chromosome Segregation (GO:0098813) 88 (BADGE; pvalue: 3 x10-07) 70 (BPAF; pvalue: 12 x10-05) 45 % (BADGE) 57 % (BPAF) Smc4, Espl1, Cit, Ccne1, Psrc1, Bub1b, Pttg1, Kif4, Mlh1, Dync1h1, Zw10, Zwint, Cep63, Hecw2, Spag5, Pogz, Mcmdc2, Top2b, Bub1, Anapc1, Atm, Pibf1, Msto1, Mau2, Cdc20, Ndc1, ancd2, Tpr, Ago4, Ncapd3, Sun1, Nipbl, Ddx11, Hira, Lrrk1, Smc1b, Ino80, Dmc1, Hfm1 DNA Recombination (GO:0006310) 79 (BADGE, pvalue < 0.001) 5 (BPAF, pvalue: 0.04) 40 % (BADGE) 58 % (BPAF) Cntd1, Nabp1, Gins4, Ubr2, Wrn, Uchl5, Rag1, Mms19, Prkdc, Pms2, Pif1, Rad51, Mlh1, Cdc45, Cep63, Thoc1, Mcmdc2, Top2b, Atm, Rad52, Rbbp8, Setx, Psmc3ip, Nipbl, Rtel1, Rif1, Cdc7, Ino80, Dmc1, Hfm1, Gm960, Helq Meiotic Cell Cycle process (GO:0051321) 99 (BADGE; pvalue: 2 x10-06) 73 (BPAF; pvalue <0.001) 11 % (BADGE) 15% (BPAF) Tdrd9, Cntd1, Topaz1, Ubr2, Smc4, Espl1, Ccne1, Bub1b, Pttg1, Rad51, Mlh1, Marf1, Cep63, Plcb1, Nsun2, Mcmdc2, Top2b, Bub1, Atm, Cdc20, Ndc1, Fancd2, Psmc3ip, Ago4, Ncapd3, Sun1, Lrrk1, Aspm, Dmc1, Hfm1, Gm960, Sycp2 . CC-BY 4.0 International license made available under a List of common genes between BADGE and BPAF conditions showing a e spllcing relative to control germ cell. 15 15 . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted August 20, 2020. BADGE or BPAF fetal exposures alter gene expression and mRNA splicing in ge CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted August 20, 2020. BADGE or BPAF fetal exposures alter gene expression and mRNA splicing in ge Gene Symbol Probes Splicing event estimation name Splicing pvalue (p<0.01) target location Isoform name BADGE BPAF Ago4 EEJ EEJ Exons 11-12 Exons 9-10 Ago4-201 Ago4-201 - - ns 0.0093 0.0028 ns Aspm EEJ E Exons 1-2 Exon 18 Aspm-201 Aspm-201 - ND 0.0007 ns ns 0.0044 Atm EEJ EEJ EEJ EEJ E Exons 56-57 Exons 52-53 Exons 47-48 Exons 49-50 Exon 29 Atm-201 Atm-201 Atm-201 Atm-201 Atm-201 - - - - Cassette Exon 0.0061 ns 0.0039 0.002 0.01 0.0093 0.0061 ns 0.0017 ns Bub1b EEJ E Exons 9-10 Exon1 Bub1-201 Bub1-203 - Alt 3' Acceptor Site 0.0002 ns 0.0044 0.0064 Ccne1 EEJ E Exons 6-7 Exon 1 Ccne-201 Ccne-201 - Cassette Exon ns 0.0035 0.0026 ns Cdc7 EEJ Exons 6-8 Cdc7-201 - 0.0044 0.0366 Cdc20 EEJ E Exons 6-7 Exon 1 Cdc20-201 Cdc20-201 - Cassette Exon 0.0041 ns ns 0.004 Cdc45 EEJ EEJ EEJ Exons 19-20 Exons 11-12 Exons 1-2 Cdc45-201 Cdc45-201 Cdc45-201 - - - ns 0.009 0.0094 0.0089 ns ns Cep63 E Exon 7 Cep63-201 Cassette Exon 0.0024 0.0047 Cntd1 E E E Exon 1 (-5’) Exon 1 (-3’) Exon 3 Cntd1-201 Cntd1-201 Cntd1-203 Cassette Exon Cassette Exon Cassette Exon ns ns 0.0012 0.007 0.0074 ns Dmc1 EEJ EEJ Exons 9-10 Exons 7-8 Dmc1-201 Dmc1-201 - - 0.0004 0.0013 0.0069 ns Espl1 EEJ Exons 1-2 Espl1-201 - 0.003 0.0039 Fancd2 EEJ EEJ Exons 22-23 Exons 31-32 Fancd2-201 Fancd2-201 - - 0.0019 0.0096 0.0088 ns Gins4 EEJ E Exons 2-3 Exon 8 Gins4-201 Gins4-201 - Alt 5' Donor Site 0.0061 ns ns 0.0045 Gm960 (Top6bl) EEJ E E Exons 3-4 Exon 12 Exon 11 Gm960-201 Gm960-202 Gm960-202 - Cassette Exon Cassette Exon ns 0.0033 0.0076 0.0039 ns ns Helq EEJ EEJ Exons 16-17 Exons 12-13 Helq-201 Helq-201 - - 0.0000157 0.0088 0.0038 ns Hfm1 EEJ E E E Exons 7-8 Exon 22 Exon 21 Exon 4 Hfm1-201 Hfm1-201 Hfm1-201 Hfm1-204 - Cassette Exon Cassette Exon Cassette Exon ns 0.0031 0.0087 0.0005 0.0076 ns ns ns 16 . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted August 20, 2020. ; https://doi.org/10.1101/2020.08.17.253724 doi: bioRxiv preprint . BADGE or BPAF fetal exposures alter gene expression and mRNA splicing in ge It is The copyright holder for this preprint this version posted August 20, 2020. ; https://doi.org/10.1101/2020.08.17.253724 doi: bioRxiv preprint > Smc4 EEJ EEJ > E E Exons 9-10 Exons 19-20 Exon 1 Exon 8 Smc4-201 Smc4-201 Smc4-201 Smc4-201 - - Alt 3' Acceptor Site Alt 5' Donor Site ns ns 0.0039 0.0034 0.0072 0.0061 0.0024 0.0049 > Sycp2 EEJ > E E Exons 36-37 Exon 38 Exon 36 Sycp2-201 Sycp2-201 Sycp-201 - Alt 3' Acceptor Site Cassette Exon 0.0022 0.0027 0.0000384 ns ns 0.0003 Sun1 EEJ EEJ EEJ EEJ E E E E E E Exons 8-10 Exons 13-14 Exons 2-3 Exons 6-8 Exon 1 Exon 4 Exon 15 Exon 17 Exon 18 Exon 23 Sun1-201 Sun1-201 Sun1-218 Sun1-201 Sun1-215 Sun1-201 Sun1-201 Sun1-201 Sun1-201 Sun1-201 - - - - ND Alt 3' Acceptor Site Cassette Exon Cassette Exon Alt 3' Acceptor Site Alt 5' Donor Site 0.0094 0.0089 0.0063 0.0055 0.0003 0.0094 0.0076 0.0007 0.0081 ns ns ns ns ns 0.01 ns ns 0.01 ns 0.01 Tdrd9 E E E Exon 5 Exon 6 Exon 24 Tdrd9-201 Tdrd9-201 Tdrd9-201 Cassette Exon Cassette Exon Cassette Exon ns ns 0.005 0.0026 0.0077 ns Thoc1 EEJ E Exons 3-4 Exon 7 Thoc1-201 Thoc1-201 - Cassette Exon ns 0.0054 0.01 ns Top2b EEJ EEJ EEJ Exons 6-7 Exons 22-23 Exons 25-26 Top2b-201 Top2b-201 Top2b-201 - - - ns 0.0096 0.008 0.009 ns ns Topaz EEJ EEJ E Exons 4-5 Exons 9-10 Exon 10 Topaz1-201 Topaz1-201 Topaz1-201 - - Cassette Exon 0.0038 ns 0.0014 ns 0.0047 ns Ubr2 E EEJ EEJ E E Exon 1 Exons 44-45 Exons 27-28 Exon 5 Exon 1 Ubr-201 Ubr-201 Ubr-201 Ubr-201 Ubr-205 ND - - Cassette Exon Intron Retention 0.007 ns ns ns 0.0074 ns 0.005 0.0029 0.0075 ns Uchl5 EEJ E Exons 1-2 Exon 11 Uchl5-201 Uchl5-201 - Alt 5' Donor Site 0.0083 ns ns 0.0008 Wrn EEJ E Exons 12-13 Exon 21 Wrn-201 Wrn-201 - Cassette Exon 0.0061 ns ns 0.0057 Some of the differentially spliced meiotic genes retrieved in both bisphenols exhibit the same splicing events such as Smc4, a condensin protein (Table 2 in red, Figure 5C). The presence of an alternative 3' acceptor sites in the first exon allows the production of different forms of Smc4 transcripts containing or not a truncated exon 1 as observed in the Smc4-202 and Smc4-201 variants respectively. BADGE or BPAF fetal exposures alter gene expression and mRNA splicing in ge ; https://doi.org/10.1101/2020.08.17.253724 doi: bioRxiv preprint Ino80e EEJ Exons 2-3 Ino80e-203 - 0.0007 0.0034 Lrrk1 E Exon 28 Lrrk1-201 Cassette Exon 0.0077 0.0042 Marf1 EEJ EEJ Exons 24-25 Exons 9-10 Marf1-201 Marf1-201 - - ns 0.0023 0.0084 0.0069 Mcmdc2 E E E Exon 10 Exon 14 Exon 16 Mcmdc2-201 Mcmdc2-203 Mcmdc2-204 - Cassette Exon Alt 5' Donor Site ns 0.0047 0.0018 0.0096 n ns Mlh1 EEJ E E Exons 15-17 Exon 18 Exon 18 Mlh1-201 Mlh1-206 Mlh1-201 - ND ND ns 0.0056 0.0035 0.0091 ns ns Mms19 EEJ E E E Exons 4-6 Exon 28 Intron 6 Exon 6 Mms19-201 Mms19-201 Mms19-201 Mms19-201 - Cassette Exon Intron Retention Cassette Exon 0.0008 ns 0.0066 0.0094 0.007 0.0022 0.0021 ns Nabp1 EEJ E E Exons 5-6 Exon 1 Exon 6 Nabp1-204 Nabp1-208 Nabp1-201 - Alt 5' Donor Site ND 0.0088 0.004 ns ns ns 0.0056 Ncapd3 EEJ E E E Exons 29-30 Exon 4 Exon 26 Exon 29 Ncapd3-201 Ncapd3-201 Ncapd3-201 Ncapd3-201 - Cassette Exon Cassette Exon Cassette Exon ns 0.0069 0.0047 0.0041 0.0065 ns ns ns Ndc1 EEJ Exons 1-2 Ndc1-205 - 0.0018 0.0032 Nipbl EEJ E E Exons 25-26 Exon 28 Exon 16 Nipbl-201 Nipbl-201 Nipbl-201 - Cassette Exon Cassette Exon 0.001 ns ns ns 0.004 0.0055 Nsun2 EEJ EEJ Exons 7-8 Exons1-2 Nsun2-201 Nsun2-203 - - 0.002 ns ns 0.0045 Plcb1 E Exon 32 Plcb1-201 Cassette Exon 0.0017 0.0079 Prkdc EEJ Exons 52-53 Prkdc-201 - 0.0039 0.0079 Psmc3ip EEJ Exons 2-3 Psmc3ip-201 - 0.0025 0.0059 Pttg1 E Exon 1 Pttg1-201 Alt 5' Donor Site 0.0026 0.0026 Rad51 EEJ EEJ Exons 1-2 Exons 7-8 Rad51-201 Rad51-201 - - 0.0064 0.0023 ns 0.0012 Rag1 E E Exon 6 Exon 1 Rag1-201 Rag1-207 Cassette Exon Cassette Exon 0.01 ns ns 0.0085 Rif1 EEJ E E E Exons 24-25 Exon 5 Exon 18 Exon 22 Rif1-201 Rif1-201 Rif1-201 Rif1-201 - Cassette Exon Cassette Exon ND 0.003 0.0068 ns ns 0.0058 ns 0.0083 0.0027 Rbbp8 EEJ Exons 15-16 Rbbp8-201 - 0.000084 1 0.0002 Rtel1 EEJ E Exons 6-7 Exon 18 Rtel1-201 Rtel1-201 - Cassette Exon ns 0.005 0.0025 ns Setx EEJ E Exons 17-18 Exon 4 Setx-201 Setx-201 - Cassette Exon 0.0082 0.0067 ns 0.0041 17 17 . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. BADGE or BPAF fetal exposures alter gene expression and mRNA splicing in ge Using specific primers of the truncated/spliced region of the first exon and of the core/unspliced region of the transcript, we quantified by QPCR the relative abundance of the 335 336 337 338 339 340 18 18 . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted August 20, 2020. ; https://doi.org/10.1101/2020.08.17.253724 doi: bioRxiv preprint . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted August 20, 2020. ; https://doi.org/10.1101/2020.08.17.253724 doi: bioRxiv preprint mRNAs containing a long first exon. In BADGE as in BPAF -treated germ cells, we observed a significant decrease in the abundance of the long form with a corresponding increase of the truncated form of this first exon (Figure 5C). While we retrieved the same affected genes in BADGE and BPAF treated germ cells, the precise differential splicing events differs between conditions as observe for meiotic gene set (Table 2 in black). In BADGE -treated germ cells, we identified a differentially specific event occurring in the exon 38 of Sycp2, a component of the synaptonemal complex, due to alternative 3' acceptor sites (Table 2). These sites lead to the production of two types of variants with a complete or a truncated exon as observed in Sycp2-201 and Sycp2-202 transcripts. QPCR quantification revealed a significant increase in the relative abundance of the truncated form of the exon 38 only after BADGE exposure (Figure 5D). 339 340 341 342 343 344 345 346 347 348 Genes differentially spliced in BPAF or BADGE conditions were not preferentially differentially expressed compared to others genes (Supplementary Figure 4). As observed with non-spliced genes, less than 8 % of spliced genes were also differentially expressed in BPAF or BADGE conditions. Interestingly, we observed closer transcription behaviors after BPAF or BADGE exposure of differentially spliced genes in comparison to non-differentially spliced genes randomly chosen (Supplementary Figure 4). BADGE or BPAF fetal exposures alter gene expression and mRNA splicing in ge Meiob Snx33 Trip6 Gm23844 Gm16103 Ccdc84 Mir5128 Gm26501 Tubb2b Gm26127 Gm22112 Gm22798 Gm25829 Gm24584 Gm23355 Cldn8 Gm25817 Hddc3 Gm23606 1700086D15Rik Gm21742 Gm25256 Cpeb4 Tc2n Klf4 Gm25966 Gm24693 Tst Mir3074−1 Gm22468 Gm10462 Gnrh1 Gm26239 Gm14654 4930518I15Rik Uck2 Gm16091 Gm25337 Gm26738 Gm26054 D130012P04Rik Gm24339 Gm22067 Pol Ahsp Tspan7 AC132444.6 Anxa8 Gm25670 Acta2 Gm24870 Gm21833 Pten.1 Mettl24 Gm24630 Ybx2 Gm23815 Gm22358 Murc Gm22502 Mir3074−1.1 Erbb2 Gm22502.1 Gm11639 Gm14734 Gm24713 Pgm5 Trpc1 E230008O15Rik Gm15246 Krt8 Gm25632 2410089E03Rik Gm3173 Gm26501.1 Rbm25.1 Sox9 Ybx2.1 Pank1 Gm24427 Gm24492 Gm24392 Cited2 Gm22997 Tex18 Gm25484 4921507P07Rik Tgfbi Rgs2 Gm3317 Gm11578 Gm25021 LOC432823 Tc2n.1 Gm26331 Acsl5 Gm3317.1 Ccdc41 Gm23115 BC030307 Gm23095 Alms1 Odf2l Lyst Col3a1.1 Brca2 Msh4 Sycp1 Mei1 Spo11 Dmc1 Rad21l Sycp2 Msh5 Prdm9 Spata22 Hmga2 Rev3l Helq.1 Uimc1.1 Uvssa Parp6 Stxbp5 Plxnb2.1 Sparcl1 Cdh1 Mycbp2.1 Snrnp48 Pkd1 Hk2 Mmp14 Ace2 Kmt2a Ash1l Aldob 4932438A13Rik Slc26a8 Ccnb3 Zkscan3 Herc1.1 Micu3 Csrp2 Slc4a7.1 Grk4 Aldh1a2.1 Stk38 Kcnq1ot1 Dmxl1 Sfi1 Mir296 Nktr Rel Strip2 Gldn Cmtm7 Nicn1 Eml5 Catsperg1 Catsperg2 Dennd4a.1 Syt14 Capns1 Lyst Chuk Col3a1 Gfra2 Pdgfra Nrp2 Crmp1 Plxnb2 Mycbp2 Mapk8ip3 Stxbp5 Stard9 AI481877 Dmc14930447C04Rik Paxbp1 Ccnb3 Mybl1 Sycp2 Uimc1 Helq Aldh1a2 Slc4a7 Kmt2c Anxa5 Cdh1 Cdh17 Stk38 Ash1l Ace2 Herc1 Zkscan3 Gldn Krt19 Susd2 Nktr Enpp3 Golgb1 Sfxn1 Taf7l Grk4 Nlrp9b Rmdn2 Eml5 Mga Pcsk7 Lgals4 Catsperg1 Dennd4a NON-DIFFERENTIALLY SPLICED GENES DIFFERENTIALLY SPLICED GENES IN BPAF CONDITION −1.0 −0.5 0.0 0.5 1.0 −1.0 −0.5 0.0 0.5 1.0 −1.0 −0.5 0.0 0.5 1.0 −1 0 1 1 4 16 count DIFFERENTIALLY SPLICED GENES IN BADGE CONDITION BPAF (Log2FC) 3543 genes (random sampling) 3543 genes 2678 genes 5.5 % DEGs 5.1 % DEGs 4.9 % DEGs 355 nal response and RNA splicing after BADGE and BPAF exposure. transcriptional response and RNA splicing after BADGE and BPAF exposure. Gm10462 Rad21l Sycp2 Msh5 Spata22 NON-DIFFERENTIALLY SPLICED GENES DIFFERENTIALLY SPLI DIFFERENTIALLY SPLICED GENES IN BADGE CONDITION 3543 genes (random sampling) 3543 genes 2678 genes 5.5 % DEGs 5.1 % DEGs 4.9 % DEGs 355 DIFFERENTIALLY SPLICED GENES IN BADGE CONDITION Supplementary Figure 4: 2d density plot between BADGE and BPAF cond BADGE (Log2FC) 356 Supplementary Figure 4: 2d density plot between BADGE and BPAF condition of the differential expression (Log2FC) of differentially spliced genes in BADGE or BPAF condition. The plot area is divided in 200 hexagons and the color of the hexagon correspond to number of genes inside the hexagon. BADGE or BPAF fetal exposures alter gene expression and mRNA splicing in ge Meiob Snx33 Trip6 Gm23844 Gm16103 Ccdc84 Mir5128 Gm26501 Tubb2b Gm26127 Gm22112 Gm22798 Gm25829 Gm24584 Gm23355 Cldn8 Gm25817 Hddc3 Gm23606 1700086D15Rik Gm21742 Gm25256 Cpeb4 Tc2n Klf4 Gm25966 Gm24693 Tst Mir3074−1 Gm22468 Gm10462 Gnrh1 Gm26239 Gm14654 4930518I15Rik Uck2 Gm16091 Gm25337 Gm26738 Gm26054 D130012P04Rik Gm24339 Gm22067 Pol Ahsp Tspan7 AC132444.6 Anxa8 Gm25670 Acta2 Gm24870 Gm21833 Pten.1 Mettl24 Gm24630 Ybx2 Gm23815 Gm22358 Murc Gm22502 Mir3074−1.1 Erbb2 Gm22502.1 Gm11639 Gm14734 Gm24713 Pgm5 Trpc1 E230008O15Rik Gm15246 Krt8 Gm25632 2410089E03Rik Gm3173 Gm26501.1 Rbm25.1 Sox9 Ybx2.1 Pank1 Gm24427 Gm24492 Gm24392 Cited2 Gm22997 Tex18 Gm25484 4921507P07Rik Tgfbi Rgs2 Gm3317 Gm11578 Gm25021 LOC432823 Tc2n.1 Gm26331 Acsl5 Gm3317.1 Ccdc41 Gm23115 BC030307 Gm23095 Alms1 Odf2l Lyst Col3a1.1 Brca2 Msh4 Sycp1 Mei1 Spo11 Dmc1 Rad21l Sycp2 Msh5 Prdm9 Spata22 Hmga2 Rev3l Helq.1 Uimc1.1 Uvssa Parp6 Stxbp5 Plxnb2.1 Sparcl1 Cdh1 Mycbp2.1 Snrnp48 Pkd1 Hk2 Mmp14 Ace2 Kmt2a Ash1l Aldob 4932438A13Rik Slc26a8 Ccnb3 Zkscan3 Herc1.1 Micu3 Csrp2 Slc4a7.1 Grk4 Aldh1a2.1 Stk38 Kcnq1ot1 Dmxl1 Sfi1 Mir296 Nktr Rel Strip2 Gldn Cmtm7 Nicn1 Eml5 Catsperg1 Catsperg2 Dennd4a.1 Syt14 Capns1 Lyst Chuk Col3a1 Gfra2 Pdgfra Nrp2 Crmp1 Plxnb2 Mycbp2 Mapk8ip3 Stxbp5 Stard9 AI481877 Dmc14930447C04Rik Paxbp1 Ccnb3 Mybl1 Sycp2 Uimc1 Helq Aldh1a2 Slc4a7 Kmt2c Anxa5 Cdh1 Cdh17 Stk38 Ash1l Ace2 Herc1 Zkscan3 Gldn Krt19 Susd2 Nktr Enpp3 Golgb1 Sfxn1 Taf7l Grk4 Nlrp9b Rmdn2 Eml5 Mga Pcsk7 Lgals4 Catsperg1 Dennd4a NON-DIFFERENTIALLY SPLICED GENES DIFFERENTIALLY SPLICED GENES IN BPAF CONDITION −1 0 1 1 4 16 count DIFFERENTIALLY SPLICED GENES IN BADGE CONDITION BPAF (Log2FC) 3543 genes (random sampling) 3543 genes 2678 genes 5.5 % DEGs 5.1 % DEGs 4.9 % DEGs 355 transcriptional response and RNA splicing after BADGE and BPAF exposure. BADGE or BPAF fetal exposures alter gene expression and mRNA splicing in ge This observation suggests a common alteration impacting the t i ti l d RNA li i ft BADGE d BPAF 349 350 351 352 353 354 355 transcriptional response and RNA splicing after BADGE and BPAF exposure. Supplementary Figure 4: 2d density plot between BADGE and BPAF condition of the differential expression (Log2FC) of differentially spliced genes in BADGE or BPAF condition. The plot area is divided in 200 hexagons and the color of the hexagon correspond to number of genes inside the hexagon. A simple random sampling of 3543 non differentially spliced genes (maximal number of differentially spliced genes) was used to represent transcriptionnal behavior after BADGE or BPAF exposure of non differentially spliced genes. BADGE or BPAF fetal exposures alter gene expression and mRNA splicing in ge Meiob Snx33 Trip6 Gm23844 Gm16103 Ccdc84 Mir5128 Gm26501 Tubb2b Gm26127 Gm22112 Gm22798 Gm25829 Gm24584 Gm23355 Cldn8 Gm25817 Hddc3 Gm23606 1700086D15Rik Gm21742 Gm25256 Cpeb4 Tc2n Klf4 Gm25966 Gm24693 Tst Mir3074−1 Gm22468 Gm10462 Gnrh1 Gm26239 Gm14654 4930518I15Rik Uck2 Gm16091 Gm25337 Gm26738 Gm26054 D130012P04Rik Gm24339 Gm22067 Pol Ahsp Tspan7 AC132444.6 Anxa8 Gm25670 Acta2 Gm24870 Gm21833 Pten.1 Mettl24 Gm24630 Ybx2 Gm23815 Gm22358 Murc Gm22502 Mir3074−1.1 Erbb2 Gm22502.1 Gm11639 Gm14734 Gm24713 Pgm5 Trpc1 E230008O15Rik Gm15246 Krt8 Gm25632 2410089E03Rik Gm3173 Gm26501.1 Rbm25.1 Sox9 Ybx2.1 Pank1 Gm24427 Gm24492 Gm24392 Cited2 Gm22997 Tex18 Gm25484 4921507P07Rik Tgfbi Rgs2 Gm3317 Gm11578 Gm25021 LOC432823 Tc2n.1 Gm26331 Acsl5 Gm3317.1 Ccdc41 Gm23115 BC030307 Gm23095 Alms1 Odf2l Lyst Col3a1.1 Brca2 Msh4 Sycp1 Mei1 Spo11 Dmc1 Rad21l Sycp2 Msh5 Prdm9 Spata22 Hmga2 Rev3l Helq.1 Uimc1.1 Uvssa Parp6 Stxbp5 Plxnb2.1 Sparcl1 Cdh1 Mycbp2.1 Snrnp48 Pkd1 Hk2 Mmp14 Ace2 Kmt2a Ash1l Aldob 4932438A13Rik Slc26a8 Ccnb3 Zkscan3 Herc1.1 Micu3 Csrp2 Slc4a7.1 Grk4 Aldh1a2.1 Stk38 Kcnq1ot1 Dmxl1 Sfi1 Mir296 Nktr Rel Strip2 Gldn Cmtm7 Nicn1 Eml5 Catsperg1 Catsperg2 Dennd4a.1 Syt14 Capns1 Lyst Chuk Col3a1 Gfra2 Pdgfra Nrp2 Crmp1 Plxnb2 Mycbp2 Mapk8ip3 Stxbp5 Stard9 AI481877 Dmc14930447C04Rik Paxbp1 Ccnb3 Mybl1 Sycp2 Uimc1 Helq Aldh1a2 Slc4a7 Kmt2c Anxa5 Cdh1 Cdh17 Stk38 Ash1l Ace2 Herc1 Zkscan3 Gldn Krt19 Susd2 Nktr Enpp3 Golgb1 Sfxn1 Taf7l Grk4 Nlrp9b Rmdn2 Eml5 Mga Pcsk7 Lgals4 Catsperg1 Dennd4a NON-DIFFERENTIALLY SPLICED GENES DIFFERENTIALLY SPLICED GENES IN BPAF CONDITION −1.0 −0.5 0.0 0.5 1.0 −1.0 −0.5 0.0 0.5 1.0 −1.0 −0.5 0.0 0.5 1.0 −1 0 1 1 4 16 count DIFFERENTIALLY SPLICED GENES IN BADGE CONDITION BADGE (Log2FC) BPAF (Log2FC) 3543 genes (random sampling) 3543 genes 2678 genes 5.5 % DEGs 5.1 % DEGs 4.9 % DEGs 355 356 357 358 359 360 361 transcriptional response and RNA splicing after BADGE and BPAF exposure. BADGE or BPAF fetal exposures alter gene expression and mRNA splicing in ge A simple random sampling of 3543 non differentially spliced genes (maximal number of differentially spliced genes) was used to represent transcriptionnal behavior after BADGE or BPAF exposure of non differentially spliced genes. ( g ) 356 357 358 359 360 361 19 19 . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted August 20, 2020. ; https://doi.org/10.1101/2020.08.17.253724 doi: bioRxiv preprint . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted August 20, 2020. ; https://doi.org/10.1101/2020.08.17.253724 doi: bioRxiv preprint BADGE or BPAF fetal exposures increase oxidative DNA damages impairing meiosis initiation. Supplementary Figure 5. Genome mapping on selected chromosomes (Chr 3, 4, 7, 10, 14, 17) of the DEGs in BPAF (red line) and BADGE (green line) conditions. The sum of the filtered genes per megabase (1000000 bases) was reported according to their location on the chomosome. Grey dotted line represent the sum of the total genes referenced in the murine genome. Purple line correspond to the mapping of the OxiDIP-Seq signal r profiles retrieved in the Amente et al publication (Amente et al., 2019). condition BADGE BPAF transcripts position 11.5dpc 13.5dpc 0 20 40 60 counts_normalized 0 20 40 60 chr3 0 5.0 x107 1.0 x108 1.5 x108 chr4 0 5.0 x107 1.0 x108 1.5 x108 chr7 0 5.0 x107 1.0 x108 1.5 x108 0 20 40 60 0 20 40 60 0 20 40 60 0 20 40 60 chr10 0 5.0 x107 1.0 x108 position position chr14 0 5.0 x107 1.0 x108 position position chr17 0 2.5 x107 5.0 x108 7.5 x107 1.0 x108 position 11.5dpc 13.5dpc 11.5dpc 13.5dpc counts_normalized counts_normalized 8-OdG 8oxodG (OxidipSeq, MEF, Amente et al , 2019) 8-OdG 8-OdG 62 63 64 65 66 67 68 69 BADGE or BPAF fetal exposures increase oxidative DNA damages impairing meiosis initiation. BADGE or BPAF fetal exposures alter gene expression and mRNA splicing in ge condition position 11.5dpc 13.5dpc 0 20 40 60 counts_normalized 0 20 40 60 chr3 0 5.0 x107 1.0 x108 1.5 x108 chr4 0 5.0 x107 1.0 x108 1.5 x108 chr7 0 5.0 x107 1.0 x108 1.5 x108 0 20 40 60 0 20 40 60 0 20 40 60 0 20 40 60 chr10 0 5.0 x107 1.0 x108 position position chr14 0 5.0 x107 1.0 x108 position position chr17 0 2.5 x107 5.0 x108 7.5 x107 1.0 x108 position 11.5dpc 13.5dpc 11.5dpc 13.5dpc counts_normalized counts_normalized 8-OdG 8-OdG 8-OdG 362 363 BADGE or BPAF fetal exposures increase oxidative DNA damages impairing meiosis initiation. 362 363 BADGE or BPAF fetal exposures increase oxidative DNA damages impairing meiosis 62 375 376 Therefore, we analyzed the induction of oxidative DNA damages in response to bisphenols in 12.5 dpc proliferative oogonia by detection of 8-OdG (Figure 6A-B). As a positive control, we added the pro-oxidant potassium bromate (KBrO3) in drinking water from 10.5 dpc. As observed after KBrO3 exposure, bisphenols-exposed PGCs showed a significant increase in 8-OdG when compared to the vehicle-treated germ cells (Figure 6B). 377 378 379 380 381 F in m re (8 12 ce ve (B K (B of ar n= oo ex m id im ex (0 B w sh in ** W m ce w 0. W sy m id in (+ (O fe W A B 8OdG DAPI MERGE ETOH KRBO3 BADGE BPAF ETOH KBrO3 BADGE BPAF 0 20 40 60 80 100 8OdG area/cell area (%) C BADGE BPAF BADGE BPAF KBrO3 ETOH 100 95 90 85 0 +NAC SYCP3 (%) ** ** ** D OGG1+/+ OGG1-/- 0 SYCP3 (%) 40 60 50 E 0 20 40 60 80 100 OGG1+/+ OGG1-/- > 3 MLH1 foci / synapsis (%) *** ** ** *** 55 45 35 382 383 384 385 386 387 388 389 390 391 392 393 394 395 396 397 398 399 400 401 402 403 404 405 406 407 408 409 410 411 412 413 414 415 416 417 418 419 420 421 Figure 6: Oxidative DNA damages induced by bisphenols impact meiosis initiation and meiotic recombination. (A) 8-oxoguanine (8OdG, green) immunostaining in 12.5 dpc germ cells (TRA98 positive cells) from ovaries exposed to vehicle (ETOH), bisphenols (BADGE, BPAF) or the pro-oxidant KbrO3 (0.15 mM). Scale bar, 10µm. (B) Quantification of the total area of 8OdG normalized by the nucleus area. n= 60 (ETOH), n=90 (KrBO3), n=94 (BADGE) and 64 (BPAF) oogonia from 3-4 independant exposures. (C) Percentage of meiotic oocytes in germ cells identified by SYCP3 and TRA98 immunostaining in 14.5 dpc ovaries exposed to vehicle (ETOH), KrBO3 (0.15 mM) and bisphenols (BADGE, BPAF) supplemented with or without NAC (4 mM). Error bars show mean±s.e.m. n=3-5 mice from independant exposures; ** p<0.01; *** p<0.001; **** p<0.0001 (Mann- Whitney’s test). (D) Percentage of meiotic oocytes in 14.5 dpc germ cells from OGG1 deficient (-/-) and wild-type (+/+) mice. n=3-4 mice. p= 0.0832 determined with a Mann- Whitney’s test. BADGE or BPAF fetal exposures increase oxidative DNA damages impairing meiosis 62 362 Supplementary Figure 5. Genome mapping on selected chromosomes (Chr 3, 4, 7, 10, 14, 17) of the DEGs in BPAF (red line) and BADGE (green line) conditions. The sum of the filtered genes per megabase (1000000 bases) was reported according to their location on the chomosome. Grey dotted line represent the sum of the total genes referenced in the murine genome. Purple line correspond to the mapping of the OxiDIP-Seq signal r profiles retrieved in the Amente et al publication (Amente et al., 2019). 364 365 366 367 368 369 As BPA is a well-known oxidative stress generator in germ cells and other cell types we speculated that the formation of oxidative DNA damage such as 8-OdG might explain common transcriptional responses and RNA splicing after BADGE or BPAF exposure. Indeed, it is well known that the formation of oxidative DNA damages such as 8-OdG alters gene expression (Ba et Boldogh 2018). Using Oxidip-seq data obtained by Amente et al (Amente et al. 2019) on mouse embryonic 370 371 372 373 374 As BPA is a well-known oxidative stress generator in germ cells and other cell types we speculated that the formation of oxidative DNA damage such as 8-OdG might explain common transcriptional responses and RNA splicing after BADGE or BPAF exposure. Indeed, it is well known that the formation of oxidative DNA damages such as 8-OdG alters gene expression (Ba et Boldogh 2018). Using Oxidip-seq data obtained by Amente et al (Amente et al. 2019) on mouse embryonic 370 371 372 373 374 20 . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted August 20, 2020. ; https://doi.org/10.1101/2020.08.17.253724 doi: bioRxiv preprint . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted August 20, 2020. ; https://doi.org/10.1101/2020.08.17.253724 doi: bioRxiv preprint fibroblasts (MEF), we observed that DEGs genes could overlap with DNA regions susceptible to be oxidized (Supplementary Figure 5). BADGE or BPAF fetal exposures increase oxidative DNA damages impairing meiosis 62 (E) Percentage of synaptonemal complexes with 3 or more MLH1 foci in pachytene cells identified on the basis of the SYCP3 in OGG1 deficient (-/-) and wild-type (+/+) 18.5 dpc fetuses. n= 64 (OGG+/+), n=80 (OGG-/-) from 3 fetuses. *** p<0,001 (Mann- Whitney’s test). A B 8OdG DAPI MERGE ETOH KRBO3 BADGE BPAF ETOH KBrO3 BADGE BPAF 0 20 40 60 80 100 8OdG area/cell area (%) C BADGE BPAF BADGE BPAF KBrO3 ETOH 100 95 90 85 0 +NAC SYCP3 (%) ** ** ** D OGG1+/+ OGG1-/- 0 SYCP3 (%) 40 60 50 E 0 20 40 60 80 100 OGG1+/+ OGG1-/- > 3 MLH1 foci / synapsis (%) *** ** ** *** 55 45 35 382 383 384 385 386 387 388 389 390 391 392 393 394 395 396 397 398 399 400 401 402 403 404 405 406 407 408 409 410 411 412 413 414 415 416 417 418 419 420 421 Figure 6: Oxidative DNA damages induced by bisphenols impact meiosis initiation and meiotic recombination. (A) 8-oxoguanine (8OdG, green) immunostaining in 12.5 dpc germ cells (TRA98 positive cells) from ovaries exposed to vehicle (ETOH), bisphenols (BADGE, BPAF) or the pro-oxidant KbrO3 (0.15 mM). Scale bar, 10µm. (B) Quantification of the total area of 8OdG normalized by the nucleus area. n= 60 (ETOH), n=90 (KrBO3), n=94 (BADGE) and 64 (BPAF) A B 8OdG DAPI MERGE ETOH KRBO3 BADGE BPAF ETOH KBrO3 BADGE BPAF 0 20 40 60 80 100 8OdG area/cell area (%) *** ** ** 382 383 384 385 386 387 388 389 390 391 392 393 394 395 B ETOH KBrO3 BADGE BPAF 0 20 40 60 80 100 8OdG area/cell area (%) D *** ** ** Figure 6: Oxidative DNA damages induced by bisphenols impact meiosis initiation and meiotic recombination. (A) 8-oxoguanine (8OdG, green) immunostaining in 12.5 dpc germ cells (TRA98 positive cells) from ovaries exposed to vehicle (ETOH), bisphenols (BADGE, BPAF) or the pro-oxidant KbrO3 (0.15 mM). Scale bar, 10µm. (B) Quantification of the total area of 8OdG normalized by the nucleus area. n= 60 (ETOH), n=90 (KrBO3), n=94 (BADGE) and 64 (BPAF) oogonia from 3-4 independant exposures. (C) Percentage of meiotic oocytes in germ cells identified by SYCP3 and TRA98 immunostaining in 14.5 dpc ovaries exposed to vehicle (ETOH), KrBO3 (0.15 mM) and bisphenols (BADGE, BPAF) supplemented with or without NAC (4 mM). Error bars show mean±s.e.m. BADGE or BPAF fetal exposures increase oxidative DNA damages impairing meiosis 62 n=3-5 mice from independant exposures; ** p<0.01; *** p<0.001; **** p<0.0001 (Mann- Whitney’s test). (D) Percentage of meiotic oocytes in 14.5 dpc germ cells from OGG1 deficient (-/-) and wild-type (+/+) mice. n=3-4 mice. p= 0.0832 determined with a Mann- Whitney’s test. (E) Percentage of synaptonemal complexes with 3 or more MLH1 foci in pachytene cells identified on the basis of the SYCP3 in OGG1 deficient (-/-) and wild-type (+/+) 18.5 dpc fetuses. n= 64 (OGG+/+), n=80 (OGG-/-) from 3 fetuses. *** p<0,001 (Mann- Whitney’s test). B ETOH KBrO3 BA C BADGE BPAF BADGE BPAF KBrO3 ETOH 100 95 90 85 0 +NAC SYCP3 (%) ** ** ** D OGG1+/+ OGG1-/- 0 SYCP3 (%) 40 60 50 E 0 20 40 60 80 100 OGG1+/+ OGG1-/- > 3 MLH1 foci / synapsis (%) *** 55 45 35 395 396 397 398 399 400 401 402 403 404 405 406 407 408 409 410 411 412 413 414 415 416 417 418 419 420 421 ETOH KBrO3 BA C BADGE BPAF BADGE BPAF KBrO3 ETOH 100 95 90 85 0 +NAC SYCP3 (%) ** ** ** D OGG1+/+ OGG1-/- 0 SYCP3 (%) 40 60 50 E 55 45 35 395 396 397 398 399 400 401 402 403 404 405 406 407 408 409 410 D E 0 20 40 60 80 100 OGG1+/+ OGG1-/- > 3 MLH1 foci / synapsis (%) *** 409 410 411 412 413 414 415 416 417 418 419 420 421 21 21 . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted August 20, 2020. ; https://doi.org/10.1101/2020.08.17.253724 doi: bioRxiv preprint . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted August 20, 2020. ; https://doi.org/10.1101/2020.08.17.253724 doi: bioRxiv preprint To explore the relationship between meiosis initiation delay and oxidative stress, we quantified meiosis initiation in KBrO3 treated ovaries at 14.5 dpc. KBrO3 exposure significantly decreased the proportion of meiotic oocytes (SYCP3 positive cells; Figure 6C). BADGE or BPAF fetal exposures increase oxidative DNA damages impairing meiosis 62 OGG +/+ OGG -/- 100 75 50 25 0 8 oxodG area/cell area (%) *** 438 439 440 441 BADGE or BPAF fetal exposures increase oxidative DNA damages impairing meiosis 62 422 423 424 On the contrary, when we treated pregnant mice with bisphenols supplemented with the antioxidant N-acetylcysteine (NAC) to pregnant mice, we restored the percentage of oocytes to that of vehicle treated (ETOH) 14.5 dpc ovaries. To further asses the impact of oxidative lesions, we used mice deficient for OGG1, a DNA glycosylase involved in the removal of oxidative DNA damage through the Base Excision Repair (BER). Ogg1-/- mice showed increased levels of 8-OdG in proliferative 12.5 dpc oogonia in comparison to Ogg1+/+ mice (Supplementary Figure 6). The lack of OGG1 also impaired meiosis initiation as we observed a decreasing trend of oocyte number at 14.5 dpc (48.0% ± 3.5, Ogg1+/+ vs 35.7% ± 1.8, Ogg1-/-, p=0.0832; Figure 6D). To link oxidative DNA damage and recombination events, we quantified the number of MLH1 foci at 18.5 dpc in OGG1 deficient mice. Interestingly, as observed for bisphenols treated mice, the number of MLH1 foci was significantly increased in the mutant when compared to wild type mice (Figure 6E). Taken together these data reveal that bisphenols induce oxidative DNA damages which in turn could delay fetal meiosis and induce recombination defects. 425 426 427 428 429 430 431 432 433 434 435 436 437 On the contrary, when we treated pregnant mice with bisphenols supplemented with the antioxidant N-acetylcysteine (NAC) to pregnant mice, we restored the percentage of oocytes to that of vehicle treated (ETOH) 14.5 dpc ovaries. To further asses the impact of oxidative lesions, 425 426 427 Supplementary Figure 6: Quantification of the total area of 8OdG normalized by the nucleus area in 12.5 dpc OGG +/+ and -/- oogonia .n= 51 (OGG+/+), n=58 (OGG-/-). *** p<0.001 (Mann- Whitney’s test). OGG +/+ OGG -/- 100 75 50 25 0 8 oxodG area/cell area (%) *** 438 439 440 441 Supplementary Figure 6: Quantification of the total area of 8OdG normalized by the nucleus area in 12.5 dpc OGG +/+ and -/- oogonia .n= 51 (OGG+/+), n=58 (OGG-/-). *** p<0.001 (Mann- Whitney’s test). OGG +/+ OGG -/- 100 75 50 25 0 8 oxodG area/cell area (%) *** 438 439 440 441 Supplementary Figure 6: Quantification of the total area of 8OdG normalized by the nucleus area in 12.5 dpc OGG +/+ and -/- oogonia .n= 51 (OGG+/+), n=58 (OGG-/-). *** p<0.001 (Mann- Whitney’s test). BADGE or BPAF fetal exposures alter DNA demethylation in PGCs. 442 BADGE or BPAF fetal exposures alter DNA demethylation in PGCs. 442 BPA has been reported to alter DNA methylation in various cell types and removal of DNA methylation is a mandatory event in proliferative PGC to allow later the proper expression of the meiotic program(Anderson et al., 2012; Chao et al., 2012; Hargan-Calvopina et al., 2016b; Yamaguchi et al., 2012). For this reason, we quantified the proportion of methylated/pluripotent 443 444 445 446 22 22 . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted August 20, 2020. ; https://doi.org/10.1101/2020.08.17.253724 doi: bioRxiv preprint . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted August 20, 2020. ; https://doi.org/10.1101/2020.08.17.253724 doi: bioRxiv preprint PGC compared to unmethylated/differentiating PGC by immunodetection in 12.5 dpc bisphenols exposed and unexposed ovaries of the 5-hydroxymethylCytosine (5hmC), the first intermediate form during active demethylation (Figure 7A). After bisphenols exposure, we observed an enrichment of methylated germ cells with a global staining (homogeneous staining) of 5hmC in the nucleus in TRA98 cells (Figure 7B). This result suggests that bisphenols exposure alters or delays the DNA demethylation in PGC increasing the proportion of methylated germ cells compared to unmethylated ones. 447 448 449 450 451 452 453 Figure 7: BADGE or BPAF fetal exposures alters DNA methylation in proliferative PGC. Immunodetection of 5hmC (green) in 12.5 dpc TRA98 positive PGC (red) counterstained with DAPI (blue). (A) Representative photomicrograph of methylated (homogeneous nuclear staining) and unmethylated (unstained) TRA98 PGC. (B) percentage of methylated and unmethylated PGC. n= 5 from 3 different exposures. * p<0.05 (Mann- Whitney’s test). 0 5 10 15 ETOH BADGE BPAF germ cells (%) homogeneous unstained B * * ns * Figure 7: BADGE or BPAF fetal exposures alters DNA methylation in proliferative PGC. Immunodetection of 5hmC (green) in 12.5 dpc TRA98 positive PGC (red) counterstained with DAPI (blue). (A) Representative photomicrograph of methylated (homogeneous nuclear staining) and unmethylated (unstained) TRA98 PGC. (B) percentage of methylated and unmethylated PGC. n= 5 from 3 different exposures. * p<0.05 (Mann- Whitney’s test). Discussion 469 The concentration of total BPAF detected in the plasma of the pregnant mice was of the a same order of magnitude (5.96 ± 0.78 ng/ml) than the one we previously observed for BPA (Eladak et al., 470 471 472 473 474 475 476 477 478 479 480 481 482 483 484 485 486 487 488 489 490 491 492 493 494 495 Woman's reproductive pathologies include the ovarian dysgenesis syndrome that is defined as a precocious alteration of ovarian function and mostly associated with premature ovarian insufficiency, anovulation and/or pregnancy loss in the adulthood (Buck Louis et al., 2011). Oocyte aneuploidy is strongly associated with these reproductive failures and growing evidences suggest that fetal exposure to endocrine disruptors such as Bisphenol A (BPA) can cause female reproductive defect (Johansson et al., 2017). In Vertebrates and Invertebrates species, exposure to BPA induces transcription defect of meiotic genes, alters frequency of MLH1 foci and chiasmata leading to chromosome missegregation during meiotic resumption (Allard & Colaiácovo, 2010a; Brieno-Enriquez et al., 2012; Brieño-Enríquez et al., 2011; P. A. Hunt et al., 2012; Patricia A. Hunt et al., 2003; Lawson et al., 2011; Susiarjo et al., 2007). Because BPA has been restricted and regulated in many countries, BPA alternatives have been introduced into industrial products increasing the exposure to other bisphenols such as BPAF and BADGE. Exposure to BADGE and BPAF can be comparable to BPA exposure. A recent study has reported comparable median concentrations of BPA and BADGE hydrated derivatives in human plasma collected from New York City individuals (7.15 ng/ml for BADGE·2H20 and 2.26 ng/ml for BADGE·H20 versus 1.77 ng/ml for BPA) likely due to similar exposure source to BPA (ie. from epoxy coatings into canned food) (L. Wang et al., 2015). In China, BPAF mean concentration in human plasma is 0.073 ng/mL versus 0.4 ng/ml for BPA in the same samples from 81 individuals in general population (Jin et al., 2018). For this reason, we investigated the consequences of BPAF and BADGE exposure on female germ cell differentiation. Although BPAF concentration detected in human samples is lower than the one of BADGE or BPA, we chose to expose mice by drinking water to BADGE or BPAF at the same concentrations (ie 10µM). This allows to compare BPAF and BADGE and to compare our results to previous studies, including ours, that used BPA at environmentally doses (Eladak et al., 2018). BADGE or BPAF fetal exposures alter DNA demethylation in PGCs. 442 0 5 10 15 ETOH BADGE BPAF germ cells (%) homogeneous unstained B * * ns * 0 5 10 15 ETOH BADGE BPAF germ cells (%) homogeneous B * * homogeneous unstained TRA98 5hMC E A Figure 7: BADGE or BPAF fetal exposures alters DNA methylation in proliferative PGC. Immunodetection of 5hmC (green) in 12.5 dpc TRA98 positive PGC (red) counterstained with DAPI (blue). (A) Representative photomicrograph of methylated (homogeneous nuclear staining) and unmethylated (unstained) TRA98 PGC. (B) percentage of methylated and unmethylated PGC. n= 5 from 3 different exposures. * p<0.05 (Mann- Whitney’s test). 0 5 10 15 ETOH BADGE BPAF germ cells (%) homogeneous unstained homogeneous unstained TRA98 5hMC ME R GE A B * * ns * 454 455 456 457 458 459 460 461 462 463 464 465 466 467 468 homogeneous unstained TRA98 5hMC ME R GE A 454 455 456 457 458 459 460 461 462 463 464 465 466 467 468 B A Figure 7: BADGE or BPAF fetal exposures alters DNA methylation in proliferative PGC. Immunodetection of 5hmC (green) in 12.5 dpc TRA98 positive PGC (red) counterstained with DAPI (blue). (A) Representative photomicrograph of methylated (homogeneous nuclear staining) and unmethylated (unstained) TRA98 PGC. (B) percentage of methylated and unmethylated PGC. n= 5 from 3 different exposures. * p<0.05 (Mann- Whitney’s test). Figure 7: BADGE or BPAF fetal exposures alters DNA methylation in proliferative PGC. Immunodetection of 5hmC (green) in 12.5 dpc TRA98 positive PGC (red) counterstained with DAPI (blue). (A) Representative photomicrograph of methylated (homogeneous nuclear staining) and unmethylated (unstained) TRA98 PGC. (B) percentage of methylated and unmethylated PGC. n= 5 from 3 different exposures. * p<0.05 (Mann- Whitney’s test). 1 germ cells (%) 23 23 . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted August 20, 2020. ; https://doi.org/10.1101/2020.08.17.253724 doi: bioRxiv preprint . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted August 20, 2020. ; https://doi.org/10.1101/2020.08.17.253724 doi: bioRxiv preprint Discussion 469 Woman's reproductive pathologies include the ovarian dysgenesis syndrome that is defined as a precocious alteration of ovarian function and mostly associated with premature ovarian insufficiency, anovulation and/or pregnancy loss in the adulthood (Buck Louis et al., 2011). Oocyte aneuploidy is strongly associated with these reproductive failures and growing evidences suggest that fetal exposure to endocrine disruptors such as Bisphenol A (BPA) can cause female reproductive defect (Johansson et al., 2017). In Vertebrates and Invertebrates species, exposure to BPA induces transcription defect of meiotic genes, alters frequency of MLH1 foci and chiasmata leading to chromosome missegregation during meiotic resumption (Allard & Colaiácovo, 2010a; Brieno-Enriquez et al., 2012; Brieño-Enríquez et al., 2011; P. A. Hunt et al., 2012; Patricia A. Hunt et al., 2003; Lawson et al., 2011; Susiarjo et al., 2007). Because BPA has been restricted and regulated in many countries, BPA alternatives have been introduced into industrial products increasing the exposure to other bisphenols such as BPAF and BADGE. Exposure to BADGE and BPAF can be comparable to BPA exposure. A recent study has reported comparable median concentrations of BPA and BADGE hydrated derivatives in human plasma collected from New York City individuals (7.15 ng/ml for BADGE·2H20 and 2.26 ng/ml for BADGE·H20 versus 1.77 ng/ml for BPA) likely due to similar exposure source to BPA (ie. from epoxy coatings into canned food) (L. Wang et al., 2015). In China, BPAF mean concentration in human plasma is 0.073 ng/mL versus 0.4 ng/ml for BPA in the same samples from 81 individuals in general population (Jin et al., 2018). For this reason, we investigated the consequences of BPAF and BADGE exposure on female germ cell differentiation. Although BPAF concentration detected in human samples is lower than the one of BADGE or BPA, we chose to expose mice by drinking water to BADGE or BPAF at the same concentrations (ie 10µM). This allows to compare BPAF and BADGE and to compare our results to previous studies, including ours, that used BPA at environmentally doses (Eladak et al., 2018). In the present study, we exposed pregnant mice to bisphenols from 10.5 to 18.5 dpc. Discussion 469 In the present study, we exposed pregnant mice to bisphenols from 10.5 to 18.5 dpc. The concentration of total BPAF detected in the plasma of the pregnant mice was of the a same order of magnitude (5.96 ± 0.78 ng/ml) than the one we previously observed for BPA (Eladak et al., 470 471 472 473 474 475 476 477 478 479 480 481 482 483 484 485 486 487 488 489 490 491 492 493 494 495 24 . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted August 20, 2020. ; https://doi.org/10.1101/2020.08.17.253724 doi: bioRxiv preprint . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted August 20, 2020. ; https://doi.org/10.1101/2020.08.17.253724 doi: bioRxiv preprint As observed for BPA, fetal exposure to BPAF or BADGE induces subtle oocyte defects during fetal life leading to aneuploidy at adulthood. We observed changes in expression levels of meiotic genes during meiosis initiation, and a modification of the frequency of MLH1 foci during pachynema and of chiasmata in diplonema leading to oocyte aneuploidy after meiotic division. Thus, we demonstrate that BPAF or BADGE seem as much harmful as BPA with regard to their deleterious effect on oogenesis. 497 498 499 500 501 502 Until now the modality of the action of BPA on meiosis remains unclear. Numerous evidences highlight a temporal window of oocyte vulnerability during fetal life, specifically before and during meiotic prophase I (Brieno-Enriquez et al., 2012; P. A. Hunt et al., 2012; Lawson et al., 2011; Susiarjo et al., 2007). Because consequences are mostly detectable in later steps it was difficult to determine the precise cellular target (ie. PGC or oocyte) of BPA. This questionned whether the bisphenol-induced meiotic failure originated from alterations that occurred during the differentiation of the PGC or during meiotic prophase I. In this study, we showed that exposure to bisphenols before meiotic prophase I affected crossover frequency. Therefore, this observation demonstrates that alterations induced by BPAF and BADGE in PGC alters the establishment of the meiotic program. Discussion 469 503 504 505 506 507 508 509 510 511 512 Proper establishment and orchestration of the meiotic program are essential to ensure a correct regulation of crossover distribution [57]. The establishment of prophase I requires two events: acquisition of meiotic competence and initiation of the meiotic program (Soh et al., 2015). Meiotic competence is acquired after PGCs colonization. Migratory PGCs have a genomic program associated with stemness and express pluripotent genes. In the gonad, at 10.5 dpc, PGCs switch off the pluripotency program and activate genes involved in gametogenesis. DAZL, a germ cell- specific RNA binding protein, binds germ cell specific mRNA and promotes the stabilization of meiotic mRNA allowing the initiation of the meiotic program (Atala, 2012; Hu et al., 2015; Kato et al., 2016; Nicholls et al., 2019). Our transcriptomic analyses revealed that bisphenol exposure alters the acquisition of the gametogenic competence and the initiation of the meiotic program. First, bisphenol exposure seems to induce a failure or a delay on the switch from pluripotency to 513 514 515 516 517 518 519 520 521 522 523 25 . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted August 20, 2020. ; https://doi.org/10.1101/2020.08.17.253724 doi: bioRxiv preprint . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted August 20, 2020. ; https://doi.org/10.1101/2020.08.17.253724 doi: bioRxiv preprint gametogenesis/meiosis competence. This is illustrated by a global down-regulation of meiosis associated genes such as Sycp1&3 and Hormad2 and an up-regulation of pluripotency associated genes such as Msx1 and Gata4. Second, bisphenols exposure also impacts the initiation of the meiotic program at 13.5 dpc as illustrated by the up-regulation of meiotic genes. These observations are consistent with previous studies on the effect of BPA on human and murine meiosis initiation (Houmard et al., 2009; Lawson et al., 2011). However, it is unclear whether the up-regulation of meiotic genes induced by bisphenols is the consequence of the PGC differentiation delay observed earlier, or a real induction of gene expression and/or RNA stabilization. Discussion 469 Indeed, numerous meiotic genes such as Stra8 or Rec8 are drastically down- regulated just after meiosis onset. Therefore, a delay of meiosis initiation would lead us to observe a higher level of expression of some meiotic transcripts (Soh et al., 2015). Moreover, bisphenol exposure also induced a global alteration of splicing events during this step. Recent studies have highlighted the role of mRNA splicing on the mitosis to meiosis transition in male and female germ cells and lack of splicing regulators in male germ cells leads to meiotic defects (Liu et al., 2017; Naro et al., 2017; Schmid et al., 2013; J. Wang et al., 2019). In consequences, minor modifications of the splicing of meiotic genes after bisphenols exposure could impact meiosis. As observed for BPA exposure, changes in level of gene expression were subtle but, interestingly, we observed common transcriptional signature between BPAF and BADGE suggesting common bisphenol molecular targets. 524 525 526 527 528 529 530 531 532 533 534 535 536 537 538 539 540 541 542 Transcriptional modifications observed after bisphenol exposure impacted the transition from mitosis to meiosis and were certainly the origin of the observed delay of meiosis initiation and progression, the abnormal distribution of MLH1 foci but also the cause of folliculogenesis alterations (ie.MOF, follicle number). Indeed, the initiation of follicle assembly requires completion of meiotic prophase and any delay in meiotic progression could interfere with this process (Chao et al., 2012; A. Paredes et al., 2005; H.-Q. Zhang et al., 2012). Thus all these events may be related forming a cascade resulting from the early alteration of the establishment of the meiotic program in PGC. 543 544 545 546 547 548 549 550 26 . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted August 20, 2020. ; https://doi.org/10.1101/2020.08.17.253724 doi: bioRxiv preprint . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted August 20, 2020. Discussion 469 ; https://doi.org/10.1101/2020.08.17.253724 doi: bioRxiv preprint Interestingly, common cellular (ie MLH1 and chiasmata distribution and MOF) and transcriptional meiotic signatures observed after bisphenols exposure and other pollutants such as phthalates in multiple organisms suggest a common mechanism of action of these molecules (Allard & Colaiácovo, 2010b; Cuenca et al., 2020; Gely-Pernot et al., 2017; Parodi et al., 2015; Shin et al., 2019; Susiarjo et al., 2007; Tu et al., 2019). In this study, we propose oxidative stress as an new player involved in bisphenol response that could impair the PGC differentiation. One of the major targets of reactive oxygen species is the DNA, and 8OdG is the most prominent lesion in the genome (Ba & Boldogh, 2018). We showed that bisphenol exposure quickly induces the formation of this oxidative DNA lesion in proliferative germ cells. Interestingly, analyzing the genomic landscape of oxidative DNA damage revealed a specific susceptibility to oxidation in genomic regions harboring genes differentially expressed after bisphenol exposure. It is well known that oxidative DNA damages can modulate gene expression directly or undirectly. First, oxidative DNA damage induce antioxidant and inflammatory transcriptional responses and recruiting DNA repair proteins (Hörandl & Hadacek, 2013; Pan et al., 2016). Second, oxidized guanine biases the recognition of methylated CpG dinucleotides and alters the dynamic of DNA demethylation (Gruber et al., 2018; Pan et al., 2016). Interestingly, acquisition of the meiotic competence is completely dependent on an intensive germinal DNA demethylation (Hargan-Calvopina et al., 2016b; Yamaguchi et al., 2012). In this study, we observed that bisphenols exposure interferes with DNA demethylation and could be the consequence of the presence of oxidative DNA damages. The presence of 8OdG has also post-transcriptional consequences and could alter splicing events. Pre- mRNA splicing requires the identification of specific 5' donor and 3' acceptor sites. The 5' and 3' splicing sites mostly begin with dinucleotide GT(U) and end with dinucleotide AG that allows the major 5'_3' combination GT-AG (Calvello et al., 2013). Incorporation of the wrong base within these splicing signals would lead to an alteration of the fidelity of mRNA splicing. In mammalian cells, unrepaired DNA lesions such as 8OdG on the transcribed strand is a source of transcriptional mutagenesis. Unrepaired 8OdG leads to a single-nucleotide substitution in the 3' or 5' splice site subsequently resulting mostly in exon skipping and altering the protein product as it has been observed in the context of Ogg1 deficiency (J. A. Discussion 469 Paredes et al., 2017). For these reasons, we 551 552 553 554 555 556 557 558 559 560 561 562 563 564 565 566 567 568 569 570 571 572 573 574 575 576 577 578 Interestingly, common cellular (ie MLH1 and chiasmata distribution and MOF) and transcriptional meiotic signatures observed after bisphenols exposure and other pollutants such as phthalates in multiple organisms suggest a common mechanism of action of these molecules (Allard & Colaiácovo, 2010b; Cuenca et al., 2020; Gely-Pernot et al., 2017; Parodi et al., 2015; Shin et al., 2019; Susiarjo et al., 2007; Tu et al., 2019). In this study, we propose oxidative stress as an new player involved in bisphenol response that could impair the PGC differentiation. One of the major targets of reactive oxygen species is the DNA, and 8OdG is the most prominent lesion in the genome (Ba & Boldogh, 2018). We showed that bisphenol exposure quickly induces the formation of this oxidative DNA lesion in proliferative germ cells. Interestingly, analyzing the genomic landscape of oxidative DNA damage revealed a specific susceptibility to oxidation in genomic regions harboring genes differentially expressed after bisphenol exposure. It is well known that oxidative DNA damages can modulate gene expression directly or undirectly. First, oxidative DNA damage induce antioxidant and inflammatory transcriptional responses and recruiting DNA repair proteins (Hörandl & Hadacek, 2013; Pan et al., 2016). Second, oxidized guanine biases the recognition of methylated CpG dinucleotides and alters the dynamic of DNA demethylation (Gruber et al., 2018; Pan et al., 2016). Interestingly, acquisition of the meiotic competence is completely dependent on an intensive germinal DNA demethylation (Hargan-Calvopina et al., 2016b; Yamaguchi et al., 2012). In this study, we observed that bisphenols exposure interferes with DNA demethylation and could be the consequence of the presence of oxidative DNA damages. The presence of 8OdG has also post-transcriptional consequences and could alter splicing events. Pre- mRNA splicing requires the identification of specific 5' donor and 3' acceptor sites. The 5' and 3' splicing sites mostly begin with dinucleotide GT(U) and end with dinucleotide AG that allows the major 5'_3' combination GT-AG (Calvello et al., 2013). Incorporation of the wrong base within these splicing signals would lead to an alteration of the fidelity of mRNA splicing. In mammalian cells, unrepaired DNA lesions such as 8OdG on the transcribed strand is a source of transcriptional mutagenesis. Discussion 469 Unrepaired 8OdG leads to a single-nucleotide substitution in the 3' or 5' splice site 551 552 553 554 555 556 557 558 559 560 561 562 563 564 565 566 567 568 569 570 571 572 573 574 575 576 27 . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted August 20, 2020. ; https://doi.org/10.1101/2020.08.17.253724 doi: bioRxiv preprint . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted August 20, 2020. ; https://doi.org/10.1101/2020.08.17.253724 doi: bioRxiv preprint speculated that induction of oxidative stress by bisphenols could be responsible of observed transcriptional alteration as well as RNA splicing modification during meiosis initiation leading to prophase I defects. This is confirmed after specific induction of oxidative DNA damages in proliferative germ cell. Indeed, in KrBO3-treated ovaries and the timing of meiosis initiation is restored in presence of the antioxidant NAC. Moreover, in Ogg1 deficient mice, we also observed a delay of meiosis initiation and modification of MLH1 distribution in pachynema. 579 580 581 582 583 584 This study highlights the central role of oxidative DNA damage in the meiotic response after bisphenol exposure. Numerous past studies proposed a role for estrogen signaling in the meiotic responses (ie delay of meiotic progression, alteration of crossover distribution and aneuploidy) due to the effect of molecules with a xenoestrogenic potential (Cuenca et al., 2020; Susiarjo et al., 2007; Tu et al., 2019). Our hypothesis is in agreement with an involvement of estrogen signaling during this process as DNA binding by the estrogen receptor drives the local production of oxidative DNA damages via LSD1, a Lysine-specific histone demethylase 1, activity in promoter and enhancer regions (Perillo et al., 2008). 585 586 587 588 589 590 591 592 Current knowledge on the potential toxicological effects of BPA analogs is limited. We provide here proofs that endocrine disruptors such as bisphenols negatively impact the female germline causing oocyte defects with dramatic consequences such as aneuploïdy. We also reveal that bisphenols effects are mediated by DNA oxidation. Discussion 469 Numerous toxicological studies have linked prophase I alterations induced by pollutants, aneuploidy and folliculogenesis defect and, here, we demonstrated the central role of oxidative DNA damages in these ovarian reproductive failures (Cuenca et al., 2020; Gely-Pernot et al., 2017; P. A. Hunt et al., 2012; Susiarjo et al., 2013). This opens new research avenues considering DNA oxidation in the developing germline as the cause of adult reproductive defects. Such mechanism remains to be investigated in the human germline and could also be invoked for numerous pollutants, either considered or not as endocrine disruptors, whose reprotoxic potential has poorly been studied despite a strong oxidative potential. 593 594 595 596 597 598 599 600 601 602 603 28 . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted August 20, 2020. ; https://doi.org/10.1101/2020.08.17.253724 doi: bioRxiv preprint . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted August 20, 2020. ; https://doi.org/10.1101/2020.08.17.253724 doi: bioRxiv preprint Animals and gonads collection 605 All animal care protocols and experiments were reviewed and approved by the ethics committee of CETEA–CEA DSV (France, APAFIS 18515-2019011615312443v1) and followed the guidelines for the care and use of laboratory animals of the French Ministry of Agriculture. Mice were maintained in used polypropylene cage in standard and controlled photoperiod conditions (lights on from 8 a.m. to 8 p.m.) and had free access to tap water incubated in glass bottle and food. Males and females were caged together overnight (one male for 2 females), and the presence of vaginal plug was examined the following morning. The day following overnight mating is counted as 0.5 day post conception (dpc). All mice were killed by cervical dislocation at 11.5 dpc, 12.5 dpc, 14.5 dpc, 18.5 dpc and 8 dpp or at 3 months after birth. For pregnant mice, fetuses were removed after euthanasia from uterine horns before gonad isolation under a binocular microscope . The mice used in this study were NMRI mice (Naval Maritime Research Institute) and OGG1 deficient C57/Bl6 mice obtained from the production colony at our laboratory (Klungland et al., 2002). 606 607 608 609 610 611 612 613 614 615 616 617 BrdU incorporation and detection in fetal ovaries 635 For BrdU incorporation, 14.5 dpc ovaries were cultured in hanging drops with BrdU (1%). After three hours, ovaries were fixed in 4% paraformaldehyde and processed for histology. 636 637 Exposition protocol 618 The exposition protocol of this study is shown in Figure 2C and Supplementary Figure 2. BADGE (CAS number 1675-54-3, Sigma D3415) and BPAF (CAS number 1478-61-1, Sigma 257591) were dissolved in absolute ethanol (puriss, ≥99.8%). Final concentration of 10 µM (diluted in ethanol 0.1%) were provided in drinking water to isolated pregnant females from 10.5 dpc to 14.5 dpc for short-term exposure (Supplementary Figure 2) or to 18.5 dpc for long-term exposure (Figure 2C). The control group was given drinking water added with 0.1% ethanol. For transcriptomic analyses, pregnant mice were euthanasied at 11.5 dpc and 13.5 dpc to collect gonad for germ cell sorting. As one adult mouse drinks 150 ml per kg body weight and per day, the evaluated daily intake of BPAF and BADGE by treated mice is ~500 µg/kg/day. Internal BPAF concentration was evalueted. Total BPAF was measured by gas chromatography coupled to tandem mass spectrometry (GC-MS/MS) in the plasma of 18.5 dpc pregnant mice as previoulsy described (Eladak et al., 2018) The mean ± sem values in the plasma of BPAF group were 5.96 ± 0.78 ng/ml (n=4). As a comparison, he 619 620 621 622 623 624 625 626 627 628 629 630 29 . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted August 20, 2020. ; https://doi.org/10.1101/2020.08.17.253724 doi: bioRxiv preprint . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted August 20, 2020. ; https://doi.org/10.1101/2020.08.17.253724 doi: bioRxiv preprint ’European Food Safety Authority (EFSA) stated that the NOAELs for BADGE and BPAF is 15 and 30 mg/kg/d respectively https://www.anses.fr/fr/system/files/CHIM2009sa0331Ra-1.pdf. N- acetylcystein (NAC; Sigma A9165, 4 mM) and potassium bromate (KBrO3; Merck 1.04212.0250, 0.15 mM) were also provided in water drink from day 10.5 dpc to the end of experiment. 631 632 633 634 Histology and immunofluorescence on ovarian sections 638 Protocols used have been described previously (Abby et al., 2016, Poulain et al., 2014). Briefly, fetal and adult ovaries were fixed overnight in Bouin’s fluid. After being dehydrated and embedded in paraffin, gonads were cut into 5-μm-thick sections. Sections were then mounted on slides for haematoxilin and eosin coloration. Germ cells on each section were identified on the basis of their histological features, as previously described and oogonia were identified as small cells with high nucleocytoplasmic ratio and the presence of prominent nucleoli. Meiotic cells displayed markedly condensed chromatin, forming distinct fine threads with a beaded appearance at the leptotene stage, and a characteristic criss-cross of coiled chromosome threads at the zygotene stage, while oocytes reaching the diplotene stage (naked or enclosed in follicle) had an increased size with the reformation of a single nucleolus. The Histolab analysis software (Microvision Instruments, Evry, France) was used for counting. Immunostaining were performed on sections from fetal gonads (12.5 and 14.5 dpc) fixed overnight in 4% paraformaldehyde (PFA). Sections were submitted to antigen retrieval with citrate buffer (pH 6) and then blocked in Normal Horse Serum (Impress HRP Reagent Kit MP-7402) or 2% gelatin, 0.05% tween, 0.2% BSA for one hour before adding antibodies. Primary antibodies used in this study were as follows: monoclonal mouse anti-5mC (abcam ab10805, 1:100), monoclonal rabbit anti-Stra8 (abcam ab49602; 1:1000), monoclonal rat anti-TRA98 (abcam ab82527, 1:500), and monoclonal mouse anti-SYCP3 (abcam ab97672, 1:500) antibodies were used. Specific donkey secondary antibodies conjugated with either Alexa Fluor 639 640 641 642 643 644 645 646 647 648 649 650 651 652 653 654 655 656 30 . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted August 20, 2020. ; https://doi.org/10.1101/2020.08.17.253724 doi: bioRxiv preprint . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted August 20, 2020. ; https://doi.org/10.1101/2020.08.17.253724 doi: bioRxiv preprint 488 or 594 (1:500). BrdU detection in histological sections was performed with the Cell Proliferation kit (RPN20, GE Healthcare). Slides were mounted with Vectashield medium. Histology and immunofluorescence on ovarian sections 638 Images acquisition was accomplished with a Leica DM5500 B epifluorescence microscope (Leica Microsystems) equipped with a CoolSNAP HQ2camera (Photometrics) and ImageJ Software. Images were analyzed with the Image J software. 657 658 659 660 661 Immunofluorescence on chromosome spreads 662 CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted August 20, 2020. ; https://doi.org/10.1101/2020.08.17.253724 doi: bioRxiv preprint then dried and stored at −20 °C. Slides were washed three times in PBS and then incubated in blocking solution (PBS with 2% gelatin, 0.05% tween, 0.2% BSA) before adding monoclonal human anti-CREST (1:1000) antibody overnight. After 3 washes, goat anti-human secondary antibodies conjugated with Alexa Fluor 488 (1:500) were added. Slides were mounted with Vectashield with or without DAPI medium. Images were processed and specific structures were quantified with the ImageJ software (Cell Counter plugin). 684 685 686 687 688 689 Germ cell isolation 690 11.5 dpc and 13.5 dpc germ cell isolation using SSEA-1 antigen was performed as previously described (Guerquin et al, 2015). Briefly, fetal ovaries were dissociated using trypsin solution. Cells are incubated with anti-SSEA (1/5, anti SSEA1 monoclonal antibody DSHB) and then incubated with a microbead-linked donkey anti-mouse IgM antibody (Miltenyi Biotec). Then, cells were applied onto an MS+ column (Miltenyi Biotec) and the positive fraction was flushed after the removal of the magnet. 691 692 693 694 695 696 Immunofluorescence on chromosome spreads 662 Chromosome spreads were prepared using fetal gonads (12.5 and 18.5 dpc). Fetal ovaries were lacerated on precleaned/ready-to-use superfrost slides in 1X PBS, then they were supplemented with 0.2% sucrose before adding 1% paraformaldehyde/1% Triton. Slides were incubated for 1 h at room temperature in a humid chamber and then dried under a hood and then washed two times for 10 min with 0.4% H2O/Photoflow (Sigma-Aldrich). Slides were stained immediately or dried and stored at −20 °C. Spreaded 12.5 dpc oogonia were rehydrated in PBS, denaturated with 2N HCl for 45 minutes and then neutralized with a 50 mM Tris-HCl (pH8.8) solution. Slides were washed three times in PBS-0.1% Triton and then incubated in blocking solution (PBS with 0.1% triton and 5% donkey serum) before adding antibodies. Monoclonal mouse anti-8-OHdG (Eurobio MOG-020P, 1:200) and monoclonal rat anti-TRA98 antibodies were diluted in blocking solution and incubated overnight. Spreaded 18.5 dpc oocytes were blocked in blocking solution before adding antibodies. Monoclonal mouse anti-MLH1 (BD Bio sciences, G16815, 1:50) and monoclonal rabbit anti-SYCP3 (Novus, NB300232, 1:500) antibodies were diluted in blocking solution and incubated overnight. After 3 washes, donkey secondary antibodies conjugated with either Alexa Fluor 488 or 594 (1:500) were added. Metaphase I and II oocytes were obtained with adult ovaries (3 months old females). Ovaries were lacerated in M2 medium (Sigma M7167) and oocytes at germinal vesicle stage were collected and cultured for five hours (for metaphase I) or sixteen hours (for metaphase II) in M16 medium, covered with mineral oil. Oocytes at Germinal Vesicle Breakdown (GVBD) stage (for metaphase I) or with the first polar globule expulsion (for metaphase II) were transferred into a drop of Tyrode solution for zona pellucida removal. They were spread on Teflon printed diagnostic slides in water with 3mM DTT, 0.15% Triton and 0.6% paraformaldehyde. Slides were 663 664 665 666 667 668 669 670 671 672 673 674 675 676 677 678 679 680 681 682 683 31 . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted August 20, 2020. ; https://doi.org/10.1101/2020.08.17.253724 doi: bioRxiv preprint . Microarray analysis 707 For gene expression analyses (11.5 dpc or 13.5 dpc), R oligo package 1.42.0 (Carvalho & Irizarry, 2010) was used for probeset annotation with expression summarized at the transcript level and robust multi-array averaging-normalized. Differential expression testing was conducted by ANOVA after linear model fitting of expression intensities with the limma R package. Microarray expression values are represented as log(2) normalized intensities. Differentially expressed genes (DEG) was filtered with a logFC ≥ 0.5 and a pvalue < 0.05. For 13.5 dpc RNA splicing analyses, the expression was summarized at the exon level. Samples were analyzed using default parameters. An alternative splicing event was defined as a differentially expressed exon with at least a 1 Log fold change in expression, at a pvalue of < 0.05.Downstream analyses were performed with R version 3.5.0 on a CentOS Linux 7 system (64-bit).Enrichment analyses were performed using ClusterProfiler, a R package for comparing biological themes among gene clusters (G. Yu et al., 2012). 708 709 710 711 712 713 714 715 716 717 718 719 Splicing variant detection by quantitative PCR 720 Total RNA from isolated germ cells was extracted using the RNeasy minikit (QIAGEN, Valencia, CA, USA). cDNA was obtained by reverse transcription using the high capacity kit (Applied Biosystems, Foster City, CA, USA) according to the manufacturer’s instructions. Set of primers specific to the truncated/spliced region and reference primers located to the core/unspliced of the transcript were used to quantify the region of splicing using 2 delta delta CT methods. 721 722 723 724 725 Affymetrix sample preparation 697 After isolation, cells were centrifuged and resuspended in RNeasy lysis buffer. RNA was extracted using Qiagen Rneasy miniKit as recommended by the manufacturer. Total RNA concentration and RNA integrity was monitored by electrophoresis (Agilent Bioanalyzer; RNA 6000 Pico Assay). Three pools of cells from 20-30 fetus (5 independent exposures) were used for differential expression analyses. Gene expression analysis was conducted using Mouse Clariom S array (Thermo Fisher) at 11.5 dpc and a Mouse Clariom D array at 13.5 dpc (Thermo Fisher). 500 µg of total RNA were processed according to the manufacturer. Raw data were generated and controlled with Expression console (Affymetrix) at the Genomic’s platform (Institut Cochin, Paris) for 11.5 dpc analyses and at the CEA for 13.5 dpc analyses. 698 699 700 701 702 703 704 705 706 32 32 . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted August 20, 2020. ; https://doi.org/10.1101/2020.08.17.253724 doi: bioRxiv preprint . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted August 20, 2020. ; https://doi.org/10.1101/2020.08.17.253724 doi: bioRxiv preprint Statistical analysis 726 All data are presented as means ± s.e.m. Statistical analyses were performed using Graphpad software and the R version 3.5.0. All individual biological replicates were randomly sampled from 3 independent exposures (for bisphenols exposure). The statistical significance in the difference between control and bisphenols-treated data were evaluated using the non-parametric test Mann- Whitney. Statistical significance was set as p<0.05. 727 728 729 730 731 33 . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted August 20, 2020. ; https://doi.org/10.1101/2020.08.17.253724 doi: bioRxiv preprint . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted August 20, 2020. ; https://doi.org/10.1101/2020.08.17.253724 doi: bioRxiv preprint Acknowledgments 736 We are grateful to Katja Wassmann, Damien Cladière and Eulalie Buffin for discussion and suggestions and technical help for metaphase I and II preparation. We also thank A. Gouret and A. Leliard for her skillful secretarial assistance. We also thank the team within the animal housing facility at the iRCM. For affymetrix data, we thank Sébastien Jacques and Angeline Duché from Genom'ic (institut Cochin). For BPAF concentration in serum samples, we thank jean-Philippe Antignac from LABERCA (Oniris, INRAE, Nantes). This research was supported by the ADEME (French Environment & Energy Management Agency), ANSES (French Agency for Food, Environmental and Occupational Health & Safety) Université de Paris and INSERM. S.A. were supported by a fellowship from the Ministère de l’Enseignement et Recherche. 737 738 739 740 741 742 743 744 745 Data and Code Availability 732 Datasets are available at the European Bioinformatics Institute under accession n° (E-MTAB- 9344). The authors declare that the data supporting the findings of this study and custom code generated for the manuscript are available from the Lead Contact upon request. 733 734 735 34 . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted August 20, 2020. ; https://doi.org/10.1101/2020.08.17.253724 doi: bioRxiv preprint . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted August 20, 2020. ; https://doi.org/10.1101/2020.08.17.253724 doi: bioRxiv preprint Author Contributions 746 Conceptualization: M.J.G, G.L., V.R.F, A.C, J.P.R, E.M and R.H ; Methodology: S.A., M.J.G, A.C, J.P.R, E.M; Validation: S.A., D.M., M.W and S.M.; Formal Analysis: M.J.G and S.A. Investigation: S.A., D.M., M.W and S.M; Resources: A.C. and J.P.R; Data Curation: M.J.G.; Writing –Original Draft: S.A. and M.J.G.; Writing –Review & Editing: S.A., G.L., V.R.F, A.C, J.P.R, E.M and R.H. Visualization: S.A. and M.J.G. Supervision: M.J.G, V.R.F and G.L. Project Administration: M.J.G, V.R.F and G.L. Funding Acquisition: M.J.G, V.R.F and G.L. 747 748 749 750 751 752 Declaration of Interests 753 The authors declare no competing interests 754 References 755 Allard, P., & Colaiácovo, M. P. (2010a). Bisphenol A impairs the double-strand break repair machinery in the germline and causes chromosome abnormalities. 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It is The copyright holder for this preprint this version posted August 20, 2020. ; https://doi.org/10.1101/2020.08.17.253724 doi: bioRxiv preprint Wang, J., Tian, G. References 755 Di-(2-ethylhexyl) phthalate and bisphenol A exposure impairs mouse primordial follicle assembly in vitro : DEHP and BPA Impairs Mouse Primordial Follicle Assembly. Environmental and Molecular Mutagenesis, 55(4), 343-353. https://doi.org/10.1002/em.21847 Zhang, T., Zhou, Y., Li, L., Zhao, Y., De Felici, M., Reiter, R. J., & Shen, W. (2018). Melatonin protects prepuberal testis from deleterious effects of bisphenol A or diethylhexyl phthalate by preserving H3K9 methylation. Journal of Pineal Research, 65(2), e12497. References 755 https://doi.org/10.1111/jpi.12497 40 ETOH BADGE BPAF ETOH BADGE BPAF primordial primary secondary antral * * C multi-oocyte follicle 0.0 multi-oocyte follicle (%) ETOH BADGE BPAF 1.0 2.0 3.0 4.0 5.0 ETOH BADGE BPAF * * * * 8 dpp 3 months 3 months A H BADGE BPAF multi-oocyte follicle BADGE ETOH BPAF C 0.0 multi-oocyte follicle (%) ETOH BADGE BPAF 1.0 2.0 3.0 4.0 5.0 ETOH BADGE BPAF * * * * 8 dpp 3 months B ETOH BADGE BPAF primordial primary secondary antral 0 2000 4000 6000 8000 total follicle count per ovary * * C 0.0 multi-oocyte follicle (%) ETOH BADGE BPAF 1.0 2.0 3.0 4.0 5.0 ETOH BADGE BPAF * * * * 8 dpp 3 months 3 months B ETOH BADGE BPAF primordial primary secondary antral 0 2000 4000 6000 8000 total follicle count per ovary * * 3 th B 8 3 3 months A euploid aneuploid aneuploid oocytes (%) 0 20 40 60 * * ETOH BADGE BPAF adult oocytes B metaphase I 0 10 20 30 40 50 > 3 chiasmata / bivalent (%) ETOH BADGE BPAF **** bivalents adult oocytes C long term exposure 18.5 (dpc) 14.5 10.5 meiotic prophase I *** CREST / DAPI CREST / DAPI A A euploid aneuploid aneuploid oocytes (%) 0 20 40 60 * * ETOH BADGE BPAF adult oocytes CREST / DAPI aneuplo aneup 0 ETOH BADGE BPAF adult oocytes B metaphase I 0 10 20 30 40 50 > 3 chiasmata / bivalent (%) ETOH BADGE BPAF **** bivalents adult oocytes C 0 ETOH 5 10 15 20 > 3 MLH1 foci / bivalent (%) ETOH BADGE BPAF ETOH BADGE 18.5 dpc oocytes long term exposure 18.5 (dpc) 14.5 10.5 meiotic prophase I *** *** ** CREST / DAPI MLH1 / SYCP3 B metaphase I 0 10 20 30 40 50 > 3 chiasmata / bivalent (%) ETOH BADGE BPAF **** bivalents adult oocytes *** CREST / DAPI B adult oocytes C 0 ETOH 5 10 15 20 > 3 MLH1 foci / bivalent (%) ETOH BADGE BPAF ETOH BADGE 18.5 dpc oocytes long term exposure 18.5 (dpc) 14.5 10.5 meiotic prophase I *** ** MLH1 / SYCP3 C 18 5 d t MLH1 / SYCP3 0 ETOH 5 10 15 20 > 3 MLH1 foci / bivalent (%) ETOH BADGE BPAF *** ** ETOH 18.5 dpc oocytes A ETOH BADGE BPAF 0 20 40 60 80 100 germ cells (%) early pachynema late pachynema diplonema * * * A B C 0 20 40 germ ce early pachynema late pachynema diplonema BRDU TRA98 SYCP3 MERGE oogonia pre PL post PL 0 5 10 15 100 95 90 85 0 BrdU+/ SYCP3+ (%) ETOH BADG BPAF BrdU-/ SYCP3+ (%) preleptotene post-preleptotene * ** * * * B BRDU TRA98 SYCP3 MERGE oogonia pre PL post PL B C C 0 5 10 15 100 95 90 85 0 BrdU+/ SYCP3+ (%) ETOH BADGE BPAF BrdU-/ SYCP3+ (%) preleptotene post-preleptotene * ** * * A BADGE BPAF BADGE BPAF 1817 733 886 1376 11.5dpc 13.5dpc DEG(LFC>0.5, pvalue <0.05) 802 (44.2%) 243 (34.6%) 640 (72.2%) 846 (61.4%) upregulated downregulated 1015 (55.8 %) 490 (65.4%) 247 (27.8%) 528 (38.6%) A DEG(LFC>0.5, pvalue <0.05) upregulated downregulated B p.adjust (Log) 11.5 dpc 13.5 dpc 0 25 50 75 100 transmembrane receptor protein serine/threonine kinase signaling pathway Notch signaling pathway negative regulation of Wnt signaling pathway retinoic acid receptor signaling pathway stem cell differentiation promoter DNA−binding tactivator activity, RNA polymerase II−specific DNA recombination meiotic cell cycle meiotic nuclear division synapsis BADGE BPAF 0 25 50 75 100 B Meiosis Stem Cell Differentiation ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● BADGE (Log2FC) BPAF (Log2FC) Gpm6a Msx1 Snai2 Jag1 Sox9 Lin28a Nrp1 Erbb4 Shh Hnf1b Pdgfra Runx2 Bmp4 Hes5 Alx1 Mir99a Mir130a Setd6 Lbh Rbm24 Fgf15 Hnrnpu Wnt7b Folr1 Pef1 Pax6 Cfl1 Frzb Gbx2 Gata4 Aldh1a2 Efnb1 Foxc2 Foxa1 Foxc1 Sox21 Kitl Sema6d Hoxd4 Fzd1 Htr2b Hoxa7 Cdk6 Sox18 Sema5b Pax2 Ptn Sema6a Gsk3b r=0.31 pvalue=0.005648 −1.0 −0.5 0.0 0.5 1.0 −1.0 −0.5 0.0 0.5 1.0 ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● BPAF (Log2FC) Sfrp1 Tgfb2 Ednra Tcf7l1 Myocd Gpm6a Nrp2 Snai2 Jag1 Gdnf Epcam Sox9Sema3d Nrp1 Hnf1b Pdgfra Anxa6 Bmp4 Rest Hes1 Sox2 Sox11 Twist1 Tacstd2 Lbh Nanog Wnt7a Folr1 Frzb Gata4 Aldh1a2 Ednrb Prickle1 Kitl Sema3a Gata6 Pax2 Cited2 Ptn Sema6a BADGE (Log2FC) r=0.45 pvalue=5.455E-05 −1.0 −0.5 0.0 0.5 1.0 −1.0 −0.5 0.0 0.5 1.0 ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● BADGE (Log2FC) BPAF (Log2FC) Zfp318 Sycp2 Mybl1 Stag3 Brca2 Tex15Sycp1 Sycp3 Xlr Ccdc36 Prdm9 Hormad2 Dmc1 Rnf212 Ythdc2 Spo11 Mei1 Hormad1 Syce1 Gm1140 Mcmdc2 Slc26a8 Dmrtc2 Spdya Spata22 Meiob Topaz1 Boll Tex11 Ccne2 Xlr5b Syce3 r=0.87 pvalue<2.2E-16 −1.0 −0.5 0.0 0.5 1.0 −1.0 −0.5 0.0 0.5 1.0 BADGE (Log2FC) BPAF (Log2FC) ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● Smc1b Sycp3 Hormad2Mei4 Cpeb1 Brca2 Stag3 Mael Hormad1 Syce1 Tex15 Hfm1 Tex12 Fanca Mybl1 Stra8 Topaz1 Tex11 Sycp1 1700013H16Rik Xlr4a M1ap Lfng Fmn2 Cdc25b r=0.37 pvalue= 1.408E-09 −1.0 −0.5 0.0 0.5 1.0 −1.0 −0.5 0.0 0.5 1.0 C 11.5 dpc 13.5 dpc C Meiosis ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● BADGE (Log2FC) BPAF (Log2FC) Zfp318 Sycp2 Mybl1 Stag3 Brca2 Tex15Sycp1 Sycp3 Xlr Ccdc36 Prdm9 Hormad2 Dmc1 Rnf212 Ythdc2 Spo11 Mei1 Hormad1 Syce1 Gm1140 Mcmdc2 Slc26a8 Dmrtc2 Spdya Spata22 Meiob Topaz1 Boll Tex11 Ccne2 Xlr5b Syce3 r=0.87 pvalue<2.2E-16 −1.0 −0.5 0.0 0.5 1.0 −1.0 −0.5 0.0 0.5 1.0 BADGE (Log2FC) BPAF (Log2FC) ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● Smc1b Sycp3 Hormad2Mei4 Cpeb1 Brca2 Stag3 Mael Hormad1 Syce1 Tex15 Hfm1 Tex12 Fanca Mybl1 Stra8 Topaz1 Tex11 Sycp1 1700013H16Rik Xlr4a M1ap Lfng Fmn2 Cdc25b r=0.37 pvalue= 1.408E-09 −1.0 −0.5 0.0 0.5 1.0 −1.0 −0.5 0.0 0.5 1.0 C 11.5 dpc 13.5 dpc BPAF (Log2FC) ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● BADGE BPAF homologous recombination chromosome organization involved in meiotis meiotic chromosome segregation meiosis I meiosis I cell cycle process meiotic nuclear division meiotic cell cycle process DNA recombination nuclear chromosome segregation meiotic cell cycle chromosome segregation regulation of chromosome organization DNA repair 0.000 0.025 0.050 0.075 0.100 p.adjust GeneRatio ● ● ● 0.01 0.02 0.03 Alternative 3' Acceptor Site Alternative 5' Donor Site Cassette Exon Intron Retention Unclassified event BADGE (n=5085) BPAF (n=3879) splicing events all transcripts meiotic transcripts Gene ratio A B reciprocal DNA recombination C Smc4 splicing site primers reference primers 1 reference primers 2 Smc4-201 ification 0.8 1.2 * * * ETOH Smc4-202 Sycp2-201 Sycp2-202 reference primers 1 reference primers 2 Sycp2 alternative 3' acceptor site splicing site primers ification 1.0 1.5 * * ETOH alternative 3' acceptor site 5' UTR 1 2 3 4 5 2 3 4 5 1 5' UTR 18 17 16 15 14 40 39 37 36 38 5' UTR D ratio Smc4 201/ total Smc4 ratio Sycp2-202/ total Sycp2 Alternative 3' Acceptor Site Alternative 5' Donor Site Cassette Exon Intron Retention Unclassified event BADGE (n=5085) BPAF (n=3879) splicing events all transcripts meiotic transcripts B B A ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● BADGE BPAF homologous recombination hromosome organization involved in meiotis meiotic chromosome segregation meiosis I meiosis I cell cycle process meiotic nuclear division meiotic cell cycle process DNA recombination nuclear chromosome segregation meiotic cell cycle chromosome segregation regulation of chromosome organization DNA repair 0.000 0.025 0.050 0.075 0.100 p.adjust GeneRatio ● ● ● 0.01 0.02 0.03 A A C I BADGE (n=5085) BPAF (n=3879) splicing events all tr Gene ratio B reciprocal DNA recombination Unclassified event C Smc4 splicing site primers reference primers 1 reference primers 2 Smc4-201 relative quantification 0.0 0.4 0.8 1.2 BADGE BPAF * relative to: primers 1 primers 2 BADGE BPAF * * ETOH Smc4-202 Sycp2-201 Sycp2-202 reference primers 1 reference primers 2 Sycp2 alternative 3' acceptor site splicing site primers relative quantification 0.0 0.5 1.0 1.5 BADGE BPAF * relative to: primers 1 primers 2 BADGE BPAF * ETOH alternative 3' acceptor site 5' UTR 1 2 3 4 5 2 3 4 5 1 5' UTR 18 17 16 15 14 40 39 37 36 38 5' UTR D ratio Smc4 201/ total Smc4 ratio Sycp2-202/ total Sycp2 C Smc4 splicing site primers reference primers 1 reference primers 2 Smc4-201 Smc4-202 Sycp2-201 Sycp2-202 reference primers 1 reference primers 2 Sycp2 alternative 3' acceptor site splicing site primers alternative 3' acceptor site 5' UTR 1 2 3 4 5 2 3 4 5 1 5' UTR 18 17 16 15 14 40 39 37 36 38 5' UTR D Sycp2-201 Sycp2-202 reference primers 1 reference primers 2 Sycp2 alternative 3' acceptor site splicing site primers 18 17 16 15 14 40 39 37 36 38 D D C Smc4 splicing site primers reference primers 1 reference primers 2 Smc4-201 Smc4-202 alternative 3' acceptor site 5' UTR 1 2 3 4 5 2 3 4 5 1 5' UTR 5' UTR C alternative 3' acceptor site relative quantification 0.0 0.4 0.8 1.2 BADGE BPAF * relative to: primers 1 primers 2 BADGE BPAF * * ETOH relative quantification 0.0 0.5 1.0 1.5 BADGE BPAF * relative to: primers 1 primers 2 BADGE BPAF * ETOH ratio Smc4 201/ total Smc4 ratio Sycp2-202/ total Sycp2 relative quantification 0.0 0.4 0.8 1.2 BADGE BPAF * relative to: primers 1 primers 2 BADGE BPAF * * ETOH relative quantification 0.0 0.5 1.0 1.5 BADGE BPAF * relative to: primers 1 primers 2 BADGE BPAF * ETOH ratio Smc4 201/ total Smc4 ratio Sycp2-202/ total Sycp2 ratio Smc4 201/ total Smc4 * ratio Sycp2-202/ total Sycp2 relative quantification 0.0 0.4 0.8 1.2 BADGE BPAF * relative to: primers 1 primers 2 BADGE BPAF * * ETOH ratio Smc4 201/ total Smc4 relative quantification 0.0 0.5 1.0 1.5 BADGE BPAF * relative to: primers 1 primers 2 BADGE BPAF * ETOH ratio Sycp2-202/ total Sycp2 primers 2 BADGE BPAF A B 8OdG DAPI MERGE ETOH KRBO3 BADGE BPAF ETOH KBrO3 BADGE BPAF 0 20 40 60 80 100 8OdG area/cell area (%) C BADGE BPAF BADGE BPAF KBrO3 ETOH 100 95 90 85 0 +NAC SYCP3 (%) ** ** ** D OGG1+/+ OGG1-/- 0 SYCP3 (%) 40 60 50 E *** ** ** 55 45 35 A B 8OdG DAPI MERGE ETOH KRBO3 BADGE BPAF ETOH KBrO3 BADGE BPAF 0 20 40 60 80 100 8OdG area/cell area (%) *** ** ** B A DAP MERGE ETOH KBrO3 BAD 0 20 40 8OdG area/c C BADGE BPAF BADGE BPAF KBrO3 ETOH 100 95 90 85 0 +NAC SYCP3 (%) ** ** ** D OGG1+/+ OGG1-/- 0 SYCP3 (%) 40 60 50 E 0 20 40 60 80 100 OGG1+/+ OGG1-/- > 3 MLH1 foci / synapsis (%) *** 55 45 35 ETOH KBrO3 BADGE BPAF 0 C BADGE BPAF BADGE BPAF KBrO3 ETOH 100 95 90 85 0 +NAC SYCP3 (%) ** ** ** D OGG1+/+ OGG1-/- 0 SYCP3 (%) 40 60 50 E 55 45 35 ETOH KBrO3 BADGE BPAF C D +NAC E 0 20 40 60 80 100 OGG1+/+ OGG1-/- > 3 MLH1 foci / synapsis (%) *** E 0 20 40 60 80 100 OGG1+/+ OGG1-/- > 3 MLH1 foci / synapsis (%) *** 0 5 10 15 ETOH BADGE BPAF germ cells (%) homogeneous unstained omogeneous unstained B * * ns * 0 5 10 15 germ cells (%) homogeneous unstained TRA98 5hMC MERGE A B B A homogeneous unstained MERGE 0 ETOH BADGE BPAF 0 20 40 60 STRA8 positive (%) B ns ns A ETOHBADGE BPAF SYCP3 negative (%) ns * ETOH BADGE BPAF 0 20 40 60 STRA8 positive (%) B ns ns B ETOHBADGE BPAF short term exposure 18.5 (dpc) 14.5 10.5 meiotic prophase I 10 20 30 40 50 3 MLH1 foci / bivalent (%) MLH1 / SYCP3 ETOH BADGE ** * short term exposure 18.5 (dpc) 14.5 10.5 meiotic prophase I 0 10 20 30 40 50 > 3 MLH1 foci / bivalent (%) MLH1 / SYCP3 ETOH BADGE 18.5 dpc oocytes E 0 10 20 30 40 50 > 3 MLH1 foci / bivalent (%) MLH1 / SYCP3 ETOH BADGE 18.5 dpc oocytes E 11.5 dpc 13.5 dpc cell−cell junction organization cell−cell signaling by wnt cell−matrix adhesion chromosome organization involved in meiotic cell cycle chromosome segregation cofactor metabolic process collagen−containing extracellular matrix condensed chromosome condensed nuclear chromosome developmental cell growth developmental growth involved in morphogenesis DNA recombination drug catabolic process embryonic appendage morphogenesis embryonic organ development embryonic organ morphogenesis endocrine system development extracellular matrix female gamete generation gland development gland morphogenesis homologous chromosome segregation hormone metabolic process hormone secretion male meiotic nuclear division meiosis I meiosis I cell cycle process meiotic cell cycle meiotic cell cycle process meiotic chromosome segregation meiotic nuclear division mesenchymal cell development mesenchymal cell differentiation mesenchyme development mesonephric epithelium development mesonephric tubule development mesonephros development negative regulation of canonical Wnt signaling pathway negative regulation of cell development negative regulation of response to wounding negative regulation of Wnt signaling pathway negative regulation of wound healing Notch signaling pathway nuclear chromosome segregation nuclear division oogenesis organelle fission organic acid binding organic hydroxy compound metabolic process oxidoreductase activity, acting on CH−OH group of donors oxidoreductase activity, acting on peroxide as acceptor oxidoreductase activity, acting on the CH−OH group of donors, NAD or NADP as acceptor oxygen binding oxygen carrier activity positive regulation of epithelial cell proliferation positive regulation of ERK1 and ERK2 cascade positive regulation of reproductive process proximal promoter DNA−binding transcription activator activity, RNA polymerase II−specific regulation of developmental growth regulation of epithelial to mesenchymal transition regulation of stem cell differentiation regulation of transforming growth factor beta receptor signaling pathway regulation of transmembrane receptor protein serine/threonine kinase signaling pathway regulation of Wnt signaling pathway regulation of wound healing renal system process renal system vasculature development retinoic acid receptor signaling pathway sex differentiation SMAD protein signal transduction small molecule catabolic process smooth muscle cell proliferation stem cell development stem cell differentiation steroid biosynthetic process steroid metabolic process synapsis synaptonemal complex synaptonemal complex assembly synaptonemal complex organization synaptonemal structure transforming growth factor beta receptor signaling pathway transmembrane receptor protein kinase activity transmembrane receptor protein serine/threonine kinase signaling pathway Wnt signaling pathway wound healing BADGE Meiob Snx33 Trip6 Gm23844 Gm16103 Ccdc84 Mir5128 Gm26501 Tubb2b Gm26127 Gm22112 Gm22798 Gm25829 Gm24584 Gm23355 Cldn8 Gm25817 Hddc3 Gm23606 1700086D15Rik Gm21742 Gm25256 Cpeb4 Tc2n Klf4 Gm25966 Gm24693 Tst Mir3074−1 Gm22468 Gm10462 Gnrh1 Gm26239 Gm14654 4930518I15Rik Uck2 Gm16091 Gm25337 Gm26738 Gm26054 D130012P04Rik Gm24339 Gm22067 Pol Ahsp Tspan7 AC132444.6 Anxa8 Gm25670 Gm24870 Gm21833 Pten.1 Mettl24 Gm24630 Ybx2 Gm23815 Gm22358 Murc Gm22502 Mir3074−1.1 Erbb2 Gm22502.1 Gm11639 Gm14734 Gm24713 Pgm5 Trpc1 E230008O15Rik Gm15246 Krt8 Gm25632 2410089E03Rik Gm3173 Gm26501.1 Rbm25.1 Sox9 Ybx2.1 Pank1 Gm24427 Gm24492 Gm24392 Cited2 Gm22997 Tex18 Gm25484 4921507P07Rik Tgfbi Rgs2 Gm3317 Gm11578 Gm25021 LOC432823 Tc2n.1 Gm26331 Acsl5 Gm3317.1 Ccdc41 Gm23115 BC030307 Gm23095 Alms1 Odf2l Lyst Col3a1.1 Brca2 Msh4 Sycp1 Mei1 Spo11 Dmc1 Rad21l Sycp2 Msh5 Prdm9 Spata22 Hmga2 Rev3l Helq.1 Uimc1.1 Uvssa Parp6 Stxbp5 Plxnb2.1 Sparcl1 Cdh1 Mycbp2.1 Snrnp48 Pkd1 Hk2 Mmp14 Ace2 Kmt2a Ash1l Aldob 4932438A13Rik Slc26a8 Ccnb3 Zkscan3 Herc1.1 Micu3 Csrp2 Slc4a7.1 Grk4 Aldh1a2.1 Stk38 Kcnq1ot1 Dmxl1 Sfi1 Mir296 Nktr Rel Strip2 Gldn Cmtm7 Nicn1 Eml5 Catsperg1 Catsperg2 Dennd4a.1 Syt14 Capns1 Lyst Chuk Col3a1 Gfra2 Pdgfra Nrp2 Crmp1 Plxnb2 Mycbp2 Mapk8ip3 Stxbp5 Stard9 AI481877 Dmc1 4930447C04Rik Paxbp1 Ccnb3 Mybl1 Sycp2 Uimc1Helq Aldh1a2 Slc4a7 Kmt2c Anxa5 Cdh1 Cdh17 Stk38 Ash1l Ace2 Herc1Zkscan3 Gldn Krt19 Susd2 Nktr Enpp3 Golgb1 Sfxn1 Taf7l Grk4 Nlrp9b Rmdn2 Eml5 Mga Pcsk7 Lgals4 Catsperg1 Dennd4a NON-DIFFERENTIALLY SPLICED GENES DIFFERENTIALLY SPLICED GENES IN BPAF CONDITION −1.0 −0.5 0.0 0.5 1.0 −1.0 −0.5 0.0 0.5 1.0 −1.0 −0.5 0.0 0.5 1.0 co DIFFERENTIALLY SPLICED GENES IN BADGE CONDITION BADGE (Log2FC) genes (random sampling) 3543 genes 2678 genes DEGs 5.1 % DEGs 4.9 % DEGs Meiob nx33 Trip6 Gm23844 Gm16103 Ccdc84 Mir5128 Gm26501 Tubb2b Gm26127 Gm22112 Gm22798 Gm25829 Gm24584 Gm23355 Cldn8 Gm25817 Hddc3 Gm23606 1700086D15Rik Gm21742 Gm25256 Cpeb4 Tc2n Klf4 Gm25966 Gm24693 Tst Mir3074−1 Gm22468 Gm10462 Gnrh1 Gm26239 Gm14654 4930518I15Rik Uck2 Gm16091 Gm25337 Gm26738 Gm26054 D130012P04Rik Gm24339 Gm22067 Pol Ahsp Tspan7 AC132444.6 8 Gm25670 Gm24870 Gm21833 Pten.1 Mettl24 Gm24630 Ybx2 Gm23815 Gm22358 Murc Gm22502 Mir3074−1.1 Erbb2 Gm22502.1 Gm11639 Gm14734 Gm24713 Pgm5 Trpc1 E230008O15Rik Gm15246 Krt8 Gm25632 2410089E03Rik Gm3173 Gm26501.1 Rbm25.1 Sox9 Ybx2.1 Pank1 Gm24427 Gm24492 Gm24392 Cited2 Gm22997 Tex18 Gm25484 4921507P07Rik gfbi Rgs2 Gm3317 Gm11578 Gm25021 LOC432823 Tc2n.1 Gm26331 Acsl5 Gm3317.1 Ccdc41 Gm23115 BC030307 Gm23095 Alms1 Odf2l Lyst Col3a1.1 Brca2 Msh4 Sycp1 Mei1 Spo11 Dmc1 Rad21l Sycp2 Msh5 Prdm9 Spata22 Hmga2 Rev3l Helq.1 Uimc1.1 Uvssa Parp6 Stxbp5 Plxnb2.1 Sparcl1 Cdh1 Mycbp2.1 Snrnp48 Pkd1 Hk2 Mmp14 Ace2 Kmt2a Ash1l Aldob 4932438A13Rik Slc26a8 Ccnb3 Zkscan3 Herc1.1 Micu3 Csrp2 Slc4a7.1 Grk4 Aldh1a2.1 Stk38 Kcnq1ot1 Dmxl1 Sfi1 Mir296 Nktr Rel Strip2 Gldn Cmtm7 Nicn1 Eml5 Catsperg1 Catsperg2 Dennd4a.1 Syt14 Capns1 Lyst Chuk Col3a1 Gfra2 Pdgfra Nrp2 Crmp1 Plxnb2 Mycbp2 Mapk8ip3 Stxbp5 Stard9 AI481877 Dmc1 4930447C04Rik Paxbp1 Ccnb3 Mybl1 Sycp2 Uimc1Helq Aldh1a2 Slc4a7 Kmt2c Anxa5 Cdh1 Cdh17 Stk38 Ash1l Ace2 Herc1Zkscan3 Gldn Krt19 Susd2 Nktr Enpp3 Golgb1 Sfxn1 Taf7l Grk4 Nlrp9b Rmdn2 Eml5 Mga Pcsk7 Lgals4 Catsperg1 Dennd4a NON-DIFFERENTIALLY SPLICED GENES DIFFERENTIALLY SPLICED GENES IN BPAF CONDITION −0.5 0.0 0.5 1.0 −1.0 −0.5 0.0 0.5 1.0 −1.0 −0.5 0.0 0.5 1.0 1 4 16 count DIFFERENTIALLY SPLICED GENES IN BADGE CONDITION BADGE (Log2FC) andom sampling) 3543 genes 2678 genes 5.1 % DEGs 4.9 % DEGs DIFFERENTIALLY SPLICED GENES IN BADGE CONDITION DIFFERENTIALLY SPLICED GENES IN BADGE CONDITION DIFFERENTIALLY SPLICED GENES IN BPAF CONDITION NON-DIFFERENTIALLY SPLICED GENES NON-DIFFERENTIALLY SPLICED GENES BPAF (Log2FC) condition position 11.5dpc 13.5dpc chr3 0 5.0 x107 1.0 x108 1.5 x108 chr4 0 5.0 x107 1.0 x108 1.5 x108 chr7 0 5.0 x107 1.0 x108 1.5 x108 chr10 0 5.0 x107 1.0 x108 position position chr14 0 5.0 x107 1.0 x108 position position chr17 0 2.5 x107 5.0 x108 7.5 x107 1.0 x108 position 11.5dpc 13.5dpc 11.5dpc 13.5dpc 8-OdG 8-OdG 8-OdG chr10 0 5.0 x107 1.0 x108 position 11.5dpc 13.5dpc 8-OdG chr10 counts_normalized condition position 11.5dpc 13.5dpc 0 5.0 x107 1.0 x108 1.5 x108 chr4 0 5.0 x107 1.0 x108 1.5 x108 chr7 0 5.0 x107 1.0 x108 1.5 x108 0 5.0 x107 1.0 x108 position position chr14 0 5.0 x107 1.0 x108 position position chr17 0 2.5 x107 5.0 x108 7.5 x107 1.0 x108 position 11.5dpc 13.5dpc dpc 8-OdG 8-OdG counts_normalized counts_normalized 100 100 75 50 25 0 8 oxodG area/cell area (%) Table 1: List of common genes between BADGE and BPAF conditions showing an alternative spllcing relative to control germ cell. References 755 Table 1: List of common genes between BADGE and BPAF conditions showing an alternative spllcing relative to control germ cell. Go term Differentially Spliced genes count (adj pvalue) Common genes between bisphenols % common genes Chromosome Segregation (GO:0007059) 105 (BADGE ; pvalue 9 x10-8) 85 (BPAF; pvalue 2 x10-6) 46 % (BADGE) 56% (BPAF) Smc4, Espl1, Mms19, Srpk1, Cit, Ccne1, Psrc1, Bub1b, Pttg1, Kif4, Mlh1, Dync1h1, Zw10, Zwint, Cep63, Hecw2, Spag5, Pogz, Pum1, Mcmdc2, Top2b, Bub1, Setdb2, Anapc1, Atm, Pibf1, Msto1, Mau2, Cdc20, Ndc1, Fancd2, Usp9x,Top3b , Tpr, Ago4, Ncapd3, Sun1, Cenph, Nipbl, Ddx11, Hira, Csnk2a1, Lrrk1, Smc1b, Ino80, Dmc1, Hfm1 DNA repair (GO:0006281) 151 (BADGE; pvalue: 5 x10-10) 108 (BPAF; pvalue: 7 x10-06) 44 % (BADGE) 61 % (BPAF) Nhej1, Nabp1, Pold2, Gins4, Wrn, Uchl5, Smc4, Hdac9, Npas2, Usp47, Mms19, Prkdc, Pms2, Fanci, Pif1, Upf1, Pttg1, Rad51, Mlh1, Cdc45, Kdm2a, Usp3, Otub1, Abl1, Dclre1a, Shprh, Parp9, Cep164, Neil1, Marf1, Rbm17, Mcmdc2, Npm1, Top2b, Taok3, Ticrr, Cdc5l, Supt16, Xpc, Atm, Rad52, Uimc1, Poli, Ube2t, Taok1, Rbbp8, Fancd2, Setx, Sprtn, Ercc6l2, Gtf2h1, Nipb, Atr, Alkbh1, Ddx11, Usp28, Eya1, Rtel1, Rif1, Rev1, Cdc7, Ino80, Dmc1, Huwe1, Helq Nuclear Chromosome Segregation (GO:0098813) 88 (BADGE; pvalue: 3 x10-07) 70 (BPAF; pvalue: 12 x10-05) 45 % (BADGE) 57 % (BPAF) Smc4, Espl1, Cit, Ccne1, Psrc1, Bub1b, Pttg1, Kif4, Mlh1, Dync1h1, Zw10, Zwint, Cep63, Hecw2, Spag5, Pogz, Mcmdc2, Top2b, Bub1, Anapc1, Atm, Pibf1, Msto1, Mau2, Cdc20, Ndc1, ancd2, Tpr, Ago4, Ncapd3, Sun1, Nipbl, Ddx11, Hira, Lrrk1, Smc1b, Ino80, Dmc1, Hfm1 DNA Recombination (GO:0006310) 79 (BADGE, pvalue < 0.001) 5 (BPAF, pvalue: 0.04) 40 %(BADGE) 58 %(BPAF) Cntd1, Nabp1, Gins4, Ubr2, Wrn, Uchl5, Rag1, Mms19, Prkdc, Pms2, Pif1, Rad51, Mlh1, Cdc45, Cep63, Thoc1, Mcmdc2, Top2b, Atm, Rad52, Rbbp8, Setx, Psmc3ip, Nipbl, Rtel1, Rif1, Cdc7, Ino80, Dmc1, Hfm1, Gm960, Helq Meiotic Cell Cycle process (GO:0051321) 99 (BADGE; pvalue: 2 x10-06) 73 (BPAF; pvalue <0.001) 11 % (BADGE) 15% (BPAF) Tdrd9, Cntd1, Topaz1, Ubr2, Smc4, Espl1, Ccne1, Bub1b, Pttg1, Rad51, Mlh1, Marf1, Cep63, Plcb1, Nsun2, Mcmdc2, Top2b, Bub1, Atm, Cdc20, Ndc1, Fancd2, Psmc3ip, Ago4, Ncapd3, Sun1, Lrrk1, Aspm, Dmc1, Hfm1, Gm960, Sycp2 p g j y p , y probes. References 755 Only PSRs are assigned Event Estimation Name Gene Symbol Probes Splicing event estimation name Splicing pvalue (p≤0.01) target location Isoform name BADGE BPAF Ago4 EEJ EEJ Exons 11-12 Exons 9-10 Ago4-201 Ago4-201 - - ns 0.0093 0.0028 ns Aspm EEJ E Exons 1-2 Exon 18 Aspm-201 Aspm-201 - ND 0.0007 ns ns 0.0044 Atm EEJ EEJ EEJ EEJ E Exons 56-57 Exons 52-53 Exons 47-48 Exons 49-50 Exon 29 Atm-201 Atm-201 Atm-201 Atm-201 Atm-201 - - - - Cassette Exon 0.0061 ns 0.0039 0.002 0.01 0.0093 0.0061 ns 0.0017 ns Bub1b EEJ E Exons 9-10 Exon1 Bub1-201 Bub1-203 - Alt 3' Acceptor Site 0.0002 ns 0.0044 0.0064 Ccne1 EEJ E Exons 6-7 Exon 1 Ccne-201 Ccne-201 - Cassette Exon ns 0.0035 0.0026 ns Cdc7 EEJ Exons 6-8 Cdc7-201 - 0.0044 0.0366 Cdc20 EEJ E Exons 6-7 Exon 1 Cdc20-201 Cdc20-201 - Cassette Exon 0.0041 ns ns 0.004 Cdc45 EEJ EEJ EEJ Exons 19-20 Exons 11-12 Exons 1-2 Cdc45-201 Cdc45-201 Cdc45-201 - - - ns 0.009 0.0094 0.0089 ns ns Cep63 E Exon 7 Cep63-201 Cassette Exon 0.0024 0.0047 Cntd1 E E E Exon 1 (-5’) Exon 1 (-3’) Exon 3 Cntd1-201 Cntd1-201 Cntd1-203 Cassette Exon Cassette Exon Cassette Exon ns ns 0.0012 0.007 0.0074 ns Dmc1 EEJ EEJ Exons 9-10 Exons 7-8 Dmc1-201 Dmc1-201 - - 0.0004 0.0013 0.0069 ns Espl1 EEJ Exons 1-2 Espl1-201 - 0.003 0.0039 Fancd2 EEJ EEJ Exons 22-23 Exons 31-32 Fancd2-201 Fancd2-201 - - 0.0019 0.0096 0.0088 ns Gins4 EEJ E Exons 2-3 Exon 8 Gins4-201 Gins4-201 - Alt 5' Donor Site 0.0061 ns ns 0.0045 Gm960 (Top6bl) EEJ E E Exons 3-4 Exon 12 Exon 11 Gm960-201 Gm960-202 Gm960-202 - Cassette Exon Cassette Exon ns 0.0033 0.0076 0.0039 ns ns Helq EEJ EEJ Exons 16-17 Exons 12-13 Helq-201 Helq-201 - - 0.0000157 0.0088 0.0038 ns Hfm1 EEJ E E E Exons 7-8 Exon 22 Exon 21 Exon 4 Hfm1-201 Hfm1-201 Hfm1-201 Hfm1-204 - Cassette Exon Cassette Exon Cassette Exon ns 0.0031 0.0087 0.0005 0.0076 ns ns ns Ino80e EEJ Exons 2-3 Ino80e-203 - 0.0007 0.0034 Lrrk1 E Exon 28 Lrrk1-201 Cassette Exon 0.0077 0.0042 Marf1 EEJ Exons 24-25 Marf1-201 - ns 0.0084 EEJ Exons 9-10 Marf1-201 - 0.0023 0.0069 Mcmdc2 E E E Exon 10 Exon 14 Exon 16 Mcmdc2-201 Mcmdc2-203 Mcmdc2-204 - Cassette Exon Alt 5' Donor Site ns 0.0047 0.0018 0.0096 n ns Mlh1 EEJ E E Exons 15-17 Exon 18 Exon 18 Mlh1-201 Mlh1-206 Mlh1-201 - ND ND ns 0.0056 0.0035 0.0091 ns ns Mms19 EEJ E E E Exons 4-6 Exon 28 Intron 6 Exon 6 Mms19-201 Mms19-201 Mms19-201 Mms19-201 - Cassette Exon Intron Retention Cassette Exon 0.0008 ns 0.0066 0.0094 0.007 0.0022 0.0021 ns Nabp1 EEJ E E Exons 5-6 Exon 1 Exon 6 Nabp1-204 Nabp1-208 Nabp1-201 - Alt 5' Donor Site ND 0.0088 0.004 ns ns ns 0.0056 Ncapd3 EEJ E E E Exons 29-30 Exon 4 Exon 26 Exon 29 Ncapd3-201 Ncapd3-201 Ncapd3-201 Ncapd3-201 - Cassette Exon Cassette Exon Cassette Exon ns 0.0069 0.0047 0.0041 0.0065 ns ns ns Ndc1 EEJ Exons 1-2 Ndc1-205 - 0.0018 0.0032 Nipbl EEJ E E Exons 25-26 Exon 28 Exon 16 Nipbl-201 Nipbl-201 Nipbl-201 - Cassette Exon Cassette Exon 0.001 ns ns ns 0.004 0.0055 Nsun2 EEJ EEJ Exons 7-8 Exons1-2 Nsun2-201 Nsun2-203 - - 0.002 ns ns 0.0045 Plcb1 E Exon 32 Plcb1-201 Cassette Exon 0.0017 0.0079 Prkdc EEJ Exons 52-53 Prkdc-201 - 0.0039 0.0079 Psmc3ip EEJ Exons 2-3 Psmc3ip-201 - 0.0025 0.0059 Pttg1 E Exon 1 Pttg1-201 Alt 5' Donor Site 0.0026 0.0026 Rad51 EEJ EEJ Exons 1-2 Exons 7-8 Rad51-201 Rad51-201 - - 0.0064 0.0023 ns 0.0012 Rag1 E E Exon 6 Exon 1 Rag1-201 Rag1-207 Cassette Exon Cassette Exon 0.01 ns ns 0.0085 Rif1 EEJ E E E Exons 24-25 Exon 5 Exon 18 Exon 22 Rif1-201 Rif1-201 Rif1-201 Rif1-201 - Cassette Exon Cassette Exon ND 0.003 0.0068 ns ns 0.0058 ns 0.0083 0.0027 Rbbp8 EEJ Exons 15-16 Rbbp8-201 - 0.0000841 0.0002 Rtel1 EEJ E Exons 6-7 Exon 18 Rtel1-201 Rtel1-201 - Cassette Exon ns 0.005 0.0025 ns Setx EEJ E Exons 17-18 Exon 4 Setx-201 Setx-201 - Cassette Exon 0.0082 0.0067 ns 0.0041 Smc4 EEJ EEJ E E Exons 9-10 Exons 19-20 Exon 1 Exon 8 Smc4-201 Smc4-201 Smc4-201 Smc4-201 - - Alt 3' Acceptor Site Alt 5' Donor Site ns ns 0.0039 0.0034 0.0072 0.0061 0.0024 0.0049 Sycp2 EEJ E E Exons 36-37 Exon 38 Exon 36 Sycp2-201 Sycp2-201 Sycp-201 - Alt 3' Acceptor Site Cassette Exon 0.0022 0.0027 0.0000384 ns ns 0.0003 Sun1 EEJ EEJ EEJ EEJ E E E E E E Exons 8-10 Exons 13-14 Exons 2-3 Exons 6-8 Exon 1 Exon 4 Exon 15 Exon 17 Exon 18 Exon 23 Sun1-201 Sun1-201 Sun1-218 Sun1-201 Sun1-215 Sun1-201 Sun1-201 Sun1-201 Sun1-201 Sun1-201 - - - - ND Alt 3' Acceptor Site Cassette Exon Cassette Exon Alt 3' Acceptor Site Alt 5' Donor Site 0.0094 0.0089 0.0063 0.0055 0.0003 0.0094 0.0076 0.0007 0.0081 ns ns ns ns ns 0.01 ns ns 0.01 ns 0.01 Tdrd9 E E E Exon 5 Exon 6 Exon 24 Tdrd9-201 Tdrd9-201 Tdrd9-201 Cassette Exon Cassette Exon Cassette Exon ns ns 0.005 0.0026 0.0077 ns Thoc1 EEJ E Exons 3-4 Exon 7 Thoc1-201 Thoc1-201 - Cassette Exon ns 0.0054 0.01 ns Top2b EEJ EEJ EEJ Exons 6-7 Exons 22-23 Exons 25-26 Top2b-201 Top2b-201 Top2b-201 - - - ns 0.0096 0.008 0.009 ns ns Topaz EEJ EEJ E Exons 4-5 Exons 9-10 Exon 10 Topaz1-201 Topaz1-201 Topaz1-201 - - Cassette Exon 0.0038 ns 0.0014 ns 0.0047 ns Ubr2 E EEJ EEJ E E Exon 1 Exons 44-45 Exons 27-28 Exon 5 Exon 1 Ubr-201 Ubr-201 Ubr-201 Ubr-201 Ubr-205 ND - - Cassette Exon Intron Retention 0.007 ns ns ns 0.0074 ns 0.005 0.0029 0.0075 ns Uchl5 EEJ E Exons 1-2 Exon 11 Uchl5-201 Uchl5-201 - Alt 5' Donor Site 0.0083 ns ns 0.0008 Wrn EEJ E Exons 12-13 Exon 21 Wrn-201 Wrn-201 - Cassette Exon 0.0061 ns ns 0.0057

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An enriched meshless finite volume method for the modeling of material discontinuity problems in 2D elasticity

Latin American Journal of Solids and Structures

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1 INTRODUCTION The finite-volume method ሺFVMሻ is a numerical technique that provides approximate solutions to boundary value problems with partial differential equations. In this method, the computational domain is divided into smaller sub-domains, called as finite volumes, each containing one node point on the discretized geometry and the partial differential equation is satisfied in the integral sense over each finite volume. A distinctive feature of the method is the use of boundary integral instead of the domain integral, for satisfying partial differential equa- tion. The mesh-based formulation of FVM is a well-established technique in a wide range of problems concerned with the mechanics of solids and structures, see ሺTaylor et al. 2003, Fallah 2004, Cardiff et al. 2014, Escarpini Fil- ho and Marques 2016, Cavalcante and Pindera 2016ሻ. However, for certain classes of problems, the heavy and rigid reliance of the mesh-based FVM on a mesh leads to some difficulties. Some of these difficulties are as follows ሺLiu 2009ሻ: ሺLiu 2009ሻ: • High computational cost and human-labor in creation of a quality mesh for domain with complex geometry, ሻ computational cost and human-labor in creation of a quality mesh for domain with complex geometry, • Additional computation as well as a degradation of accuracy in remeshing approach to deal with evolution problems, and • Discontinuous nature in calculating stress fields, due to the element-wise continuous nature of the displacement field assumed in the mesh- based formulation. • Discontinuous nature in calculating stress fields, due to the element-wise continuous nature of the displacement field assumed in the mesh- based formulation. These difficulties could be decreased by using a mesh independent formulation. For this purpose, meshless formulation of FVM, which is also named as MLPG5 by Atluri and Shen ሺ2002ሻ, has been introduced. This method has been accepted by many researchers and widely used for solving problems in solid and structural mechanics, see ሺBatra et al. 2004, Han et al. 2005, Sladek et al. 2008, Hosseini et al. 2011, Soares et al. 2012, Ebrahimnejad et al. 2014, 2015, 2017ሻ. Interpolation of field variables by moving least squares approximation and integrating weak form of the equilibrium equation over local finite volumes in meshless FVM eliminates the need for mesh generation. Instead, it relies on background local finite volumes where each finite volume is independent of the others, i.e. they can be of any geometric shape and size and even using overlapped finite volumes are allowed. Original Article Original Article Keywords finite volume method, meshless method, material discontinuity, enrich- ment technique, Voronoi tessellation. Abstract Abdullah Davoudi-Kia a N. Fallah a,b,* Abdullah Davoudi-Kia a N. Fallah a,b,* A 2D formulation for incorporating material discontinuities into the mesh- less finite volume method is proposed. In the proposed formulation, the moving least squares approximation space is enriched by local continuous functions that contain discontinuity in the first derivative at the location of the material interfaces. The formulation utilizes space-filling Voronoi- shaped finite volumes in order to more intelligently model irregular geom- etries. Numerical experiments for elastostatic problems in heterogeneous media are presented. The results are compared with the corresponding so- lutions obtained using the standard meshless finite volume method and el- ement free Galerkin method in order to highlight the improvements achieved by the proposed formulation. It is demonstrated that the enriched meshless finite volume method could alleviate the expecting oscillations in derivative fields around the material discontinuities. The results have re- vealed the potential of the proposed method in studying the mechanics of heterogeneous media with complex micro-structures. N. Fallah a Department of Civil Engineering, University of Guilan, P.O. Box 3756, Rasht, Iran. E- mail: [email protected] b Faculty of Civil Engineering, Babol Noshirvani University of Technology, Babol, Iran. E- mail: [email protected] *Corresponding author http://dx.doi.org/10.1590/1679-78254121 Received: June 14 2017 Received: June 14, 2017 In Revised Form: December 01, 2017 Accepted: December 01, 2017 Available online: March 20, 2018 Latin American Journal of Solids and Structures, 2018, 15ሺ2ሻ, e13 1 INTRODUCTION However, they should locate entirely inside the boundaries and their union should cover the whole domain. In addition, the high order of continuity inherits from moving least squares approximation, provides solutions with smooth derivatives Latin American Journal of Solids and Structures, 2018, 15ሺ2ሻ, e13 Abdullah Davoudi-Kia et al. Abdullah Davoudi Kia et al. An enriched meshless finite volume method for the modeling of material discontinuity problems in 2D elasticity throughout the domain. As a result, negligible pre-processing and post-processing costs of meshless FVM are the main attractive features over the prevalent mesh-based methods ሺAtluri and Shen 2002ሻ. Atluri and Shen ሺ2002ሻ have demonstrated that meshless FVM can be a simple and efficient alternative to the all-purpose FEM, due to its speed, accuracy and response stability. In this paper the meshless FVM is applied to the material discontinuity problem which is an important issue in numerical modeling of heterogeneous media. It should be noted that heterogeneity is a ubiquitous property of solids and structures associated with features such as holes or inclusions that have an important role in the fail- ure ሺGdoutos 2005ሻ. ሺ ሻ The solution of problems in heterogeneous media typically involves discontinuity in gradient fields. In order to capture non-smooth property of the solutions, there are two essentially different techniques. The first tech- nique applies smooth polynomial approximation spaces and trusts in domain discretization that conforms to ma- terial discontinuities. The polynomial approximation spaces can be constructed by using either a mesh-based shape function or meshless approximation function. The alternative technique is based on ‘enrichment’ of smooth approximation space such that the domain discretization being independent of the material discontinuities. The enrichment will be accomplished by adding special weak discontinuous functions to the approximation space with unknown parameters that control the strength of the discontinuity. The first above mentioned technique has been used by Cavalcante et al. ሺ2007ሻ within the mesh-based FVM framework for functionally graded materials and also by Li et al. ሺ2003ሻ in the 3D formulation of the MLPG meth- od to problems with singularities and material discontinuities. In the latter, the heterogeneous medium is sepa- rated into isolated, homogeneous parts and then continuity constraints are enforced at the interface to ‘reconnect’ the pieces. To accomplish the ‘separation’, meshless approximation function applied separately within each ho- mogeneous part and therefore domains of influence are truncated at the material interfaces. 1 INTRODUCTION Also, local sub- domains, which partially cut by the interface, are redefined to locate entirely inside the boundaries. In spite of the simple concept of this technique, the presence of truncated domains of influence could introduce some mesh de- pendency into the solution ሺCordes and Moran 1996, Li et al. 2000ሻ; Furthermore, re- definition of local sub- domains and domains of influence could be difficult and computationally expensive ሺde Borst 2008ሻ. The so- called enrichment technique has been applied both in the area of mesh-based and meshless methods. Melenk and Babuška ሺ1996ሻ enriched the standard finite element approximation, Belytschko and Black ሺ1999ሻ set up the frame of the extended FEM ሺXFEMሻ and Belytschko et al. ሺ1996ሻ implemented it within the EFG method for intro- ducing discontinuous derivatives into the solution. Batra et al. ሺ2004ሻ utilized this technique in the 1D meshless formulation of FVM to analyze heat conduction in a bimetallic circular disk. Recently, Yoon and Song ሺ2014ሻ and Hu et al. ሺ2015ሻ applied this technique in order to handle discontinuities in heterogeneous media. Using the en- richment technique is interesting since a fix and regular domain discretization can be applied and domain dis- cretization cost is reduced to a minimum ሺAn et al. 2011ሻ. In this paper, based on the enrichment technique used already by Krongauz and Belytschko ሺ1998ሻ, a 2D formulation within the meshless FVM framework is proposed to handle material discontinuity. The structure of the present paper is as follows. In the next section, the basic concepts including standard meshless FVM, moving least squares approximation and enrichment of approximation space are briefly described. A 2D formulation in modeling heterogeneous material within the meshless FVM framework is presented in sec- tion 3. Numerical examples and results to verify the capability of the formulation are presented in section 4 and concluding remarks are given in the last section. Latin American Journal of Solids and Structures, 2018, 15ሺ2ሻ, e13 2.1 Standard meshless FVM Utilizing weighted residual method, weak form of the Equations ሺ1ሻ-ሺ2ሻ over I-th local finite volume s  enclosed by s , can be written as ( ) ( ) , 0, s su I I ij j i i i b v d u u v d s a W G + W - - G = ò ò ሺ3ሻ ( ) ( ) , 0, s su I I ij j i i i b v d u u v d s a W G + W - - G = ò ò ሺ3ሻ where I v is the test function for I-th local finite volume and 1 >> is a penalty parameter which is applied to impose the essential boundary conditions. In meshless formulations, a local approximation scheme will be applied to approximate the trial function; therefore, the essential boundary conditions cannot be imposed directly, a priori, but superimposed in a weak form. In this paper, the penalty method is applied to superimpose the essential boundary conditions, as the second term in Equation ሺ3ሻ. Let s denotes a part of the s  located on the global boundary, then su s u is a part of the s that coincides with the global essential boundary. By applying the divergence theorem, Equation ሺ3ሻ could be rewritten as where I v is the test function for I-th local finite volume and 1 >> is a penalty parameter which is applied to impose the essential boundary conditions. In meshless formulations, a local approximation scheme will be applied to approximate the trial function; therefore, the essential boundary conditions cannot be imposed directly, a priori, but superimposed in a weak form. In this paper, the penalty method is applied to superimpose the essential boundary conditions, as the second term in Equation ሺ3ሻ. Let s denotes a part of the s  located on the global boundary, then su s u is a part of the s that coincides with the global essential boundary. 2.1 Standard meshless FVM It should be mentioned that Equations ሺ1ሻ-ሺ2ሻ would be satisfied, a posteriori, in the global domain  and on its boundary , respectively, if the union of all local finite volumes covers the global domain ሺAtluri and Zhu 1998ሻ. 2.1 Standard meshless FVM Most of the meshless methods, e.g. element free Galerkin ሺEFGሻ and smoothed particle hydrodynamics ሺSPHሻ, are based on global weak forms and required the use of background mesh in order to integrate the weak form of the equilibrium equation. On the other hand, the meshless local Petrov-Galerkin ሺMLPGሻ method satisfies the weak form over the local sub-domains. As a special case, MLPG5 ሺAtluri and Shen 2002ሻ chose the Heaviside step function as the test function. Consequently, domain integrals are vanished and integration is performed along the boundary of local sub-domains. Satisfying local weak form denotes the momentum balance law over the local sub- domains that resemble the concept of FVM. Therefore, the MLPG5 method could be recognized as the meshless FVM ሺAtluri et al. 2004ሻ.  be a domain bounded by . Momentum balance principles for static problems are given b Let  be a domain bounded by . Momentum balance principles for static problem ሺ1ሻ , 0 , ij j ib for all x s + = Î W Latin American Journal of Solids and Structures, 2018, 15ሺ2ሻ, e13 2/22 Abdullah Davoudi-Kia et al. An enriched meshless finite volume method for the modeling of material discontinuity problems in 2D elasticity Abdullah Davoudi-Kia et al. An enriched meshless finite volume method for the modeling of material discontinuity problems in 2D elasticity Abdullah Davoudi-Kia et al. Abdullah Davoudi-Kia et al. An enriched meshless finite volume method for the modeling of material discontinuity problems in 2D elasticity Abdullah Davoudi-Kia et al. An enriched meshless finite volume method for the modeling of material discontinuity problems in 2D elasticity where σ is the stress tensor and b is the body force measured per unit volume. The essential and natural boundary conditions are as follows i i u u u on = G , ሺ2aሻ i i t t t on = G , ሺ2bሻ i i u u u on = G , i i t t t on = G , i i u u u on = G , i i t t t on = G , ሺ2b i i t t t on = G , ሺ2bሻ where u and t are defined as the prescribed displacements and tractions, respectively. u is the global essential boundary and t is the global natural boundary. 2.1 Standard meshless FVM By applying the divergence theorem, Equation ሺ3ሻ could be rewritten as ( ) ( ) , 0 s s su I I I I ij j ij j i i i n v d v b v d u u v d s s a ¶W W G G - - W - - G = ò ò ò , ሺ4ሻ ሺ4ሻ where n is the unit outward normal vector to the boundary s . By using the Heaviside step function as the test function, imposing natural boundary conditions and doing some algebraic operations, one can obtain the following expression 0 s su su st s su i i i i i i t d t d u d t d b d u d G G G G W G G G a G G W a G - - - = + - ò ò ò ò ò ò , ሺ5ሻ ሺ5ሻ ሺ5ሻ where 0 s  is a part of the s  which is totally inside the global domain and st s t  is a part of the s that coincides with the global natural boundary. Equation ሺ5ሻ represents a weak form of the balance law in the local finite volume s , with the boundary conditions being enforced. It can be seen that the left hand side of Equation ሺ5ሻ, which leads to the stiffness matrix, does not involve domain integration. In addition, in the absence of body force, domain integration is totally eliminated. It should be mentioned that Equations ሺ1ሻ-ሺ2ሻ would be satisfied, a posteriori, in the global domain  and on its boundary , respectively, if the union of all local finite volumes covers the global domain ሺAtluri and Zhu 1998ሻ. where 0 s  is a part of the s  which is totally inside the global domain and st s t  is a part of the s that coincides with the global natural boundary. Equation ሺ5ሻ represents a weak form of the balance law in the local finite volume s , with the boundary conditions being enforced. It can be seen that the left hand side of Equation ሺ5ሻ, which leads to the stiffness matrix, does not involve domain integration. In addition, in the absence of body force, domain integration is totally eliminated. Latin American Journal of Solids and Structures, 2018, 15ሺ2ሻ, e13 2.2 Moving least squares approximation T J x P a x U W P a x U é ù é ù = - - ê ú ê ú ë û ë û , ሺ9ሻ where ( ) ( ) ( ) ˆ ˆ . . . . T x P a x U W P a x U é ù é ù = - - ê ú ê ú ë û ë û , ሺ9ሻ ( ) ( ) ( ) ˆ ˆ . . . . T J x P a x U W P a x U é ù é ù = - - ê ú ê ú ë û ë û , ሺ9ሻ where ( ) ( ) ( ) 1 2 , , , T T T T n P p x p x p x é ù = ¼ ê ú ë û , ሺ10ሻ ( ) ( ) 1 0 0 n w x x W w x x é ù - ¼ ê ú ê ú = ¼ ¼ ¼ ê ú ê ú ê ú ¼ - ë û , ሺ11ሻ ሺ10ሻ ሺ11ሻ 1 2 , ˆ ˆ ˆ ˆ , , T n u U u u é ù = ¼ ê ú ë û . 1 2 , ˆ ˆ ˆ , , T n u u u é ù = ¼ ê ú ë û . ሺ12ሻ 1 2 , ˆ ˆ ˆ ˆ , , T n u U u u é ù = ¼ ê ú ë û . 2.2 Moving least squares approximation In Meshless methods, a local partition of unity approximation is applied to approximate unknown field func- tions with the nodal parameters of unknown variable at some randomly scattered nodes. Among available local partition of unity approximation schemes, moving least squares ሺMLSሻ approximation has been utilized in mesh- less FVM due to its reasonable accuracy and simplicity of extension to 3D problems ሺAtluri and Zhu 1998ሻ. Let the neighborhood of a point x, where   1 2 , T x x x  contains space coordinates, be a sub-domain x  and called the domain of definition of MLS approximation of the field function  u x . The MLS approximation  h u x of the function  u x can be defined over a given set of nodes   1 2 , , ,  n x x x as ( ) ( ) ( ) ( ) ( ) 1 , m h J J T x J u x p x a x p x a x x W = = = " Î å , ሺ6ሻ ) ( ) ( ) ( ), J T x a x p x a x x W = " Î , ሺ6ሻ ሺ6ሻ ( ) ( ) ( ) ( ) ( ) 1 , m h J J T x J u x p x a x p x a x x W = = = " Î å , Latin American Journal of Solids and Structures, 2018, 15ሺ2ሻ, e13 3/22 Abdullah Davoudi-Kia et al. Abdullah Davoudi-Kia et al. An enriched meshless finite volume method for the modeling of material discontinuity problems in 2D elasticity where  p x is the complete polynomial basis function of order m and  a x is a vector containing associated unknown coefficients. In two dimensions,  p x can be expressed as where  p x is the complete polynomial basis function of order m and  a x is a vector containing associated unknown coefficients. Latin American Journal of Solids and Structures, 2018, 15ሺ2ሻ, e13 2.2 Moving least squares approximation ሺ12ሻ ሺ12ሻ The unknown  a x coefficients can be found by minimizing of  J x with respect to  a x The unknown  a x coefficients can be found by minimizing of  J x with respect to  a x ( ) ( ) ( ) ( ) ( ) ˆ 0 J x A x a x V x U a x ¶ = - = ¶ , ሺ13ሻ ( ) ( ) ( ) ( ) ( ) ˆ 0 J x A x a x V x U a x = - = , ሺ13ሻ ሺ13ሻ where the matrices A and V are defined as where the matrices A and V are defined as ( ) ( ) ( ) ( ) 1 n I I T I T I A x w x x p x p x P WP = = - = å , ሺ14ሻ ( ) ( ) ( ) ( ) ( ) ( ) ( ) 1 1 2 2 , , , n n T V x w x x p x w x x p x w x x p x P W é ù = - - ¼ - = ê ú ë û . ሺ15ሻ ሺ15ሻ Solving for  a x from Equation ሺ13ሻ and substituting in Equation ሺ6ሻ, the MLS approximation can be de- fined as Solving for  a x from Equation ሺ13ሻ and substituting in Equation ሺ6ሻ, the MLS approximation can be de- fined as ( ) ( ) 1 ˆ n I I I x x u f = = å , ሺ16ሻ ( ) ( ) 1 ˆ n h I I I u x x u f = = å , ሺ16ሻ where the shape function of the MLS approximation corresponding to I-th node defined by ( ) ( ) ( ) ( ) 1 1 JI m I J J x p x A x V x f - = é ù = ê ú ë û å . ሺ17ሻ where the shape function of the MLS approximation corresponding to I-th node defined by ( ) ( ) ( ) ( ) 1 1 JI m I J J x p x A x V x f - = é ù = ê ú ë û å . 2.2 Moving least squares approximation In two dimensions,  p x can be expressed as 1 2 ( ) [1, , ] , 3 T p x x x linear basis m = = , ሺ7aሻ ( ) ( ) ( ) 2 2 1 2 1 1 2 2 1, , , , , , 6 T p x x x x x x x quadratic basis m é ù = = ê ú ë û . ሺ7bሻ 1 2 ( ) [1, , ] , 3 T p x x x linear basis m = = , ( ) ( ) ( ) 2 2 1 2 1 1 2 2 1, , , , , , 6 T p x x x x x x x quadratic basis m é ù = = ê ú ë û . ሺ7bሻ In the MLS approximation, the coefficient vector  a x can be determined by minimizing the weighted re- sidual  J x In the MLS approximation, the coefficient vector  a x can be determined by minimizing the weighted re- sidual  J x ( ) ( ) ( ) ( ) 1 2 ˆ I T I I n I J x w x x p x u a x = é ù = - - ê ú ë û å , ሺ8ሻ ሺ8ሻ Where n is the number of nodes in the domain of definition for which the weight function   w  I x x associated with the I-th node is positive and ˆ I u refers to the fictitious nodal values and not the nodal values of MLS approximant  h u x at the I-th node. Equation ሺ8ሻ can be rewritten in the form Where n is the number of nodes in the domain of definition for which the weight function   w  I x x associated with the I-th node is positive and ˆ I u refers to the fictitious nodal values and not the nodal values of MLS approximant  h u x at the I-th node. Equation ሺ8ሻ can be rewritten in the form ( ) ( ) ( ) ˆ ˆ . . . . 2.2 Moving least squares approximation where I I c r r   I x x  and  I x x denotes the distance from I-th node to point x and I cr is the radius of the circular support for the weight function for I-th node. The cubic spline weight function has 2nd order continuity over the entire domain; therefore, MLS approximant  h u x is C2 continuous over the entire domain. 2.3 Enrichment of approximation space According to the 'reproduction' property of the MLS approximation, it can reproduce any functions that are included in the basis. Clearly Equation ሺ16ሻ is just able to reproduce continuous fields; therefore, only if the basis enriched by a special approximation function, including discontinuity in the derivative, MLS approximation will reflect the discontinuity in the gradient fields ሺLiu and Gu 2005ሻ. The enrichment can be accomplished by adding a special function to the approximation space, extrinsically. In addition, most non-smooth phenomena in solid mechanics have a local character; therefore, it may be useful to employ the enrichment in local regions ሺFries and Belytschko 2010ሻ. There are two conditions for the special approximation function which is customized for material interface. It should be continuous in the approximation field with a discontinuity in the first derivative at the location of the material interfaces ሺBelytschko et al. 1996ሻ. In addition, it should have compact support to maintain discrete equations banded and sparse ሺKrongauz and Belytschko 1998ሻ. There are several ways to construct such an en- richment approximation function. An et al. ሺ2011ሻ introduced a piecewise linear function with a cusp at the loca- tion of material interface. Krongauz and Belytschko ሺ1998ሻ employed a higher-order polynomial function with more constraints and also a localized ramp function. More different enrichment approximation functions could be defined by using the level set function which used within the XFEM framework, see ሺSukumar et al. 2001, Moës et al. 2003ሻ. In this paper, the spline function introduced already by Krongauz and Belytschko ሺ1998ሻ is applied to enrich MLS approximation space. In a two dimensional domain with dn lines of material discontinuity, the en- richment approximation function for J-th line of material discontinuity is defined as: ( ) ( ) ( ) 3 2 1 1 1 1 1 6 2 2 6 0 1 J J J J J J J r r r r r r y ìïï- + - + £ ïï = íïï ³ ïïî , ሺ19ሻ ሺ19ሻ where J J J c r r r   and Jr is the distance to the closest point on the J-th line of material discontinuity and J cr is the length of the support of J , i.e. the distance over which 0 J . 2.2 Moving least squares approximation ሺ17ሻ ሺ17ሻ ( ) ( ) ( ) ( ) 1 1 JI m I J J x p x A x V x f - = é ù = ê ú ë û å . The MLS approximation would be well-defined if matrix A in Equation ሺ14ሻ be invertible, therefore, do- mains of definition should be large enough to cover at least m nodes ሺi.e. n m  ሻ for each point of interest to pre- vent the singularity of the matrix A. These nodes are also not allowed to lie on a straight line. On the other hand, domains of definition should be small enough in order to preserve the local character of the MLS approximation ሺAtluri and Zhu 1998ሻ. Latin American Journal of Solids and Structures, 2018, 15ሺ2ሻ, e13 4/22 Abdullah Davoudi-Kia et al. Abdullah Davoudi Kia et al. An enriched meshless finite volume method for the modeling of material discontinuity problems in 2D elasticity It should be noted that according to Equation ሺ17ሻ, continuity of the MLS approximation depends on the con- tinuity of the basis function and also the smoothness of the weight function. Thus, the weight function has a signif- icant role in the efficiency of the MLS approximation. The exponential function, cubic and quartic spline functions are common useful examples of weight functions ሺLiu and Gu 2005ሻ. In this paper, the cubic spline weight func- tion is used where defined as ( ) ( ) ( ) ( ) ( ) 2 3 2 3 2 3 4 4 0.5 4 3 4 4 4 3 0.5 1 0 1 I I I I I I I I I r r r w x x r r r r r ìïï - + £ ïïïï - = - + - < < íïïï ³ ïïïî , ሺ18ሻ ሺ18ሻ where I I c r r   I x x  and  I x x denotes the distance from I-th node to point x and I cr is the radius of the circular support for the weight function for I-th node. The cubic spline weight function has 2nd order continuity over the entire domain; therefore, MLS approximant  h u x is C2 continuous over the entire domain. 2.3 Enrichment of approximation space In 2D problems, q is expressed in term of the arc length of the material discontinuity and then is discretized over the discontinuity line as follows ( ) 1 ˆ K K q s f = , ሺ21ሻ ( ) ( ) 1 1 ˆ n K K K q q s s f = = å , ሺ21 ( ) ( ) 1 1 ˆ n K K K q q s s f = = å , ሺ21ሻ e K  are one dimensional approximation function, 1 n is the number of enrichment nodes i imensional approximation function, 1 n is the number of enrichment nodes in the support K domain of 1D approximation and ˆK q are fictitious nodal values that are considered as unknowns in the discretized equations. In contrast to field nodes, which are employed to define fictitious nodal values of displacement fields, enrichment nodes are distributed along material discontinuities in order to discretize the amplitude parameters; they have no displacement degree of freedom. The definition of r and s in a typical two-dimensional domain is illustrated in Figure 2. It should be noted that, at the points far away from discontinuities the second term on the right-hand side of Equation ሺ20ሻ vanishes due to the compact support property of enrichment approximation function and this equation degenerates to Equation ሺ16ሻ. As a result, using enrichment approximation function, Equation ሺ20ሻ led to a local enriched formulation. L K r s r s Figure 2: Illustration of r and s values for a material discontinuity. Line of material discontinuity is illustrated by a solid line; solid and open circles are denoted the field nodes and the enrichment nodes, respectively and squares are arbi- trary points. The values of r and s for point K are shown by dashed lines and those for point L are illustrated by dash- dot lines. Figure 2: Illustration of r and s values for a material discontinuity. Line of material discontinuity is illustrated by a solid line; solid and open circles are denoted the field nodes and the enrichment nodes, respectively and squares are arbi- trary points. The values of r and s for point K are shown by dashed lines and those for point L are illustrated by dash- dot lines. 2.3 Enrichment of approximation space The plot of the enrichment approximation function ሺEquation 19ሻ and its derivative in one dimension are illustrated in Figure 1, where the kink of the enrichment function and the jump in its derivative across the material discontinuity are clearly seen. where J J J c r r r   and Jr is the distance to the closest point on the J-th line of material discontinuity and J cr is the length of the support of J , i.e. the distance over which 0 J . The plot of the enrichment approximation function ሺEquation 19ሻ and its derivative in one dimension are illustrated in Figure 1, where the kink of the enrichment function and the jump in its derivative across the material discontinuity are clearly seen. 5/22 Latin American Journal of Solids and Structures, 2018, 15ሺ2ሻ, e13 Abdullah Davoudi-Kia et al. An enriched meshless finite volume method for the modeling of material discontinuity problems in 2D elasticity oudi Kia et al. meshless finite volume method for the modeling of material discontinuity problems in 2D elasticity Figure 1: Enrichment function and its derivative in one dimension. Figure 1: Enrichment function and its derivative in one dimension. The enriched approximation  h u x for the function  u x , is then given by ( ) ( ) ( ) 2 1 1 ˆ d n n h I I J J J I J u x x u q r f y = = = + å å , ሺ20ሻ ( ) ( ) ( ) 2 1 1 ˆ d n n h I I J J J I J u x x u q r f y = = = + å å , ሺ20ሻ where the first term on the right-hand side of this equation represents the standard meshless FVM approximation, see Equation ሺ16ሻ, 2n is the number of field nodes in the support domain of 2D MLS approximation, dn is the number of lines of material discontinuity and Jq are amplitude parameters that govern the strength of the discontinuity across the material interfaces. 3.1 Domain discretization A promised feature of enriched formulations is the domain discretization independent of the geometry and location of the heterogeneities ሺSukumar et al. 2001ሻ; therefore a regular discretization can be employed in het- erogeneous media, but these regular sub-domains may be intersected by irregular-shaped external boundaries. In the proposed formulation, the Voronoi tessellation is applied for constructing local background finite volumes for a set of considered field nodes. The resulted finite volumes can be regular inside the domain and irregular for nodes next to the external boundaries. It should be mentioned that in mesh-based methods usually mesh refinement is applied where there are de- tails. In work presented by Cavalcante et al. ሺ2007ሻ, the need for extensive mesh refinement at the arbitrarily shaped geometries has been eliminated using a parametric mapping. Li et al. ሺ2003ሻ, which applied pure spherical local finite volumes in their meshless FVM formulation, reduced the size of the finite volumes that are close to the weak or strong boundaries so as not to cross over the boundaries; therefore, a dense cloud of nodes is required to completely represent the irregularities. As mentioned above, the inherent flexibility of the meshless FVM enables the use of the Voronoi tessellation for the domain discretization. In mathematics, for a given set of nodes, partitioning of a domain into sub-domains based on distance to the considered nodes called Voronoi tessellation ሺOkabe et al. 2009ሻ. These sub-domains, so called as finite volumes are space-filling polygons in 2D space and can be constructed corresponding to each node by forming the perpendicular bisector lines between the nodes and connecting the lines around each node. A Vo- ronoi tessellation and the local background finite volume associated with an arbitrary node are illustrated in Fig- ure 3. Figure 3: Voronoi shaped finite volumes. Voronoi tessellation for a given set of nodes ሺdotsሻ is illustrated by solid lines and the bold polygon is represented the local background finite volume associated with an arbitrary node. Figure 3: Voronoi shaped finite volumes. Voronoi tessellation for a given set of nodes ሺdotsሻ is illustrated by solid lines and the bold polygon is represented the local background finite volume associated with an arbitrary node. 3 THE PROPOSED FORMULATION BASED ON THE MESHLESS FVM FRAMEWORK 3.1 Domain discretization Abdullah Davoudi-Kia et al. An enriched meshless finite volume method for the modeling of material discontinuity problems in 2D elasticity Abdullah Davoudi Kia et al. An enriched meshless finite volume method for the modeling of material discontinuity problems in 2D elasticity 2.3 Enrichment of approximation space Latin American Journal of Solids and Structures, 2018, 15ሺ2ሻ, e13 Latin American Journal of Solids and Structures, 2018, 15ሺ2ሻ, e13 6/22 Abdullah Davoudi-Kia et al. 3.2 Enrichment of approximation space In order to discretize the governing equations, formulation of the meshless FVM can be used. It satisfies weak form of the balance law over local finite volumes which are formed around the field nodes, see Equation ሺ5ሻ. For a linear elastic solid within the range of infinitesimal deformations, the traction in Equation ሺ5ሻ can be written as follows i ij j ijkl kl j t n C e n s = = , ሺ22 i ij j ijkl kl j t n C e n s = = , ሺ22ሻ where C is the 4th order elasticity tensor, n is the unit outward normal vector and e is the strain tensor which is defined as where C is the 4th order elasticity tensor, n is the unit outward normal vector and e is the strain tensor which is defined as where C is the 4th order elasticity tensor, n is the unit outward normal vector and e is the strain tensor which is defined as ( ) , , 2 kl k l l k e u u = + . ሺ23ሻ Latin American Journal of Solids and Structures, 2018, 15ሺ2ሻ, e13 Latin American Journal of Solids and Structures, 2018, 15ሺ2ሻ, e13 7/22 Abdullah Davoudi-Kia et al. Abdullah Davoudi Kia et al. An enriched meshless finite volume method for the modeling of material discontinuity problems in 2D elasticity In heterogeneous media that contains material interfaces, enriched approximation space can be applied in order to make domain discretization independent of the material discontinuities; to this end, the enriched ap- proximation Equation ሺ20ሻ is utilized as displacement trial function. In order to obtain the discrete equations, approximated displacement function and its derivatives are needed. 3.2 Enrichment of approximation space By differentiating this equation one can obtain ( ) ( ) ( ) ( ) ( ) ( ) 2 1 1 , , , , 1 1 1 1 1 ˆ ˆ ˆ d d n n n n n h I I K J J K K J J K i j j i j i j i I J K J K u x x u s r q q s r f f y f y = = = = = = + + å å å å å ሺ25ሻ ( ) ( ) ( ) ( ) ( ) ( ) 2 1 1 , , , , 1 1 1 1 1 ˆ ˆ ˆ d d n n n n n h I I K J J K K J J K i j j i j i j i I J K J K u x x u s r q q s r f f y f y = = = = = = + + å å å å å ሺ25ሻ ( ) ( ) ( ) ( ) ( ) ( ) 2 1 1 , , , 1 1 1 1 1 ˆ ˆ ˆ d d n n n n n h I I K J J K K J J K j j i j i j i I J K J K x x u s r q q s r f f y f y = = = = = = + + å å å å å ሺ25ሻ where , I j  and , K j are the derivatives of 2D and 1D MLS approximation functions, respectively, and , J j  are derivative of the enrichment approximation functions. Using the chain rule of differentiation where , I j  and , K j are the derivatives of 2D and 1D MLS approximation functions, respectively, and , J j  are derivative of the enrichment approximation functions. Using the chain rule of differentiation ative of the enrichment approximation functions. 3.2 Enrichment of approximation space Using the chain rule of differentiation ( ) ( ) ( ) , K K K j j j s s s s x s x f f f ¶ ¶ ¶ = = ¶ ¶ ¶ , ( ) ( ) ( ) , J J J j j j r r r r x r x y y y ¶ ¶ ¶ = = ¶ ¶ ¶ . ( ) ( ) ( ) , K K K j j j s s s s x s x f f f ¶ ¶ ¶ = = ¶ ¶ ¶ , ( ) ( ) ( ) , K K K j j j s s s s x s x f f f ¶ ¶ ¶ = = ¶ ¶ ¶ , ሺ26ሻ ( ) ( ) ( ) , J J J j j j r r r r x r x y y y ¶ ¶ ¶ = = ¶ ¶ ¶ . ሺ27ሻ ሺ26ሻ ሺ27ሻ By substituting Equations ሺ22ሻ-ሺ27ሻ into Equation ሺ5ሻ, finally one obtains the linear algebraic discretized equations for a local finite volume as follows By substituting Equations ሺ22ሻ-ሺ27ሻ into Equation ሺ5ሻ, finally one obtains the linear algebraic discretized equations for a local finite volume as follows ˆ KU F = , ሺ28ሻ ˆ KU F = , ሺ28ሻ where ˆU is the vector of unknowns, K and F are the equivalent local stiffness matrix and load vector, respectively, and can be written as follows 0 s su su K nDBd nDBd Nd G G G G G a G = - - - ò ò ò   , ሺ29ሻ st s su F td bd ud G W G G W a G = + - ò ò ò , ሺ30ሻ st s su F td bd ud G W G G W a G = + - ò ò ò , ሺ30ሻ where t , b and u are the prescribed tractions, the body force measured per unit volume and the prescribed displacements, respectively. In Equation ሺ29ሻ, D is the elasticity matrix and n contains the entries of the unit outward normal vector and defined as where t , b and u are the prescribed tractions, the body force measured per unit volume and the prescribed displacements, respectively. 3.2 Enrichment of approximation space The enriched approximation is given as ( ) ( ) ( ) 1 1 1 1 ˆ ˆ d n n I I K J K J i i J K x s u q r f f y = = + å å , ሺ24ሻ ( ) ( ) ( ) ( ) 2 1 1 1 1 ˆ ˆ d n n n h I I K J K J i i i I J K u x x s u q r f f y = = = = + å å å , ሺ24 ሺ24ሻ where ˆI iu and ˆ J K iq are the unknown fictitious nodal values for displacement fields and discontinuities amplitude parameters. By differentiating this equation one can obtain where ˆI iu and ˆ J K iq are the unknown fictitious nodal values for displacement fields and discontinuities amplitude parameters. 3.2 Enrichment of approximation space In Equation ሺ29ሻ, D is the elasticity matrix and n contains the entries of the unit outward normal vector and defined as 1 2 2 1 0 0 n n n n n é ù ê ú = ê ú ê ú ë û  . 1 2 2 1 0 0 n n n n n é ù ê ú = ê ú ê ú ë û  . Accordingly, the vector of unknowns, ˆU , is expressed as 1 2 1 2 ˆ ˆ ˆ ˆ ˆ I I J K J K U u u q q é ù = ê ú ë û . ሺ36ሻ 1 2 1 2 ˆ ˆ ˆ ˆ ˆ I I J K J K U u u q q é ù = ê ú ë û . ሺ36ሻ It should be noted that both N and B are composed of the standard part and the enriched part, where the standard part is computed at every node but the enriched part is only computed at enriched nodes. It should be noted that both N and B are composed of the standard part and the enriched part, where the standard part is computed at every node but the enriched part is only computed at enriched nodes. Due to the non-polynomial form of the both MLS approximation and enrichment approximation function, ex- act integration of the weak form of the balance law is difficult. So, efficient numerical integration of the weak form may have an important role in the accuracy and computational efficiency of the method ሺNguyen et al. 2008; Ven- tura et al. 2009ሻ. Hence, weak form of the balance law can be numerically integrated by Gaussian quadrature rule over the finite volumes boundaries. Ventura et al. ሺ2009ሻ have demonstrated that the boundary integration scheme is much more accurate than the domain integration for the same computational cost and it is especially efficient when the integrand has discontinuous and/or non-polynomial form. 3.3 Treatment of additional unknowns Although in enriched Galerkin-based methods assembling all local equations to the global system leads to as many discrete equations as the number of unknowns, but; the proposed formulation faces to a different situation. Although in enriched Galerkin-based methods assembling all local equations to the global system leads to as many discrete equations as the number of unknowns, but; the proposed formulation faces to a different situation. Consider a discretized 2D problem with 1n enrichment nodes and 2n field nodes. There are two unknown fictitious nodal values associated with every field node and every enrichment node. The unknowns associated with the field nodes are fictitious displacement fields and with the enrichment nodes are fictitious nodal disconti- nuities amplitude parameters. Therefore, the vector of unknowns has   1 2 2 n n  entries. On the other hand, Equation ሺ28ሻ provides two discrete equations for each individual field node or corresponding finite volume, whereas there are no equations corresponding to enrichment nodes, because they have no displacement degrees of freedom. In order to satisfy the momentum balance law over the entire domain, Equation ሺ28ሻ should be ap- plied over all finite volumes. The resulted equations are assembled to form the global system equations. By doing so, we have only   2 2 n discrete equations. This is due to the stack form assembly procedure in finite volume methods. In order to have the required   1 2 n discrete equations, we need 1n additional local finite volumes. Thanks to the inherent flexibilities of the meshless FVM, arbitrarily overlapping local finite volumes can be defined, which resolves this difficulty. To this end, we define 1n new local finite volumes corresponding to the selected field nodes. By doing so, Equation ሺ28ሻ provides two additional discrete equations for each newly-defined local finite volume. These equations are independent from the previous set one. 4 NUMERICAL EXPERIMENTS AND RESULTS In order to assess the performance and accuracy of the proposed formulation, a set of numerical experiments are performed. The considered test problems are linear elastostatic within the range of infinitesimal defor- mations. A one-dimensional bi-material bar and also a two-dimensional infinite plate with a central inclusion both subjected to the prescribed displacement fields are studied. The results are compared with the corresponding solutions obtained by the standard meshless FVM in order to demonstrate the improvements achieved by the proposed formulation. In these experiments, both numerical methods utilized similar domain discretization, but different local approximations. The meshless FVM takes Equation ሺ16ሻ, whereas the proposed formulation takes Equation ሺ20ሻ as displacement trial function. The computations are performed using a domain discretization which is independent of the shape and location of the interface with the appropriate material property at each quadrature point. In order to further highlight the capability of the proposed formulation, some comparisons are also made with the results from the EFG method which has been introduced by Belytschko et al. ሺ1994ሻ. 3.2 Enrichment of approximation space ሺ31ሻ ሺ31ሻ Also, the element contribution to N and strain–displacement matrix B are as follows I J K std enr N N N é ù = ê ú ë û , I J K std enr B B B é ù = ê ú ë û , I J K std enr N N N é ù = ê ú ë û , I J K std enr N N N é ù = ê ú ë û , ሺ32ሻ I J K std enr B B B é ù = ê ú ë û , ሺ33ሻ ሺ32ሻ ሺ33ሻ with their entries being ( ) ( ) ( ) ( ) ( ) ( ) 0 0 , 0 0 I K J I J K std enr I K J x s r N N x s r f f y f f y é ù é ù ê ú ê ú = = ê ú ê ú ê ú ê ú ë û ë û , ሺ34ሻ ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) 0 0 ,1 ,1 ,1 0 , 0 ,2 ,2 ,2 ,2 ,1 ,2 ,2 ,1 ,1 I K J K J x s r s r I I J K K J K J B x B s r s r std enr I I K J K J K J K J x x s r s r s r s r f f y f y f f y f y f f f y f y f y f y + = = + + + é ù é ù ê ú ê ú ê ú ê ú ê ú ê ú ê ú ê ú ë û ë û .ሺ35ሻ ሺ34ሻ ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) 0 0 ,1 ,1 ,1 0 , 0 ,2 ,2 ,2 ,2 ,1 ,2 ,2 ,1 ,1 I K J K J x s r s r I I J K K J K J B x B s r s r std enr I I K J K J K J K J x x s r s r s r s r f f y f y f f y f y f f f y f y f y f y + = = + + + é ù é ù ê ú ê ú ê ú ê ú ê ú ê ú ê ú ê ú ë û ë û .ሺ Latin American Journal of Solids and Structures, 2018, 15ሺ2ሻ, e13 8/22 s, 2018, 15ሺ2ሻ, e13 8/22 Latin American Journal of Solids and Structures, 2018, 15ሺ2ሻ, e13 8/22 8/22 Abdullah Davoudi-Kia et al. 3.2 Enrichment of approximation space Abdullah Davoudi-Kia et al. An enriched meshless finite volume method for the modeling of material discontinuity problems in 2D elasticity Abdullah Davoudi Kia et al. An enriched meshless finite volume method for the modeling of material discontinuity problems in 2D elasticity Abdullah Davoudi Kia et al. An enriched meshless finite volume method for the modeling of material discontinuity problems in 2D elasticity Accordingly, the vector of unknowns, ˆU , is expressed as Latin American Journal of Solids and Structures, 2018, 15ሺ2ሻ, e13 4.1 1D Bi-material bar Consider a one-dimensional bar of length L and constant cross sectional area, A, composed of two different materials, i.e. 1 2 . Young's moduli in   1 0,   and   2 , L  are denoted by 1E and 2 E , respec- tively. There is a material discontinuity at x . The bar is fixed at one end and the other end is subjected to a prescribed displacement u . The problem is depicted in Figure 4. Latin American Journal of Solids and Structures, 2018, 15ሺ2ሻ, e13 9/22 Abdullah Davoudi-Kia et al. Abdullah Davoudi-Kia et al. An enriched meshless finite volume method for the modeling of material discontinuity problems in 2D elasticity dullah Davoudi-Kia et al. enriched meshless finite volume method for the modeling of material discontinuity problems in 2D elasticity Abdullah Davoudi Kia et al. An enriched meshless finite volume method for the modeling of material discontinuity problems in 2D elasticity x = L Ω1 Ω2 x = Γ x = 0 u = 0 u = uത Figure 4: 1D bi-material bar. Problem description. The bar is composed of two different materials, i.e. 1 2 , with a material discontinuity at x . The bar is fixed at one end and the other end is subjected to a prescribed dis- placement u . Figure 4: 1D bi-material bar. Problem description. The bar is composed of two different materials, i.e. 1 2 , with a material discontinuity at x . The bar is fixed at one end and the other end is subjected to a prescribed dis- placement u . In the computations, let L ൌ 10, A ൌ 1,  ൌ 5, 1E ൌ 1, 2E ൌ 0.5 and u ൌ 1. The computation is performed using 22 uniformly distributed field nodes. One-dimensional finite volumes are constructed with faces located at the middle of the field nodes. In the proposed formulation one enrichment node at the location of the material discontinuity is required. The domain discretization and corresponding finite volumes used in this test are shown in Figure 5. It should be noted that the computations of the EFG method are also performed by using the similar nodal arrangement applied in the standard meshless FVM. D i di i i d di fi i l Bl Figure 5: 1D bi-material bar. Domain discretization and corresponding finite volumes. Blue and red dots are denoted the enrichment node and field nodes, respectively. 1D local background finite volumes are illustrated by red lines. A linear basis and the cubic spline weight function are used in the MLS approximation. The essential bounda- ry conditions are imposed by the penalty method using a value of 1.00E൅07 as penalty parameter. The exact ana- lytical solutions for this problem are as follows ሺKrongauz and Belytschko 1998ሻ ( ) ( ) ( ) 2 1 2 1 2 5 1 5 5 5 5 E x x u x E x E x E E ì £ ïï = íï - + ³ + ïî , ሺ37ሻ ( ) ( ) 1 2 1 2 5 E E x for all x E E s = + . ሺ38ሻ ሺ37ሻ ሺ38ሻ The nodal displacement and stress values are obtained from the proposed formulation and compared with the exact analytical solutions in Figures 6-7, respectively. In addition, the numerical solutions obtained from the meshless FVM and EFG method are also shown in the same figures for comparison purposes. Figure 6 shows a very good agreement between the nodal displacements obtained from the proposed formulation and the exact analytical solution. In Figure 7, all these numerical methods result in a stress concentration in the vicinity of the material discontinuity. In order to compare local accuracy of these methods, a local relative error norm is defined as the maximum value of   2 / numerical exact exact     which is calculated at each node. According to Figure 7, the the maximum value of   2 / numerical exact exact     which is calculated at each node. Accordin as the maximum value of   2 / numerical exact exact     which is calculated at each node. According to Figure 7, the EFG method yields a local error of 4.95%, the meshless FVM produces the local error 47.53% where the proposed formulation improved it to 17.24%.   EFG method yields a local error of 4.95%, the meshless FVM produces the local error 47.53% where the proposed formulation improved it to 17.24%. EFG method yields a local error of 4.95%, the meshless FVM produces the local error 47.53% where the proposed formulation improved it to 17.24%. Latin American Journal of Solids and Structures, 2018, 15ሺ2ሻ, e13 Latin American Journal of Solids and Structures, 2018, 15ሺ2ሻ, e13 D i di i i d di fi i l Bl 10/22 Latin American Journal of Solids and Structures, 2018, 15ሺ2ሻ, e13 Latin American Journal of Solids and Structures, 2018, 15ሺ2ሻ, e13 Abdullah Davoudi-Kia et al. Abdullah Davoudi-Kia et al. An enriched meshless finite volume method for the modeling of material discontinuity problems in 2D elasticity An enriched meshless finite volume method for the modeling of material discontinuity problems in 2D elasticity An enriched meshless finite volume method for the modeling of material discontinuity problems in 2D elasticity Figure 6: 1D bi-material bar. Displacement variation along the bar. Figure 7: 1D bi-material bar. Stress variation along the bar. In order to further compare the proposed formulation with the meshless FVM, a convergence study ducted using 22, 34 and 50 uniformly distributed field nodes. As mentioned above, in these tests, the pro formulation employed one enrichment node at the location of the material discontinuity. The results of t merical nodal stresses are obtained and a magnified view around the interface is presented in Figure 8. It i that the maximum error in nodal values has remained roughly at the same level while the node number incr Figure 6: 1D bi-material bar. Displacement variation along the bar. Figure 6: 1D bi-material bar. Displacement variation along the bar. Figure 6: 1D bi-material bar. Displacement variation along the bar. Figure 7: 1D bi-material bar. Stress variation along the bar. Figure 7: 1D bi-material bar. Stress variation along the bar. Figure 7: 1D bi-material bar. Stress variation along the bar. In order to further compare the proposed formulation with the meshless FVM, a convergence study is con- ducted using 22, 34 and 50 uniformly distributed field nodes. As mentioned above, in these tests, the proposed formulation employed one enrichment node at the location of the material discontinuity. The results of the nu- merical nodal stresses are obtained and a magnified view around the interface is presented in Figure 8. It is clear that the maximum error in nodal values has remained roughly at the same level while the node number increases; therefore, the accuracy of the proposed formulation can't be achieved by the meshless FVM even with a dense nodal distribution. 11/22 Latin American Journal of Solids and Structures, 2018, 15ሺ2ሻ, e13 Abdullah Davoudi-Kia et al. Abdullah Davoudi-Kia et al. An enriched meshless finite volume method for the modeling of material discontinuity problems in 2D elasticity h Davoudi Kia et al. D i di i i d di fi i l Bl hed meshless finite volume method for the modeling of material discontinuity problems in 2D elasticity Figure 8: 1D bi-material bar. Convergence results. ሺaሻ Meshless FVM, ሺbሻ Proposed formulation. n the calculation of error, let  num and  exact represent approximated and exact analytical so vely. Then, the relative error norms in displacement and stress can be defined as follows Figure 8: 1D bi-material bar. Convergence results. ሺaሻ Meshless FVM, ሺbሻ Proposed formulation. Figure 8: 1D bi-material bar. Convergence results. ሺaሻ Meshless FVM, ሺbሻ Proposed formulation. In the calculation of error, let  num and  exact represent approximated and exact analytical solutions, re- spectively. Then, the relative error norms in displacement and stress can be defined as follows In the calculation of error, let  num and  exact represent approximated and exact analytical solutions, re- spectively. Then, the relative error norms in displacement and stress can be defined as follows ( ) ( ) 2 2 num exact d exact u u d e u d W W W W - = ò ò , ( ) ( ) 2 2 num exact exact d e d W s W s s W s W - = ò ò , ( ) ( ) 2 2 num exact d exact u u d e u d W W W W - = ò ò , ሺ39ሻ ( ) ( ) 2 2 num exact exact d e d W s W s s W s W - = ò ò , ሺ40ሻ ሺ39ሻ ሺ40ሻ Latin American Journal of Solids and Structures, 2018, 15ሺ2ሻ, e13 12/22 Abdullah Davoudi-Kia et al. Abdullah Davoudi Kia et al. An enriched meshless finite volume method for the modeling of material discontinuity problems in 2D elasticity where u and  are the displacement and stress at each node, respectively, and  is the computational domain of the bar. Table 1 represents the numerical results for the relative error norms obtained with 50 uniform field nodes and one enrichment node. where u and  are the displacement and stress at each node, respectively, and  is the computational domain of the bar. Table 1 represents the numerical results for the relative error norms obtained with 50 uniform field nodes and one enrichment node. Table 1: 1D bi-material bar. The relative error norms. D i di i i d di fi i l Bl Error norms Proposed formulation Meshless FVM EFG method de 0.37% 0.38% 0.05% es 3.12% 6.96% 1.03% It is clear that both of the proposed formulation and the meshless FVM give almost equal relative error norms in displacement. On the other hand, the proposed formulation yields to a more accurate solution for stress; the relative error norm in stress obtained from the proposed formulation is about half of the meshless FVM result. In addition, both error norms obtained from the EFG method are significantly smaller than those of other meth- ods. Although in this numerical experiment, the EFG method demonstrates good results, however, it should be mentioned that the observed performance of EFG method is due to the well-adjusted quadrature points which cannot be accommodated in 1D formulation of FV-based methods. It is worth mentioning that in the 1D formula- tion of FV-based methods quadrature points are restricted to be located at two endpoints of each 1D local finite volume. However, as demonstrated, the effect of this drawback has been reduced by the proposed formulation through the enrichment of approximation space. 4.2 2D infinite plate with an inclusion Consider a two-dimensional infinite plate with a circular inclusion subjected to a prescribed displacement field. In the numerical model, a bi-material square domain of length L with a centered circular inclusion of radius 1 r is considered, i.e. 1 2 . Young's modulus and Poisson's ratio of the inclusion 1  are denoted by 1E and 1  and those of the matrix 2  are denoted by 2E and 2 , respectively. There is a material discontinuity across the interface 1 ሺ 1 r r  ሻ where tractions and displacements are continuous across it. The problem is de- picted in Figure 9. Ω2    Ω1 r1 θ r Γ1 Γ2 Γ2: ur = r , uθ = 0 x2 x1 L L Figure 9: 2D infinite plate with an inclusion. Problem description. The plate is composed of a square domain of length L with a centered circular inclusion of radius 1 r , i.e. 1 2 Ω Ω Ω   , with a material discontinuity across the interface 1 Γ. The model is subjected to a prescribed displacement field on the external boundary 2 Γ . In the polar coordinate system, the essential boundary conditions on the external boundary 2 are given by Ω2    Ω1 r1 θ r Γ1 Γ2 Γ2: ur = r , uθ = 0 x2 x1 L Ω2    Ω1 r1 θ r Γ1 Γ2 Γ2: ur = r , uθ = 0 x2 x1 L L L Figure 9: 2D infinite plate with an inclusion. Problem description. The plate is composed of a square domain of length L with a centered circular inclusion of radius 1 r , i.e. 1 2 Ω Ω Ω   , with a material discontinuity across the interface 1 Γ. The model is subjected to a prescribed displacement field on the external boundary 2 Γ . In the polar coordinate system, the essential boundary conditions on the external boundary  i b L Figure 9: 2D infinite plate with an inclusion. Problem description. The plate is composed of a square domain of length L with a centered circular inclusion of radius 1 r , i.e. 1 2 Ω Ω Ω   , with a material discontinuity across the interface 1 Γ. The model is subjected to a prescribed displacement field on the external boundary 2 Γ . L Figure 9: 2D infinite plate with an inclusion. 4.2 2D infinite plate with an inclusion Problem description. The plate is composed of a square domain of length L with a centered circular inclusion of radius 1 r , i.e. 1 2 Ω Ω Ω   , with a material discontinuity across the interface 1 Γ. with a centered circular inclusion of radius 1 r , i.e. 1 2 Ω Ω Ω   , with a material discontinuity across the interface 1 Γ The model is subjected to a prescribed displacement field on the external boundary 2 Γ . In the polar coordinate system, the essential boundary conditions on the external boundary Latin American Journal of Solids and Structures, 2018, 15ሺ2ሻ, e13 Latin American Journal of Solids and Structures, 2018, 15ሺ2ሻ, e13 13/22 Abdullah Davoudi-Kia et al. Abdullah Davoudi-Kia et al. An enriched meshless finite volume method for the modeling of material discontinuity problems in 2D elasticity Abdullah Davoudi-Kia et al. An enriched meshless finite volume method for the modeling of material discontinuity problems in 2D elasticity , 0 ru r uq = = . ሺ41ሻ ons, L ൌ 2, 1r ൌ 0.4, 1E ൌ 2, 1  ൌ 0.2, 2E ൌ 10, 2 ൌ 0.3 and the plane strain state are as- In the computations, L ൌ 2, 1r ൌ 0.4, 1E ൌ 2, 1  ൌ 0.2, 2E ൌ 10, 2 ൌ 0.3 and the plane strain state are as- sumed. The computation is made using 452 field nodes. The proposed formulation added 36 enrichment nodes along the line of material discontinuity. The Voronoi tessellation is employed to construct finite volumes. The domain discretization and corresponding finite volumes used in this test are shown in Figure 10. It should be not- ed that similar nodal arrangement used in the standard meshless FVM is also utilized in the EFG method. In the computations, L ൌ 2, 1r ൌ 0.4, 1E ൌ 2, 1  ൌ 0.2, 2E ൌ 10, 2 ൌ 0.3 and the plane strain state are as- sumed. The computation is made using 452 field nodes. The proposed formulation added 36 enrichment nodes along the line of material discontinuity. The Voronoi tessellation is employed to construct finite volumes. The domain discretization and corresponding finite volumes used in this test are shown in Figure 10. It should be not- ed that similar nodal arrangement used in the standard meshless FVM is also utilized in the EFG method. 4.2 2D infinite plate with an inclusion 2001ሻ 2 2 1 2 2 1 1 2 2 1 1 0 r L L r r r r r u L L r r r L r r b b ìéæ ö ù ï ÷ ç ïê ú ÷ ç ï - + £ £ ÷ çê ú ï ÷ ç ÷ ï è ø ïê ú ë û = íæ ö ï ÷ ïç ÷ ïç - + £ £ ÷ ïç ÷÷ ç ïè ø ïî , 2 2 1 2 2 1 1 2 2 1 1 0 r L L r r r r r u L L r r r L r r b b ìéæ ö ù ï ÷ ç ïê ú ÷ ç ï - + £ £ ÷ çê ú ï ÷ ç ÷ ï è ø ïê ú ë û = íæ ö ï ÷ ïç ÷ ïç - + £ £ ÷ ïç ÷÷ ç ïè ø ïî , ሺ42ሻ ሺ42ሻ 0 uq = , ሺ43 0 uq = , ሺ43ሻ 0 uq = , ሺ43ሻ 0 uq = , ሺ43ሻ and the exact analytical stress fields are given by the following equations where the shear stress field is zero ሺSukumar et al. 2001ሻ. and the exact analytical stress fields are given by the following equations where the shear stress field is zero ሺSukumar et al. 2001ሻ. 4.2 2D infinite plate with an inclusion A four-point Gaussian quadrature rule is applied in order to integrate the weak form of governing equation. A quadratic basis, circular domain of influence and the cubic spline weight function are used in the MLS approxima- tion. The essential boundary conditions are imposed by the penalty method using a value of 1.00E൅07 as penalty parameter. tion. The essential boundary conditions are imposed by the penalty method using a value of 1.00E൅07 as pena parameter. ሺaሻ ሺbሻ Figure 10: 2D infinite plate with an inclusion Domain discretization and corresponding finite volumes Blue and re ሺaሻ ሺbሻ Figure 10: 2D infinite plate with an inclusion. Domain discretization and corresponding finite volumes. Blue and red dots are denoted the enrichment nodes and field nodes, respectively. 1D and 2D local background finite volumes are illustrated by blue and red lines, respectively. Due to symmetry, only a quarter of the domain is shown. ሺaሻ Meshless FVM, ሺbሻ Proposed formulation. ሺaሻ ሺbሻ ሺaሻ ሺbሻ ሺሻ ሺbሻ ሺbሻ Figure 10: 2D infinite plate with an inclusion. Domain discretization and corresponding finite volumes. Blue and red dots are denoted the enrichment nodes and field nodes, respectively. 1D and 2D local background finite volumes are illustrated by blue and red lines, respectively. Due to symmetry, only a quarter of the domain is shown. ሺaሻ Meshless FVM, ሺbሻ Proposed formulation. Latin American Journal of Solids and Structures, 2018, 15ሺ2ሻ, e13 Latin American Journal of Solids and Structures, 2018, 15ሺ2ሻ, e13 14/22 Abdullah Davoudi-Kia et al. Abdullah Davoudi-Kia et al. Abdullah Davoudi-Kia et al. An enriched meshless finite volume method for the modeling of material discontinuity problems in 2D elasticity In the polar coordinate system, the exact analytical solution for displacements is as follows ሺSukumar et al. 2001ሻ In the polar coordinate system, the exact analytical solution for displacements is as follows ሺSukumar et al. 4.2 2D infinite plate with an inclusion ( ) ( ) 2 2 1 2 2 1 1 2 2 1 2 2 1 2 2 0 1 2 2 rr L L r r r r L L r r L r r b m l s b m lb ìéæ ö ù ï ÷ ç ïê ú ÷ ç ï - + + £ £ ÷ çê ú ï ÷ ç ÷ ï è ø ïê ú ë û = íéæ ö ù ï ÷ ï çê ú ÷ ï ç + - + £ £ ÷ ï çê ú ÷÷ ç ï è ø ê ú ïîë û , ሺ44ሻ ( ) ( ) 2 2 1 2 2 1 1 2 2 1 2 2 1 2 2 0 1 2 2 L L r r r r L L r r L r r qq b m l s b m lb ìéæ ö ù ï ÷ ç ïê ú ÷ ç ï - + + £ £ ÷ çê ú ï ÷ ç ÷ ï è ø ïê ú ë û = íéæ ö ù ï ÷ ï çê ú ÷ ï ç - + + £ £ ÷ ï çê ú ÷÷ ç ï è ø ê ú ïîë û , ሺ45ሻ ሺ44ሻ ( ) ( ) 2 2 1 2 2 1 1 2 2 1 2 2 1 2 2 0 1 2 2 L L r r r r L L r r L r r b m l b m lb ìéæ ö ù ï ÷ ç ïê ú ÷ ç ï - + + £ £ ÷ çê ú ï ÷ ç ÷ ï è ø ïê ú ë û = íéæ ö ù ï ÷ ï çê ú ÷ ï ç - + + £ £ ÷ ï çê ú ÷÷ ç ï è ø ê ú ïîë û , ሺ45ሻ ሺ45ሻ where and are appropriate Lame constants at each material and is defined as where where where  and  are appropriate Lame constants at each material and  is defined as ( ) ( ) ( )( ) 2 1 1 2 2 2 2 2 2 2 1 1 1 1 2 L r L r L l m m b l m l m m + + = + + + - + . 4.2 2D infinite plate with an inclusion ሺ46ሻ ሺ46ሻ The analytical solutions for this problem are given in the polar coordinate system, in which they can be trans- formed to the Cartesian coordinate system using equations provided in Appendix A. The analytical solutions for this problem are given in the polar coordinate system, in which they can be trans- formed to the Cartesian coordinate system using equations provided in Appendix A. The solutions obtained from the proposed formulation, meshless FVM, EFG method and also exact analytical one are shown in Figures 11-12 for the horizontal displacement xu and the normal stress xx  both along the line y x  , respectively. A magnified view around the discontinuities is presented in Figure 12 for better observa- tion. Figure 11: 2D infinite plate with an inclusion. Variation of the x u along the line y x  . Figure 11: 2D infinite plate with an inclusion. Variation of the x u along the line y x  . Latin American Journal of Solids and Structures, 2018, 15ሺ2ሻ, e13 Latin American Journal of Solids and Structures, 2018, 15ሺ2ሻ, e13 15/22 Abdullah Davoudi-Kia et al. An enriched meshless finite volume method for the modeling of material discontinuity problems in 2D elasticity Abdullah Davoudi-Kia et al. Abdullah Davoudi-Kia et al. An enriched meshless finite volume method for the modeling of material discontinuity problems in 2D elasticity Figure 12: 2D infinite plate with an inclusion. Variation of the xx  along the line y x  . A magnified around the dis- continuities view. Figure 12: 2D infinite plate with an inclusion. Variation of the xx  along the line y x  . A magnified around the di continuities view. Figure 12: 2D infinite plate with an inclusion. Variation of the xx  along the line y x  . A magnified around the dis- continuities view. It is clear from Figure 11 that the nodal displacement curve obtained from the proposed formulation agrees well with the exact analytical solution, especially in the vicinity of the material discontinuity. In addition, it is seen in Figure 12 that a significantly higher accuracy in the nodal stress curve is achieved by the proposed formulation as compared with the other meshless methods. The meshless FVM and EFG method fail to reproduce the expected jumps in the nodal stress curve. 4.2 2D infinite plate with an inclusion However, oscillations around the material discontinuity are vanished in the solu- tion obtained from the proposed formulation. Another notable observation is that normal stress xx  is constant within the inclusion. The L2 relative error norm of the predicted xx  inside the inclusion for meshless FVM and EFG method is 0.012 and 0.008, respective- ly, while the proposed formulation reduces it to 0.002. In the calculation of error, the numerical solutions are compared to the exact analytical ones. Let  num and  exact represent approximated numerical and exact analytical solutions, respectively, then the relative error norms in displacement and stress could be defined as follows ( ) ( ) . . num exact num exact d exact exact u u u u d e u u d W W W W - - = ò ò , ሺ47ሻ ( ) ( ) . . num exact num exact exact exact d e d W s W s s s s W s s W - - = ò ò , ሺ48ሻ ( ) ( ) . . num exact num exact d exact exact u u u u d e u u d W W W W - - = ò ò , ሺ47ሻ ( ) ( ) . . num exact num exact exact exact d e d W s W s s s s W s s W - - = ò ò , ሺ48ሻ ሺ47ሻ ሺ48ሻ where u and σ are the vectors of displacement and stress at each point, respectively, and  is the computational domain of the problem. Table 2 represents the results for the relative error norms. Clearly, proposed formulation yields to more accurate results than both meshless FVM and EFG method. Table 2: 2D infinite plate with an inclusion. The relative error norms. Error norms Proposed formulation Meshless FVM EFG method de 0.48% 2.56% 1.50% es 1.96% 20.20% 8.05% Table 2: 2D infinite plate with an inclusion. The relative error norms. Latin American Journal of Solids and Structures, 2018, 15ሺ2ሻ, e13 Latin American Journal of Solids and Structures, 2018, 15ሺ2ሻ, e13 16/22 Abdullah Davoudi-Kia et al. Abdullah Davoudi Kia et al. An enriched meshless finite volume method for the modeling of material discontinuity problems in 2D elasticity In order to further evaluate the proposed formulation, a convergence study is conducted using 452, 1068 and 1548 field nodes. 4.2 2D infinite plate with an inclusion In the above mentioned tests, the proposed formulation employs 36, 84 and 140 enrichment nodes along the material interface, respectively. Computations of the meshless FVM are carried out using similar field nodes. The numerical results for the relative error norm in displacement predictions are plotted against the field node number in Figure 13. Figure 13: 2D infinite plate with an inclusion. Convergence results. Figure 13: 2D infinite plate with an inclusion. Convergence results. The proposed formulation yields to solutions with less error than meshless FVM and the error of the pro- posed formulation falls below 0.2% when the node number increases to 1688 ሺincluding 1548 field nodes and 140 enrichment nodesሻ. Abdullah Davoudi-Kia et al. Latin American Journal of Solids and Structures, 2018, 15ሺ2ሻ, e13 4.3 2D infinite plate with an extremely soft inclusion In order to demonstrate the capability of the proposed formulation in the extreme cases, above mentioned two-dimensional infinite plate is considered in a different state regarding to inclusion material property. In this case we let 1E ൌ 0.02 while 2E remains equal to 10. Similar geometry, boundary conditions, nodal arrangement and numerical techniques are used as presented in the previous test. The solutions for the horizontal displacement xu and the normal stress xx σ both along the line y x  , ob- tained from the proposed formulation, meshless FVM, EFG method and also exact analytical one are shown in Figures 14-15, respectively. 17/22 Latin American Journal of Solids and Structures, 2018, 15ሺ2ሻ, e13 Latin American Journal of Solids and Structures, 2018, 15ሺ2ሻ, e13 Abdullah Davoudi-Kia et al. Abdullah Davoudi-Kia et al. An enriched meshless finite volume method for the modeling of material discontinuity problems in 2D elasticity Figure 14: 2D infinite plate with an extremely soft inclusion. Variation of the x u along the line y x  . Figure 14: 2D infinite plate with an extremely soft inclusion. Variation of the x u along the line y x  . Figure 15: 2D infinite plate with an extremely soft inclusion. Variation of the xx  along the line y x  . A magnified around the discontinuities view. Figure 15: 2D infinite plate with an extremely soft inclusion. Variation of the xx  along the line y x  . A magnified around the discontinuities view. Figure 14 shows a very good agreement between the solution of the proposed formulation and the exact ana- lytical one. The kinks across the material discontinuities are accurately captured. In Figure 15, a magnified view around the discontinuities is presented. As can be seen in Figure 15, while nodal stress curves obtained from meshless FVM and EFG method show oscillations in the vicinity of the material discontinuity, they are alleviated in the proposed formulation results. As mentioned already, the normal stress xx σ is constant within the inclu- sion. The L2 relative error norm of the predicted xx σ inside the extremely soft inclusion for the meshless FVM, EFG method and proposed formulation are 0.019, 0.024 and 0.001, respectively. sion. The L2 relative error norm of the predicted xx σ inside the extremely soft inclusion for the meshless FVM, EFG method and proposed formulation are 0.019, 0.024 and 0.001, respectively. 5 CONCLUSIONS The aim of this paper is to propose a 2D formulation based on the meshless FVM framework for solving prob- lems with material discontinuity. In order to capture the discontinuity in the gradient fields, the MLS approxima- tion space has been enriched by a continuous function, having discontinuity in its derivatives. In contrast to the standard meshless methods, in the proposed formulation, the domain discretization task is done independently of the material discontinuities and also displacement and traction continuity constraints at the interface is automati- cally satisfied without additional treatment. Utilizing enrichment and also space-filling Voronoi-shaped local finite volumes, greatly simplifies the domain discretization task. Several numerical experiments have been performed. The comparisons with the analytical solutions have shown an excellent agreement in both displacement and stress fields predictions. It has demonstrated that the proposed formulation could alleviate the expecting oscillations in derivative fields in the vicinity of the material discontinuities appeared in the standard meshless methods. The formulation has been also successfully applied to the case of extremely soft inclusion problem. In comparison with the Galerkin-based methods, the proposed for- mulation is particularly attractive as it employs boundary integration over the finite volumes boundaries instead of domain integration. It should be mentioned that, as stated in Ventura et al. ሺ2009ሻ, the boundary integration scheme is much more accurate than the domain integration for the same computational cost and it is especially efficient when the integrand has non-polynomial form. g p y The results indicate the potential and promise feathers of the proposed formulation for studies of the me- chanics of heterogeneous media. 4.3 2D infinite plate with an extremely soft inclusion p p p y In addition, the results for the relative error norms calculated using Equations ሺ47ሻ-ሺ48ሻ are reported in Ta- ble 3. results for the relative error norms calculated using Equations ሺ47ሻ-ሺ48ሻ are reported in Ta- Table 3: 2D infinite plate with an extremely soft inclusion. The relative error norms. : 2D infinite plate with an extremely soft inclusion. The relative error norms. Table 3: 2D infinite plate with an extremely soft inclusion. The relative error norms. Error norms Proposed formulation Meshless FVM EFG method de 0.97% 3.77% 3.23% es 3.40% 31.09% 7.00% Again, the convergence study is conducted where three different tests with the same nodal arrangement as the problem of 2D infinite plate with an inclusion are considered. The numerical results for the relative error norm in displacement predictions are plotted against the field node number in Figure 16. It is clear that the pro- posed formulation yields to more accurate results as compared to the meshless FVM. Latin American Journal of Solids and Structures, 2018, 15ሺ2ሻ, e13 18/22 Abdullah Davoudi-Kia et al. Abdullah Davoudi-Kia et al. An enriched meshless finite volume method for the modeling of material discontinuity problems in 2D elasticity Figure 16: 2D infinite plate with an extremely soft inclusion. Convergence results. Figure 16: 2D infinite plate with an extremely soft inclusion. Convergence results. Latin American Journal of Solids and Structures, 2018, 15ሺ2ሻ, e13 Abdullah Davoudi-Kia et al. References An, X., Ma, G., Cai, Y., Zhu, H. ሺ2011ሻ. A new way to treat material discontinuities in the numerical manifold meth- od. Computer Methods in Applied Mechanics and Engineering, 200ሺ47ሻ: 3296-3308. An, X., Ma, G., Cai, Y., Zhu, H. ሺ2011ሻ. A new way to treat material discontinuities in the numerical manifold meth- od. Computer Methods in Applied Mechanics and Engineering, 200ሺ47ሻ: 3296-3308. Atluri, S., Han, Z., Rajendran, A. ሺ2004ሻ. A new implementation of the meshless finite volume method, through the MLPG “mixed” approach. CMES: Computer Modeling in Engineering & Sciences, 6ሺ6ሻ: 491-513. Atluri, S., Han, Z., Rajendran, A. ሺ2004ሻ. A new implementation of the meshless finite volume method, through the MLPG “mixed” approach. CMES: Computer Modeling in Engineering & Sciences, 6ሺ6ሻ: 491-513. Atluri, S.N., Shen, S. ሺ2002ሻ. The Meshless Local Petrov-Galerkin ሺMLPGሻ Method: A Simple\ & Less-costly Alterna- tive to the Finite Element and Boundary Element Methods. CMES: Computer Modeling in Engineering & Sciences, 3ሺ1ሻ: 11-52. Atluri, S.N., Zhu, T. ሺ1998ሻ. A new meshless local Petrov-Galerkin ሺMLPGሻ approach in computational mechanics. Computational mechanics, 22ሺ2ሻ: 117-127. Batra, R., Porfiri, M., Spinello, D. ሺ2004ሻ. Treatment of material discontinuity in two meshless local Petrov- Galerkin ሺMLPGሻ formulations of axisymmetric transient heat conduction. International Journal for Numerical Methods in Engineering, 61ሺ14ሻ: 2461-2479. Latin American Journal of Solids and Structures, 2018, 15ሺ2ሻ, e13 19/22 Latin American Journal of Solids and Structures, 2018, 15ሺ2ሻ, e13 19/22 Abdullah Davoudi-Kia et al. An enriched meshless finite volume method for the modeling of material discontinuity problems in 2D elasticity Belytschko, T., Black, T. ሺ1999ሻ. Elastic crack growth in finite elements with minimal remeshing. International journal for numerical methods in engineering, 45ሺ5ሻ: 601-620. Belytschko, T., Krongauz, Y., Organ, D., Fleming, M., Krysl, P. ሺ1996ሻ. Meshless methods: an overview and recent developments. Computer methods in applied mechanics and engineering, 139ሺ1ሻ: 3-47. Belytschko, T., Lu, Y.Y., Gu, L. ሺ1994ሻ. Element-Free Galerkin Methods. International journal for numerical meth- ods in engineering, 37ሺ2ሻ: 229-256. Cardiff, P., Karač, A., Ivanković, A. ሺ2014ሻ. A large strain finite volume method for orthotropic bodies with general material orientations. Computer Methods in Applied Mechanics and Engineering, 268 318-335. Cavalcante, M.A., Marques, S.P., Pindera, M.-J. ሺ2007ሻ. Parametric formulation of the finite-volume theory for func- tionally graded materials—part I: analysis. Journal of Applied Mechanics, 74ሺ5ሻ: 935-945. Cavalcante, M.A., Pindera, M.-J. ሺ2016ሻ. Generalized FVDAM theory for elastic–plastic periodic materials. Interna- tional Journal of Plasticity, 77 90-117. Cordes, L., Moran, B. ሺ1996ሻ. Latin American Journal of Solids and Structures, 2018, 15ሺ2ሻ, e13 References Treatment of material discontinuity in the element-free Galerkin method. Computer Methods in Applied Mechanics and Engineering, 139ሺ1ሻ: 75-89. de Borst, R. ሺ2008ሻ. Challenges in computational materials science: multiple scales, multi-physics and evolving discontinuities. Computational Materials Science, 43ሺ1ሻ: 1-15. Ebrahimnejad, M., Fallah, N., Khoei, A. ሺ2014ሻ. New approximation functions in the meshless finite volume method for 2D elasticity problems. Engineering Analysis with Boundary Elements, 46 10-22. Ebrahimnejad, M., Fallah, N., Khoei, A. ሺ2015ሻ. Adaptive refinement in the meshless finite volume method for elas- ticity problems. Computers & Mathematics with Applications, 69ሺ12ሻ: 1420-1443. Ebrahimnejad, M., Fallah, N., Khoei, A.R. ሺ2017ሻ. Three types of meshless finite volume method for the analysis of two-dimensional elasticity problems. Computational and Applied Mathematics, 36ሺ2ሻ: 971-990. Escarpini Filho, R.d.S., Marques, S.P.C. ሺ2016ሻ. A model for homogenization of linear viscoelastic periodic compo- site materials with imperfect interface. Latin American Journal of Solids and Structures, 13ሺ14ሻ: 2706-2735. Fallah, N. ሺ2004ሻ. A cell vertex and cell centred finite volume method for plate bending analysis. Computer meth- ods in applied Mechanics and Engineering, 193ሺ33ሻ: 3457-3470. Fries, T.-P., Belytschko, T. ሺ2010ሻ. The extended/generalized finite element method: an overview of the method and its applications. International Journal for Numerical Methods in Engineering, 84ሺ3ሻ: 253-304. Gdoutos, E.E. ሺ2005ሻ. Fracture mechanics: an introduction. Springer Science & Business Media, Netherlands. Han, Z., Rajendran, A., Atluri, S. ሺ2005ሻ. Meshless Local Petrov-Galerkin ሺMLPGሻ Approaches for Solving Nonlinear Problems with Large Deformations and Rotations. CMES: Computer Modeling in Engineering & Sciences, 10ሺ1ሻ: 1- 12. Hosseini, S.M., Sladek, J., Sladek, V. ሺ2011ሻ. Meshless local Petrov–Galerkin method for coupled thermoelasticity analysis of a functionally graded thick hollow cylinder. Engineering Analysis with Boundary Elements, 35ሺ6ሻ: 827- 835. Hu, M., Wang, Y., Rutqvist, J. ሺ2015ሻ. An effective approach for modeling fluid flow in heterogeneous media using numerical manifold method. International Journal for Numerical Methods in Fluids, 77ሺ8ሻ: 459-476. Latin American Journal of Solids and Structures, 2018, 15ሺ2ሻ, e13 20/22 Latin American Journal of Solids and Structures, 2018, 15ሺ2ሻ, e13 20/22 Abdullah Davoudi-Kia et al. An enriched meshless finite volume method for the modeling of material discontinuity problems in 2D elasticity voudi-Kia et al. meshless finite volume method for the modeling of material discontinuity problems in 2D elasticity Krongauz, Y., Belytschko, T. ሺ1998ሻ. EFG approximation with discontinuous derivatives. International Journal for Numerical Methods in Engineering, 41ሺ7ሻ: 1215-1233. Krongauz, Y., Belytschko, T. ሺ1998ሻ. EFG approximation with discontinuous derivatives. Yoon, Y.-C., Song, J.-H. ሺ2014ሻ. Extended particle difference method for weak and strong discontinuity problems: part I. Derivation of the extended particle derivative approximation for the representation of weak and strong discontinuities. Computational Mechanics, 53ሺ6ሻ: 1087-1103. Latin American Journal of Solids and Structures, 2018, 15ሺ2ሻ, e13 References An enriched meshless finite volume method for the modeling of material discontinuity problems in 2D elasticity Appendix A Transformation of field variables Two-dimensional displacement and stress components in the polar coordinate system can be transformed to the Cartesian coordinate system through the equations provided here. The coordinate systems are illustrated in Figure A.1. Abdullah Davoudi-Kia et al. An enriched meshless finite volume method for the modeling of material discontinuity problems in 2D elasticity Latin American Journal of Solids and Structures, 2018, 15ሺ2ሻ, e13 References International Journal for Numerical Methods in Engineering, 41ሺ7ሻ: 1215-1233. Li, Q., Shen, S., Han, Z., Atluri, S. ሺ2003ሻ. Application of meshless local Petrov-Galerkin ሺMLPGሻ to problems with singularities, and material discontinuities, in 3-D elasticity. Computer Modeling in Engineering and Sciences, 4ሺ5ሻ: 571-586. Li, S., Hao, W., Liu, W.K. ሺ2000ሻ. Mesh-free simulations of shear banding in large deformation. International Jour- nal of solids and structures, 37ሺ48ሻ: 7185-7206. Liu, G.-R. ሺ2009ሻ. Meshfree methods: moving beyond the finite element method. CRC Press, Taylor & Francis, Boca Raton. Liu, G.-R., Gu, Y.-T. ሺ2005ሻ. An introduction to meshfree methods and their programming. Springer Science & Business Media, Netherlands. Melenk, J.M., Babuška, I. ሺ1996ሻ. The partition of unity finite element method: basic theory and applications. Com- puter methods in applied mechanics and engineering, 139ሺ1ሻ: 289-314. Moës, N., Cloirec, M., Cartraud, P., Remacle, J.-F. ሺ2003ሻ. A computational approach to handle complex microstruc- ture geometries. Computer methods in applied mechanics and engineering, 192ሺ28ሻ: 3163-3177. Nguyen, V.P., Rabczuk, T., Bordas, S., Duflot, M. ሺ2008ሻ. Meshless methods: a review and computer implementation aspects. Mathematics and computers in simulation, 79ሺ3ሻ: 763-813. Okabe, A., Boots, B., Sugihara, K., Chiu, S.N. ሺ2009ሻ. Spatial tessellations: concepts and applications of Voronoi dia- grams. John Wiley & Sons, England. Sladek, J., Sladek, V., Solek, P., Saez, A. ሺ2008ሻ. Dynamic 3D axisymmetric problems in continuously non- homogeneous piezoelectric solids. International Journal of Solids and Structures, 45ሺ16ሻ: 4523-4542. Soares, D., Sladek, V., Sladek, J. ሺ2012ሻ. Modified meshless local Petrov–Galerkin formulations for elastodynamics. International Journal for Numerical Methods in Engineering, 90ሺ12ሻ: 1508-1528. Sukumar, N., Chopp, D.L., Moës, N., Belytschko, T. ሺ2001ሻ. Modeling holes and inclusions by level sets in the ex- tended finite-element method. Computer methods in applied mechanics and engineering, 190ሺ46ሻ: 6183-6200. Taylor, G., Bailey, C., Cross, M. ሺ2003ሻ. A vertex-based finite volume method applied to non-linear material prob- lems in computational solid mechanics. International journal for numerical methods in engineering, 56ሺ4ሻ: 507- 529. Ventura, G., Gracie, R., Belytschko, T. ሺ2009ሻ. Fast integration and weight function blending in the extended finite element method. International Journal for Numerical Methods in Engineering, 77ሺ1ሻ: 1-29. Yoon, Y.-C., Song, J.-H. ሺ2014ሻ. Extended particle difference method for weak and strong discontinuity problems: part I. Derivation of the extended particle derivative approximation for the representation of weak and strong discontinuities. Computational Mechanics, 53ሺ6ሻ: 1087-1103. 21/22 Latin American Journal of Solids and Structures, 2018, 15ሺ2ሻ, e13 Abdullah Davoudi-Kia et al. Appendix A Transformation of field variables Displacement transformation Displacement transformation p cos sin x r u u uq q q = - ሺA.1ሻ sin cos y r u u uq q q = + ሺA.2ሻ Stress transformation ( ) ( ) ( ) 1 1 cos2 1 cos2 2 sin 2 2 x r r q q s q s q s q s é ù = + + - - ë û ሺA.3ሻ p cos sin x r u u uq q q = - ሺA.1ሻ sin cos y r u u uq q q = + ሺA.2ሻ Stress transformation cos sin x r u u uq q q = - ሺA.1ሻ sin cos y r u u uq q q = + ሺA.2ሻ Stress transformation cos sin x r u u uq q q = - cos sin x r u u uq q q = - sin cos y r u u uq q q = + sin cos y r u u uq q q = + ሺA.2ሻ Stress transformation ( ) ( ) ( ) 1 1 cos2 1 cos2 2 sin 2 2 x r r q q s q s q s q s é ù = + + - - ë û ሺA.3ሻ ( ) ( ) ( ) 1 1 cos2 1 cos2 2 sin2 2 y r r q q s q s q s q s é ù = - + + + ë û ሺA.4ሻ ( ) ( ) ( ) 1 sin 2 sin 2 2 cos2 2 xy r r q q s q s q s q s é ù = - + ë û ሺA.5ሻ ሺA.3ሻ ( ) ( ) ( ) 1 sin 2 sin 2 2 cos2 2 xy r r q q s q s q s q s é ù = - + ë û 22/22 Latin American Journal of Solids and Structures, 2018, 15ሺ2ሻ, e13 http://dx.doi.org/10.1590/1679-78255239 Erratum Erratum In the article An enriched meshless finite volume method for the modeling of material discontinuity problems in 2D elasticity, doi number: http://dx.doi.org/10.1590/1679-78254121, published in journal Latin American Journal of Solids and Structures, 15(2):e13, page 1: Where it reads: http://dx.doi.org/10.1590/1679-78255239 Erratum In the article An enriched meshless finite volume method for the modeling of material discontinuity problems in 2D elasticity, doi number: http://dx.doi.org/10.1590/1679-78254121, published in journal Latin American Journal of Solids and Structures, 15(2):e13, page 1: Where it reads: In the article An enriched meshless finite volume method for the modeling of material discontinuity problems in 2D elasticity, doi number: http://dx.doi.org/10.1590/1679-78254121, published in journal Latin American Journal of Solids and Structures, 15(2):e13, page 1: Where it reads: Abdullah Davoudi-Kia a N. Fallah a,b,* a Department of Civil Engineering, University of Guilan, P.O. Box 3756, Rasht, Iran. b Faculty of Civil Engineering, Babol Noshirvani University of Technology, Babol, Iran. [email protected] *[email protected] It should read: Abdullah Davoudi-Kia a N. Fallah a,b,* a Department of Civil Engineering, University of Guilan, P.O. Box 3756, Rasht, Iran. E-mail: [email protected] b Faculty of Civil Engineering, Babol Noshirvani University of Technology, Babol, Iran. E-mail: [email protected] *Corresponding author Appendix A Transformation of field variables Two-dimensional displacement and stress components in the polar coordinate system can be transformed to the Cartesian coordinate system through the equations provided here. The coordinate systems are illustrated in Figure A.1. Two-dimensional displacement and stress components in the polar coordinate system can be transformed to the Cartesian coordinate system through the equations provided here. The coordinate systems are illustrated in Figure A.1. x2 x1 θ r Figure A.1: Cartesian and polar coordinate systems. Figure A.1: Cartesian and polar coordinate systems. Abdullah Davoudi-Kia a Abdullah Davoudi-Kia a Abdullah Davoudi-Kia a N. Fallah a,b,* a Department of Civil Engineering, University of Guilan, P.O. Box 3756, Rasht, Iran. b Faculty of Civil Engineering, Babol Noshirvani University of Technology, Babol, Iran. b Faculty of Civil Engineering, Babol Noshirvani University of Technology, Babol, Iran. It should read: Abdullah Davoudi-Kia a N. Fallah a,b,* a Department of Civil Engineering, University of Guilan, P.O. Box 3756, Rasht, Iran. E-mail: [email protected] b Faculty of Civil Engineering, Babol Noshirvani University of Technology, Babol, Iran. E-mail: [email protected] *Corresponding author Latin American Journal of Solids and Structures, 2018, 15(9), e126 Latin American Journal of Solids and Structures, 2018, 15(9), e126 Latin American Journal of Solids and Structures, 2018, 15(9), e126 1/1 1/1

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Subjektive Belastung durch Sichtkontrollen bei Urinabgaben im Rahmen von Abstinenzüberprüfungen

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Rechtsmedizin Originalien Rechtsmedizin https://doi.org/10.1007/s00194-024-00694-9 Angenommen: 1. März 2024 © The Author(s) 2024 Subjektive Belastung durch Sichtkontrollen bei Urinabgaben im Rahmen von Abstinenzüberprüfungen A. Holzer1 · M. Graw2 · A. Hameder2 · M. Eppler2 · S. Schick2 1 2 Institut für Rechtsmedizin, Universitätsklinikum Essen, Universität Duisburg-Essen, Essen, Deutschland Institut für Rechtsmedizin, Ludwig-Maximilians-Universität München, München, Deutschland Zusammenfassung Hintergrund: Laut einem Beschluss des Bundesverfassungsgerichts (Aktenzeichen: 2 BvR 1630/21) stellt eine Urinabgabe unter Sichtkontrolle in einer Justizvollzugsanstalt einen schwerwiegenden Eingriff in das allgemeine Persönlichkeitsrecht dar, der die Intimsphäre eines Inhaftierten berührt. Auch bei Dopingkontrollen fühlen sich immer wieder Athleten durch das beobachtete Urinieren beeinträchtigt. Es liegen jedoch bislang kaum Daten vor, die die psychische Belastung einer Urinabgabe im Rahmen von Abstinenzüberprüfungen, z. B. bei Fahreignungsbegutachtungen oder Bewährungsauflagen, untersuchen. Material und Methoden: Es wurden 100 Personen (davon 84 % männlich), die sich in 5 Entnahmestellen in Bayern, Thüringen und Baden-Württemberg zur Abstinenzkontrolle mittels Urinprobe vorstellten, fragebogenbasiert zu ihrer subjektiven Wahrnehmung der Sichtkontrolle und dem Wunsch nach einem nicht näher definierten, kosten- und zeitintensiveren alternativen Verfahren befragt. Ergebnisse: Es gaben 37 % der Probanden (n = 31) und 44 % der Probandinnen (n = 7) an, die Sichtkontrollen nicht als unangenehm zu empfinden, wohingegen 12 % der männlichen (n = 10) und 31 % der weiblichen Untersuchten (n = 5) diese Methode als „sehr unangenehm“ oder sogar „unerträglich“ wahrnehmen. Während 75 % der Beteiligten kein alternatives Verfahren wünschen, sind 32 Personen bereit, einen zunächst begrenzten zeitlichen und/oder finanziellen Mehraufwand in ein solches Verfahren zu investieren. Diskussion: Die Ergebnisse der Untersuchung legen nahe, dass die Urinabgaben unter Sichtkontrollen mit keiner wesentlichen psychischen Belastung für 85 % der untersuchten Personen einhergehen und offenbar kein vermehrtes Interesse an einem mit Zeit- und Kostenmehraufwand verbundenen alternativen Verfahren vorliegt. Schlüsselwörter Probennahme · Urinproben · Kontrollprogramm · Manipulation · Fragebogen Einleitung QR-Code scannen & Beitrag online lesen Im Juli 2022 kam das Bundesverfassungsgericht zu dem Ergebnis, dass eine Urinabgabe unter Sichtkontrolle das allgemeine Persönlichkeitsrecht verletzt (Aktenzeichen: 2 BvR 1630/21). Ein Inhaftierter einer Justizvollzugsanstalt (JVA) hatte Beschwerde eingelegt, da er die Entblößung seines Genitales im Zuge von Drogenscree- nings gemäß § 65 Abs. 1 StVollzG NRW als „entwürdigend und beschämend“ empfand [5]. Die Abgabe einer Urinprobe unter Sicht wird inverschiedenenBereichengefordert. Neben der Überprüfung einer Alkohol-/ Drogen-/Medikamentenabstinenz, u. a. im Zusammenhang mit einer Fahreignungsbegutachtung oder mit Bewährungsauflagen, wird sie bei Dopingkontrollen im Rechtsmedizin 1 Originalien Abb. 1 8 Grad der Belastung durch die Sichtkontrolle. Zur besseren Veranschaulichung wurden folgende Gruppengebildet:diejenigen,die das Verfahrennicht odernurleicht unangenehm empfanden, wurden der Gruppe „eher nicht unangenehm“ zugeordnet; diejenigen, die das Verfahren als sehr unangenehm oder unerträglich wahrnehmen, wurden der Gruppe „sehr unangenehm“ zugeordnet, p = 0,047 Leistungssport oder zur Feststellung von Suchtmittelkonsum, u. a. in JVA, durchgeführt. Ziel der Kontrollen ist es, Suchtmittelmissbrauch bzw. den Gebrauch von körperfremden Substanzen zur Leistungssteigerung aufzudecken, wobei sich Urin aufgrund der längeren Nachweisdauer und der weniger invasiven Probengewinnung als Probenmaterial besser eignet als Blut [3, 11]. Um etwaige positive Untersuchungsergebnisse zu verschleiern, ist der Versuch einer Probenmanipulation durch die Betroffenen denkbar. Um Manipulationen möglichst zu vermeiden, wird im forensischen Setting von den anerkannten Untersuchungsstellen eine Urinabgabe unter Sichtkontrolle gefordert, wodurch gewährleistet werden soll, dass die Probe eindeutig einem Probanden zugeordnet und die Zusammensetzung der Probe nachträglich nicht verändert werden kann. Der Einsatz von sog. Markerverfahren, bei denen bestimmte Stoffe (sog. Marker) nach dem Zufallsprinzip vom Probanden vor der Uringewinnung eingenommen und in der Urinprobe bestimmt werden müssen, ist nach Auffassung der Gesellschaft für Toxikologische und Forensische Chemie (GTFCh), der Deutschen Gesellschaft für Verkehrsmedizin (DGVM), der Deutschen Gesellschaft für Verkehrspsychologie (DGVP) und der Deutschen Gesellschaft für Rechtsmedizin (DGRM) nicht geeignet, Manipulationen 2 Rechtsmedizin auszuschließen und Sichtkontrollen zu ersetzen [14]. Franz et al. beobachteten jährlich bis zu 8 Manipulationsversuche bei ca. 7000 Urinabgaben zu Abstinenzkontrollzwecken [13], während in der Arbeit von Kluge et al. Täuschungen in 6 der 550 untersuchten Urinproben zur Abstinenzkontrolle identifiziert werden konnten [17]. Auch bei Dopingkontrollen wird wiederholt von Manipulationen der Urinproben berichtet, wie beispielsweise im Jahr 1992, als die Leichtathletinnen Katrin Krabbe, Grit Breuer und Silke Möller jeweils identische Urinproben vorwiesen [8]. Die Möglichkeiten der Manipulation sind mannigfaltig und umfassen neben Methoden der In-vivo-Probenverfälschung – z. B. durch exzessive Flüssigkeitsaufnahme oder Einnahme von Diuretika zur Verdünnung des Urins – auch in vitro durchgeführte Täuschungsversuche durch Beimengung von Fremdsubstanzen nach der Blasenentleerung wie beispielsweise Essig, Waschmittel oder anderen Chemikalien, um eine Substanzdetektion in den chemisch-toxikologischen Analysen zu stören oder zu verhindern. Darüber hinaus kann der Urin durch Flüssigkeiten wie beispielsweise Fremd- oder „Fakeurin“ substituiert werden. Die Vorgehensweisen reichen in diesem Zusammenhang von Applikation der Fremdflüssigkeit durch Katheterisierung der Harnblase im Vorfeld der Probennahme bis hin zu kommerziell verfügbaren Penisprothesen, wie z. B. „The Whizzinator“, der in verschiedenen Hauttönen und mit einem Heizkissen zur Erwärmung einer als Urin vorgewiesenen Fremdflüssigkeit auf Körpertemperatur erhältlich ist [9, 16, 19]. Um Täuschungsversuche möglichst auszuschließen, wird als Standardverfahren bei Urinkontrollen zur Alkohol-/ Drogen-/Medikamentenabstinenz im forensischen Setting eine Sichtkontrolle in Anlehnung an die Bestimmungen der World Anti-Doping Agency (WADA) [6] und der Stiftung Nationale Anti Doping Agentur Deutschland (NADA) [4] gefordert. Die Vorgaben sind in den Beurteilungskriterien zur Fahreignungsbegutachtung niedergelegt und beinhalten u. a., dass für die Kontrollperson eine ungehinderte Sicht auf die Körperaustrittsöffnung während der Probenabgabe gesichert sein muss, was zwingend mit einer Freilegung des Genitales und der Unterarme verbunden [3]. Vorgaben für bauliche Vorrichtungen in den Entnahmestellen existieren zwar nicht, es muss jedoch gewährleistet sein, dass die Toiletten für beide Geschlechter so ausgestattet sind, dass die Gewinnung der Urinprobe direkt oder indirekt (z. B. durch geneigte Spiegel) kontrolliert werden kann [2]. Laut Bundesverfassungsgericht stellen beaufsichtigte und mit einer Entkleidung verbundene Urinprobenahmen einen schwerwiegenden Eingriff in das allgemeine Persönlichkeitsrecht dar und berühren die Intimsphäre der betroffenen Personen und bedürfen daher besonderer Rücksichtnahme und Abwägung [5]. Zwar erfolgen die Abstinenzkontrollen teils im eigenen Interesse der Betroffenen – z. B. um einen Führerschein wiederzuerlangen oder der Vollstreckung einer Strafe zu entgehen –, allerdings ist nicht davon auszugehen, dass sich hierdurch das Schamgefühl der Probanden und Probandinnen reduziert. Die psychischen Auswirkungen einer beobachteten Urinprobenabgabe auf die Betroffenen wurden bislang lediglich im Kontext von Dopingtests, die jedoch für die Athleten verpflichtend sind, um an Wettkämpfen teilnehmen zu dürfen, untersucht. Die negativen Wahrnehmungen reichen hierbei Abb. 2 9 Wunsch nach einem alternativen Verfahren unter Berücksichtigung des Grades der empfundenen Belastung, p < 0,001 von „peinlich berührt“ über „gedemütigt“ [7] bis hin zur Verletzung der persönlichen Integrität [9] und können im ungünstigsten Fall einen psychogenen Harnverhalt (Paruresis) zur Folge haben [9, 10, 15]. Alternative Methoden zur Sichtkontrolle existieren bislang kaum. Im Zuge der Pandemie wurden virtuelle, kontaktlose Verfahren z. B. via Videotelefonie [12], die jedoch bislang ebenso wenig in den Bestimmungen der WADA oder der Fahreignungsbegutachtung Berücksichtigung fanden wie sog. Markerverfahren, erprobt [1, 14]. Ziel der Erhebung war es, einen Einblick zu gewinnen, wie beeinträchtigt sich Betroffene durch die Sichtkontrollen bei Abstinenzüberprüfungen fühlen, ob ein grundsätzliches Interesse an alternativen Verfahren besteht und welche Mehrkosten die Betroffenen bereit wären, hierfür in Kauf zu nehmen. Methodik Zur Abschätzung der individuellen Belastung einer Sichtkontrolle wurden Probandinnen und Probanden, die 2019 an einem Abstinenzkontrollprogramm mit Urinscreenings in insgesamt 5 Entnahmestellen in Bayern, Baden-Württemberg und Thüringen teilnahmen, nach dem Zufallsprinzip unter Verwendung eines standardisierten Fragebogens untersucht. Der Fragebogen wurde von den Autoren entwickelt und umfasst neben soziodemografischen Variablen wie Alter und Geschlecht den Grund der Probennahme. Die teilnehmenden Personen wurden gebeten, anhand einer 4-stufigen Skala einzuschätzen, wie unangenehm sie die praktizierte Abgabe der Urinprobe empfinden, wobei die 4-stufige Skala von „nicht unangenehm“ über „etwas unangenehm“ und „sehr unangenehm“ bis zu „unerträglich“ reichte. Zudem sollten sie abwägen, ob sie an nicht näher definierten, alternativen Verfahren interessiert wären, die die Urinprobenahme unter Beobachtung zwar ersetzen würden, jedoch mit einem finanziellen und zeitlichen Mehraufwand verbunden wären. Bei Interesse an einem alternativen Verfahren sollte in einer offenen Frage der maximale finanzielle und zeitliche Zusatzaufwand, den sie zu tragen bereit wären, angeben werden. Die Teilnahme an der Umfrage war für die Probandinnen und Probanden freiwillig, verbunden mit der Aufklärung, dass hierdurch weder Vor- noch Nachteile für sie entstehen. Von den Entnahmestellen wurden in der zweiten Jahreshälfte 100 vollständig ausgefüllte Fragebogen zurückgeschickt, was einer Rücklaufquote von 100 % entspricht. Der Fragebogen wurde anonymisiert erfasst und die Einträge zunächst deskriptiv ausgewertet. Bivariat wurde analysiert, inwieweit sich Alter, Geschlecht, der Anlass der Urinprobe und das Ausmaß eines unangenehmen Gefühls auf den Wunsch nach einer Alternative auswirken; zusätzlich wurden dafür auch die Einträge „sehr“ und „unerträglich“ unangenehm zur Kategorie „sehr unangenehm“ und die Einträge „nicht“ und „wenig“ unangenehm zu „eher nicht unangenehm“ zusammengefasst. Es wurden Kreuztabellen erstellt und der Chi2-Test angewendet, für das Alter wurde mittels Mann-Whitney-UTest verglichen. Die Assoziation zwischen dem Ausmaß des unangenehmen Gefühls und Geschlecht wurde ebenso überprüft. Für die Fälle mit Wunsch nach Alternativen wurde die Höhe der maximalen Mehrkosten in Euro und des maximalen Zeitaufwands in Minuten dafür nach Ausmaß des unangenehmen Gefühls und nach Geschlecht getrennt ausgewertet. Die Statistik und die Diagramme wurden mit IBM SPSS Version 23 erstellt. Boxplots stellen als Box den Bereich vom 25. bis zum 75. Perzentilwert dar, der Median wird angezeigt. DieWhiskers dokumentierendenhöchsten bzw. niedrigsten Wert, der innerhalb des 1,5-fachen Interquartilsbereichs (IQR) ab der oberen bzw. ab der unteren Boxgrenze in den Daten auftritt. Ausreisser liegen zwischen dem 1,5-fachen und dem dreifachen IQR und werden mit einem Kreis gekennzeichnet, Extremwerte liegen außerhalb des dreifachen IQR und werden mit einem Sternchen markiert. ® Rechtsmedizin 3 Originalien Abb. 3 9 Höhe des maximalen finanziellen (p = 0,098) (a) und zeitlichen (b) Mehraufwandes (p = 0,894) derjenigen, die ein alternatives Verfahren befürworten unter Berücksichtigung des Grads der empfundenen Belastung durch die Sichtkontrollen Ergebnisse Das Untersuchungskollektiv umfasste überwiegend männliche Probanden (n = 84, 84 %); der Altersdurchschnitt betrug bei ihnen 36 Jahre (Range 18 bis 62 Jahre, Median 35 Jahre); die Frauen waren tendenziell etwas älter (23 bis 67 Jahre, Median 41,5 Jahre). Es unterzogen sich 67 Probandinnen und Probanden (67 %) einem Abstinenzkontrollprogramm im Rahmen einer Fahreignungsbegutachtung, und in 26 Fällen (26 %, darunter eine Frau) wurden Urinkontrollen im Zusammenhang von Bewährungsauflagen 4 Rechtsmedizin durchgeführt. Dass eine Urinprobe sowohl wegen einer Bewährungsauflage als auch wegen einer Fahreignungsbegutachtung gefordert sei, kreuzten 3 männliche Teilnehmer (3 %) an, während 4 männliche Probanden sonstige Gründe wie beispielsweise Führungsaufsicht oder eine freiwillige Untersuchung angaben. Es gaben 38 % der Probandinnen und Probanden an, keine subjektive Belastung durch eine Urinabgabe unter Sichtkontrolle zu empfinden. Knapp die Hälfte des Untersuchungskollektivs (47 %) fühlte sich durch das Urinieren unter Beobachtung leicht beeinträchtigt, wohingegen das Vor- gehen für 12 der untersuchten Personen (12 %) sehr unangenehm und für 3 (3 %) gar unerträglich war. Zwar nahmen 43,8 % der untersuchten Frauen (n = 7) keine Belastung durch die praktizierte Entnahmemethode wahr, allerdings ist der Anteil derer, die die Urinkontrolle unter Beobachtung als „sehr unangenehm“ bzw. „unerträglich“ empfanden (Frauen: n = 5, Männer n = 10) gegenüber denjenigen, die dadurch keine oder nur eine geringe Belastung wahrnahmen (Frauen: n = 11, Männer: n = 74), bei den Frauen signifikant (p = 0,047) höher, . Abb. 1. Abb. 4 9 Höhe des maximalen finanziellen (p = 0,389) (a) und zeitlichen (b) (p = 0,495) Mehraufwandes derjenigen, die ein alternatives Verfahren befürworten, unter Berücksichtigung des Geschlechts An einem alternativen Verfahren ohne Sichtkontrolle waren insgesamt 25 Personen interessiert. Während keine altersspezifischen Unterschiede festzustellen sind (Mediane bei 35 bzw. 36 Jahren, p = 0,711), scheinen Frauen einer Alternative gegenüber eher aufgeschlossen zu sein (43,8 % (7 von 16) der Frauen im Gesamtkollektiv, 21,4 % der Männer (18 von 84), p = 0,059). Auch der Grad der individuellen Belastung durch die Sichtkontrollen scheint Einfluss auf das Interesse zu haben. So waren 13 von 15 (87 %) Teilnehmenden mit einer subjektiven starken bzw. unerträglichen Belastung an einem alternati- ven Verfahren interessiert, während lediglich 12 der 85 (14 %) Probanden und Probandinnen ohne subjektive Belastung den Wunsch nach einer Alternative äußerten (p < 0,001), . Abb. 2. Der Wunsch nach einer Alternative zeigt keine statistische Assoziation mit dem Grund der Urinabgabe (p = 0,759); die 3, die als Grund Bewährung und eine Fahreignungsbegutachtung angaben, wünschten alle keine Alternative. Die 25 Personen mit dem Wunsch nach einer Alternative würden im Median 30 min zusätzlich dafür in Kauf nehmen; die Spannweite reichte von keinem zusätzlichen Zeitaufwand bis zu 180 min; dafür würden sie zwischen 0 und 110  mehr ausgeben, im Median 20 . Ein Unterschied zwischen den Geschlechtern oder dem Grad der individuellen Belastung zeigte sich hierbei nicht, . Abb. 3 und 4. Nur ein Proband würde weder Zeit noch Geld investieren; zwei weitere wären bereit, Zeit (männlich: 10 min, weiblich: 30 min), aber kein Geld zu investieren; ein Proband würde nur 80  Zusatzkosten, aber keinen zeitlichen Mehraufwand tragen. Sieben weitere Personen (6 Männer und eine Frau) waren zwar an keiner Alternative interessiert, trugen aber Geld- und Zeitbeträge ein, die sie maximal zu investieren Rechtsmedizin 5 Originalien Tab. 1 Lfd. Nr. 42 71 87 Teilnehmende, die eine sehr hohe Belastung („unerträglich“) durch die Sichtkontrolle empfinden GeAlter Grund für AbstiInteresse an alternatiMax. finanzieller Max. zeitlicher schlecht nenzkontrolle vem Verfahren Mehraufwand Mehraufwand w 48 J Fahreignungsbegut- Ja 20  40 min achtung m 26 J Fahreignungsbegut- Nein – – achtung m 19 J Sonstiges Ja 40  30 min bereit wären. Diese lagen im Median bei 5  (0–150 ) bzw. 15 min (0–60 min). Diejenigen, die Interesse an einem alternativen Verfahren bekundeten, würden nicht signifikant mehr Geld oder Zeit investieren als diejenigen, die kein Interesse geltend machen, aber dennoch bereit wären, einen zeitlichen oder finanziellen Mehraufwand zu investieren. Statistisch signifikante alters- oder geschlechtsspezifische Unterschiede bzw. Unterschiede in der individuell wahrgenommenen Belastung durch die Sichtkontrolle sind zwar nicht zu verzeichnen, allerdings würden Männer sowie Probandinnen und Probanden, die die Sichtkontrolle als sehr unangenehm bis unerträglich empfinden, tendenziell auch mehr Zeit und/oder Geld aufwenden. Bei den 3 Teilnehmenden, die die Sichtkontrolle als „unerträglich“ empfanden, war lediglich bei 2 Personen der Wunsch nach einer Alternative, für die sie einen Mehraufwand von 30 min bzw. 40 min sowie 40  bzw. 20  investieren würden, zu erkennen (. Tab. 1). Diskussion Die Ergebnisse der Untersuchung legen nahe, dass Sichtkontrollen bei der Abgabe von Urinproben insbesondere für den Großteil der männlichen Betroffenen offenbar keine wesentliche psychische Belastung darstellen. Lediglich 15 % der befragten Personen empfanden die Sichtkontrollen als „sehr unangenehm“ oder gar „unerträglich“, während 38 % der Befragten keinerlei Unannehmlichkeiten angaben. Zu einem ähnlichen Ergebnis kamen bereits 2014 Elbe et al. [9], die dänische Leistungssportlerinnen und Leistungssportler im Rahmen einer Fragebogenstudie zu ihrer Einstellung zu Dopingtests befragten. Es gaben 15,3 % der befragten Athletinnen und Athleten an, dass durch die Be- 6 Rechtsmedizin obachtung beim Urinieren ihre persönliche Integrität verletzt werde, während sich 33,4 % der Sportlerinnen und Sportler wegen Schwierigkeiten beim Wasserlassen „gestresst“ fühlten. In Deutschland tätiges Doping-Kontrollpersonal vermutet in ca. 42 % der Kontrollen Verzögerungen bei der Urinabgabe, was es vornehmlich auf die psychische Belastung in der Kontrollsituation zurückführt [18]. Diese Vermutung konnten Elbe et al. bestätigen, indem 60 % der 222 befragten deutschsprachigen Sportlerinnen und Sportler angaben, mindestens einmal einen psychogenen Harnverhalt bei Dopingkontrollen erfahren zu haben [10]. Die höhere Belastung der Sportlerinnen und Sportler lässt sich möglicherweise dadurch erklären, dass Probandinnen und Probanden im Vorfeld der Abstinenzkontrolle das Probenmaterial (Haar oder Urin) selbst wählen können und in der Regel keine sportlichen Höchstleistungen vor dem Toilettengang erbringen müssen. Auch besteht bei einer erfolglosen Urinprobenabgabe nicht die Gefahr, dass die Testung als positiv gewertet wird, was ggf. mit dem Ende einer Sportkarriere verbunden sein kann [9], vielmehr kann bei einer Abstinenzkontrolle eine Probe u. U. auch wiederholt werden [3]. Dass die kontrollbedingte Belastung subjektiv sehr unterschiedlich wahrgenommen wird, unterstreicht eine Untersuchung unter nordamerikanischen Athletinnen und Athleten, von denen 71,4 % angaben, dass ein Dopingtest für sie „keine große Sache“ darstelle [7]. Im Gegensatz zu Untersuchungen unter Sportlerinnen und Sportlern [9, 18], die keine geschlechtsspezifischen Unterschiede feststellen konnten, empfanden die Frauen der gegenständlichen Untersuchung eine beobachtete Urinprobenahme unangenehmer als Männer. Anders als in den Dopingbestimmungen der WADA [6] und NADA [4] ist bei Urinkontrollen Sonstiges – – Vorschlag: Anwendung einer Kamera im Zusammenhang mit Fahreignungsbegutachtungen gemäß CTU-Kriterien keine geschlechtsidentische Kontrollperson gefordert [3], was zu einer unangenehmeren Atmosphäre für Frauen beitragen kann. Lediglich 25 der befragten Personen (davon 28 % weiblich) waren an einem alternativen Verfahren ohne Sichtkontrolle bei zusätzlichem finanziellen und/oder zeitlichen Mehraufwand interessiert. Das Ergebnis unserer Untersuchung suggeriert ein deutlich geringeres Bestreben nach alternativen Verfahren im Kontext von Abstinenzüberprüfungen als unter Sportlerinnen und Sportlern, die typischerweise die Kosten für die Dopingkontrollen nicht selbst tragen müssen. In der Arbeit von Elbe et al. wurde die Haltung hinsichtlich Markerverfahren im Rahmen von Dopingkontrollen unter Athletinnen und Athleten untersucht. Während ein Teil der Studienteilnehmenden seine Einstellung anhand eines Fragebogens theoretisch einschätzen sollte, unterzog sich ein anderer Teil der Studienteilnehmenden zunächst einer Urinkontrolle mittels Markerverfahren und wurde im Anschluss daran standardisiert befragt. 47,6 % der hypothetisch befragten und 85,3 % der die Alternative testenden und in Dopingtests erfahrenen Athletinnen und Athleten würden demnach ein Markerverfahren gegenüber den Sichtkontrollen bevorzugen [11]. Nur 2 der 3 Personen, die die Sichtkontrollen als „unerträglich“ wahrnahmen, waren einer Alternative gegenüber positiv aufgeschlossen, wobei der akzeptierte Mehraufwand nicht überdurchschnittlich war. Die gegenständliche Erhebung lässt somit vermuten, dass eine sehr hohe subjektive Belastung nicht zwingend mit einer erhöhten Bereitschaft für alternative Methoden einhergeht, oder dass in diesen Einzelfällen die finanziellen und zeitlichen Möglichkeiten eher begrenzt sind. Die eingangs erwähnte Entscheidung des Bundesverfassungsgerichts ist nur eingeschränkt auf unser Untersuchungskollektiv übertragbar, da die Voraussetzungen des § 65 StVollzG NRW mit einer Teilnahmeaneinem Abstinenzkontrollprogramm nicht vergleichbar sind. Die Richterinnen und Richter stellten zwar fest, dass staatliche Maßnahmen, bei denen sich Betroffene entkleiden müssen, einen schwerwiegenden Eingriff in das allgemeine Persönlichkeitsrecht darstellen. Da sie von besonderem Gewicht und im Strafvollzug nicht immer vermeidbar seien, habe der Gefangene Anspruch auf besondere Rücksichtnahme. Neben der Frequenz der anlasslosen Drogenscreenings wurde im Urteil u. a. auch das Versäumnis der JVA, dem Häftling eine Kapillarblutprobe als milderes Mittel anzubieten, kritisiert [5]. Diese Kritikpunkte treffen auf ein Abstinenzkontrollprogramm jedoch nicht vollumfänglich zu. Zwar erfolgt die Überprüfung der Abstinenz im Auftrag der Betroffenen selbst, auch ist die Anzahl der Proben gemäß der Richtlinien innerhalb eines bestimmten Zeitraums vorab festgelegt und die Teilnehmerinnen und Teilnehmer können zwischen einer Urin- und Haaranalytik wählen, allerdings begründen sich auch die forensischen Urinkontrollprogramme nicht gänzlich auf der Freiwilligkeit der Probanden und können bei Nichtkooperation mit teils erheblichen negativen Folgen für die Betroffenen (z. B. Widerruf einer Bewährung) einhergehen. Limitierung Bei 100 Teilnehmenden ist das Ergebnis der gegenständlichen Stichprobe nicht repräsentativ und bedarf der weiteren, umfassenderen Erhebung in einem größeren Untersuchungskollektiv. Da die psychogene Belastung einer Methode interund intraindividuell unterschiedlich ist und von multiplen Faktoren wie beispielsweise der Persönlichkeitsstruktur der Probanden und Probandinnen oder den individuellen Erfahrungen mit Urinkontrollen abhängt, wäre die Erhebung zusätzlicher, persönlichkeitsassoziierter Variablen in zukünftigen Untersuchungen anzustreben, um die Aussagekraft zu erhöhen. Fazit für die Praxis Die Ergebnisse der Befragung legen nahe, dass sich Probandinnen und Probanden, die sich aufgrund eines Abstinenznachweises einer Urinprobenabgabe unterziehen, durch die Sichtkontrolle nicht wesentlich belastet fühlten, wenngleich sich Frauen mit der Maßnahme unwohler fühlten als Männer. Ein alternatives Verfahren, das ohne Sichtkontrolle, jedoch mit einem finanziellen und/ oder zeitlichen Mehraufwand verbunden ist, befürworteten lediglich 21 % der befragten Männer und 44 % der Frauen. Ein dringender Handlungsbedarf hinsichtlich der Anpassung der gängigen Methoden zur Probengewinnung im Zusammenhang mit Abstinenzkontrollen ist somit aus Sicht der überwiegend männlichen Betroffenen nicht zu erkennen. Korrespondenzadresse Dr. med. A. Holzer Institut für Rechtsmedizin, Universitätsklinikum Essen, Universität Duisburg-Essen Hufelandstr. 55, 45147 Essen, Deutschland [email protected] Funding. Open Access funding enabled and organized by Projekt DEAL. Datenverfügbarkeit. Die erhobenen Datensätze sind in anonymisierter Form beim korrespondierenden Autor auf Anfrage verfügbar. Einhaltung ethischer Richtlinien Interessenkonflikt. A. Holzer, M. Graw, A. Hameder, M. Eppler und S. Schick geben an, dass kein Interessenkonflikt besteht. Für diesen Beitrag wurden von den Autor/-innen keine Studien an Menschen oder Tieren durchgeführt. Für die aufgeführten Studien gelten die jeweils dort angegebenen ethischen Richtlinien. Open Access. Dieser Artikel wird unter der Creative Commons Namensnennung 4.0 International Lizenz veröffentlicht, welche die Nutzung, Vervielfältigung, Bearbeitung, Verbreitung und Wiedergabe in jeglichem Medium und Format erlaubt, sofern Sie den/die ursprünglichen Autor(en) und die Quelle ordnungsgemäß nennen, einen Link zur Creative Commons Lizenz beifügen und angeben, ob Änderungen vorgenommen wurden. Die in diesem Artikel enthaltenen Bilder und sonstiges Drittmaterial unterliegen ebenfalls der genannten Creative Commons Lizenz, sofern sich aus der Abbildungslegende nichts anderes ergibt. Sofern das betreffende Material nicht unter der genannten Creative Commons Lizenz steht und die betreffende Handlung nicht nach gesetzlichen Vorschriften erlaubt ist, ist für die oben aufgeführten Weiterverwendungen des Materials die Einwilligung des jeweiligen Rechteinhabers einzuholen. Weitere Details zur Lizenz entnehmen Sie bitte der Lizenzinformation auf http://creativecommons.org/ licenses/by/4.0/deed.de. Literatur 1. Bundesanstalt für Straßenwesen (BASt) (2017) Stellungnahme: Sichtkontrolle und Markerverfahren bei Urinabgaben im Zusammenhang mit der Fahreignungsbegutachtung. https://www.bast.de/DE/Verkehrssicherheit/ Fachthemen/U1-MPU/stellungnahme-marker. pdf?__blob=publicationFile&v = 2, aufgerufen am 5. Jan. 2024 2. Deutsche Gesellschaft für Verkehrsmedizin (DGVM) Häufige Fragen zu „Urteilsbildung in der Fahreignungsbegutachtung – Beurteilungskriterien“ https://dgvm-verkehrsmedizin.de/h aeufige-fragen-zu-den-beurteilungskriterien/, aufgerufen am 25. Febr. 2024 3. Deutsche Gesellschaft für Verkehrspsychologie (DGVP) & Deutsche Gesellschaft für Verkehrsmedizin (DGVM) (2022) Urteilsbildung in der Fahreignungsbegutachtung Beurteilungskriterien, 4. Aufl. Kirschbaum Verlag, Bonn 4. Nationale Anti Doping Agentur Deutschland (NADA) (2023) Standard für Dopingkontrollen und Ermittlungen. https://www.nada.de/ fileadmin/nada/SERVICE/Downloads/Standards/ 2023_Standard_fuer_Dopingkontrollen_und_ Ermittlungen_Version_8.pdf, aufgerufen am 5. Jan. 2024 5. Bundesverfassungsgericht (2022) Beschluss der 1. Kammer des Zweiten Senats vom 22. Juli. BvR 1630(21):2022–2022 6. World Anti-Doping Agency (WADA) (2023) International Standard for Testing and Investigations. https://www.wada-ama.org/sites/default/files/ 2022-12/isti_2023_w_annex_k_final_clean.pdf, aufgerufen am 5. Jan. 2024 7. Coombs RH, Coombs CJ (1991) The impact of drug testingonthemoraleandwell-beingofmandatory participants. Int J Addict 26:981–992 8. Dahlke K (2003) Analyse der Berichterstattung ausgesuchter Printmedien über die Dopingfälle Krabbe und Baumann. diplom.de 9. Elbe AM, Overbye M (2014) Urine doping controls: the athletes’ perspective. Int J Sport Policy Polit 6:227–240 10. Elbe AM, Schlegel MM, Brand R (2012) Psychogenic urine retention during doping controls: Consequences for elite athletes. Perform Enhanc Health 1:66–74 11. Elbe AM, Jensen SN, Elsborg P, Wetzke M, Woldemariam GA, Huppertz B, Keller R, Butch AW (2016) The Urine Marker Test: An Alternative Approach to Supervised Urine Collection for Doping Control. Sports Med 46:15–22 12. Fedoruk MN (2020) Virtual drug testing: redefining samplecollectioninaglobalpandemic.Bioanalysis 12:715–718 13. Franz S, Skopp G, Musshoff F (2020) Manipulation bei der Abgabe einer Urinprobe im Rahmen von Abstinenzprogrammen – Erfahrungen mit der Sichtkontrolle. Z Verkehrssicherheit 66:18–20 14. Gesellschaft für Toxikologische und Forensische Chemie (GTFCh) Gemeinsame Stellungnahme der Gesellschaft für Toxikologische und Forensische Chemie (GTFCh), der Deutschen Gesellschaft für Verkehrsmedizin (DGVM), der Deutschen Gesellschaft für Verkehrspsychologie (DGVP) und der Deutschen Gesellschaft Rechtsmedizin 7 Abstract für Rechtsmedizin (DGRM) zum Einsatz von Markerverfahren als Alternative zur Sichtkontrolle bei Urinkontrollen für Untersuchungen für die Beurteilung der Eignung zum Führen von Kraftfahrzeugen. https://www.gtfch.org/cms/ index.php/en/news/534-stellungnahme-zumeinsatz-von-markerverfahren-als-alternativezur-sichtkontrolle-bei-urinkontrollen, aufgerufen am 8. Jan. 2024 15. Hammelstein P (2002) Paruresis: Bemerkungen zur Ätiologie und verhaltenstherapeutischen Behandlung. Verhaltenstherapie 12:224–227 16. Jaffee WB, Trucco E, Levy S, Weiss RD (2007) Is this urine really negative? A systematic review of tampering methods in urine drug screening and testing. J Subst Abuse Treat 33:33–42 17. Kluge J, Rentzsch L, Remane D, Peters FT, Wissenbach DK (2018) Systematic investigations of novel validity parameters in urine drug testing and prevalence of urine adulteration in a two-year cohort. Drug Test Anal 10:1536–1542 18. Strahler K, Elbe AM (2007) Wollen, aber nicht Können-DasProblemDopingkontrolle. Leistungssport 37:35–38 19. Wissenbach DK, Steuer AE (2023) Advances in testing for sample manipulation in clinical and forensic toxicology—Part A: urine samples. Anal Bioanal Chem 415:5101–5115 Hinweis des Verlags. Der Verlag bleibt in Hinblick auf geografische Zuordnungen und Gebietsbezeichnungen in veröffentlichten Karten und Institutsadressen neutral. 8 Rechtsmedizin Subjective burden of urine sample collection under direct observation as part of drug testing Background: According to a decision by the Federal Constitutional Court (2 BvR 1630/21), directly observed urine sample collection in correctional facilities constitutes a serious infringement of the general personal rights, which affects the intimate sphere of an inmate. Athletes frequently feel burdened by the observed urination during doping tests, too; however, there are hardly any data available that examine the psychological stress of urine sample collection in the context of drug testing, e.g., for driving fitness assessment or probation conditions. Material and methods: A total of 100 subjects (84% male) who presented for urine drug monitoring at 5 collection points in Bavaria, Thuringia and Baden-Württemberg were asked via a questionnaire about their individual perception of the monitoring method and their desire for a not further specified alternative nonobservational but more cost-intensive and time-intensive procedure. Results: Overall, 37% of the male (n = 31) and 44% (n = 7) of the female participants stated that they did not feel any discomfort urinating under supervision, whereas 12% of the males (n = 10) and 31% of the females (n = 5) perceived this method as very unpleasant or unbearable. While 75% of the subjects did not desire an alternative procedure, 32 subjects were willing to invest additional time and/or money. Discussion: The results suggest that supervised urine sample collection is not associated with a significant burden for 85% of the subjects and that there is no increased interest in an alternative procedure associated with additional time and costs. Keywords Sampling · Urine screening · Substance control · Manipulation · Questionnaire 

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Female rap in Arab countries. The case of Mayam Mahmoud

Miscelánea de estudios árabes y hebraicos. Sección árabe-Islam/Miscelánea de estudios árabes y hebraicos.Sección árabe-Islam

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Recibido: 31/05/2021 Aceptado: 15/07/2021 DOI: https://doi.org/10.30827/meaharabe.v71.21485 Abstract: Female rappers in the Arab world have increased in both number and promi- nence in recent years. Yet, while there is extensive bibliography on female rap in the Western world, few studies have examined female rap in the Arab world. This paper repre- sents a preliminary approach to this phenomenon, offering a general overview of the fe- male rappers in various Arab countries and the different issues they address. The paper then goes on to analyze a corpus consisting of the texts of the young Egyptian rapper Ma- yam Mahmoud, who has become internationally famous, highlighting linguistic aspects as well as the themes. The topics preferred by female Arab rappers include political problems in their country and social themes such as the situation of women, their day-to-day reality, the struggle against sexism and solidarity among women. The language used is direct, straightforward and free of social conventions. These aspects are in line with “conscious” rap created by women around the world. Rap music has become a vehicle through which women seek to secure a share of the power, affirm their choices and build alternative vi- sions of their identity. Resumen: Las raperas en el mundo árabe han ido aumentando y ganando notoriedad en los últimos años. Si bien encontramos una extensa bibliografía sobre el rap femenino en el mundo occidental, existen pocos estudios sobre el rap femenino en el mundo árabe. Esta investigación constituye un acercamiento preliminar a este fenómeno, ofreciendo un pano- rama general de los nombres de las raperas en los distintos países árabes y los temas abor- dados. A continuación, el trabajo se centra en el análisis de un corpus formado por los tex- tos de la joven rapera egipcia Mayam Mahmoud, que se ha hecho famosa internacional- mente, y destaca tanto los aspectos lingüísticos como los temáticos. Los temas de las rape- ras árabes consisten en problemas políticos vinculados a su país y cuestiones sociales co- mo la condición de la mujer, la denuncia del sexismo, la realidad vivida a diario, la solida- ridad femenina. El lenguaje utilizado es directo, desafilado y libre de convenciones socia- les. Estos aspectos están en consonancia con el rap “consciente” femenino en el mundo. La música rap se convierte en un vehículo a través del cual las mujeres buscan asegurarse una parte del poder, afirmar sus elecciones y reconstruir visiones alternativas de su identidad. Key words: Female Arab rap. FEMALE RAP IN ARAB COUNTRIES. THE CASE OF MAYAM MAHMOUD Rap femenino en países árabes. El caso de Mayam Mahmoud Emanuela DE BLASIO [email protected] Universidad de Tuscia Recibido: 31/05/2021 Aceptado: 15/07/2021 DOI: https://doi.org/10.30827/meaharabe.v71.21485 INTRODUCTION In the Arab world the rap scene, although dominated by men, is not comple- tely hostile to the idea of the possibility of a female presence within the genre. Famous singers or rap groups such as DAM, Y Crew, Arabian Knightz or El Général do not contemplate misogynist or macho ideas in their music; on the con- trary, mixed collaborations are frequent, such as the one between Arabian Kni- ghtz and Shadia Mansour in the realization of the famous song il-Sigīn “The pri- soner”. We find attitudes of solidarity on the part of some male artists as in the lyric il-Ḥurriyye unta “Freedom is female” by DAM, in which the contribution of women in the Egyptian revolution is celebrated. Noteworthy is the case of the famous Moroccan rapper Muslim who was the first to make a song about the vio- lence suffered by women in Morocco: his text La la “No, no” (2019) caused a sensation among the public. In recent years, therefore, women have been making their way into the Arab music scene of hip hop, with all the difficulties due both to rap itself considered as an import and “rebellious” genre and to the difficulty of seeing this genre sui- table for a woman. Female artists use their performances as a platform to reject, destroy or re- construct alternative views of their identity. So rap music becomes a vehicle through which women seek to secure a share of power, affirm their choices and create spaces for themselves and other women. The panorama is quite varied, but most of the female artists make a socially and politically committed rap, addressing in their songs themes considered taboo or in any case linked to the condition of women. Studies on music and gender on western female rap are numerous, but re- search on female rap in the Arab world is scant if not non-existent. This work aims to make a contribution in this area, through an overview of the state of female rap in the Arab world and then through the examination of a speci- fic singer, the young Egyptian Mayam Mahmoud, a very interesting case for the issues addressed and for being one of the few artists to become internationally known, thanks to the Arabs Got Talent program. The singer's production is still small; in this work three songs, chosen for the relevance of their issues, have been analyzed. Recibido: 31/05/2021 Aceptado: 15/07/2021 DOI: https://doi.org/10.30827/meaharabe.v71.21485 Mayam Mahmoud. Sociolinguistics. Egyptian Arabic. Ara- bic language. Key words: Female Arab rap. Mayam Mahmoud. Sociolinguistics. Egyptian Arabic. Ara- bic language. Palabras clave: Rap árabe femenino. Mayam Mahmoud. Sociolingüística. Árabe egipcio. Idioma árabe. Idioma árabe. MEAH, SECCIÓN ÁRABE-ISLAM [ISSN 1696-5868, e-ISSN 2341-0906] 71 (2022), 85-108 86 EMANUELA DE BLASIO MEAH, SECCIÓN ÁRABE-ISLAM [ISSN 1696-5868, e-ISSN 2341-0906] 71 (2022), 85-108 MEAH, SECCIÓN ÁRABE-ISLAM [ISSN 1696-5868, e-ISSN 2341-0906] 71 (2022), 85-108 1. The bibliography is very extensive. Among the many studies we mention: Gaunt. “Translating double-dutch”; Koskoff. “An introduction to women, music, and culture”. 2. El-Tayeb. European others; Hill Collins. From Black power to hip hop. 3. For further information: Keyes. “Empowering self”. 4. Queen Kenya, a member of the Zulu Nation, was the first female MC to use the title “Queen”. 5. Term born from the fusion of the two English words black and exploitation: it was a genre of film that was born in the United States, in the early seventies, when many films were made at low cost ha- ving African Americans as their target audience. 6. Regarding studies about black lesbian identity and culture: Walker. In search of our mothers' gar- dens; Omosupe. “Black/lesbian/bulldagger”. 7. Ryan & Calhoun. “Gender or genre?”, p. 144. INTRODUCTION A content and linguistic analysis was carried out on these three texts. MEAH, SECCIÓN ÁRABE-ISLAM [ISSN 1696-5868, e-ISSN 2341-0906] 71 (2022), 85-108 FEMALE RAP IN ARAB COUNTRIES. THE CASE OF MAYAM MAHMOUD 87 MEAH, SECCIÓN ÁRABE-ISLAM [ISSN 1696-5868, e-ISSN 2341-0906] 71 (2022), 85-108 PREVIOUS STUDIES ON FEMALE RAP Gender studies in ethnomusicology emerged as academic research in the early 1980s1. About the rap genre it is often channeled into gender studies2. Critics and aca- demics have often associated rap music with urban male culture. Although rap has often been presented as a male-dominated art form, women have been part of the scene since the years of commercial rap. The spread of a successful female rap has taken place starting from the 1990s. Specific studies on women and rap, have identified four categories of female rappers: “Queen Mother”, “Fly Girl”, “Sista with Attitude” and “Lesbian”3. The first category includes female rappers who present themselves as African- American icons, an image often suggested by their clothes and reflect the African cultural tradition. These include singers such as Queen Kenya4, Queen Latifah, Sister Souljah. “Fly Girl” describes the kind of woman rapper who dresses fashionably, sometimes provocatively, who wears jewels and flashy makeup, a style that spread in the wake of the Blaxploitation films5 in the late 1960s and mid-1970s. From the mid-1980s, many female MCs began to contest the image of the “fly girl”, aiming for an audience that would focus more on their rapping skills, rather than their looks. The third category “Sista with Attitude” includes those MC wo- men who consider the attitude to aggression, arrogance, rebellion as a manifesta- tion of power and present themselves in such attitudes. The last type of female rapper is the “Lesbian” who emerged later than the others and precisely towards the end of the Nineties6. Some research on hip hop has focused attention on language and genre in rap music, but it still need to be expanded and deepened. These studies are based on the emotional models present in rap and illustrate how the dominant emotional sphere in the songs of male rappers is linked to strength and skills7. Studies of rap songs in the United States highlight how the lyrics of male singers support pa- triarchal superiority with sexist terms. Regarding terms used by male rappers and 88 EMANUELA DE BLASIO female rappers, linguistic differences are identified: in menʼs texts, women are often referred to with unflattering nouns such as “bitch”, “slut”, “whore” and are considered as sexual objects8. 9. Rose. Black noise, pp. 32-33. 8. Motschenbacher. Language gender, pp. 51-52. MEAH, SECCIÓN ÁRABE-ISLAM [ISSN 1696-5868, e-ISSN 2341-0906] 71 (2022), 85-108 10. Historically “gangsta rap” describes the life of the ghetto from the point of view of a criminal fi- gure. 11. For further information: Haugen. “Unladylike divas”. 13. Adams & Fuller. “The words have changed”, p. 950. 12. Stenström. “It’s not that I really care”, pp. 115-116. 8. Motschenbacher. Language gender, pp. 51-52. 9. Rose. Black noise, pp. 32-33. 10. Historically “gangsta rap” describes the life of the ghetto from the point of view of a criminal fi- gure. 11. For further information: Haugen. “Unladylike divas”. 12. Stenström. “It’s not that I really care”, pp. 115-116. 13. Adams & Fuller. “The words have changed”, p. 950. PREVIOUS STUDIES ON FEMALE RAP The male- dominated attitude on the part of some rappers explains the marginalization of women within the rap music scene, particularly when the latter do not conform to the normative roles assigned to them within hip hop14. Rapper Roxanne Shante became the symbol of feminist hip hop when she re- leased the song “Roxanneʼs Revenge” in 1984, in response to a UTFO song in which she was referred to with offensive nicknames as “bitch”. In the late 1980s and early 1990s, female rappers such as Queen Latifah, Mo- nie Love, MC Lyte emerged and were considered the feminist pioneers of hip hop. Latifah promoted womenʼs rights in the song “Ladies First” on the album “All Hail the Queen” (1989). MC Lyte, who debuted with the album “Lyte as a Rock”, included the adapted song “Iʼm woman” by Helen Reddy from 1972, a text considered one of the anthems of female emancipation. Although references to misogyny in the hip hop world are fundamental for an in-depth analysis on gender and language issues, it should be emphasized that on- ly a part of rap possesses these discriminatory characteristics towards women; in fact some groups that carry out a politically committed type of hip hop include women15. Also in the Arab world there are musical collaborations between the two se- xes, such as the one between the Arabian Knightz and Shadia Mansour in the rea- lization of the famous song il-Sigīn “The prisoner” (2011). A novelty in the Arab rap scene is the introduction in the historic and famous DAM group of a young Palestinian female rapper, Maysa Daw: a rare case of a mixed group in the world of hip hop. In one of the last songs, Mīn inta? “Who are you?” (2015) together with the new female member of the group, the DAM take sides against all kinds of misogyny in society. This concept is expressed, not only in this one, but also in other previous songs such as Law iržaʽ bə-l-zamān (“If I went back in time”) (2012) in which the group declares itself against the crime of honor. Despite these cases, misogynist rappers are also present in the Arab world; about the language used in their texts, the tones are moderate compared to the language used in many American songs. PREVIOUS STUDIES ON FEMALE RAP The female singers, on the other hand, through a feminist language, try to transform the image of the black woman, sometimes however, through a contradictory message deriving from the exposure of their bodies and their own sexuality9. In the language of female rappers of the “gangsta” genre10, the derogatory terms used by men, such as “bitch” and “ho” (from “whore”) to refer to women, are also used but in a different way: women rappers can use these terms with ne- gative meanings, but also with positive connotations, as an affirmation of solida- rity, in the same way that male rappers use the terms “nigga” or “nigger” 11. The gender influences communication, choice of topics, way of interaction and vocabulary. Some studies12 researched the language of adolescents and found that girls use language to entertain relationships and create feelings of closeness and equality, while boys use language to establish and maintain their positions and supremacy. The speeches of the girls appear to have neither hierarchical nor competitive traits, but reflect cooperation, intimacy, loyalty and dedication, while the masculine speeches are characterized by hierarchical and strength structures, by elements that express competition and lack of cooperation. These characteristics are also found in the language and themes of rap lyrics: menʼs songs tend to be more self-celebrating and individualistic, while womenʼs songs focus more on relationality even between men and women and on the con- dition of women in society. Female rappers, albeit sometimes using a very aggressive language, never reach the threatening and violent tones of male rap- pers. Women also tend to a genre of rap more linked to the social and political dimension, touching, from time to time, personal issues; men, in many cases, even when they face social issues often express themselves with violent images. Although women rappers constitute a strong presence in the hip hop scene, they remain fewer than male artists: this imbalance is both cause and effect of the marked misogyny in rap. Misogynistic hip hop shows a conception of woman as an object that man can dispose of, use, abuse13. FEMALE RAP IN ARAB COUNTRIES. THE CASE OF MAYAM MAHMOUD 89 Rap music and hip hop culture in American society are often associated with forms of transmission and reproduction of sexism and misogyny. MEAH, SECCIÓN ÁRABE-ISLAM [ISSN 1696-5868, e-ISSN 2341-0906] 71 (2022), 85-108 14. There is a significant amount of feminist research on hip hop in the United States: Durham. “The stage hip-hop feminism built”; Pough. Home girls make some noise; Rose. Black noise. 15. To name one example, rapper Lin Que, known as Isis, was a prominent member of the rap group Blackwatch, known for her Afrocentrism and militant activism. 16. Some verses are reported below: Tell me what’s on you mind I’ll freak you like that / Lemme know what’s on your mind I’ll freak you like that / You’re going to like that I’m hot like that. 17. Literature offers many studies about Maghrebi rap: Guerrero. “Zanka Flow”; Meouak y Aguadé. “La Rhorhomanie et les beurs”; Caubet. “Génération darija!”; Langone. “Facteur D (Darija) et nouvelle génération marocaine”. Many authors have turned their attention to rap in Palestine: McDon- ald. My voice is my weapon; Lovatt. Palestinian hip hop culture; Maira & Magid. Hip hop from ’48 Palestine; Massad. Liberating songs; Orr. Legitimating narratives in rhyme. For further information on Lebanese rap: Fischione. “A critique of religious sectarianism through satire”. The following stu- dies are some researches emerged in recent years on hip hop during the Arab revolutions: Filiu. La révolution arabe; Guerrero. “Rap y revolución en el mundo árabe”; Procházka. “The voice of free- dom”. For more information on rap in the Arab world and some hints on female rap: De Blasio. Il rap nel mondo arabo. PREVIOUS STUDIES ON FEMALE RAP In the Moroccan reality, the rapper Yas- sine, in his song entitled “I’ll freak you like that”16, refers to women in an inele- 90 EMANUELA DE BLASIO gant way, but it is interesting that these kind of texts are produced in French or English. gant way, but it is interesting that these kind of texts are produced in French or English. 19. Besides the female rappers that I quote in this paragraph we find on the web: Medusa TN (Tuni- sia), Selma Rosa (Algeria/France), MC Meera (Jordan), Raja Meziane (Algeria/Czechia), Krtas Nssa (Morocco), Ily (Morocco), N1yah (United Arab Emirates), Dania DN Closer (Syria), Leesa A (Saudi Arabia), Queen Nesrin (Tunisia), Miss Moone (Bahrain), Whezzy (Libya), Ettijah (Palestine), Haifa 18. The reference sites are: www.revolutionaryarabrap.blogspot.com; www.reverbanation.com; www.genius.com and the You Tube channel for videos. MEAH, SECCIÓN ÁRABE-ISLAM [ISSN 1696-5868, e-ISSN 2341-0906] 71 (2022), 85-108 A LOOK AT FEMALE RAP IN THE ARAB WORLD While rap studies in the Arab world17 are on the rise, specific research on fe- male Arab rap is scarce. The development of hip hop in the Middle East and North Africa has also fa- vored female voices: in the Arab world, in recent years, we are witnessing the success of several female rappers. Taking into consideration what the web offers us, an overview of the female rap scene in the Arab world will be presented be- low18, but there is also an underground reality known only locally. From the data provided by the web, the phenomenon of female rap in Arab countries dates back to just over ten years ago with diffusion and developments that differ from country to country19. The countries showing a more active scene are Morocco and Tunisia in the Maghreb and Egypt and the Palestinian territories in the Mashreq. One of the dif- ficulties in analyzing the songs of the artists is that often the videos are removed in a short time, presumably for censorship or for reasons still unknown. The artists are generally released from the record companies, so the songs are traceable only online and are not distributed on CD: this also as a result of ideological choices of the authors themselves, in order to have autonomy and freedom, not negotiable for commercial purposes in their own artistic production. In addition to individual ideological choices, it is also important to underline that hip hop in general is affected by the controversial relationship between protest music and the Arab music industry. One of the problems for artists is to access FEMALE RAP IN ARAB COUNTRIES. THE CASE OF MAYAM MAHMOUD 91 recording studios and find record labels willing to sponsor them. Furthermore, despite the popularity of some channels dedicated to Arabic music, it is not easy for rap to conquer a space on radio or television. Starting from the Maghreb, in Morocco one of the most successful rappers is surely Soultana; this Moroccan artist is not only the first female rapper in Morocco, she is also a staunch supporter for women’s rights and an activist that uses her lyrical prowess to tackle social issues in home country. MEAH, SECCIÓN ÁRABE-ISLAM [ISSN 1696-5868, e-ISSN 2341-0906] 71 (2022), 85-108 Beseisso (Palestine), Popytirz (Tunisia), Khtek (Morocco). For further information: https://madamerap.com/. 20. https://www.kulturevulturez.com/top-tunisian-rappers/. Beseisso (Palestine), Popytirz (Tunisia), Khtek (Morocco). For further information: https://madamerap.com/. 20. https://www.kulturevulturez.com/top-tunisian-rappers/. MEAH, SECCIÓN ÁRABE-ISLAM [ISSN 1696-5868, e-ISSN 2341-0906] 71 (2022), 85-108 A LOOK AT FEMALE RAP IN THE ARAB WORLD Her international hit, Ṣawt nsa “The Voice of Women” (2011) is an effort to combat sexual ha- rassment in Morocco; in this lyric the artist represents the life conditions of some Moroccan women, through a very frank and direct language. In recent years in Morocco Manal Benchlikha became very famous for her texts that highlight the Moroccan manʼs vision of women. One of her most popu- lar songs is Tāž “Crown” (2018) in which she denounces the male-dominated and arrogant attitude of her peers and responds to such behaviors with aggression: in the video the artist holds guns and sticks and uses vulgar gestures, apparently re- flecting the typology of the rapper “Sista with Attitude” who considers the attitu- de to violence as a manifestation of power; only at the end of the video we disco- ver that the guns are not real and that the singer has made fun of her molesters, getting her revenge. Another Moroccan rapper is known under the pseudonym of Rikamora and she is author of Ma-ši ana “Not me” (2018), and Ġī lli bġāw “Just what they want” (2018), texts that mainly deal with the condition of young people in Mo- roccan society. On the web we can also come across the Tigresse Flow group made up of four girls from Casablanca; among the best known lyrics we mention Kīfāš? “How?” (2010) whose text denounces corruption in Morocco and Maġribiyya “Moroccan” (2008) song that expresses the strength of the Moroccan woman and the pride for her country. Tania Chanel is a Spanish-Moroccan trap rapper. The texts of the young sin- ger, who broke into the traditionally male dominated rap scene in Spain, reflects her everyday experiences. Among the Tunisian rappers we mention Ruka with the song Fawḍā “Chaos” (2017), F.B. K with the lyric Kaʽāb-i “My heels” (2020) and Tuny Girl who has produced several singles but has made herself known above all through her song ʽAksa “Bad luck”20 (2014). Boutheina El Alouadi is one of the few professional MEAH, SECCIÓN ÁRABE-ISLAM [ISSN 1696-5868, e-ISSN 2341-0906] 71 (2022), 85-108 92 EMANUELA DE BLASIO and politically courageous female rappers in Tunisia: in her songs she denounces sexism and criticizes Salafism21. Meryem Saci is a Montreal-based songwriter and hip-hop artist who escaped Algeria to Montreal during the civil war. A LOOK AT FEMALE RAP IN THE ARAB WORLD Her music ranges in different genres: soul, R&B, jazz, reggae and hip-hop and her newest track, “On My Way” (2017), is a perfect example of her eclectic sound. The music of Meryem Saci was used on the Netflix series “Iron Fist”. In Saudi Arabia Asayel Slay became famous with her debut song, Bint Makka “Mecca Girl” (2020) in which she celebrates the Meccan women. The song drew criticism from conservatives, who accused the rapper of undermining the customs and traditions of Mecca. The contents of the text led to the singerʼs arrest, but she was released shortly after. A famous artist of Palestinian origin is Shadia Mansour know as “the first la- dy of Arabic hip hop” who started composing rap music in 2003 and has gained recognition in the Middle East, Europe and the United States for her songs and for the various collaborations with other artists. Although she was born and raised in England, she sings in Arabic, since this language brings her closer to her roots and represents a tool through which to re- present in a more incisive way the issues dear to her such as that of the Palestini- an cause. The lyrics of Shadia fall into the genre of political, non-commercial rap, which is why the singer is not linked to any record company. Her first single is il- Kūfiyyi ʽarabiyyi “The keffiyeh is Arab” (2011), a song performed together with the rapper M-1 of Dead Prez, in which the singer emphasizes the role of the kef- fiyeh, symbol of Arab nationalism. As previously mentioned Shadia collaborated with the Egyptian group of Arabian Knightz on the song il-Sigīn “The prisoner” that has become internation- ally famous; the words of the refrain express the denunciation of contingent reali- ty and the desire for a better future: ana ʽāyiz balad ḥurra min il-ẓulm, ʽāyiz balad ḥurra min il-qahr, ʽāyiz balad ḥurra min il-šarr “I want a country free from injus- tice, I want a country free from oppression, I want a country free from evil”. In the Palestinian territories other women have had a significant role in the underground scene: Sabreena Da Witch who also wrote a song to all those who lost their lives before their time and the Arapyat (ʽArabiyyāt) formed by the duo Nahawa Abed Alaal and Safaah Hathot of Acre. 21 https://en.qantara.de/content/tunisian-rapper-boutheina-el-alouadi-rapping-against-sexism-and- salafists A LOOK AT FEMALE RAP IN THE ARAB WORLD In Lebanon, Lynn Fattouh famous as Malikah (from the Arabic malika “queen”) has increasingly established herself; she was born in France and precise- MEAH, SECCIÓN ÁRABE-ISLAM [ISSN 1696-5868, e-ISSN 2341-0906] 71 (2022), 85-108 FEMALE RAP IN ARAB COUNTRIES. THE CASE OF MAYAM MAHMOUD 93 ly in Marseille in 1986, but raised in Beirut under the shadow of civil war. Mali- kah made her musical debut in the Lebanese hip hop scene at the age of sixteen. She worked with EMI Arabia in 2003, after winning a hip-hop contest. Since then, Malikah has been known as the “Arab queen of hip-hop”, attracting fans throughout the Arab world but also in Europe. Many texts of this singer have as topics events and situations inherent to her country, therefore, her rap is part of the “conscious rap”22 trend. The choice of the nickname Malikah follows the style of the American rappers (“Queen Mothers”) who have distinguished themselves for their political rap. Furthermore, female rappers often appeal to each other “queens”, as a sign of solidarity, in fact they associate an idea of power and res- pect to this term. ly in Marseille in 1986, but raised in Beirut under the shadow of civil war. Mali- kah made her musical debut in the Lebanese hip hop scene at the age of sixteen. She worked with EMI Arabia in 2003, after winning a hip-hop contest. Since then, Malikah has been known as the “Arab queen of hip-hop”, attracting fans throughout the Arab world but also in Europe. Many texts of this singer have as topics events and situations inherent to her country, therefore, her rap is part of the “conscious rap”22 trend. The choice of the nickname Malikah follows the style of the American rappers (“Queen Mothers”) who have distinguished themselves for their political rap. Furthermore, female rappers often appeal to each other “queens”, as a sign of solidarity, in fact they associate an idea of power and res- pect to this term. ly in Marseille in 1986, but raised in Beirut under the shadow of civil war. Mali- kah made her musical debut in the Lebanese hip hop scene at the age of sixteen. In Malikahʼs lyrics, in addition to political issues, there is a particular interest in questions concerning women: in Ya imraʼa “Oh woman” (2008) the singer be- comes the spokesperson for the topic of emancipation of women in Lebanon. 22 The “conscious rap” focuses on social issues. The Messag di Grandmaster Flash (1982) was the first “conscious” song; the themes of the text are poverty, violence and the problems of black youth. MEAH, SECCIÓN ÁRABE-ISLAM [ISSN 1696-5868, e-ISSN 2341-0906] 71 (2022), 85-108 A LOOK AT FEMALE RAP IN THE ARAB WORLD The texts of Egyptian ar- tist know as Taffy are about her daily life and experience; her rhymes and lyrics address the issues of modern love and contemporary materialism, accompanied by fresh sounds and catchy tunes. Sometimes these artists have even managed to reach great audience, as in the case of the young Egyptian rapper Mayam Mahmoud who deals in her texts with the thorny issue of sexual harassment of women in Egypt and who arrived at the semifinals of the Arabs Got Talent television program (2013) with the lyric Ana miš sigāra “I am not a cigarette”. In the hip hop scenario, female rappers tend to follow a male jargon and style, characterized by direct language and strong expressions, traits however inherent to this musical genre. Generally the language, expressed in their songs, is more aggressive than that used by girls within society and in everyday life, in contrast to the general stereotype of what is expected of a female language. Presumably this could be the result of male dominance in the rap scene in which the female artists work, but also a consequence of the possibilities and freedom offered by rap in terms of communication, providing women with a public space in which to express themselves to assert their identity and their own thought. As with global hip hop, we can observe that female artists of Arab origin use terms referring to concepts of solidarity and positivity in general as the topics of their lyrics show. The themes of their texts focus on social issues linked above all to the question of women, for example Malikah in her song Ya imraʼa (“Wom- an”) addresses the issue of wo-menʼs emancipation in Lebanon, Shadia Mansour in Rūḥa bala rižʽa (“One way only”) deals with a sensitive issue, namely that of women in prison and Soultana in Ṣawt nsa “The Voice of Women” draws atten- tion to issues related to women. A recurring theme is the condition of women in the various realities and local contexts in which each singer finds herself, such as for example in Maġribiyya of Tigresse Flow, Bint Makka of Asayel Slay and Bint maṣriyya of Sawsan Adel. The rap produced by the women artists can be defined “conscious rap” for the issues addressed, for the interest related to current issues of local contemporary society, for the deep knowledge of contingent political dynamics. MEAH, SECCIÓN ÁRABE-ISLAM [ISSN 1696-5868, e-ISSN 2341-0906] 71 (2022), 85-108 A LOOK AT FEMALE RAP IN THE ARAB WORLD Among other lyrics we also mention: Heyk ṣāyra blēd-na “So is our country”, Ya Ləbnēn “Lebanon!”, Šū ʽam-biṣīr “What is happening?”, ʽAm-əbḥāreb “I am fighting”, Samm bə-l-damm “Poison in the blood” (all written between 2007 and 2008). The American-Syrian rapper Mona Haydar is known for her protest music, and is powerfully challenging misconceptions about veiled women around the world. Since 2017 the singer has become an outspoken role model for young Muslim women. With her protest anthem, “Wrap my Hijab” (2017), sung in En- glish, the artist has become famous as not only a talented female rapper, but one who isn’t afraid to unapologetically stand up for what she believes in. Haydar uses her platform to promote equality and cultural awareness through rap, dis- cussing everything from religion to gender to sexuality. A particular case is represented by Nayomiʼs multilingualism: the Iraqi-Syrian artist, who resides in Sweden, raps in Swedish, English and Arabic. The female rap scene in Egypt is quite prolific. In Egypt the first female rap- per was the Anglo-Egyptian Princess Emmanuelle, who sings mostly in English. Among the most recent rap singers in Egypt we find: the cairote Sawsan Adel known as “Soska Girl” who has composed countless lyrics including ʽĀfārīt “Brats” (2018), Hugūm ʽa-l-sawra “Attack on the revolution” (2017), Bint maṣriyya “Egyptian girl” (2011), Alya Alee author of ʼAsfa, “Iʼm sorry” (2013), Ana mīn “Who I am?” (2014) and Yukka Shahin originally from Alexandria and currently a law student in Copenhagen, who has composed texts such as Risāla MEAH, SECCIÓN ÁRABE-ISLAM [ISSN 1696-5868, e-ISSN 2341-0906] 71 (2022), 85-108 94 EMANUELA DE BLASIO EMANUELA DE BLASIO “Letter” (2018), Ma-fī-š fayda “There is no hope” (2011), Il-Sabab “The reason” (2017). “Letter” (2018), Ma-fī-š fayda “There is no hope” (2011), Il-Sabab “The reason” (2017). The Egyptian rapper Felukah is one of the most up-and-coming artists on the scene today, she is a prolific performer who draws a great deal of inspiration from her cultural heritage; her lyrics deal with various issues, ranging from the Arab Spring, all the way to gender roles in Egyptian society. The texts of Egyptian ar- tist know as Taffy are about her daily life and experience; her rhymes and lyrics address the issues of modern love and contemporary materialism, accompanied by fresh sounds and catchy tunes. g ; y , g g Spring, all the way to gender roles in Egyptian society. A LOOK AT FEMALE RAP IN THE ARAB WORLD A difference to be noted for what concerns the themes, compared to the Arab male rapper world, MEAH, SECCIÓN ÁRABE-ISLAM [ISSN 1696-5868, e-ISSN 2341-0906] 71 (2022), 85-108 FEMALE RAP IN ARAB COUNTRIES. THE CASE OF MAYAM MAHMOUD 95 is that the lyrics of the female singers rarely contemplate the theme of love, a to- pic instead treated more often by male rappers. MEAH, SECCIÓN ÁRABE-ISLAM [ISSN 1696-5868, e-ISSN 2341-0906] 71 (2022), 85-108 23. https://www.theguardian.com/world/2013/dec/01/egypt-rapper-mayam-mahmoud. 24. From 2016 to today there is no news of new songs. 25. The etymologically long vowels, in post-tonic open syllable, are transcribed, only when they are pronounced as such (therefore according to a phonetic and non-phonemic transcription), so the final vowels, which in classical Arabic are long, are made short. Note that /q/ is transcribed q to make the 23. https://www.theguardian.com/world/2013/dec/01/egypt-rapper-mayam-mahmoud. 23. https://www.theguardian.com/world/2013/dec/01/egypt-rapper-mayam-mahmoud. 24. From 2016 to today there is no news of new songs. 25. The etymologically long vowels, in post-tonic open syllable, are transcribed, only when they are pronounced as such (therefore according to a phonetic and non-phonemic transcription), so the final vowels, which in classical Arabic are long, are made short. Note that /q/ is transcribed q to make the MEAH, SECCIÓN ÁRABE-ISLAM [ISSN 1696-5868, e-ISSN 2341-0906] 71 (2022), 85-108 24. From 2016 to today there is no news of new songs. MAYAM MAHMOUD AND HER TEXTS miš ḥa-mūt la-ḥadd ma afham mafhūm il-ḥurriyya ḥurriyyet-i wāgeb ʽalē-k qabil ma tibqa haqq leyya ya abū-ya willa aḫū-ya willa gūz-i hterem-ni fakker w-isʼal nafs-ak marra ēh taʽrīf il-ʽayša l-murra tirodd tiqūl-li iḥna šarq miš ʽayšīn barra il-ḫuṣūṣiyya bardo ḥaqq-i w-lāzem tiḥterem ana miš milkiyya ʽamma bitzīdu ʽalē-ha l-alam sībū-ni aʽīš ḥayyat-i w-būš-i l-ḥaqīqa miš min w-ana lissa ṣaġīra bitḫaṭṭaṭu fi ṭarīq-i ana gism-i bass milk-i miš min ḥaqq-ak tašwī-(h) da ana lissa ḥuqūq-i ktīr fal-yisqoṭ is-safīh il-ḫaṭwa l-ūla bitqūl min ḥaqq-i inn-i ḫtār w-ana lē-k ʽalayya tḥammel masʼuliyyet l-qarār min ḥaqq-i akammel taʽlīm-i la-ḥadd ma ktifi min ḥaqq-i abān li-l-ʽālam miš ʽayza ḫtifi min ḥaqq-i ḫtār šarīk miš šarīk la-ʽaylt-i yimši maʽāya l-ṭarīq w-yikammel-li maṣirat-i min ḥaqq-i ḫū-ya yiḥterem-ni ma-yihāgem-š miš barra yiqūl ḥurriyya w-ʽand-i yigi yiqūl hēš miš ḥa-mūt la-ḥadd ma afham mafḥum il-ḥurriyya ḥurriyyat-i wāgeb ʽalē-k qabil ma tibqa haqq leyya occlusive laryngeal [ʔ], according to the method used, among others, by Kassab (1987). In some terms, the pronunciation of this consonant coincides with that of the classic and is transcribed q. MEAH, SECCIÓN ÁRABE-ISLAM [ISSN 1696-5868, e-ISSN 2341-0906] 71 (2022), 85-108 MAYAM MAHMOUD AND HER TEXTS About the life of Mayam Mahmoud there is not much news from the web; she was born in Giza (a city twenty kilometers from Cairo) in 1995. The artist graduated with a degree in Economics from Cairo. Her mother played an important role in Mayamʼs path to rap, in fact she introduced her to po- etry at the age of twelve and encouraged her to write lyrics. When the poetry of Mayam turned into rap, her parents were initially skeptical but gradually suppor- ted and allowed her to record the first track in Alexandria23. At just eighteen through her participation on Arabs Got Talent, the variety show that became a prime-time hit across the Middle East, Mayam is been one of the few female rappers to attract mainstream attention. In fact, she reached the semifinals of the program with the song Ana miš sigāra “I am not a cigarette” (2013). Her appearances on television were her first public performances. The fact that Mayam wears the hiġāb in her performances has aroused various reac- tions in the audience, challenging the expectations of some Egyptians. The interest in her music grew rapidly and the artist performed several con- certs. All her rap lyrics were written between 2013 and 201624. Beyond the lyrics Min ḥaqq-i “Itʼs my right”, Il-unūṯa fikr w-ʽaql “Feminity is intelligence and inte- llect” and Ana miš sigāra “I am not a cigarette” analyzed below, we also mention Il-kāmel inta “You are perfect”, Ḥurriyyat-i, “My freedom” Sakte w-miš sakte “I am silent or I am not silent”. The three texts taken into consideration are transcribed25 and translated below: Text 1 Text 1 ḥa-bqa guzʼ min il-ḥaqīqa miš bass min il-sirāb lissa fakra l-tabrīqa w-l-ġīma w-l-ḍabāb ḥa-qūl ḥāgāt ktīr qālū-li šibh mistaḥīla min haqq-i agri l-ṣubḥ badri min ġēr taʽlīqāt faṣīla fī-ha ēh arkab agla w-ma-yitqāl-š di bit habla EMANUELA DE BLASIO 96 ṭab lēh ʽalašān albes fusṭān yibqa lāzem munāsaba? “Itʼs My Right” I will be a part of reality, not only part of the mirage. I’m still thinking of the gasp, the cloud, and the fog. I will say many things that they told me that are almost impossible. I have the right to go running early in the morning without silly comments. What will happen if I ride a bike without it being said: “This stupid girl!” Why, when I wear a dress, does it need to be a special occasion? I won't die until I know the definition of freedom. Even before it’s my right, my freedom is your duty. My father, my brother, or my husband: Respect me! Just ask yourself once: what is the definition of the bitter life? You’ll respond by telling me that we’re Middle Easterners, not foreigners. My privacy is my right and you must respect it. I will be a part of reality, not only part of the mirage. FEMALE RAP IN ARAB COUNTRIES. THE CASE OF MAYAM MAHMOUD 97 I am not a public possession. You increase my pain. Let me live my life with my true face. You’ve been planning out the path of my life since I was a little girl. My body is mine alone and it isnʼt yours to disfigure. I still have a lot of rights, so down with the stupid! The first step, you say, is that I have the right to choose and that I will carry the responsibility for my decision. I have the right to complete my education until I’m fully satisfied. I have the right to show myself to the world. I don't want to disappear. I have the right to choose my own partner and not my familyʼs choice to share the road with me and to help me complete my journey. I have the right to have my brother respect me and not attack me. Outside he talks about freedom and comes to me he says “shut up” I won't die until I know the definition of freedom. Even before it’s my right, my freedom is your duty. MEAH, SECCIÓN ÁRABE-ISLAM [ISSN 1696-5868, e-ISSN 2341-0906] 71 (2022), 85-108 MEAH, SECCIÓN ÁRABE-ISLAM [ISSN 1696-5868, e-ISSN 2341-0906] 71 (2022), 85-108 “Itʼs My Right” Text 2 Il-unūsa fikr w-ʽaql Il-banāt fi mugtamʽ-na mitqassemīn fī l-higāb w-fī l-niqāb fī bēn il-bēnēn fī qaḍāyā ktīr hāwālē-na mitwaqqafa ʽala l-bint ʽala libs-ha w ʽala šakl-ha w-da aṣlan miš šarṭ izzāy btiḥkom ʽalayya min šaʽr-i willa ṭarḥat-i law yōm baṣṣēt ʽalayya miš ana lli ḥa-dāri kasfet-i bitʽākes w-btitḥarraš šāyef ann-ak ṣaḥḥ miš ġalaṭ law ḥatta bass bi-l-kalām di miš miʽāksa da zalaṭ miš libsi-na l-ġalaṭ bass il-fikr illi zāġ aywa saʽāt byibqa ziyāda bass il-rākk ʽa-l-dimāġ bi-naẓra wāḥda bitingereḥ ḥatta l-naẓra miš min ḥaqq-ak btibqa miḥtāg titfaḍḍaḥ w-tiḍreb qalamēn ʽa-wušṣ-ak mafḥūm il-ʼunūṯa fi Maṣr mitqassem la-ḥāgtēn ʽand il-rāgel ġēr il-sitt w-il-tnēn ġalṭānēn mīn illi qāl fi yōm anna l-unūsa fi l-fasāṭīn il-unūsa fikr w-ʽaql kamān tarbiyya w-dīn waṣalt anna l-bint ma-ʽand-ha-š siqqa fi nafs-ha badaʼt tiḥoṭṭ full makup tilbes ilwān fōq baʽḍ-ha il-miškila miš fi bint il-mawḍūʽ ḥāga tānya fi l-mugtamaʽ illi byiʼtor ʽalē-na fi kull sānya law saʼalna hal il-banāt ʽand-ha zōq fi-l-libs 98 EMANUELA DE BLASIO aywa ṭabaʽan ʽanda-hom bass il-ḥayāt ma-titwaṣṣef-š il-ḥayāt baqit mādiyya w-kull-o ʽāyiz hāga tiʽīš il-ġāli taman-o fī w-kamān aḥla min il-raḫīṣ. “Feminity is intelligence and intellect” Girls in our society are divided Into those who wear the niqab, those who wear the veil and those who are in between There are a lot of cases that depend on the girl How she dresses and how she look but this is not the rule How can you judge me by my hair or by my veil If one day you look at me I'm not the one who will hide my shame You harass and provoke thinking this is right not wrong Even if these are words this is not just a harassment, these are stones It is not her clothing that is wrong but it is the thought that has deviated from the truth Sometimes it's too much but it all depends on the brain With just one look you hurt, even the look is not your right You deserve to be dishonored and give two slaps in the fase Did you understand that femininity in Egypt is divided into two parts There is a difference between what men and women consider and both are wrong Who said one day that femininity is in clothes? “Itʼs My Right” Femininity is about intelligence and intellect and also education and reli- gión I’ve become a girl who doesn’t have self-confidence I’ve started to wear full make-up and wear colors on top of each other The problem is not with the girl, the point is an other The problem is with the society that influences the girl every second If you ask girls if they have good taste in dressing, they will say yes we have But our lives can not be described, our lives have become very materialistic And everyone wants something that would endure What is expensive has its value and is better than what is cheap 3 “Feminity is intelligence and intellect” Girls in our society are divided Into those who wear the niqab, those who wear the veil and those who are in between There are a lot of cases that depend on the girl How she dresses and how she look but this is not the rule How can you judge me by my hair or by my veil If one day you look at me I'm not the one who will hide my shame You harass and provoke thinking this is right not wrong Even if these are words this is not just a harassment, these are stones It is not her clothing that is wrong but it is the thought that has deviated from the truth And everyone wants something that would endure What is expensive has its value and is better than what is cheap Text 3 Text 3 MEAH, SECCIÓN ÁRABE-ISLAM [ISSN 1696-5868, e-ISSN 2341-0906] 71 (2022), 85-108 99 FEMALE RAP IN ARAB COUNTRIES. THE CASE OF MAYAM MAHMOUD 99 ʽadam il-tafʽīl maʽ il-wugūd zay l-šaḫṣ il-ṣamm ana miš sigāra miš zay ma bitqūl ʽalayya miš aḫr-i dōsa willa ḥa-asīb-ek tahīn fī-yya miš karāfat-o tiḫnoq bass tiḫallī-k miḥtaram w-lēh aṣlan ana ḥa-ʽaref-ak law kunt ʽadīm il-iḥtirām ʽomr-ak fakkart tiḥoṭṭ nafs-ak makān bint fi l-šāreʽ ḥāssa anna-ha farīsa w-ʽammāla fi ḫaṭwet-a ti-sāreʽ la w-kamān law ḥadd ʽākes-a bitgību l-ġalaṭ miš miḥtarama law raddēti ḥatta karāmt-ak ḥa-tihīni-ha šwayye min-na šayfīn il-ḥall fi l-ġaṭa w-an law l-bint iddārit miš ḥa-tkūn mitmarmaṭa māši ḫallīna maʽā-hom li-l-aḫer w-ʽayza asʼāl-hom suʼāl mīn aktar balad fī-ha ḥirmān? MEAH, SECCIÓN ÁRABE-ISLAM [ISSN 1696-5868, e-ISSN 2341-0906] 71 (2022), 85-108 “Itʼs My Right” You speak that you have raised your voice for some time Break down the wall, you who are the source of all protection You who sacrificed your aspiration to raise the family (You who) to show up for work you have to be beautiful You who have your life divided between sacrifice and exploitation They give you in marriage to those who have money in exchange for the one you love Since she is little she has kept her image She must be beautiful and everyone must be jealous of her The girl grows up, she becomes good at studying But itʼs still not enough “we want to find you a husband” Concepts that change if she is respectful fails If she is sociable and she plays a role immediately her nature is libertine Tell me that you leave me and go away why did you put me as an orna- ment? And when I ask for my rights, you take me to the doctor And when I ask for my rights, you take me to the doctor And when I ask for my rights, you take me to the doctor y g , y Tell me who you are, why donʼt you see yourself as you are? Tell me who you are, why donʼt you see yourself as you are? “Itʼs My Right” Afġānistān itḥakkem inta fi nafs-ak ma titḥakkem-š fī-yya law nūr il-šams ḥabs-ak qurb-ak min-ni aziyya di-l-waqti bān le-na mīn illi ʽalē-h il-ḥaqq w-mīn lamma iqūl ana ṣāḥḥ ḥa-niqūl-o la itkallemi ma-nti ṣōt-ek ʽāli min zamān heddi l-ḥīṭān da inti maṣdar kull il-amān inti lli ḍaḥḥēti bi-ṭumūḥ-ek ʽšān tarbī l-ʽiyāl ašān taqdīm il-waẓīfa ikūn fī-h šarṭ il-gamāl inti lli ḥayāt-ek mitqassema taḍhiya w-stiġlāl bigawwizū-ki ṣāḥeb il-māl badal illi qalb-ek li-h mayyāl min w-hiyye ṣġayyaṛa bitḥāfeẓ ʽala šakl-ha ʽalašān lāzem tibqa ḥēlwa w kill-o iġīr min-ha w-btikber il-bannūta tibqa šāṭra fi dirāsa bardo lissa miš kifāya iḥna ʽayzīn-lek gawāza miṣṭalaḥāt titqalleb law miḥtarema tibqa ḫayba law igtimāʽiyya w-lē-ha dōr tibqa ʽala-ṭūl qūli yalli sāyeb-ni w-māši ʽāyiz-ni lēh ḥattēt dīkūr w-yōm ma bṭāleb b-ḥaqq-i tiwaddī-ni li-l-duktūr ma tiqūl-li inta mīn willa šāyef nafs-ak lēh “I am not a cigarette” We want laws that bring order out of chaos We want laws that keep our girls away from worries You will say that there is already a law, but where, uncle Even if exisite is not performed and is like the deaf person Iʼm not a cigarette, Iʼm not like what you say about me 100 EMANUELA DE BLASIO In the end I cannot be trampled and I wonʼt let you offend me Iʼm not a tie that strangles but makes you look respectable Why should I know you if you are not worthy of respect Have you ever thought about putting yourself in the shoes of a girl on the street? The feeling is of being a prey, her pace is quickening And even if it comes to harass her, blame her for the mistake You are not respectful if you answer, even your dignity will offend Some of us see the solution in covering (with the veil) If the girl hides (behind a veil) she will not be in trouble Come on, letʼs follow them to the end and I want to ask them a question Which country has the most illegal acts? Afghanistan Control yourself and don't control me If the sunlight is holding you back, being close to me is bad Now it has appeared to us who is wrong and to whom when he says I am right we will say no! 26. Harassment is an endemic problem in Egypt: According to a United Nations survey published in April 2013, 99.3% of Egyptian women reported having experienced sexual harassment, with 91% saying they feel insecure on the street. 26. Harassment is an endemic problem in Egypt: According to a United Nations survey published in April 2013, 99.3% of Egyptian women reported having experienced sexual harassment, with 91% saying they feel insecure on the street. 27. On Egyptian Arabic there is an extensive bibliography of studies and manuals including: Ba- dawi Mustawayyāt al ʽarabiyya al muʽāṣira fī Miṣr; Haeri “Synchronic variation in Cairene Arabic”; 27. On Egyptian Arabic there is an extensive bibliography of studies and manuals including: Ba- dawi. Mustawayyāt al-ʽarabiyya al-muʽāṣira fī Miṣr; Haeri. “Synchronic variation in Cairene Arabic”; Woidich. “Egypt”; Jomier et Khouzam. Manuel d'arabe égyptien. 26. Harassment is an endemic problem in Egypt: According to a United Nations survey published in April 2013, 99.3% of Egyptian women reported having experienced sexual harassment, with 91% saying they feel insecure on the street. 27. On Egyptian Arabic there is an extensive bibliography of studies and manuals including: Ba- dawi. Mustawayyāt al-ʽarabiyya al-muʽāṣira fī Miṣr; Haeri. “Synchronic variation in Cairene Arabic”; Woidich. “Egypt”; Jomier et Khouzam. Manuel d'arabe égyptien. MEAH, SECCIÓN ÁRABE-ISLAM [ISSN 1696-5868, e-ISSN 2341-0906] 71 (2022), 85-108 THEMATİC AND LİNGUİSTİC ANALYSİS Mayam Mahmoud has earned many fans interested in her talent and in the themes covered in her songs: in particular the artist deals with the theme of sexual MEAH, SECCIÓN ÁRABE-ISLAM [ISSN 1696-5868, e-ISSN 2341-0906] 71 (2022), 85-108 FEMALE RAP IN ARAB COUNTRIES. THE CASE OF MAYAM MAHMOUD 101 harassment, a local taboo26. In her lyrics, the rapper condemns Egyptian society for accepting harassment as part of daily life and for blaming women rather than men and hopes her rap will encourage others to follow her example. The songs focus above all on the denunciation of the condition of the Arab woman in general and Egyptian in particular, on her rights often trampled on. Mayam claims the right of women to choose how to dress, what studies to do, what job to undertake and who to marry. In her lyrics the reference to female solidarity (a typical trait of female rap) is strong: the artist encourages other women not to submit to a certain vision imposed by men and society. The message of her songs is a push for other women to openly express the injustices suffered, with the hope that the harsh reality will change. The singer also addresses men directly, fathers, brothers, husbands, criticizing their actions and asking directly that their attitudes change. From a linguistic point of view the artist uses only colloquial Arabic, in fact we find in her songs the characteristics of the Egyptian Arabic of Cairo27 both on a phonetic and morpho-syntactic level. p p y In the analyzed texts we find: In the analyzed texts we find: In the analyzed texts we find: — the interdental consonants /ṯ/, /ḏ,/ are rendered by dentals /t/ e /d/ or sibilans /s/ and /z/. — the phoneme /ǧ/ is pronounced g [g] — the phoneme /q/ is rendered ʼ [Ɂ] except in rare cases where it is realized as uvular q with terms related to the legal sphere; — the rendering of the morpheme of the nominal feminine ﺓ is -a; in ʼiḍāfa it be- comes -et; — the etymologically long vowel in a tonic closed syllable is shortened (-CvC-). — the etymologically long vowel in a tonic closed syllable is shortened (-CvC-). THEMATİC AND LİNGUİSTİC ANALYSİS — paroxitone accent in the sequence -vCCCv (for example: madrása); aroxitone accent in the sequence -vCCCv (for examp — demonstrative themes da, di, dōl are postponed; — demonstrative themes da, di, dōl are postponed; — poslocation of interrogative adverbs; — poslocation of interrogative adverbs; — reflexive pronouns constructed with nafs (+suffix pronoun); — the negation of the conjugated verb forms is ma-faʽal-š/ ma-yifʽil-š; in the preverbal forms is miš ḥa-/ʽammāl; MEAH, SECCIÓN ÁRABE-ISLAM [ISSN 1696-5868, e-ISSN 2341-0906] 71 (2022), 85-108 102 EMANUELA DE BLASIO EMANUELA DE BLASIO — the concomitant present is rendered by ʽammāl/ʽammāla. Regarding the derived forms of the verb found in the texts we highlight: for the II form both the scheme faʽʽel- yifaʽʽel and faʽʽal- yifaʽʽal with the / a / due to the proximity of /ḍ/, /ṣ/, /ṭ/, /r/, /ġ/, /ḫ/, /ḥ/; the same thing for the V form itfaʽʽel, yitfaʽʽel and itfaʽʽal- yitfaʽʽal for the same phonetic reasons as above. At the lexical level we find the typical features of the Egyptian Arabic: badri: “soon”; badri: “soon”; aywa: “yes”; barra: “out”; ʽala ṭūl: “all straight”; di-l-waqti: “now”; bardo: “also”; fēn: “where?”; lēh: “why?”; ēh: “what?”; izzāy: “how?”; w-yōm ma: “ since when”; ḥāga, ḥāgāt with the meaning “thing” (in standard Arabic: “need”); the term ṣġīra “little” becomes ṣuġayyaṛa (from the scheme of the diminutive fuʽayyal); the term ṣġīra “little” becomes ṣuġayyaṛa (from the scheme of the diminutive fuʽayyal); the verb “to want” is rendered by ʽāyiz/ʽayza/ʽayzīn; the verb baqiya/yibqa “to become” is used with the meanig “to be”; this usage is very common among Cairo speakers; very common among Cairo speakers; We find only one case of trasglossia: the singer uses English in the expression “full makup” (text 2). Regarding the loans we find dīkūr “decor”, “ornament” (text 3), loan acclima- tized from French “décor”. Among the colloquial terms and expressions we highlight: il-bannūta “the little girl”, diminutive (scheme faʽʽūl) of bint; bit short form typical of slang from bint “girl” (text 1); hēš onomatopoeia with the sense of “shut up”(text 1); hēš onomatopoeia with the sense of “shut up”(text 1); hēš onomatopoeia with the sense of “shut up”(text 1); qalamēn lit.“two pens” (text 2), but in Egyptian colloquial Arabic it means with the meaning of “two slaps”; qalamēn lit.“two pens” (text 2), but in Egyptian colloquial Arabic it means with the meaning of “two slaps”; the expression il-rākk ʽa-l-dimāġ lit. THEMATİC AND LİNGUİSTİC ANALYSİS “the base is in the brain” (text 2) indicates that everything depends on the brain; the way of saying il-mitbaʽṭar yitlam (text 3) is very common and has the meaning of “to make order”, (lit. “the scattered is collected”), mitbaʽṭar participle of II form of the quatriconsonantal verb tabaʽtara (in standard Arabic tabaʽṯara); MEAH, SECCIÓN ÁRABE-ISLAM [ISSN 1696-5868, e-ISSN 2341-0906] 71 (2022), 85-108 FEMALE RAP IN ARAB COUNTRIES. THE CASE OF MAYAM MAHMOUD 103 mitmarmaṭa lit.: “muddy, bogged down” term used in colloquial jargon with the sense of being in a difficult situation (text 3); illi qalb-ek li-h mayyāl (text 3) has the meaning of “one that you like, that your heart loves”; the way of sayingʽāyiz-ni lēh ḥattēt dīkūr “you put me as an ornament” indicates someone who is silent and must not act, someone who is treated like an object (text 3). Regarding style, Mayamʼs language is very direct and describes images and situations of everyday life of girls often judged if they dress well, booed if they ride a bicycle or harassed in the streets. Although sometimes very harsh and blunt, the artistʼs language is not vulgar and does not resort to foul language. The rhymes that Mayam uses are for the most part final, but we also observe assonances and internal rhymes. The syntactic rhetorical figure of the anaphora is often used, in fact the author repeats at the beginning of the verses the same word. The rapper also uses the morphological rhetorical figure of alliteration, repeating the same phonemes in subsequent words. In the artistʼs texts we observe similes and metaphors, as in the verse law ḥat- ta bass bi-l-kalām di miš miʽāksa da zalaṭ “even if these are words this is not just a harassment, these are stones” (text 2), with the clear sense: even if these are just words, they hurt and are heavy as stones thrown at someone. MEAH, SECCIÓN ÁRABE-ISLAM [ISSN 1696-5868, e-ISSN 2341-0906] 71 (2022), 85-108 THEMATİC AND LİNGUİSTİC ANALYSİS The third text is all about metaphors and similes right from the title ana miš sigāra which alludes to woman as an object that is used and thrown away: ana miš sigāra “I am not a cigarette”; miš aḫr-i dōsa “in the end I cannot be trampled”, here there is always the comparison with the cigarette with the sense of something that is exploited to the end and then thrown away and trampled on; miš karāfat-o tiḫnoq bass tiḫallī-k miḥtaram “Iʼm not a tie that strangles but makes you look respectable”, with this image the artist means that the tie, a metaphor here for women, is not used to tighten the neck but to make the man look elegant; ʽadam il-tafʽīl maʽ il-wugūd zay l-šaḫṣ il-ṣamm “even if it exists it is not performed and is like the deaf per- son” (lit.:“ the lack of its implementation (of the law) even if it exists is like a deaf person”), here the comparison with the deaf person has the following mea- ning: even if the laws exist, the fact that they are not carried out makes them use- less. We encounter rhetorical questions such as ṭab lēh ʽalašān albes fusṭān yibqa lāzem munāsaba? “why, when I wear a dress, does it need to be a special occa- sion?” (text 1), mīn illi qāl fi yōm anna l-unūsa fi fasāṭīn? “who said one day that femininity is in clothes? (text 2), ḥa-tiqūl-li fī qānūn ḥa-qūl-ak fēn yāʽam? “you will say that there is already a law, but where, uncle? (text 3), ma tiqūl-li inta mīn MEAH, SECCIÓN ÁRABE-ISLAM [ISSN 1696-5868, e-ISSN 2341-0906] 71 (2022), 85-108 104 EMANUELA DE BLASIO willa šāyef nafs-ak lēh? (text 3) “tell me who you are, why donʼt you see yourself as you are?” and verses with direct speech: fī-ha ēh arkab agla w-ma-yitqāl-š dī bit habla “what will happen if I ride a bike without it being said: This stupid girl!” (text 1), miš barra yiqūl ḥurriyya w-ʽand-i yigi yiqūl hēš “outside he talks about freedom and comes to me he says: Shut up”, ya abū-ya willa aḫū-ya willa gūz-i hterem-ni “my father, my brother, or my husband: Respect me!”(text 1). bardo lissa miš kifāya iḥna ʽayzīn-lek gawāza “But itʼs still not enough: We want to find you a husband” (text 3). MEAH, SECCIÓN ÁRABE-ISLAM [ISSN 1696-5868, e-ISSN 2341-0906] 71 (2022), 85-108 CONCLUSION Female rappers are starting to be successful and are becoming quite numerous in Arab countries as well. While there is much research on the analysis of the birth and development of Western female rap, in-depth studies that investigate this reality in the Arab world from different points of view, linguistic, sociological and cultural, are lacking. From this preliminary study it appears that the themes of Arab women rappers are in line with the topics faced by most of the female rappers in the world (refe- rring to the most politically committed rap, not the commercial one): political issues related to their own country and social issues such as the condition of wo- men, the denunciation of sexism, the reality experienced daily. In general from the texts found on web, the language, expressed in their songs, is more aggressive and direct than that used within society on a daily basis, in contrast to the general stereotype of what is expected of a female language. In the Arab world we can say that in the rap musical scenario, although it re- mains mostly male prerogative, there is no total hostility towards female rappers, in fact they are often encouraged and supported by some singers and there are se- veral mixed collaborations between women and men rappers. Specifically, the work focused on three texts by the young Egyptian singer Mayam Mahmoud, who became internationally famous. The artist mainly deals with social issues related to the situation of the Arab woman in general and the Egyptian woman in particular. In her lyrics through a very frank and direct lan- guage she expresses all the difficulties and injustices suffered by the girls inclu- ding the burning issue of sexual harassment in the streets. Both the theme of the condition of women and that of solidarity can be found in many Arab women rappers but also in female rap in the world. Regarding the language the singer expresses herself in ʽāmmiyya, employing the Egyptian dialect in all her songs and does not resort to loans or trasglossia ex- cept in rare cases. Through the linguistic analysis it was considered appropiate to mention the morpho-syntactic and lexical aspects, as well as reporting the collo- FEMALE RAP IN ARAB COUNTRIES. THE CASE OF MAYAM MAHMOUD 105 quial expressios and idioms to offer further light on the contemporary Egyptian Arabic, albeit already sufficiently investigated. CONCLUSION In her texts Mayam uses rhetorical figures such as: anaphora, repetitions, a- ssonances, consonances, metaphors and similes. Puns were not found because the author prefers a very clear and unambiguous language, and uses images and des- criptions of real situations of everyday life. 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https://research.tees.ac.uk/files/9419528/Neutrosophic_Association_Rule_Mining_Algorithm_for_Big_Data_Analysis.pdf

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Neutrosophic Association Rule Mining Algorithm for Big Data Analysis

Symmetry

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Received: 5 March 2018; Accepted: 9 April 2018; Published: 11 April 2018 Abstract: Big Data is a large-sized and complex dataset, which cannot be managed using traditional data processing tools. Mining process of big data is the ability to extract valuable information from these large datasets. Association rule mining is a type of data mining process, which is indented to determine interesting associations between items and to establish a set of association rules whose support is greater than a specific threshold. The classical association rules can only be extracted from binary data where an item exists in a transaction, but it fails to deal effectively with quantitative attributes, through decreasing the quality of generated association rules due to sharp boundary problems. In order to overcome the drawbacks of classical association rule mining, we propose in this research a new neutrosophic association rule algorithm. The algorithm uses a new approach for generating association rules by dealing with membership, indeterminacy, and non-membership functions of items, conducting to an efficient decision-making system by considering all vague association rules. To prove the validity of the method, we compare the fuzzy mining and the neutrosophic mining. The results show that the proposed approach increases the number of generated association rules. Keywords: neutrosophic association rule; data mining; neutrosophic sets; big data Symmetry 2018, 10, 106; doi:10.3390/sym10040106 symmetry S S symmetry S S Article Mohamed Abdel-Basset 1,* ID , Mai Mohamed 1, Florentin Smarandache 2,* ID and Victor Ch 1 Department of Operations Research, Faculty of Computers and Informatics, Zagazig Univer Sharqiyah 44519, Egypt; [email protected] 2 Math & Science Department, University of New Mexico, Gallup, NM 87301, USA 3 International Business School Suzhou, Xi’an Jiaotong-Liverpool University, Wuzhong, Suzhou 215123, China; [email protected] * Correspondence: analyst [email protected] (M.A.-B.); [email protected] (F.S.) 1 Department of Operations Research, Faculty of Computers and Informatics, Zagazig University, Sharqiyah 44519, Egypt; [email protected] 2 Math & Science Department, University of New Mexico, Gallup, NM 87301, USA 3 International Business School Suzhou, Xi’an Jiaotong-Liverpool University, Wuzhong, Suzhou 215123, China; [email protected] * Correspondence: [email protected] (M.A.-B.); [email protected] (F.S.) (4) Smart cities designing. (4) Smart cities designing. These various applications help people to obtain better services, experiences, or be healthier, by detecting illness symptoms much earlier than before [2]. Some significant challenges of managing and analyzing big data are [4,5]: (1) Analytics Architecture: The optimal architecture for dealing with historic and real-time data at the same time is not obvious yet. (1) Analytics Architecture: The optimal architecture for dealing with historic and real-time data at the same time is not obvious yet. (2) Statistical significance: Fulfill statistical results, which should not be random. (3) Distributed mining: Various data mining methods are not fiddling to paralyze. (4) Time evolving data: Data should be improved over time according to the field of intere 5) Compression: To deal with big data, the amount of space that is needed to store is highly rele (6) Visualization: The main mission of big data analysis is the visualization of results. (7) Hidden big data: Large amounts of beneficial data are lost since modern data is unstructure Due to the increasing volume of data at a matchless rate and of various forms, we need to manage and analyze uncertainty of various types of data. Big data analytics is a significant function of big data, which discovers unobserved patterns and relationships among various items and people interest on a specific item from the huge data set. Various methods are applied to obtain valid, unknown, and useful models from large data. Association rule mining stands among big data analytics functionalities. The concept of association rule (AR) mining already returns to H’ajek et al. [6]. Each association rule in database is composed from two different sets of items, which are called antecedent and consequent. A simple example of association rule mining is “if the client buys a fruit, he/she is 80% likely to purchase milk also”. The previous association rule can help in making a marketing strategy of a grocery store. Then, we can say that association rule-mining finds all of the frequent items in database with the least complexities. From all of the available rules, in order to determine the rules of interest, a set of constraints must be determined. These constraints are support, confidence, lift, and conviction. Support indicates the number of occurrences of an item in all transactions, while the confidence constraint indicates the truth of the existing rule in transactions. 1. Introduction The term ‘Big Data’ originated from the massive amount of data produced every day. Each day, Google receives cca. 1 billion queries, Facebook registers more than 800 million updates, and YouTube counts up to 4 billion views, and the produced data grows with 40% every year. Other sources of data are mobile devices and big companies. The produced data may be structured, semi-structured, or unstructured. Most of the big data types are unstructured; only 20% of data consists in structured data. There are four dimensions of big data: (1) Volume: big data is measured by petabytes and zettabytes. (2) Velocity: the accelerating speed of data flow. (3) Variety: the various sources and types of data requiring analysis and management. (4) Veracity: noise, abnormality, and biases of generated knowledge. Consequently, Gartner [1] outlines that big data’s large volume requires cost-effective, innovative forms for processing information, to enhance insights and decision-making processes. Prominent domains among applications of big data are [2,3]: www.mdpi.com/journal/symmetry 2 of 19 Symmetry 2018, 10, 106 (1) Business domain. (1) Business domain. (2) Technology domain. (3) Health domain. (4) Smart cities designing. (4) Smart cities designing. The factor “lift” explains the dependency relationship between the antecedent and consequent. On the other hand, the conviction of a rule indicates the frequency ratio of an occurring antecedent without a consequent occurrence. Association rules mining could be limited to the problem of finding large itemsets, where a large itemset is a collection of items existing in a database transactions equal to or greater than the support threshold [7–20]. In [8], the author provides a survey of the itemset methods for discovering association rules. The association rules are positive and negative rules. The positive association rules take the form X →Y, X ⊆I, Y ⊆I and X ∩Y = ϕ, where X, Y are antecedent and consequent and I is a set of items in database. Each positive association rule may lead to three negative association rules, →Y, X →Y, and X →Y. Generating association rules in [9] consists of two problems. The first problem is to find frequent itemsets whose support satisfies a predefined minimum value. Then, the concern is to derive all of the rules exceeding a minimum confidence, based on each frequent itemset. Since the solution of the second problem is straightforward, most of the proposed work goes in for solving the first problem. An a priori algorithm has been proposed in [19], which was the basis for many of the forthcoming algorithms. A two-pass algorithm is presented in [11]. It consumes only two database scan passes, while a priori is a multi-pass algorithm and needs up to c+1 database scans, where c is the number of items (attributes). Association rules mining is applicable in numerous database communities. It has large applications in the retail industry to improve market basket analysis [7]. Streaming-Rules is an algorithm developed by [9] to report an association between pairs of elements in streams for predictive caching and detecting the previously 3 of 19 Symmetry 2018, 10, 106 undetectable hit inflation attacks in advertising networks. Running mining algorithms on numerical attributes may result in a large set of candidates. Each candidate has small support and many rules have been generated with useless information, e.g., the age attribute, salary attribute, and students’ grades. Many partitioning algorithms have been developed to solve the numerical attributes problem. The proposed algorithms faced two problems. The first problem was the partitioning of attribute domain into meaningful partitions. (4) Smart cities designing. The second problem was the loss of many useful rules due to the sharp boundary problem. Consequently, some rules may fail to achieve the minimum support threshold because of the separating of its domain into two partitions. Fuzzy sets have been introduced to solve these two problems. Using fuzzy sets make the resulted association rules more meaningful. Many mining algorithms have been introduced to solve the quantitative attributes problem using fuzzy sets proposed algorithms in [13–27] that can be separated into two types related to the kind of minimum support threshold, fuzzy mining based on single-minimum support threshold, and fuzzy mining based on multi-minimum support threshold [21]. Neutrosophic theory was introduced in [28] to generalize fuzzy theory. In [29–32], the neutrosophic theory has been proposed to solve several applications and it has been used to generate a solution based on neutrosophic sets. Single-valued neutrosophic set was introduced in [33] to transfer the neutrosophic theory from the philosophic field into the mathematical theory, and to become applicable in engineering applications. In [33], a differentiation has been proposed between intuitionistic fuzzy sets and neutrosophic sets based on the independence of membership functions (truth-membership function, falsity-membership function, and indeterminacy-membership function). In neutrosophic sets, indeterminacy is explicitly independent, and truth-membership function and falsity-membership function are independent as well. In this paper, we introduce an approach that is based on neutrosophic sets for mining association rules, instead of fuzzy sets. Also, a comparison resulted association rules in both of the scenarios has been presented. In [34], an attempt to express how neutrosophic sets theory could be used in data mining has been proposed. They define SVNSF (single-valued neutrosophic score function) to aggregate attribute values. In [35], an algorithm has been introduced to mining vague association rules. Items properties have been added to enhance the quality of mining association rules. In addition, almost sold items (items has been selected by the customer, but not checked out) were added to enhance the generated association rules. AH-pair Database consisting of a traditional database and the hesitation information of items was generated. The hesitation information was collected, depending online shopping stores, which make it easier to collect that type of information, which does not exist in traditional stores. In this paper, we are the first to convert numerical attributes (items) into neutrosophic sets. (4) Smart cities designing. While vague association rules add new items from the hesitating information, our framework adds new items by converting the numerical attributes into linguistic terms. Therefore, the vague association rule mining can be run on the converted database, which contains new linguistic terms. Research Contribution Detecting hidden and affinity patterns from various, complex, and big data represents a significant role in various domain areas, such as marketing, business, medical analysis, etc. These patterns are beneficial for strategic decision-making. Association rules mining plays an important role as well in detecting the relationships between patterns for determining frequent itemsets, since classical association rules cannot use all types of data for the mining process. Binary data can only be used to form classical rules, where items either exist in database or not. However, when classical association rules deal with quantitative database, no discovered rules will appear, and this is the reason for innovating quantitative association rules. The quantitative method also leads to the sharp boundary problem, where the item is below or above the estimation values. The fuzzy association rules are introduced to overcome the classical association rules drawbacks. The item in fuzzy association rules has a membership function and a fuzzy set. The fuzzy association rules can deal with vague rules, but not in the best manner, since it cannot consider the indeterminacy of rules. In order to overcome 4 of 19 Symmetry 2018, 10, 106 drawbacks of previous association rules, a new neutrosophic association rule algorithm has been introduced in this research. Our proposed algorithm deals effectively and efficiently with vague rules by considering not only the membership function of items, but also the indeterminacy and the falsity functions. Therefore, the proposed algorithm discovers all of the possible association rules and minimizes the losing processes of rules, which leads to building efficient and reliable decision-making system. By comparing our proposed algorithm with fuzzy approaches, we note that the number of association rules is increased, and negative rules are also discovered. The separation of negative association rules from positive ones is not a simple process, and it helps in various fields. As an example, in the medical domain, both positive and negative association rules help not only in the diagnosis of disease, but also in detecting prevention manners. The rest of this research is organized as follows. The basic concepts and definitions of association rules mining are presented in Section 2. A quick overview of fuzzy association rules is described in Section 3. The neutrosophic association rules and the proposed model are presented in Section 4. A case study of Telecom Egypt Company is presented in Section 5. 2. Association Rules Mining This problem can simply be expressed as finding all of the large itemsets, where a large itemset L is: L = {X|X ⊆I ∧σX ≥|D| × s}. L = {X|X ⊆I ∧σX ≥|D| × s}. L = {X|X ⊆I ∧σX ≥|D| × s}. Research Contribution The experimental results and comparisons between fuzzy and proposed association rules are discussed in Section 6. The conclusions are drawn in Section 7. 2. Association Rules Mining In this section, we formulate the |D| transactions from the mining association rules for a database D. We used the following notations: (i) I = {i1, i2, . . . im} represents all the possible data sets, called items. i) I = {i1, i2, . . . im} represents all the possible data sets, called items. (ii) Transaction set T is the set of domain data resulting from transactional processing such as T ⊆I. (iii) For a given itemset X ⊆I and a given transaction T, we say that T contains X if and only if X ⊆T. (iv) σX: the support frequency of X, which is defined as the number of transactions out of D that (ii) Transaction set T is the set of domain data resulting from transactional processing such as T ⊆I. (iii) For a given itemset X ⊆I and a given transaction T, we say that T contains X if and only if X ⊆T. g p g (iii) For a given itemset X ⊆I and a given transaction T, we say that T contains X if and only if X ⊆T. (iii) For a given itemset X ⊆I and a given transaction T, we say that T contains X if and only if X ⊆T. (iv) σX: the support frequency of X, which is defined as the number of transactions out of D that contain X. (v) s: the support threshold (iv) σX: the support frequency of X, which is defined as the number of transactions out of D that contain X. (v) s: the support threshold. (v) s: the support threshold. X is considered a large itemset, if σX ≥|D| × s. Further, an association rule is an implication of the form X ⇒Y , where X ⊆I, Y ⊆I and X ∩Y = ϕ. An association rule X ⇒Y is addressed in D with confidence c if at least c transactions out of D contain both X and Y. The rule X ⇒Y is considered as a large itemset having a minimum support s if: σX∪Y ≥|D| × s. For a specific confidence and specific support thresholds, the problem of mining association rules is to find out all of the association rules having confidence and support that is larger than the corresponding thresholds. 3. Fuzzy Association Rules Mining of association rules is considered as the main task in data mining. An association rule expresses an interesting relationship between different attributes. Fuzzy association rules can deal with both quantitative and categorical data and are described in linguistic terms, which are understandable terms [26]. Let T = {t1, . . . , tn} be a database transactions. Each transaction consists of a number of attributes (items). Let I = {i1, . . . , im} be a set of categorical or quantitative attributes. For each attribute ik, (k = 1, . . . , m), we consider {n1, . . . , nk} associated fuzzy sets. Typically, a domain expert determines the membership function for each attribute. The tuple < X, A > is called the fuzzy itemset, where X ⊆I (set of attributes) and A is a set of fuzzy sets that is associated with attributes from X. 5 of 19 Symmetry 2018, 10, 106 Following is an example of fuzzy association rule: Following is an example of fuzzy association rule: Following is an example of fuzzy association rule: IF salary is high and age is old THEN insurance is high Before the mining process starts, we need to deal with numerical attributes and prepare them for the mining process. The main idea is to determine the linguistic terms for the numerical attribute and define the range for every linguistic term. For example, the temperature attribute is determined by the linguistic terms {very cold, cold, cool, warm, hot}. Figure 1 illustrates the membership function of the temperature attribute. Figure 1. Linguistic terms of the temperature attribute. Figure 1. Linguistic terms of the temperature attribute. The membership function has been calculated for the following database transactions illustrated in Table 1. The membership function has been calculated for the following database transactions illustrated in Table 1. Table 1. Membership function for Database Transactions. Table 1. Membership function for Database Transactions. Transaction Temp. Membership Degree T1 18 1 cool T2 13 0.6 cool, 0.4 cold T3 12 0.4 cool, 0.6 cold T4 33 0.6 warm, 0.4 hot T5 21 0.2 warm, 0.8 cool T6 25 1 warm We add the linguistic terms {very cold, cold, cool, warm, hot} to the candidate set and calculate the support for those itemsets. After determining the linguistic terms for each numerical attribute, the fuzzy candidate set have been generated. 3. Fuzzy Association Rules Table 2 contains the support for each itemset individual one-itemsets. The count for every linguistic term has been calculated by summing its membership degree over the transactions. Table 3 shows the support for two-itemsets. The count for the fuzzy sets is the summation of degrees that resulted from the membership function of that itemset. The count for two-itemset has been calculated by summing the minimum membership degree of the 2 items. For example, {cold, cool} has count 0.8, which resulted from transactions T2 and T3. For transaction T2, membership degree of cool is 0.6 and membership degree for cold is 0.4, so the count for set {cold, cool} in T2 is 0.4. Also, T3 has the same count for {cold, cool}. So, the count of set {cold, cool} over all transactions is 0.8. Table 2 contains the support for each itemset individual one-itemsets. The count for every linguistic term has been calculated by summing its membership degree over the transactions. Table 3 shows the support for two-itemsets. The count for the fuzzy sets is the summation of degrees that resulted from the membership function of that itemset. The count for two-itemset has been calculated by summing the minimum membership degree of the 2 items. For example, {cold, cool} has count 0.8, which resulted from transactions T2 and T3. For transaction T2, membership degree of cool is 0.6 and membership degree for cold is 0.4, so the count for set {cold, cool} in T2 is 0.4. Also, T3 has the same count for {cold, cool}. So, the count of set {cold, cool} over all transactions is 0.8. 6 of 19 Symmetry 2018, 10, 106 Table 2. 1-itemset support. 1-itemset Count Support Very cold 0 0 Cold 1 0.17 Cool 2.8 0.47 Warm 1.6 0.27 Hot 0.6 0.1 Table 3. 2-itemset support. 2-itemset Count Support {Cold, cool} 0.8 0.13 {Warm, hot} 0.4 0.07 {warm, cool} 0.2 0.03 In subsequent discussions, we denote an itemset that contains k items as k-itemset. The set of all k f Table 2. 1-itemset support. Table 2. 1-itemset support. In subsequent discussions, we denote an itemset that contains k items as k-itemset. The set of all k-itemsets in L is referred as Lk. In subsequent discussions, we denote an itemset that contains k items as k-itemset. The set of all k-itemsets in L is referred as Lk. 4. Neutrosophic Association Rules In this section, we overview some basic concepts of the NSs and SVNSs over the universal set X, and the proposed model of discovering neutrosophic association rules. 4.1. Neutrosophic Set Definitions and Operations 4.1. Neutrosophic Set Definitions and Operations Definition 1 ([33]). Let X be a space of points and x∈X. A neutrosophic set (NS) A in X is definite by a truth-membership function TA(x), an indeterminacy-membership function IA(x) and a falsity-membership function FA(x). TA(x), IA(x) and FA(x) are real standard or real nonstandard subsets of ]−0, 1+[. That is TA(x): X →]−0, 1+[, IA(x): X →]−0, 1+[ and FA(x): X →]−0, 1+[. There is no restriction on the sum of TA(x), IA(x) and FA(x), so 0−≤sup TA(x) + sup IA(x) + sup FA(x) ≤3+. Neutrosophic is built on a philosophical concept, which makes it difficult to process during engineering applications or to use it to real applications. To overcome that, Wang et al. [31], defined the SVNS, which is a particular case of NS. Definition 2. Let X be a universe of discourse. A single valued neutrosophic set (SVNS) A over X is an object taking the form A = {⟨x,TA(x), IA(x), FA(x)⟩: x∈X}, where TA(x): X →[0, 1], IA(x): X →[0, 1] and FA(x): X →[0, 1] with 0 ≤TA(x) + IA(x) + FA(x) ≤3 for all x∈X. The intervals TA(x), IA(x) and FA(x) represent the truth-membership degree, the indeterminacy-membership degree and the falsity membership degree of x to A, respectively. For convenience, a SVN number is represented by A = (a, b, c), where a, b, c∈[0, 1] and a + b + c ≤3. Definition 3 (Intersection) ([31]). For two SVNSs A = ⟨TA(x), IA(x), FA(x)⟩and B = ⟨TB(x), IB(x), FB(x)⟩, the intersection of these SVNSs is again an SVNSs which is defined as C = A ∩B whose truth, indeterminacy and falsity membership functions are defined as TC(x) = min(TA(x), TB(x)), IA(x) = min(IA(x), IB(x)) and FC(x) = max(FA(x), FB(x)). Definition 3 (Intersection) ([31]). For two SVNSs A = ⟨TA(x), IA(x), FA(x)⟩and B = ⟨TB(x), IB(x), FB(x)⟩, the intersection of these SVNSs is again an SVNSs which is defined as C = A ∩B whose truth, indeterminacy and falsity membership functions are defined as TC(x) = min(TA(x), TB(x)), IA(x) = min(IA(x), IB(x)) and FC(x) = max(FA(x), FB(x)). Definition 4 (Union) ([31]). FC(x) = min(FA(x), FB(x)). 4.1. Neutrosophic Set Definitions and Operations For two SVNSs A = ⟨TA(x), IA(x), FA(x)⟩and B = ⟨TB(x), IB(x), FB(x)⟩, the union of these SVNSs is again an SVNSs which is defined as C = A ∪B whose truth, indeterminacy and falsity membership functions are defined as TC(x) = max(TA(x), TB(x)), IA(x) = max(IA(x), IB(x)) and FC(x) = min(FA(x), FB(x)). Definition 4 (Union) ([31]). For two SVNSs A = ⟨TA(x), IA(x), FA(x)⟩and B = ⟨TB(x), IB(x), FB(x)⟩, the union of these SVNSs is again an SVNSs which is defined as C = A ∪B whose truth, indeterminacy and falsity membership functions are defined as TC(x) = max(TA(x), TB(x)), IA(x) = max(IA(x), IB(x)) and FC(x) = min(FA(x), FB(x)). S t 2018 10 106 7 f 19 7 of 19 Symmetry 2018, 10, 106 Definition 5 (Containment) ([31]). A single valued neutrosophic set A contained in the other SVNS B, denoted by A ⊆B if and only if TA(x) ≤TB(x), IA(x) ≤IB(x) and FA(x) ≥FB(x) for all x in X. Definition 5 (Containment) ([31]). A single valued neutrosophic set A contained in the other SVNS B, denoted by A ⊆B if and only if TA(x) ≤TB(x), IA(x) ≤IB(x) and FA(x) ≥FB(x) for all x in X. Next, we propose a method for generating the association rule under the SVNS environmen 4.2. Proposed Model for Association Rule In this paper, we introduce a model to generate association rules of form: X →Y where X ∩Y = ϕ and X, Y are neutrosophic sets. In this paper, we introduce a model to generate association rules of form: In this paper, we introduce a model to generate association rules of form: X →Y where X ∩Y = ϕ and X, Y are neutrosophic sets. In this paper, we introduce a model to generate association rules of form: Our aim is to find the frequent itemsets and their corresponding support. Generating an association rule from its frequent itemsets, which are dependent on the confidence threshold, are also discussed here. This has been done by adding the neutrosophic set into I, where I is all of the possible data sets, which are referred as items. So I = N ∪M where N is neutrosophic set and M is classical set of items. The general form of an association rule is an implication of the form X →Y, where X ⊆I, Y ⊆I, X ∩Y = ϕ. Therefore, an association rule X →Y is addressed in Database D with confidence ‘c’ if at least c transactions out of D contains both X and Y. On the other hand, the rule X →Y is considered a large item set having a minimum support s if σX∪Y ≥|D| × s. Furthermore, the process of converting the quantitative values into the neutrosophic sets is proposed, as shown in Figure 2. Figure 2. The proposed model. The proposed model for the construction of the neutrosophic numbers is summarized in the Figure 2. The proposed model. Figure 2. The proposed model. The proposed model for the construction of the neutrosophic numbers is summarized in the following steps: The proposed model for the construction of the neutrosophic numbers is summarized in the following steps: Symmetry 2018, 10, 106 8 of 19 Step 1 Set linguistic terms of the variable, which will be used for quantitative attribute. Step 1 Set linguistic terms of the variable, which will be used for quantitative attribute. Step 1 Set linguistic terms of the variable, which will be used for quantitative attribute. Step 2 Define the truth, indeterminacy, and the falsity membership functions for each constructed linguistic term. Step 2 Define the truth, indeterminacy, and the falsity membership functions for each constructed linguistic term. 4.2. Proposed Model for Association Rule Step 3 For each transaction t in T, compute the truth-membership, indeterminacy-membership and falsity-membership degrees. Step 3 For each transaction t in T, compute the truth-membership, indeterminacy-membership and falsity-membership degrees. Step 4 Extend each linguistic term l in set of linguistic terms L into TL, IL, and FL to denote truth- membership, indeterminacy-membership, and falsity-membership functions, respectively. Step 5 For each k-item set where k = {1, 2, . . . , n}, and n number of iterations. Step 4 Extend each linguistic term l in set of linguistic terms L into TL, IL, and FL to denote truth- membership, indeterminacy-membership, and falsity-membership functions, respectively. Step 5 For each k-item set where k = {1, 2, . . . , n}, and n number of iterations. • calculate count of each linguistic term by summing degrees of membership for each transaction as Count(A) = i=t ∑ i 1 µA(x) where µA is TA, IA or FA. • calculate count of each linguistic term by summing degrees of membership for each transaction as Count(A) = i=t ∑ i=1 µA(x) where µA is TA, IA or FA. • calculate support for each linguistic term s = Count(A) No. o f trnsactions. • calculate support for each linguistic term s = Count(A) No. o f trnsactions. Step 6 The above procedure has been repeated for every quantitative attribute in the databas In order to show the working procedure of the approach, we consider the temperature as an attribute and the terms “very cold”, “cold”, “cool”, “warm”, and “hot” as their linguistic terms to represent the temperature of an object. Then, following the steps of the proposed approach, construct their membership function as below: Step 1 The attribute temperature’ has set the linguistic terms “very cold”, “cold”, “cool”, “warm”, and “hot”, and their ranges are defined in Table 4. Table 4. Linguistic terms ranges. Table 4. Linguistic terms ranges. 4.2. Proposed Model for Association Rule Linguistic Term Core Range Left Boundary Range Right Boundary Range Very Cold −∞–0 N/A 0–5 Cold 5–10 0–5 10–15 Cool 15–20 10–15 20–25 Warm 25–30 20–25 30–35 Hot 35–∞ 30–35 N/A Step 2 Based on these linguistic term ranges, the truth-membership functions of each linguistic variable are defined, as follows: Tvery−cold(x) =      1 ; f or x ≤0 (5 −x)/5 ; f or 0 < x < 5 0 ; f or x ≥5 Tcold(x) =          1 ; f or 5 ≤x ≤10 (15 −x)/5 ; f or 10 < x < 15 x/5 ; f or 0 < x < 5 0 ; f or x ≥15 or x ≤0 Tcool(x) =          1 ; f or 15 ≤x ≤20 (25 −x)/5 ; f or 20 < x < 25 (x −10)/5 ; f or 10 < x < 15 0 ; otherwise Twarm(x) =          1 ; f or 25 ≤x ≤30 (35 −x)/5 ; f or 30 < x < 35 (x −20)/5 ; f or 20 < x < 25 0 ; otherwise Tvery−cold(x) =      1 ; f or x ≤0 (5 −x)/5 ; f or 0 < x < 5 0 ; f or x ≥5 Tcold(x) =          1 ; f or 5 ≤x ≤10 (15 −x)/5 ; f or 10 < x < 15 x/5 ; f or 0 < x < 5 0 ; f or x ≥15 or x ≤0 Tcool(x) =          1 ; f or 15 ≤x ≤20 (25 −x)/5 ; f or 20 < x < 25 (x −10)/5 ; f or 10 < x < 15 0 ; otherwise Twarm(x) =          1 ; f or 25 ≤x ≤30 (35 −x)/5 ; f or 30 < x < 35 (x −20)/5 ; f or 20 < x < 25 0 ; otherwise 9 of 19 Symmetry 2018, 10, 106 Thot(x) =      1 ; f or x ≥35 (x −30)/5 ; f or 30 < x < 35 0 ; otherwise The falsity-membership functions of each linguistic variable are defined as follows: Fvery−cold(x) =      0 ; f or x ≤0 x/5 ; f or 0 < x < 5 1 ; f or x ≥5 ; Fcold(x) =          0 ; f or 5 ≤x ≤10 (x −10)/5 ; f or 10 < x < 15 (5 −x)/5 ; f or 0 < x < 5 1 ; f or x ≥15 or x ≤0 Fcool(x) =          0 ; f or 15 ≤x ≤20 (x −20)/5 ; f or 20 < x < 25 (15 −x)/5 ; f or 10 < x < 15 1 ; otherwise Fwarm(x) =          0 ; f or 25 ≤x ≤30 (x −30)/5 ; f or 30 < x < 35 (25 −x)/5 ; f or 20 < x < 25 1 ; otherwise Fhot(x) =      0 ; f or x ≥35 (35 −x)/5 ; f or 30 < x < 35 1 ; otherwise The indeterminacy membership functions of each linguistic variables are defined as follows: The indeterminacy membership functions of each linguistic variables are defined as follows: Ivery−cold(x) =          0 ; f or x ≤−2.5 (x + 2.5)/5 ; f or −2.5 ≤x ≤2.5 (7.5 −x)/5 ; f or 2.5 ≤x ≤7.5 0 ; f or x ≥7.5 Icold(x) =              (x + 2.5)/5 ; f or 2.5 ≤x ≤2.5 (7.5 −x)/5 ; f or 2.5 ≤x ≤7.5 (x −7.5)/5 ; f or 7.5 ≤x ≤12.5 (17.5 −x)/5 ; f or 12.5 ≤x ≤17.5 0 ; otherwise Icool(x) =              (x −7.5)/5 ; f or 7.5 ≤x ≤12.5 (17.5 −x)/5 ; f or 12.5 ≤x ≤17.5 (x −17.5)/5 ; f or 17.5 ≤x ≤22.5 (27.5 −x)/5 ; f or 22.5 ≤x ≤27.5 0 ; otherwise Iwarm(x) =              (x −17.5)/5 ; f or 17.5 ≤x ≤22.5 (27.5 −x)/5 ; f or 22.5 ≤x ≤27.5 (x −27.5)/5 ; f or 27.5 ≤x ≤32.5 (37.5 −x)/5 ; f or 32.5 ≤x ≤37.5 0 ; otherwise 10 of 19 Symmetry 2018, 10, 106 Ihot(x) =      (x −27.5)/5 ; f or 27.5 ≤x ≤32.5 (37.5 −x)/5 ; f or 32.5 ≤x ≤37.5 0 ; otherwise Ihot(x) =      (x −27.5)/5 ; f or 27.5 ≤x ≤32.5 (37.5 −x)/5 ; f or 32.5 ≤x ≤37.5 0 ; otherwise The graphical membership degrees of these variables are summarized in Figure 3. 4.2. Proposed Model for Association Rule 12 of 19 Symmetry 2018, 10, 106 Step 4 Now, we count the set of linguistic terms {very cold, cold, cool, warm, hot} for every element in transactions. Since the truth, falsity, and indeterminacy-memberships are independent functions, the set of linguistic terms can be extended to n Tvery−cold, Tcold, Tcool, Twarm, Thot Fvery−cold, Fcold, Fcool, Fwarm, Fhot Ivery−cold, Icold, Icool, Iwarm, Ihot o where Fwarm means not worm and Iwarm means not sure of warmness. This enhances dealing with negative association rules, which is handled as positive rules without extra calculations. Step 4 Now, we count the set of linguistic terms {very cold, cold, cool, warm, hot} for every element in transactions. Since the truth, falsity, and indeterminacy-memberships are independent functions, the set of linguistic terms can be extended to n Tvery−cold, Tcold, Tcool, Twarm, Thot Fvery−cold, Fcold, Fcool, Fwarm, Fhot Ivery−cold, Icold, Icool, Iwarm, Ihot o where Fwarm means not worm and Iwarm means not sure of warmness. This enhances dealing with negative association rules, which is handled as positive rules without extra calculations. Step 5 By using the membership degrees that are given in Table 5 for candidate 1-itemset, the count and support has been calculated, respectively. The corresponding results are summarized in Table 6. Step 5 By using the membership degrees that are given in Table 5 for candidate 1-itemset, the count and support has been calculated, respectively. The corresponding results are summarized in Table 6. Step 5 By using the membership degrees that are given in Table 5 for candidate 1-itemset, the count and support has been calculated, respectively. The corresponding results are summarized in Table 6. Table 6. Support for candidate 1-itemset neutrosophic set. Table 6. Support for candidate 1-itemset neutrosophic set. Table 6. Support for candidate 1-itemset neutrosophic set. 1-itemset Count Support Tverycold 0 0 TCold 1 0.17 TCool 2.8 0.47 TWarm 1.6 0.27 THot 0.6 0.1 Iverycold 0 0 ICold 1.8 0.3 ICool 2.6 0.43 IWarm 2.2 0.37 IHot 0.9 0.15 Fverycold 6 1 FCold 5 0.83 FCool 3.2 0.53 FWarm 4.4 0.73 FHot 5.4 0.9 Similarly, the two-itemset support is illustrated in Table 7 and the rest of itemset generation (k-itemset for k = 3, 4 . . . 8) are obtained similarly. The count for k-item set in database record is defined by minimum count of each one-itemset exists. 4.2. Proposed Model for Association Rule The graphical falsity degrees of these variables are summarized in Figure 4. Also, the graphical indeterminacy degrees of these variables are summarized in Figure 5. On the other hand, for a particular linguistic term, ‘Cool’ in the temperature attribute, their neutrosophic membership functions are represented in Figure 6. ity degrees of these variables are summarized in Figure 4. Also, the graphical indetermina rees of these variables are summarized in Figure 5. On the other hand, for a particular linguis m, ‘Cool’ in the temperature attribute, their neutrosophic membership functions are represented ure 6. Figure 3. Truth-membership function of temperature attribute. Figure 4. Falsity-membership function of temperature attribute. Figure 5. Indeterminacy-membership function of temperature attribute. Figure 3. Truth-membership function of temperature attribute. Figure 3. Truth-membership function of temperature attribute. Figure 4. Falsity-membership function of temperature attribute. Figure 5. Indeterminacy-membership function of temperature attribute. Figure 3. Truth-membership function of temperature attribute. Figure 4. Falsity-membership function of temperature attribute. Figure 4. Falsity-membership function of temperature attribute. Figure 4. Falsity-membership function of temperature attribute. Figure 5. Indeterminacy-membership function of temperature attribute. 11 of 19 11 of 19 Symmetry 2018, 10, 106 Figure 6. Cool (T, I, F) for temperature attribute. Figure 6. Cool (T, I, F) for temperature attribute. Figure 6. Cool (T, I, F) for temperature attribute. Step 3 Based on the membership grades, different transaction has been set up by taking different sets of the temperatures. The membership grades in terms of the neutrosophic sets of these transactions are summarized in Table 5. Step 3 Based on the membership grades, different transaction has been set up by taking different sets of the temperatures. The membership grades in terms of the neutrosophic sets of these transactions are summarized in Table 5. Table 5. Membership function for database Transactions. Transaction Temp. Membership Degree T1 18 Very-cold <0,0,1> cold <0,0,1> cool <1,0.1,0> warm <0,0.1,1> hot <0,0,1> T2 13 Very cold <0,0,1> cold <0.4,0.9,0.6> cool <0.6,0.9,0.4> warm <0,0,1> hot <0,0,1> T3 12 Very cold <0,0,1> cold <0.6,0.9,0.4> cool <0.4,0.9,0.6> warm <0,0,1> hot <0,0,1> T4 33 Very cold <0,0,1> cold <0,0,1> cool <0,0,1> warm <0.4,0.9,0.6> hot <0.6,0.9,0.4> T5 21 Very cold <0,0,1> cold <0,0,1> cool <0.8,0.7,0.2> warm <0.2,0.7,0.8> hot <0,0,1> T6 25 Very cold <0,0,1> cold <0,0,1> cool <0,0,1> warm <1,0.5,0> hot <0,0,1> Table 5. Membership function for database Transactions. 4.2. Proposed Model for Association Rule For example: {TCold, TCool} count is 0.8 Because they exists in both T2 and T3. In T2: TCold = 0.4 and TCool = 0.6 so, count for {TCold, TCool} in T2 = 0.4 In T3: TCold = 0.6 and TCool = 0.4 so, count for {TCold, TCool} in T2 = 0.4 Thus, count of {TCold, TCool} in (Database) DB is 0.8. Table 7. Support for candidate 2-itemset neutrosophic set. Table 7. Support for candidate 2-itemset neutrosophic set. 2-itemset Count Support 2-itemset Count Support {TCold, TCool} 0.8 0.13 {ICold, ICool} 1.8 0.30 {TCold, ICold} 1 0.17 {ICold, Fverycold} 1.8 0.30 {TCold, ICool} 1 0.17 {ICold, FCold} 1 0.17 {TCold, Fverycold} 1 0.17 {ICold, FCool} 1 0.17 {TCold, FCold} 0.8 0.13 {ICold, FWarm} 1.8 0.30 {TCold, FCool} 1 0.17 {ICold, FHot} 1.8 0.30 {TCold, FWarm} 1 0.17 {ICool, IWarm} 0.8 0.13 {TCold, FHot} 1 0.17 {ICool, Fverycold} 2.6 0.43 {TCool, TWarm} 0.2 0.03 {ICool, FCold} 1.8 0.30 {TCool, ICold} 1 0.17 {ICool, FCool} 1.2 0.20 {TCool, FCool} 1.8 0.30 {ICool, FWarm} 2.6 0.43 {TCool, IWarm} 0.8 0.13 {ICool, FHot} 2.6 0.43 {TCool, Fverycold} 2.8 0.47 {IWarm, IHot} 0.9 0.15 13 of 19 Symmetry 2018, 10, 106 Table 7. Cont. {TCool, FCold} 2.8 0.47 {IWarm, Fverycold} 2.2 0.37 {TCool, FCool} 1 0.17 {IWarm, FCold} 2.2 0.37 {TCool, FWarm} 2.8 0.47 {IWarm, FCool} 1.6 0.27 {TCool, FHot} 2.8 0.47 {IWarm, FWarm} 1.4 0.23 {TWarm, THot} 0.4 0.07 {IWarm, FHot} 1.7 0.28 {TWarm, ICool} 0.2 0.03 {IHot, Fverycold} 0.9 0.15 {TWarm, IWarm} 1.1 0.18 {IHot, FCold} 0.9 0.15 {TWarm, IHot} 0.4 0.07 {IHot, FCool} 0.9 0.15 {TWarm, Fverycold} 1.6 0.27 {IHot, FWarm} 0.6 0.10 {TWarm, FCold} 1.6 0.27 {IHot, FHot} 0.4 0.07 {TWarm, FCool} 1.6 0.27 {Fverycold, FCold} 5 0.83 {TWarm, FWarm} 0.6 0.10 {Fverycold, FCool} 3.2 0.53 {TWarm, FHot} 1.6 0.27 {Fverycold, FWarm} 4.4 0.73 {THot, IWarm} 0.6 0.10 {Fverycold, FHot} 5.4 0.90 {THot, IHot} 0.6 0.10 {FCold, FCool} 3 0.50 {THot, Fverycold} 0.6 0.10 {FCold, FWarm} 3.4 0.57 {THot, FCold} 0.6 0.10 {FCold, FHot} 4.4 0.73 {THot, FCool} 0.6 0.10 {FCool, FWarm} 1.8 0.30 {THot, FWarm} 0.6 0.10 {FCool, FHot} 2.6 0.43 {THot, FHot} 0.4 0.07 {FWarm, FHot} 4.2 0.70 5. Case Study In this section, the case of Telecom Egypt Company stock records has been studied. Egyptian stock market has many companies. One of the major questions for stock market users is when to buy or to sell a specific stock. Egyptian stock market has three indicators, EGX30, EGX70, and EGX100. Each indicator gives a reflection of the stock market. Also, these indicators have an important impact on the stock market users, affecting their decisions of buying or selling stocks. We focus in our study on the relation between the stock and the three indicators. Also, we consider the month and quarter of the year to be another dimension in our study, while the sell/buy volume of the stock per day is considered to be the third dimension. In this study, the historical data has been taken from the Egyptian stock market program (Mist) during the program September 2012 until September 2017. For every stock/indicator, Mist keeps a daily track of number of values (opening price, closing price, high price reached, low price reached, and volume). The collected data of Telecom Egypt Stock are summarized in Figure 7. Figure 7. Telecom Egypt stock records. 14 of 19 14 of 19 Symmetry 2018, 10, 106 In this study, we use the open price and close price values to get price change rate, which are defined as follows: l i i price change rate = close price −open price open price × 100 and change the volume to be a percentage of total volume of the stock with the following relation percentage of volume = volume total volume × 100 The same was performed for the stock market indicators. Now, we take the attributes as “quarter”, “month”, “stock change rate”, “volume percentage”, and “indicators change rate”. Table 8 illustrates the segment of resulted data after preparation. Table 8. Segment of data after preparation. Table 8. Segment of data after preparation. Table 8. Segment of data after preparation. Table 8. Segment of data after preparation. 5. Case Study Ts_Date Month Quarter Change Volume Change30 Change70 Change100 13 September 2012 September 3 0.64 0.03 −1.11 0.01 −0.43 16 September 2012 September 3 0.07 0.02 2.82 4.50 3.67 17 September 2012 September 3 3.47 0.12 1.27 0.76 0.81 18 September 2012 September 3 1.38 0.03 −0.08 −0.48 −0.43 19 September 2012 September 3 −1.48 0.02 0.35 −1.10 −0.64 20 September 2012 September 3 0.47 0.05 −1.41 −1.64 −1.55 23 September 2012 September 3 3.64 0.02 −0.21 1.00 0.41 24 September 2012 September 3 −0.47 0.05 0.27 −0.09 0.03 25 September 2012 September 3 −2.77 0.15 2.15 1.79 1.85 26 September 2012 September 3 1.96 0.04 0.22 0.96 0.57 27 September 2012 September 3 0.90 0.05 −1.38 −0.88 −0.92 30 September 2012 September 3 −0.14 0.00 −1.11 −0.79 −0.75 1 October 2012 October 4 −1.60 0.02 −2.95 −4.00 −3.51 Based on these linguistic terms, define the ranges under the SVNSs environment. For this, corresponding to the attribute in “change rate” and “volume” the truth membership functions by Based on these linguistic terms, define the ranges under the SVNSs environment. For this, corresponding to the attribute in “change rate” and “volume”, the truth-membership functions by defining their linguistic terms as {“high up”, “high low”, “no change”, “low down”, “high down”} corresponding to attribute “change rate”, while for the attribute “volume”, the linguistic terms are (low, medium, and high) and their ranges are summarized in Figures 8 and 9, respectively. The falsity-membership function and indeterminacy-membership function have been calculated and applied as well for change rate attribute. Figure 8. Change rate attribute truth-membership function. Figure 8. Change rate attribute truth-membership function. 15 of 19 Symmetry 2018, 10, 106 Figure 9. Volume attribute truth-membership function. Figure 9. Volume attribute truth-membership function. 6. Experimental Results We proceeded to a comparison between fuzzy mining and neutrosophic mining algorithms, and we found out that the number of generated association rules increased in neutrosophic mining. A program has been developed to generate large itemsets for Telecom Egypt historical data. VB.net has been used in creating this program. The obtained data have been stored in an access database. The comparison depends on the number of generated association rules in a different min-support threshold. It should be noted that the performance cannot be part of the comparison because of the number of items (attributes) that are different in fuzzy vs. neutrosophic association rules mining. In fuzzy mining, the number of items was 14, while in neutrosophic mining it is 34. This happens because the number of attributes increased. Spreading each linguistic term into three (True, False, Indeterminacy) terms make the generated rules increase. The falsity-generated association rules can be considered a negative association rules. As pointed out in [36], the conviction of a rule conv(X →Y) is defined as the ratio of the expected frequency that X happened without Y falsity-association rules to be used to generate negative association rules if T(x) + F(x) = 1. In Table 9, the number of generated fuzzy rules in each k−itemset using different min-support threshold are reported, while the total generated fuzzy association rule is presented in Figure 10. Figure 10. No. of fuzzy association rules with different min-support threshold. Figure 10. No. of fuzzy association rules with different min-support threshold. 16 of 19 Symmetry 2018, 10, 106 Table 9. No. of resulted fuzzy rules with different min-support. Min-Support 0.02 0.03 0.04 0.05 1-itemset 10 10 10 10 2-itemset 37 36 36 33 3-itemset 55 29 15 10 4-itemset 32 4 2 0 As compared to the fuzzy approach, by applying the same min-support threshold, we get a huge set of neutrosophic association rules. Table 10 illustrates the booming that happened to generated neutrosophic association rules. We stop generating itemsets at iteration 4 due to the noted expansion in the results shown in Figure 11, which shows the number of neutrosophic association rules. Table 10. No. of neutrosophic rules with different min-support threshold. Table 10. No. of neutrosophic rules with different min-support threshold. Min-Support 0.02 0.03 0.04 0.05 1-itemset 26 26 26 26 2-itemset 313 311 309 300 3-itemset 2293 2164 2030 1907 4-itemset 11,233 9689 8523 7768 Figure 11. No. 6. Experimental Results of neutrosophic association rules with different min-support threshold. Figure 11. No. of neutrosophic association rules with different min-support threshold. Experiment has been re-run using different min-support threshold values and the resulted neutrosophic association rules counts has been noted and listed in Table 11. Note the high values that are used for min-support threshold. Figure 12 illustrates the generated neutrosophic association rules for min-support threshold from 0.5 to 0.9. Table 11. No. of neutrosophic rules with different min-support threshold. Min-Support 0.5 0.6 0.7 0.8 0.9 1-itemset 11 9 9 6 5 2-itemset 50 33 30 11 10 3-itemset 122 64 50 10 10 4-itemset 175 71 45 5 5 5-itemset 151 45 21 1 1 6-itemset 88 38 8 0 0 Table 11. No. of neutrosophic rules with different min-support threshold. 17 of 19 Symmetry 2018, 10, 106 Figure 12. No. of neutrosophic rules for min-support threshold from 0.5 to 0.9. Figure 12. No. of neutrosophic rules for min-support threshold from 0.5 to 0.9. 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In addition, the indeterminacy-membership function has been used to prevent losing rules from boundaries problems. The proposed model is more effective in processing negative association rules. By comparing it with the fuzzy association rules mining approaches, we conclude that the proposed model generates a larger number of positive and negative association rules, thus ensuring the construction of a real and efficient decision-making system. In the future, we plan to extend the comparison between the neutrosophic association rule mining and other interval fuzzy association rule minings. Furthermore, we seized the falsity-membership function capacity to generate negative association rules. Conjointly, we availed of the indeterminacy-membership function to prevent losing rules from boundaries problems. Many applications can emerge by adaptions of truth-membership function, indeterminacy-membership function, and falsity-membership function. Future work will benefit from the proposed model in generating negative association rules, or in increasing the quality of the generated association rules by using multiple support thresholds and multiple confidence thresholds for each membership function. The proposed model can be developed to mix positive association rules (represented in the truth-membership function) and negative association rules (represented in the falsity-membership function) in order to discover new association rules, and the indeterminacy-membership function can be put forth to help in the automatic adoption of support thresholds and confidence thresholds. Finally, yet importantly, we project to apply the proposed model in the medical field, due to its capability in effective diagnoses through discovering both positive and negative symptoms of a disease. All future big data challenges could be handled by combining neutrosophic sets with various techniques. 18 of 19 18 of 19 Symmetry 2018, 10, 106 Author Contributions: All authors have contributed equally to this paper. 7. Conclusions and Future Work The individual responsibilities and contribution of all authors can be described as follows: the idea of this whole paper was put forward by Mohamed Abdel-Basset and Mai Mohamed, Victor Chang completed the preparatory work of the paper. Florentin Smarandache analyzed the existing work. The revision and submission of this paper was completed by Mohamed Abdel-Basset. Conflicts of Interest: The authors declare no conflict of interest. References A generic scheme for the design of efficient on-line algorithms for lattices. In Conceptual Structures for Knowledge Creation and Communication; Springer: Berlin/Heidelberg, Germany, 2003; pp. 282–295. 13. Verlinde, H.; de Cock, M.; Boute, R. 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Mining association rules from quantitative data. Intell. Data Anal. 1999, 3, 363–376. [CrossRef] 18. Pei, B.; Zhao, S.; Chen, H.; Zhou, X.; Chen, D. FARP: Mining fuzzy association rules from a probabilistic quantitative database. Inf. Sci. 2013, 237, 242–260. [CrossRef] 19. Siji, P.D.; Valarmathi, M.L. Enhanced Fuzzy Association Rule Mining Techniques for Prediction Analysis in Betathalesemia’s Patients. Int. J. Eng. Res. Technol. 2014, 4, 1–9. g 20. Lee, Y.-C.; Hong, T.-P.; Wang, T.-C. Multi-level fuzzy mining with multiple minimum supports. Expert Syst. Appl. 2008, 34, 459–468. [CrossRef] 19 of 19 Symmetry 2018, 10, 106 21. Chen, C.-H.; Hong, T.-P.; Li, Y. Fuzzy association rule mining with type-2 membership functions. In Proceedings of the Asian Conference on Intelligent Information and Database Systems, Bali, Indonesia, 23–25 March 2015; pp. 128–134. 2. Sheibani, R.; Ebrahimzadeh, A. An algorithm for mining fuzzy association rules. 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In Proceedings of the 5th International Conference on Computer Science and Education (ICCSE), Hefei, China, 24–27 August 2010; pp. 1344–1349. © 2018 by the authors. Licensee MDPI, Basel, Switzerland. References 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/).

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English

1-(4-Chlorophenyl)-3-phenyl-1<i>H</i>-pyrazol-5(4<i>H</i>)-one

Acta crystallographica. Section E

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organic compounds Acta Crystallographica Section E Structure Reports Online ISSN 1600-5368 Refinement R[F 2 > 2(F 2)] = 0.039 wR(F 2) = 0.111 S = 1.04 3172 reflections 172 parameters H-atom parameters constrained max = 0.23 e A˚ 3 min = 0.20 e A˚ 3 Table 1 Hydrogen-bond geometry (A˚ , ). Cg1 is the centroid of the C10–C15 ring. D—H  A D—H H  A D  A D—H  A C8—H8B  O1i 0.97 2.40 3.3115 (19) 156 C8—H8A  Cg1ii 0.97 2.76 3.5026 (17) 134 Symmetry codes: (i) x; y þ 3 2; z þ 1 2; (ii) x þ 2; y þ 1; z þ 2. Table 2 Comparison of C O and N—N bond lengths (A˚ ) between the title compound and reported pyrazolone compounds.. Compound C O N—N C13H14N2O2 a 1.313 (2) 1.395 (2) C19H16N2O2 a 1.261 (2) 1.404 (2) C15H12N2O2Sa 1.246 (2) 1.373 (2) C22H15ClN2Oc 1.228 (2) 1.405 (2) C16H11N3Oc 1.252 (3) 1.412 (4) C16H10ClN3Oc 1.250 (5) 1.420 (5) C10H8N4O5 d 1.207 (3) 1.412 (2) C15H11ClN2Oe 1.213 (2) 1.404 (2) Notes: (a) Holzer et al. (1999); (b) Bovio et al. (1974); (c) Ferretti et al. (1985); (d) Dardonville et al. (1998); (e) this work. Refinement R[F 2 > 2(F 2)] = 0.039 wR(F 2) = 0.111 S = 1.04 3172 reflections 172 parameters H-atom parameters constrained max = 0.23 e A˚ 3 min = 0.20 e A˚ 3 Table 1 Hydrogen-bond geometry (A˚ , ). Cg1 is the centroid of the C10–C15 ring. D—H  A D—H H  A D  A D—H  A C8—H8B  O1i 0.97 2.40 3.3115 (19) 156 C8—H8A  Cg1ii 0.97 2.76 3.5026 (17) 134 Symmetry codes: (i) x; y þ 3 2; z þ 1 2; (ii) x þ 2; y þ 1; z þ 2. 172 parameters H-atom parameters constrained max = 0.23 e A˚ 3 min = 0.20 e A˚ 3 Table 2 Comparison of C O and N—N bond lengths (A˚ ) between the title compound and reported pyrazolone compounds.. Key indicators: single-crystal X-ray study; T = 296 K; mean (C–C) = 0.002 A˚; R factor = 0.039; wR factor = 0.111; data-to-parameter ratio = 18.4. Compound C O N—N C13H14N2O2 a 1.313 (2) 1.395 (2) C19H16N2O2 a 1.261 (2) 1.404 (2) C15H12N2O2Sa 1.246 (2) 1.373 (2) C22H15ClN2Oc 1.228 (2) 1.405 (2) C16H11N3Oc 1.252 (3) 1.412 (4) C16H10ClN3Oc 1.250 (5) 1.420 (5) C10H8N4O5 d 1.207 (3) 1.412 (2) C15H11ClN2Oe 1.213 (2) 1.404 (2) Notes: (a) Holzer et al. (1999); (b) Bovio et al. (1974); (c) Ferretti et al. (1985); (d) Dardonville et al. (1998); (e) this work. In the crystal of the title compound, C15H11ClN2O, the molecules are linked by C—H  O and weak C—H   interactions. The chlorophenyl and phenyl rings are twisted with respect to the central pyrazolone ring, making dihedral angles of 18.23 (8) and 8.35 (8), respectively. The N—N and C O bond lengths are comparable to those reported for pyrazolone compounds. Notes: (a) Holzer et al. (1999); (b) Bovio et al. (1974); (c) Ferretti et al. (1985); (d) Dardonville et al. (1998); (e) this work. Data collection: APEX2 (Bruker, 2005); cell refinement: SAINT (Bruker, 2005); data reduction: SAINT; program(s) used to solve structure: SHELXS97 (Sheldrick, 2008); program(s) used to refine structure: SHELXL97 (Sheldrick, 2008); molecular graphics: SHELXTL (Sheldrick, 2008); software used to prepare material for publication: SHELXL97. Related literature For the properties and applications of pyrazolones and their derivatives, see: Bao et al. (2006); Bose et al. (2005); Ito et al. (2001); Li et al. (2000); Shi, et al. (2005); Whitaker (1995). For the synthesis, see: Jensen (1959). For related structures, see: Bovio et al. (1974); Dardonville et al. (1998); Ferretti et al. (1985); Holzer et al. (1999). The authors gratefully acknowledge financial support by the Scientific Research Innovation Foundation for youth teachers of Zhoukou Normal University Supplementary data and figures for this paper are available from the IUCr electronic archives (Reference: DN2522). Table 1 Cg1 is the centroid of the C10–C15 ring. Yong-Jie Dinga and Chun-Xiang Zhaob* aDepartment of Chemistry, East China Normal University, Shanghai 200062, People’s Republic of China, and bDepartment of Chemistry, Zhoukou Normal University, Zhoukou 466001, People’s Republic of China Correspondence e-mail: [email protected] Received 9 December 2009; accepted 23 December 2009 References Bao, F., Lu, X., Kang, B.-S. & Wu, Q. (2006). Eur. Polym. J. 42, 928–934. Bose, R., Murty, D. S. R. & Chakrapani, G. (2005). J. Radioanal. Nucl. Chem. 265, 115–122. Bao, F., Lu, X., Kang, B.-S. & Wu, Q. (2006). Eur. Polym. J. 42, 928–934. Bao, F., Lu, X., Kang, B. S. & Wu, Q. (2006). Eur. Polym. J. 42, 928 934. Bose, R., Murty, D. S. R. & Chakrapani, G. (2005). J. Radioanal. Nucl. Chem. 265, 115–122. Experimental Bovio, B. & Locchi, S. (1974). J. Cryst. Mol. Struct. 4, 129–140. Bruker (2005). APEX2 and SAINT. Bruker AXS Inc., Madison, Wisconsin, USA. V = 1276.31 (7) A˚ 3 Z = 4 Mo K radiation  = 0.29 mm1 T = 296 K 0.32  0.28  0.15 mm Dardonville, C., Elguero, J., Rozas, I., Ferna´ndez-Castao, C., Foces-Foces, C. & Sobrados, I. (1998). New J. Chem. 22, 1421–1430. Ferretti, V., Bertolasi, V., Gilli, G. & Borea, P. A. (1985). Acta Cryst. C41, 107– 140. Holzer, W., Mereiter, K. & Plagens, B. (1999). Heterocycles, 50, 799–818. Ito, T., Goto, C. & Noguchi, K. (2001). Anal. Chim. Acta, 443, 41–51. g ( ) Jensen, B. S. (1959). Acta Chem. Scand. 13,1668–1670. Li, J.-Z., Li, G. & Yu, W.-J. (2000). J. Rare Earth, 18, 233–236. Sheldrick, G. M. (2008). Acta Cryst. A64, 112–122. 3172 independent reflections 2578 reflections with I > 2(I) Rint = 0.021 Shi, M., Li, F.-Y., Yi, T., Zhang, D.-Q., Hu, H.-M. & Huang, C.-H. (2005). Inorg. Chem. 44, 8929–8936. , Whitaker, A. (1995). J. Soc. Dyers Colour. 111, 66–72. Ding and Zhao o709 o709 Ding and Zhao Acta Cryst. (2010). E66, o709 doi:10.1107/S1600536809055263 Experimental All reagents were obtained from commercial sources and used without further purification. 1-(4-chlorophenyl)-3-phenyl- 1H-pyrazol-5(4H)-one was synthesized according to the method proposed by Jensen (1959). (yield 84.5%; m.p. 435–436 K). Analysis required for C15H11ClN2O: C 66.55%, H 4.10%, N10.35%; found: C 66.51, H 4.08, N 10.41%. Block-like golden single crystals of CPP were grown from ethanol by slow evaporation over a period of several weeks. 1-(4-Chlorophenyl)-3-phenyl-1H-pyrazol-5(4H)-one Y.-J. Ding and C.-X. Zhao Y.-J. Ding and C.-X. Zhao Comment Pyrazolones and their derivatives constitute a group of organic compounds that have been extensively studied due to their diverse properties and applications. For example, many more applications have been devised for this group of molecules in the pharmaceutical field. Moreover, they have been applied to the solvent extraction of metal ions (Bose et al., 2005), for analytical purposes (Ito et al., 2001), in the preparation of azo colorants (Whitaker, 1995), as ligands in complexes with catalytic activity (Bao et al., 2006) and in the synthesis of rare earth metal complexes with interesting photophysical properties (Shi et al., 2005). Also, it is important in understanding the behaviour of these compounds with respect to the mechanisms of pharmacological activities (Li et al., 2000). In order to expand this field, the novel title compound (I) has been synthesized, and its crystal structure is reported herein for the first time. The asymmetric unit of the title compound is built up from a central pyrazolone ring substituted in 1,3 by a 4-chlorophenyl and a phenyl rings (Fig. 1). The chlorophenyl and phenyl rings are slightly twisted with respect to the central pyrazolone ring making dihedral angles of 18.23 (8)° and 8.35 (8)° respectively, thus indicating a high degree of conjugation and electron delocalization.The N(1)–N(2) and C(7)=O(1) distances are comparable, within experimental errors, with related pyrazolones reported in the literature (Table 2). The cohesion of the crystal is assured by weak C-H···O and C-H···π interactions (Table 1). supplementary materials supplementary materials supplementary materials Acta Cryst. (2010). E66, o709 [ doi:10.1107/S1600536809055263 ] Acta Cryst. (2010). E66, o709 [ doi:10.1107/S1600536809055263 ] supplementary materials wR(F2) = 0.111 H-atom parameters constrained S = 1.04 w = 1/[σ2(Fo 2) + (0.0526P)2 + 0.3591P] where P = (Fo 2 + 2Fc 2)/3 3172 reflections (Δ/σ)max < 0.001 172 parameters Δρmax = 0.23 e Å−3 0 restraints Δρmin = −0.20 e Å−3 wR(F2) = 0.111 H-atom parameters constrained S = 1.04 w = 1/[σ2(Fo 2) + (0.0526P)2 + 0.3591P] where P = (Fo 2 + 2Fc 2)/3 3172 reflections (Δ/σ)max < 0.001 172 parameters Δρmax = 0.23 e Å−3 0 restraints Δρmin = −0.20 e Å−3 wR(F2) = 0.111 S = 1.04 3172 reflections 172 parameters 0 restraints H-atom parameters constrained w = 1/[σ2(Fo 2) + (0.0526P)2 + 0.3591P] where P = (Fo 2 + 2Fc 2)/3 (Δ/σ)max < 0.001 Δρmax = 0.23 e Å−3 Δρmin = −0.20 e Å−3 H-atom parameters constrained H-atom parameters constrained w = 1/[σ2(Fo 2) + (0.0526P)2 + 0.3591P] where P = (Fo 2 + 2Fc 2)/3 (Δ/σ)max < 0.001 Δρmax = 0.23 e Å−3 Δρmin = −0.20 e Å−3 Refinement All H atoms were placed in calculated positions, with C—H = 0.93Å for phenyl and 0.97 Å for methylene, and treated as riding with Uiso(H) =1.2Ueq (C) . sup-1 supplementary materials Figures Fig. 1. The molecular structure of (I) (thermal ellipsoids are shown at 30% probability levels). 1-(4-Chlorophenyl)-3-phenyl-1H-pyrazol-5(4H)-one Figures Fig. 1. The molecular structure of (I) (thermal ellip 1-(4-Chlorophenyl)-3-phenyl-1H-pyrazol-5(4H)-one Fig. 1. The molecular structure of (I) (thermal ellipsoids are shown at 30% probability levels). 1-(4-Chlorophenyl)-3-phenyl-1H-pyrazol-5(4H)-one F(000) = 560.0 Dx = 1.409 Mg m−3 Mo Kα radiation, λ = 0.71073 Å Cell parameters from 8730 reflections θ = 2.5–28.4° µ = 0.29 mm−1 T = 296 K Block, yellow 0.32 × 0.28 × 0.15 mm Refinement Refinement on F2 Least-squares matrix: full R[F2 > 2σ(F2)] = 0.039 sup-2 supplementary materials Special details Geometry. All e.s.d.'s (except the e.s.d. in the dihedral angle between two l.s. planes) are estimated using the full covariance mat- rix. The cell e.s.d.'s are taken into account individually in the estimation of e.s.d.'s in distances, angles and torsion angles; correlations between e.s.d.'s in cell parameters are only used when they are defined by crystal symmetry. An approximate (isotropic) treatment of cell e.s.d.'s is used for estimating e.s.d.'s involving l.s. planes. Refinement. Refinement of F2 against ALL reflections. The weighted R-factor wR and goodness of fit S are based on F2, convention- al R-factors R are based on F, with F set to zero for negative F2. The threshold expression of F2 > σ(F2) is used only for calculating R- factors(gt) etc. and is not relevant to the choice of reflections for refinement. R-factors based on F2 are statistically about twice as large as those based on F, and R- factors based on ALL data will be even larger. Fractional atomic coordinates and isotropic or equivalent isotropic displacement parameters (Å2) Fractional atomic coordinates and isotropic or equivalent isotropic displacement parameters (Å2) x y z Uiso*/Ueq Cl1 0.49588 (4) 0.36865 (4) 0.10838 (4) 0.06295 (16) N2 0.73364 (11) 0.53865 (9) 0.68321 (12) 0.0405 (3) N1 0.75985 (10) 0.46368 (9) 0.79773 (12) 0.0396 (3) C7 0.76387 (14) 0.64516 (11) 0.72628 (15) 0.0442 (3) C8 0.82119 (14) 0.63688 (11) 0.88421 (15) 0.0434 (3) H8A 0.9060 0.6593 0.9047 0.052* H8B 0.7777 0.6811 0.9407 0.052* C1 0.67906 (12) 0.49854 (11) 0.54432 (14) 0.0380 (3) C4 0.56921 (13) 0.41944 (13) 0.27598 (14) 0.0445 (3) O1 0.74620 (13) 0.72658 (9) 0.65145 (12) 0.0620 (3) C5 0.56870 (15) 0.35700 (12) 0.39612 (16) 0.0491 (3) H5 0.5317 0.2883 0.3866 0.059* C10 0.85009 (12) 0.46390 (11) 1.05204 (14) 0.0392 (3) C9 0.80911 (12) 0.51759 (11) 0.91237 (14) 0.0375 (3) C3 0.62544 (16) 0.51956 (13) 0.28785 (16) 0.0538 (4) H3 0.6260 0.5607 0.2060 0.065* C15 0.82395 (14) 0.35388 (12) 1.07152 (17) 0.0476 (3) H15 0.7787 0.3135 0.9952 0.057* C6 0.62340 (13) 0.39676 (12) 0.53086 (15) 0.0448 (3) H6 0.6228 0.3551 0.6123 0.054* C11 0.91727 (14) 0.52294 (13) 1.16804 (15) 0.0481 (3) H11 0.9340 0.5967 1.1569 0.058* C13 0.93286 (17) 0.36390 (16) 1.31805 (18) 0.0617 (4) H13 0.9606 0.3304 1.4069 0.074* C2 0.68150 (16) 0.55939 (13) 0.42195 (16) 0.0531 (4) sup-3 supplementary materials supplementary materials supplementary materials sup-4 H2 0.7209 0.6270 0.4303 0.064* C12 0.95927 (16) 0.47258 (15) 1.29984 (16) 0.0582 (4) H12 1.0054 0.5122 1.3763 0.070* C14 0.86514 (16) 0.30462 (15) 1.20418 (18) 0.0594 (4) H14 0.8472 0.2313 1.2168 0.071* Atomic displacement parameters (Å2) U11 U22 U33 U12 U13 U23 Cl1 0.0769 (3) 0.0676 (3) 0.0378 (2) 0.0076 (2) −0.00071 (18) −0.01026 (16) N2 0.0514 (6) 0.0331 (5) 0.0348 (5) −0.0008 (5) 0.0052 (5) 0.0022 (4) N1 0.0474 (6) 0.0345 (6) 0.0352 (5) −0.0001 (5) 0.0058 (5) 0.0031 (4) C7 0.0551 (8) 0.0358 (7) 0.0416 (7) −0.0025 (6) 0.0108 (6) −0.0011 (5) C8 0.0559 (8) 0.0347 (7) 0.0392 (7) −0.0025 (6) 0.0101 (6) −0.0032 (5) C1 0.0415 (7) 0.0371 (7) 0.0347 (6) 0.0030 (5) 0.0071 (5) −0.0001 (5) C4 0.0479 (7) 0.0494 (8) 0.0342 (6) 0.0093 (6) 0.0053 (5) −0.0045 (6) O1 0.0983 (9) 0.0359 (5) 0.0487 (6) −0.0051 (5) 0.0098 (6) 0.0068 (5) C5 0.0574 (9) 0.0413 (7) 0.0435 (7) −0.0040 (6) 0.0007 (6) −0.0003 (6) C10 0.0409 (7) 0.0420 (7) 0.0354 (6) 0.0008 (5) 0.0100 (5) 0.0003 (5) C9 0.0408 (6) 0.0354 (6) 0.0369 (6) −0.0001 (5) 0.0099 (5) −0.0018 (5) C3 0.0736 (10) 0.0533 (9) 0.0353 (7) −0.0010 (7) 0.0141 (7) 0.0050 (6) C15 0.0506 (8) 0.0447 (8) 0.0452 (7) −0.0065 (6) 0.0059 (6) 0.0028 (6) C6 0.0535 (8) 0.0408 (7) 0.0370 (7) −0.0022 (6) 0.0035 (6) 0.0048 (5) C11 0.0593 (9) 0.0468 (8) 0.0380 (7) −0.0041 (6) 0.0106 (6) −0.0038 (6) C13 0.0682 (10) 0.0733 (12) 0.0418 (8) 0.0019 (8) 0.0088 (7) 0.0161 (7) C2 0.0733 (10) 0.0446 (8) 0.0422 (7) −0.0109 (7) 0.0144 (7) 0.0013 (6) C12 0.0662 (10) 0.0706 (11) 0.0355 (7) −0.0048 (8) 0.0067 (7) −0.0033 (7) C14 0.0665 (10) 0.0543 (9) 0.0559 (9) −0.0069 (8) 0.0106 (8) 0.0155 (7) Geometric parameters (Å, °) Cl1—C4 1.7406 (14) C10—C11 1.3923 (19) N2—C7 1.3806 (17) C10—C15 1.394 (2) N2—N1 1.4042 (15) C10—C9 1.4635 (18) N2—C1 1.4169 (16) C3—C2 1.382 (2) N1—C9 1.2897 (17) C3—H3 0.9300 C7—O1 1.2126 (17) C15—C14 1.384 (2) C7—C8 1.504 (2) C15—H15 0.9300 C8—C9 1.4900 (18) C6—H6 0.9300 C8—H8A 0.9700 C11—C12 1.384 (2) C8—H8B 0.9700 C11—H11 0.9300 C1—C6 1.3824 (19) C13—C12 1.377 (3) C1—C2 1.3894 (19) C13—C14 1.382 (3) C4—C3 1.367 (2) C13—H13 0.9300 C4—C5 1.378 (2) C2—H2 0.9300 C5—C6 1.383 (2) C12—H12 0.9300 C5—H5 0.9300 C14—H14 0.9300 C7—N2—N1 112.65 (11) N1—C9—C8 112.52 (11) H2 0.7209 0.6270 0.4303 0.064* C12 0.95927 (16) 0.47258 (15) 1.29984 (16) 0.0582 (4) H12 1.0054 0.5122 1.3763 0.070* C14 0.86514 (16) 0.30462 (15) 1.20418 (18) 0.0594 (4) H14 0.8472 0.2313 1.2168 0.071* supplementary materials su C7—N2—C1 128.96 (11) C10—C9—C8 125.29 (12) N1—N2—C1 118.37 (10) C4—C3—C2 119.75 (14) C9—N1—N2 107.72 (11) C4—C3—H3 120.1 O1—C7—N2 126.65 (14) C2—C3—H3 120.1 O1—C7—C8 128.47 (13) C14—C15—C10 120.14 (15) N2—C7—C8 104.88 (11) C14—C15—H15 119.9 C9—C8—C7 102.15 (11) C10—C15—H15 119.9 C9—C8—H8A 111.3 C1—C6—C5 119.88 (13) C7—C8—H8A 111.3 C1—C6—H6 120.1 C9—C8—H8B 111.3 C5—C6—H6 120.1 C7—C8—H8B 111.3 C12—C11—C10 120.47 (15) H8A—C8—H8B 109.2 C12—C11—H11 119.8 C6—C1—C2 119.68 (13) C10—C11—H11 119.8 C6—C1—N2 119.22 (12) C12—C13—C14 119.98 (15) C2—C1—N2 121.10 (12) C12—C13—H13 120.0 C3—C4—C5 120.84 (13) C14—C13—H13 120.0 C3—C4—Cl1 120.53 (11) C3—C2—C1 120.03 (14) C5—C4—Cl1 118.63 (12) C3—C2—H2 120.0 C4—C5—C6 119.79 (14) C1—C2—H2 120.0 C4—C5—H5 120.1 C13—C12—C11 120.14 (16) C6—C5—H5 120.1 C13—C12—H12 119.9 C11—C10—C15 118.94 (13) C11—C12—H12 119.9 C11—C10—C9 119.80 (13) C13—C14—C15 120.32 (16) C15—C10—C9 121.26 (13) C13—C14—H14 119.8 N1—C9—C10 122.18 (12) C15—C14—H14 119.8 C7—N2—N1—C9 −1.81 (16) C15—C10—C9—C8 173.39 (14) C1—N2—N1—C9 179.69 (11) C7—C8—C9—N1 1.93 (16) N1—N2—C7—O1 −176.48 (15) C7—C8—C9—C10 −179.50 (13) C1—N2—C7—O1 1.8 (3) C5—C4—C3—C2 −0.7 (2) N1—N2—C7—C8 2.95 (16) Cl1—C4—C3—C2 178.94 (13) C1—N2—C7—C8 −178.75 (13) C11—C10—C15—C14 −0.4 (2) O1—C7—C8—C9 176.62 (16) C9—C10—C15—C14 179.13 (14) N2—C7—C8—C9 −2.79 (15) C2—C1—C6—C5 −1.1 (2) C7—N2—C1—C6 −160.62 (14) N2—C1—C6—C5 179.36 (13) N1—N2—C1—C6 17.60 (18) C4—C5—C6—C1 −0.5 (2) C7—N2—C1—C2 19.9 (2) C15—C10—C11—C12 1.2 (2) N1—N2—C1—C2 −161.90 (13) C9—C10—C11—C12 −178.37 (13) C3—C4—C5—C6 1.4 (2) C4—C3—C2—C1 −1.0 (3) Cl1—C4—C5—C6 −178.24 (12) C6—C1—C2—C3 1.9 (2) N2—N1—C9—C10 −178.86 (11) N2—C1—C2—C3 −178.65 (14) N2—N1—C9—C8 −0.24 (15) C14—C13—C12—C11 0.6 (3) C11—C10—C9—N1 171.35 (13) C10—C11—C12—C13 −1.3 (3) C15—C10—C9—N1 −8.2 (2) C12—C13—C14—C15 0.2 (3) C11—C10—C9—C8 −7.1 (2) C10—C15—C14—C13 −0.3 (3) Hydrogen-bond geometry (Å, °) Cg1 is the centroid of the C10–C15 ring. Atomic displacement parameters (Å2) Atomic displacement parameters (Å2) sup-4 supplementary materials supplementary materials D—H···A D—H H···A D···A D—H···A sup-5 128.96 (11) C10—C9—C8 125.29 (12) 118.37 (10) C4—C3—C2 119.75 (14) 107.72 (11) C4—C3—H3 120.1 126.65 (14) C2—C3—H3 120.1 128.47 (13) C14—C15—C10 120.14 (15) 104.88 (11) C14—C15—H15 119.9 102.15 (11) C10—C15—H15 119.9 111.3 C1—C6—C5 119.88 (13) 111.3 C1—C6—H6 120.1 111.3 C5—C6—H6 120.1 111.3 C12—C11—C10 120.47 (15) 109.2 C12—C11—H11 119.8 119.68 (13) C10—C11—H11 119.8 119.22 (12) C12—C13—C14 119.98 (15) 121.10 (12) C12—C13—H13 120.0 120.84 (13) C14—C13—H13 120.0 120.53 (11) C3—C2—C1 120.03 (14) 118.63 (12) C3—C2—H2 120.0 119.79 (14) C1—C2—H2 120.0 120.1 C13—C12—C11 120.14 (16) 120.1 C13—C12—H12 119.9 118.94 (13) C11—C12—H12 119.9 119.80 (13) C13—C14—C15 120.32 (16) 121.26 (13) C13—C14—H14 119.8 122.18 (12) C15—C14—H14 119.8 −1.81 (16) C15—C10—C9—C8 173.39 (14) 179.69 (11) C7—C8—C9—N1 1.93 (16) −176.48 (15) C7—C8—C9—C10 −179.50 (13) 1.8 (3) C5—C4—C3—C2 −0.7 (2) 2.95 (16) Cl1—C4—C3—C2 178.94 (13) −178.75 (13) C11—C10—C15—C14 −0.4 (2) 176.62 (16) C9—C10—C15—C14 179.13 (14) −2.79 (15) C2—C1—C6—C5 −1.1 (2) −160.62 (14) N2—C1—C6—C5 179.36 (13) 17.60 (18) C4—C5—C6—C1 −0.5 (2) 19.9 (2) C15—C10—C11—C12 1.2 (2) −161.90 (13) C9—C10—C11—C12 −178.37 (13) 1.4 (2) C4—C3—C2—C1 −1.0 (3) −178.24 (12) C6—C1—C2—C3 1.9 (2) −178.86 (11) N2—C1—C2—C3 −178.65 (14) −0.24 (15) C14—C13—C12—C11 0.6 (3) 171.35 (13) C10—C11—C12—C13 −1.3 (3) −8.2 (2) C12—C13—C14—C15 0.2 (3) −7.1 (2) C10—C15—C14—C13 −0.3 (3) ) –C15 ring. D—H H···A D···A D—H···A sup-5 supplementary materials supplementary materials C8—H8B···O1i 0.97 2.40 3.3115 (19) 156 C8—H8A···Cg1ii 0.97 2.76 3.5026 (17) 134 Symmetry codes: (i) x, −y+3/2, z+1/2; (ii) −x+2, −y+1, −z+2. Table 2 Comparison of C═O and N—N bond lengths (Å) between the title compound and reported pyrazolone compounds. Table 2 Comparison of C═O and N—N bond lengths (Å) between the title compound and reported pyrazolone compounds. Compound C═O N—N C13H14N2O2 a 1.313 (2) 1.395 (2) C19H16N2O2 a 1.261 (2) 1.404 (2) C15H12N2O2Sa 1.246 (2) 1.373 (2) C22H15ClN2Oc 1.228 (2) 1.405 (2) C16H11N3Oc 1.252 (3) 1.412 (4) C16H10ClN3Oc 1.250 (5) 1.420 (5) C10H8N4O5 d 1.207 (3) 1.412 (2) C15H11ClN2Oe 1.213 (2) 1.404 (2) Notes: (a) Holzer et al. (1999); (b) Bovio et al. (1974); (c) Ferretti et al. (1985); (d) Dardonville et al. (1998); (e) this work. Notes: (a) Holzer et al. (1999); (b) Bovio et al. (1974); (c) Ferretti et al. (1985); (d) Dardonville et al. (1998); (e) this work. sup-6 supplementary materials Fig. 1 sup-7

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Being on the Field When the Game Is Still Under Way. The Financial Press and Stock Markets in Times of Crisis

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Introduction Global financial crises have always followed similar patterns throughout history (e.g., [1]), including the crucial role of financial press (e.g., [2]; [3]; [4]; [5]). From the bursting of the Tulip buble in 1637 in the Netherlands to the dot.com bubble in 2001 in America, the financial press has significantly influenced the stock market, often amplifying the cognitive bias and herd behaviour of investors (e.g., [6]; [7]). This behaviour may depend on the strong sensitivity of investors towards bad news. Recent studies have shown that market responses to good and bad news is asymmetric. Indeed, investors are more sensitive to negative news, especially when the market is dominated by uncertainty and unpredictability, and this is an important source of market volatility (e.g., [8]; [9]; [10]; [11]; [12]; [13]; [14]). [15] found that stock prices overreacted to bad news even in good times and underreacted to good news in bad times. Similarly [16] found that bad news has a bigger impact both in phases of market expansion and contraction. Moreover, the press may induce a ‘‘framing effect’’, according to which investors react disproportionally to negative news especially when information source is authoritative (e.g., [17]; [18]; [19]). In this case, communication research indicates that the perceived authorita- tiveness of news sources implies higher trust in the news from these sources ([20]). This effect has been empirically confirmed by a survey on 321 traders and 63 financial journalists from leading banks and financial news providers in the European foreign exchange market ([21]) and a recent case-study on three Dutch banks during the recent financial crisis ([14]). p p To look at this, we investigated the relationship between negative news in financial newspapers and volatility and correla- tion between stock markets during the recent global financial crisis. We analysed one year of front page banner headlines of three financial newspapers, such as the Wall Street Journal, Financial Times, and Il Sole24ore from 1st September 2008 to 1st September 2009, when the recent financial crisis exploded globally. We created an index of bad news per newspaper on a daily base and studied the relation between this index and the closing values of three stock market indexes, such as the DowJones, FTSE and MIB. We considered these stock markets as they were more domestically affected by these newspapers, while comparing their dynamics was essential to look at equivalences and differences across markets and between different press cultures. Received February 22, 2013; Accepted May 22, 2013; Published July 5, 2013 Received February 22, 2013; Accepted May 22, 2013; Published July 5, 2013 Received February 22, 2013; Accepted May 22, 2013; Published July 5, 2013 Copyright:  2013 Casarin, Squazzoni. 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: Roberto Casarin acknowledges financial support by a PRIN 2010–11 grant by the Italian Ministry of Education, University and Research (MIUR) and by the European Union, Seventh Framework Programme FP7/2007–2013 under grant agreement SYRTO-SSH-2012-320270. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: [email protected] recently investigated in empirical finance (e.g., [24]; [25]; [26]), with interesting parallels with recent sociological investigation on financial markets (e.g., [27]). Unlike the efficient market hypoth- esis, these studies show that investors are largely influenced by the media, rumours and gossip even in ‘normal’ market periods, where prices should contain all necessary information (e.g., [24]; [28]). If this is so, we would expect that, in times of financial crisis such as the 2008–2009 period, not only would the investors’ overreaction to bad news have drastically influenced market behaviour, it could even lead investors to overestimate the relevance of not strictly economic, general information. Indeed, in these situations, market prices and other relevant quantitative data on markets are even more variedly interpreted by investors than in normal periods ([27]). Even qualitative, big picture, subjective information, such as streamers in a newspaper, can become relevant in these cases. Roberto Casarin1*, Flaminio Squazzoni2 artment of Economics, University Ca’ Foscari, Venice, Italy, 2 Department of Economics and Business, University of Brescia, Brescia, Ita July 2013 | Volume 8 | Issue 7 | e67721 Abstract This paper looks at the relationship between negative news and stock markets in times of global crisis, such as the 2008/ 2009 period. We analysed one year of front page banner headlines of three financial newspapers, the Wall Street Journal, Financial Times, and Il Sole24ore to examine the influence of bad news both on stock market volatility and dynamic correlation. Our results show that the press and markets influenced each other in generating market volatility and in particular, that the Wall Street Journal had a crucial effect both on the volatility and correlation between the US and foreign markets. We also found significant differences between newspapers in their interpretation of the crisis, with the Financial Times being significantly pessimistic even in phases of low market volatility. Our results confirm the reflexive nature of stock markets. When the situation is uncertain and unpredictable, market behaviour may even reflect qualitative, big picture, and subjective information such as streamers in a newspaper, whose economic and informative value is questionable. Citation: Casarin R, Squazzoni F (2013) Being on the Field When the Game Is Still Under Way. The Financial Press and Stock Markets in Times of Crisis. PLoS ONE 8(7): e67721. doi:10.1371/journal.pone.0067721 Editor: Rodrigo Huerta-Quintanilla, Cinvestav-Merida, Mexico Editor: Rodrigo Huerta-Quintanilla, Cinvestav-Merida, Mexico Received February 22, 2013; Accepted May 22, 2013; Published July 5, 2013 Background and Hypotheses They found that stock messages helped to predict market volatility both on a daily base and also within the same trading day. More specifically, they found that higher message postings predicted negative subsequent returns. They also found that disagreement between the posted messages was associated with increased trading volume. More recently, [31] found that Twitter mood predicted more than 80% of daily volatility of closing values of the Dow Jones Industrial Average. This would confirm Nofsinger’s argument that social mood may cause an increase of decisions biased by optimism or pessimism that could considerably influence aggregate investment and business activity, even reflecting future economic activities ([50]). g g For instance, [29] examined Dartboard, a monthly column of the Wall Street Journal reporting analysts’ recommendations, from 1988 to 1990. Results showed that for the two days following the publication, average positive abnormal returns of 4 percent of the stock recommended were partially reversed only within 25 trading days. Similarly, [25] examined Abreast of the Market, a popular column of the Wall Street Journal, from 1984 to 1999. It is worth noting that, unlike Dartboard, which asks market analysts’ opinions, this column is closer to entertainment than information. The author found that even qualitative information, such as the fraction of negative words in this column, was incorporated in aggregate market valuations. More specifically, results showed that high level of pessimism robustly predicted downward pressure on market prices and that high or low values of pessimism helped to predict high market trading volumes. More recently, [48] examined 30 years of ‘‘Abreast of the Market’’ and showed that even specific columnists can influence stock market behaviour. [49] extended this type of analysis by addressing the impact of negative words in all Wall Street Journal and Dow Jones News Service stories about individual S&P 500 firms from 1980 to 2004. Results showed that negative words in the financial press forecasted low firm earnings and that stock market prices incorporated the information embedded in negative words only with a slight delay. This would confirm that bad news is assimilated faster than good ones in market behaviour (e.g., [19]). To our knowledge, this is the first work that extends the analysis of the impact of financial press from market volatility to market correlation. Background and Hypotheses Combining volatility and correlation was key to: (i) understanding if bad news has had an effect on the growing interdependence of markets, which is presumably correlated with crisis periods (e.g., [9]; [33]); (ii) looking at the impact of bad news not only from the point of view of risk but also from that of risk diversification. We estimated the volatility and correlation dynamics using generalized autoregressive conditional heteroske- dasticity (GARCH) models (see [34]) with dynamic conditional correlation ([35]). Secondly, we estimated the dynamic relation- ship between market volatility/correlation and bad news by using vector autoregressive models (VAR). We also performed a Granger-causality test to verify whether bad news time series had predictive value for market volatility/correlation ([36]). A strong quantitative approach to typical sociological and qualitative factors, such as investors’ mood and media pessimism, was intended to favour cross-fertilization between empirical finance (usually addressed to quantitative facts, but was little concerned with sociological aspects) and sociology of financial markets (strongly concerned with sociological aspects of markets, but poorly interested in macro quantitative market consequences). This integration is essential to understand complex institutional, socio-economic artefacts, such as financial markets. assimilated faster than good ones in market behaviour (e.g., [19]). Other studies showed that this effect was even true for unconventional, un-specialized media, whose information should be less relevant for investors. For example, [24] examined the effect of more than 1.5 million messages posted on Yahoo! Finance and Raging Bull about 45 companies in the Dow Jones Industrial Average and the Dow Jones Internet Index, by measuring bullishness. They found that stock messages helped to predict market volatility both on a daily base and also within the same trading day. More specifically, they found that higher message postings predicted negative subsequent returns. They also found that disagreement between the posted messages was associated with increased trading volume. More recently, [31] found that Twitter mood predicted more than 80% of daily volatility of closing values of the Dow Jones Industrial Average. This would confirm Nofsinger’s argument that social mood may cause an increase of decisions biased by optimism or pessimism that could considerably influence aggregate investment and business activity, even reflecting future economic activities ([50]). The rest of the paper is organized as follows. Sect. 2 illustrates the background and our research hypotheses. Sect. Introduction While the importance of these aspects has been largely underestimated in economics (e.g., [22], [23]), they have been July 2013 | Volume 8 | Issue 7 | e67721 1 PLOS ONE | www.plosone.org Financial Press and Markets in Times of Crisis Our work has important differences compared with previous work. First, unlike previous studies in empirical finance (e.g., [25]), we focused on the last financial crisis as we wanted to better understand the fabric of pessimism that dominated the last years worldwide. Secondly, rather than considering specific market information, as reported in specialized columns, we looked at the impact of general information provided by front page headlines of the financial press. Indeed, front page headlines are crucial in summarizing the meaning, tone and importance of the news but are not expected to contain true, precise and detailed information about economic facts, unlike specialized columns. This is because: (i) headline information is too succinct and (ii) front page messages heavily reflect specific information strategies of the newspapers, which are mainly concerned with impressing and attracting the reader. Furthermore, unlike [24], we did not restrict our interest to strictly speaking financial news but considered economic informa- tion in general. Unlike [29] and [25], we did not focus on precise information concerned with specific stocks but rather looked at general information, which reflects more interpretation than objective details. Thirdly, while studies on the impact of social media on stock markets have recently been carried out that focus on similar crisis periods (e.g.,[30]; [31]), our idea was that, in a situation of financial turmoil, the authoritative columns of certain influential financial newspapers and so also traditional media could have a strong impact on the investors’ mood. Finally, by comparing three newspapers and their respective stock markets, we also wanted to measure differences in interpretation of this global crisis and consider certain country-specific cultural features of the press (e.g., [32]). Background and Hypotheses Many studies in finance have shown that stock market prices incorporate financial press information (e.g., [37]; [38]; [39]). While this may be expected in cases of quantitative information on important economic statistics, such as those regularly released by important institutional agencies (e.g., [40]; [41]; [42]; [43], [44]; [45,46]), it is less likely to find a positive impact of qualitative information, such as journalists’ opinion or reports of market rumours, which is subjective ([47]; [28]). Nevertheless, empirical evidence is also growing in this area. evidence is also growing in this area. For instance, [29] examined Dartboard, a monthly column of the Wall Street Journal reporting analysts’ recommendations, from 1988 to 1990. Results showed that for the two days following the publication, average positive abnormal returns of 4 percent of the stock recommended were partially reversed only within 25 trading days. Similarly, [25] examined Abreast of the Market, a popular column of the Wall Street Journal, from 1984 to 1999. It is worth noting that, unlike Dartboard, which asks market analysts’ opinions, this column is closer to entertainment than information. The author found that even qualitative information, such as the fraction of negative words in this column, was incorporated in aggregate market valuations. More specifically, results showed that high level of pessimism robustly predicted downward pressure on market prices and that high or low values of pessimism helped to predict high market trading volumes. More recently, [48] examined 30 years of ‘‘Abreast of the Market’’ and showed that even specific columnists can influence stock market behaviour. [49] extended this type of analysis by addressing the impact of negative words in all Wall Street Journal and Dow Jones News Service stories about individual S&P 500 firms from 1980 to 2004. Results showed that negative words in the financial press forecasted low firm earnings and that stock market prices incorporated the information embedded in negative words only with a slight delay. This would confirm that bad news is assimilated faster than good ones in market behaviour (e.g., [19]). Other studies showed that this effect was even true for unconventional, un-specialized media, whose information should be less relevant for investors. For example, [24] examined the effect of more than 1.5 million messages posted on Yahoo! Finance and Raging Bull about 45 companies in the Dow Jones Industrial Average and the Dow Jones Internet Index, by measuring bullishness. Background and Hypotheses 3 presents our dataset and illustrates the bad news index that we used to measure the relationship between newspapers and markets. It also shows data on market volatility and correlation. Sect. 4 introduces the model we built to examine the impact of newspapers on markets. Sect. 5 focuses on causal statistical relationships between the press and markets. Sect. 6 provides a robustness and sensitivity analysis, while the last Sect. summarizes our main findings and discusses certain limitations. The idea that markets are influenced by reflexivity mechanisms has been explained by recent sociological investigation, which has mostly examined trading activities in specific organizational contexts (e.g., [51]; [23], [52]; [27]). Unfortunately, these studies underestimated the importance of understanding how context- specific empirical cases could result in aggregate quantitative market data. Our aim was to fill this gap by formulating and empirically testing certain hypotheses on the influence of press information on stock market behaviour comparatively, so as to July 2013 | Volume 8 | Issue 7 | e67721 PLOS ONE | www.plosone.org 2 Financial Press and Markets in Times of Crisis Figure 1. Bad news index per newspaper (in rows) from 1st September 2008 to 1st September 2009 on a daily base. Peaks of bad news are indicated with vertical dashed lines. doi:10.1371/journal.pone.0067721.g001 Figure 1. Bad news index per newspaper (in rows) from 1st September 2008 to 1st September 2009 on a daily base. Peaks of bad news are indicated with vertical dashed lines. doi:10.1371/journal.pone.0067721.g001 consider possible institutional diversity in the relation between financial press and markets. should make investors cautious of these sources ([53]). Further- more, unlike efficient market hypothesis, experimental research has shown that, in uncertainty, investors tend to overestimate their informational gap and are sensitive to any additional information, including subjective one (e.g.,[54]). Therefore, our first hypothesis (H1) is that bad news published by financial newspapers could negatively influence the daily volatility of financial markets during this period. Our research hypotheses were as follows. First, [11] and [28] showed that investors, even those following long-term strategies, are more influenced by negative news as they reduce the difficulty in predicting future outcomes by overestimating the impact of current information (see also [9]; [12]; [13]; [16]). This was also found in experimental and economic psychology (e.g., [22]). Background and Hypotheses Our hypothesis is that the importance of these psychological factors could dramatically increase during financial turmoil as investors tend to disqualify the reliability of prices and even the well- functioning of the markets and are more sensitive to other sources of information, including newspaper headlines. Indeed, the fact that information is subject to profit maximisation by newspapers Secondly, although most financial crises have had an interna- tional impact in the past, the 2008–2009 crisis was truly global as financial markets are now extremely interdependent. Indeed, modern investment technologies allow investors to make millions of operations per time unit, at any time and anywhere (e.g., [55]). In this situation, we expect that the pessimistic messages of July 2013 | Volume 8 | Issue 7 | e67721 PLOS ONE | www.plosone.org July 2013 | Volume 8 | Issue 7 | e67721 3 Financial Press and Markets in Times of Crisis Table 1. Descriptive statistics of the bad news index. Whole Sample First Sub-Sample Second Sub-Sample Mean St.D. Sk. Kurt. Mean St.D. Sk. Kurt. Mean St.D. Sk. Kurt. W 0.82 1.07 1.69 6.41 1.09 1.22 1.30 4.74 0.42 0.61 1.47 4.64 F 1.98 2.49 2.73 15.86 2.38 2.22 1.10 3.67 1.40 2.78 4.39 26.79 S 1.17 2.31 4.12 27.41 1.60 2.76 3.56 20.33 0.55 1.19 2.84 11.85 Whole Sample First Sub-Sample Second Sub-Sample W F S W F S W F S W 1.00* 0.19* 0.13* 1.00* 0.20* 0.08 1.00* 0.03 20.06 F 1.00* 0.11* 1.00* 0.06 1.00* 0.13 S 1.00* 1.00* 1.00* First panel: mean, standard deviation, skewness and kurtosis. Second panel: correlation between indexes. The symbol ‘‘*’’ indicates that the null hypothesis of zero valued Pearson’s correlation was rejected at the 5% significance level. doi:10.1371/journal.pone.0067721.t001 First panel: mean, standard deviation, skewness and kurtosis. Second panel: correlation between indexes. The symbol ‘‘*’’ indicates that the null hypothesis of zero valued Pearson’s correlation was rejected at the 5% significance level. doi:10.1371/journal.pone.0067721.t001 First panel: mean, standard deviation, skewness and kurtosis. Second panel: correlation between indexes. The symbol ‘‘*’’ indicates that the null hypothesis of zero valued Pearson’s correlation was rejected at the 5% significance level. doi:10.1371/journal.pone.0067721.t001 simplicity, we did not distinguish the degree of pessimism by raking the words used. financial newspapers could explain not only market volatility but also dependence between stock markets. Numerous previous studies showed that market interdependence tends to be highly correlated with periods of volatility (e.g., [56]; [22]). Data wk,t~ Ck,t Lk,t ð1Þ ð1Þ The Bad News Index Our dataset includes one year of front page banner headlines of three financial newspapers, namely the Wall Street Journal, Financial Times and Il Sole24ore, on a daily base. For technical constraints, i.e., the unavailability of fully accessible electronic editions or lack of front page news included in electronic versions, we collected data manually on printed versions of the newspapers. Budget, time and linguistic constraints did not permit us to consider other national newspapers or cover longer time periods. The dataset and a text file, including a full description of the variables, are available as supplementary information. We analysed any front page banner headline from 1st September 2008 to 1st September 2009 which conveyed news on the crisis (not only those expressly related to financial markets) by measuring the emphasis and the tone of the message. The emphasis was measured by counting the number of banner columns reporting an economic news compared with the total number of potentially available columns, according to standard newspaper layout. We assumed that the higher the percentage of columns assigned to the banner headline, the stronger the emphasis of the message was. The tone was measured by counting the ratio of negative words over the total words used in the headline text (all included, also verbs and conjunctions), such as ‘‘recession’’, ‘‘fear’’ and so on. We assumed that the higher was the number of negative words in the text, the stronger the pessimism of the message was. For the sake of k~F,W,S and t~1, . . . ,T. Then the journal-specific bad news index, Bk,t, at time t for the journal k, was defined as k~F,W,S and t~1, . . . ,T. Then the journal-specific bad news index, Bk,t, at time t for the journal k, was defined as Bk,t~wk,t(Tk,t(1zNk,t)) ð2Þ ð2Þ for k~F,W,S and t~1, . . . ,T. Background and Hypotheses For instance, [9] and [33] suggested that in periods of crisis and high market volatility, covariation could even include markets that do not have much in common. [57] suggested that this trend has intensified especially recently with increasing globalization of investment strategies. Our hypothesis (H2) is that in periods of turmoil, bad news could even influence market correlation and has an impact on global investment strategies. Coherently, we expected that in this period the interplay of financial markets and the press could determine a cascade of pessimism that co-influences both information and market behaviour (H3) (e.g., [58]). Our bad news index was based on three types of information. We considered the number of negative banner headlines on the crisis, Lk,t, the number of columns, Ck,t, where news were reported, and the number of negative words reported in the text, Nk,t, at time t for each journal k~F,W,S, where F stands for Financial Times, W for Wall Street Journal, and S for IlSole24ore. It is worth noting that the time index t refers to open-trading days, so time t~1, . . . 250 days. The information from the press during a non-trading day is reported together with the information of the first subsequent open-trading day. The index was build as follows. Let Tk,t be the maximum number of available columns for a banner headline in the newspaper, then the relative importance index was Descriptive Statistics Figure 1 shows the bad new index per newspaper. The vertical dashed lines correspond to certain peaks of bad news. The first peak was on 16th September 2008, the day before it was announced that Lehman Brothers filed for Chapter 11 bankruptcy protection, Merrill Lynch agreed to be sold to Bank of America for 50 billion dollars and estimates said that up to 50.000 jobs were at risk. The second peak was on 24th October 2008 after the Congressional hearing where Alan Greenspan admitted that he had put too much faith in the self-correcting power of markets. A third peak was on 6th April 2009, involving especially the Financial Times, when the Geithner plan to buy toxic assets was strongly criticised as a means to provide government ‘‘cash for trash’’ and UK analysts started to forecast that stagflation was around the corner. July 2013 | Volume 8 | Issue 7 | e67721 PLOS ONE | www.plosone.org 4 Financial Press and Markets in Times of Crisis Figure 2. Daily log-returns (first row) of the FTSE, DowJones and MIB indexes from 1st September 2008 to 1st September 2009. Daily log-volatilities (second row) and correlations (third row), evaluated sequentially over time with a rolling window of t~60 observations and a smoothing factor l~0:99. In the last row, the red lines indicate the 95% confidence band about the estimated correlations. doi:10.1371/journal.pone.0067721.g002 Financial Press and Markets in Times of Crisis Figure 2. Daily log-returns (first row) of the FTSE, DowJones and MIB indexes from 1st September 2008 to 1st September 2009. Daily log-volatilities (second row) and correlations (third row), evaluated sequentially over time with a rolling window of t~60 observations and a smoothing factor l~0:99. In the last row, the red lines indicate the 95% confidence band about the estimated correlations. doi:10.1371/journal.pone.0067721.g002 If we consider bad news dynamics, our one-year sample could be approximately divided into two sub-samples, i.e., an initial period characterized by higher concentration of bad news, and a second one after spring 2009, where bad news were generally less frequent. By comparing the three newspapers, it is evident that the Wall Street Journal was more cautious and the Financial Times published bad news more frequently, also for the second sub- sample. extreme values (kurtosis), Il Sole24Ore showed higher excess pessimism. The Wall Street Journal was more cautious throughout the entire sample, i.e., it showed both lower means and volatility. PLOS ONE | www.plosone.org Descriptive Statistics We then distinguished two sub-samples, the first from 1st September 2008 to 30th March 2009, where the market volatility was considerably higher, the second from 31st March 2009 to 1st September 2009, with less volatility. Data showed that pessimism was generally higher in the first sub-sample. The Financial Times and Wall Street Journal showed a similar level of excess pessimism, which was lower than Il Sole24Ore. In the second sub-sample, where market volatility was lower, the Financial Times showed both higher levels of pessimism and excess pessimism. This meant that the Financial Times followed a more critical stance on the extreme values (kurtosis), Il Sole24Ore showed higher excess pessimism. The Wall Street Journal was more cautious throughout the entire sample, i.e., it showed both lower means and volatility. Tab. 1 shows mean, standard deviation, skewness and kurtosis of the bad news index per newspaper. Looking at the mean value of the bad index, it is worth noting that the Financial Times was more pessimistic than the other newspapers. On the other hand, if we consider the deviation from the mean (skewness) and the July 2013 | Volume 8 | Issue 7 | e67721 PLOS ONE | www.plosone.org 5 Financial Press and Markets in Times of Crisis crisis, by reporting bad news even in periods of relatively lower k l ili Table 2. Left: the effect of the bad news indexes on volatility; Right: the effect of the bad news indexes on correlations. Descriptive Statistics Impact on volatilities h ^h t-stat p-val US n1 27.6856 2192.1191 0.0000 * y1,W 0.1332 4.6755 0.0001 * y1,F 0.0266 2.8681 0.0044 * y1,S 0.0354 2.8001 0.0551 UK n2 27.7634 2202.5992 0.0000 * y2,W 0.1198 5.0512 0.0001 * y2,F 0.0323 3.1772 0.0017 * y2,S 0.0395 3.2643 0.0012 * IT n3 27.4971 2236.6942 0.0000 * y3,W 0.0731 3.7321 0.0002 * y3,F 0.0234 2.7753 0.0059 * y3,S 0.0301 2.9962 0.0031 * Impact on correlations h ^h t-stat p-val US-IT n4 0.5818 39.6552 0.0000 * y4,W 20.0161 21.7674 0.0119 * y4,F 20.0011 20.2851 0.7762 y4,S 20.0028 20.5983 0.5502 UK-IT n5 0.8334 122.7822 0.0000 y5,W 0.0108 2.5831 0.0104 * y5,F 0.0037 2.0676 0.0398 * y5,S 0.0048 2.0397 0.0225 * UK-US n6 0.5862 38.0473 0.0000 * y6,W 20.0206 22.1631 0.0212 * y6,F 0.0021 0.5152 0.6068 y6,S 20.0020 20.4141 0.6790 Columns: the parameter h (first), estimates ^h (second), value of the t-statistics (third), p-value of the t-statistics (fourth) and :*’’ indicates significance of the parameter at the 5% significance level (last). doi:10.1371/journal.pone.0067721.t002 Table 3. Left: the effect of the bad news indexes at the first lag on volatility. Impact on volatilities h ^Y12,1 t-stat p-val US y12,1 US,W 0.0093 3.1230 0.0020 * y12,1 US,F 0.0002 0.1692 0.8663 y12,1 US,S 20.0033 22.3871 0.0177 * UK y12,1 UK,W 0.0076 2.2512 0.0253 * y12,1 UK,F 0.0002 0.1679 0.8676 y12,1 UK,S 20.0019 21.1534 0.2501 IT y12,1 IT,W 0.0073 2.1151 0.0354 * y12,1 IT,F 0.0004 0.3061 0.7602 y12,1 IT,S 20.0023 21.455 0 0.1469 Impact on newspapers h t-stat p-val W y21,1 W,US 20.32445 20.4851 0.6278 y21,1 W,UK 1.70194 2.4692 0.0142 * y21,1 W,IT 20.96905 21.8552 0.0648 F y21,1 F,US 22.0504 21.2334 0.2188 y21,1 F,UK 3.56990 2.0822 0.0384 * y21,1 F,IT 0.00951 0.0072 0.9941 S y21,1 S,US,1 22.9694 22.1252 0.03462 * y21,1 S,UK 4.58829 3.1842 0.00164 * y21,1 S,IT 0.2066 0.1892 0.8501 Right: the effect of volatility at the first lag on the bad new indexes. Columns: the parameter h (first), estimates ^h (second), value of the t-statistics (third), p- value of the t-statistics (fourth) and ‘‘*’’ indicates significance of the parameter at the 5% significance level (last). doi:10.1371/journal.pone.0067721.t003 Table 3. Left: the effect of the bad news indexes at the first lag on volatility. Right: the effect of volatility at the first lag on the bad new indexes. Descriptive Statistics H0 : V B H0 : B V US UK IT All US UK IT All W 0.0012* 0.0036* 0.0426 0.0011* 0.0000* 0.0248* 0.0086* 0.0100* F 0.0042* 0.0000* 0.0010* 0.0024* 0.1289 0.9817 0.8260 0.7534 S 0.0009* 0.0001* 0.0008* 0.0010* 0.4934 0.7773 0.8918 0.7251 All 0.0000* 0.0000* 0.0000* 0.0000* 0.7442 0.7573 0.7536 0.2550 The null hypotheses (H0) were as follows: volatility (V) did not cause (in the Granger sense) financial press pessimism (B) (V B, left panel), financial press did not cause volatility (B V, right panel). ‘‘All’’ indicates all variables included in the test and ‘‘*’’ indicates that the null is rejected at the 5% significance level. doi:10.1371/journal.pone.0067721.t004 The null hypotheses (H0) were as follows: volatility (V) did not cause (in the Granger sense) financial press pessimism (B) (V B, left panel), financial press did not cause volatility (B V, right panel). ‘‘All’’ indicates all variables included in the test and ‘‘*’’ indicates that the null is rejected at the 5% significance level. doi:10.1371/journal.pone.0067721.t004 heteroskedasticity (GARCH ) model (see [34]; [64]) with dynamic conditional correlation (see also [35]). For the sake of simplicity, we followed a non-parametric approach as follows: commentaries and academic debate. Even the tendency to blame U.S. market responsibility and critically discuss the U.S. political agenda against the crisis could explain the stronger sensitivity of the Financial Times towards the development of the crisis (see also [62]). It is worth noting that more than 80% of Financial Times front pages in the period considered included an article or commentary on the crisis, against 67% of Wall Street Journal. Si,t~ 1{l 1{lt X t{1 k~0 lk(Xi,t{k{ Xi,t)2 ð3Þ Sij,t~ 1{l 1{lt X t{1 k~0 lk(Xi,t{k{ Xi,t)(Xj,t{k{ Xj,t) ð4Þ Si,t~ 1{l 1{lt X t{1 k~0 lk(Xi,t{k{ Xi,t)2 ð3Þ ð3Þ On the other hand, the stronger concern for home investors could have lead the Wall Street Journal to follow a less critical stance and be more cautious in spreading bad news. In a recent story on the U.S. press coverage of the financial crisis, [63] suggested that American journalists were extremely cautious in reporting bad news as it was clear that, in a situation of market unpredictability and turmoil, any influential opinion or streamer could have had a dramatic influence on market behaviour. Descriptive Statistics Columns: the parameter h (first), estimates ^h (second), value of the t-statistics (third), p- value of the t-statistics (fourth) and ‘‘*’’ indicates significance of the parameter at the 5% significance level (last). doi:10.1371/journal.pone.0067721.t003 Columns: the parameter h (first), estimates ^h (second), value of the t-statistics (third), p-value of the t-statistics (fourth) and :*’’ indicates significance of the parameter at the 5% significance level (last). doi:10.1371/journal.pone.0067721.t002 times of lower market volatility was due to a few critical journalists (see more detailed analysis in Sect. 6). These findings can be explained by considering certain historical and institutional differences between the U.S. and UK financial press (e.g., [59]). [60] has argued that the higher pessimism of the Financial Times in reflecting the 2008/2009 crisis depended on a mixture of history and contingency (see also [61]). On the one hand, while the Wall Street Journal has been historically more devoted to investigation, addressed to an investor readership and focused on domestic affairs, the Financial Times has always been more concerned with economic theory and interpretation and mainly focused on international affairs. This could explain the stronger sensitivity of the British newspaper towards a general outlook of the crisis (e.g., the implications of the financial crisis for the real economy) and its stronger focus on crisis, by reporting bad news even in periods of relatively lower market volatility. Our database also included the names and amount of journalists who authored any front page leading article on the crisis. We calculated a Gini index that measured the concentration of articles per journalist. Not only did the Financial Times concentrate more articles with a few journalists (WSJ index took 0.61, while FT took 0.66), it did so especially in the low market volatility period (WSJ index for the second sub-sample took 0.68, while FT took 0.77). This meant that the stronger critical stance of Financial Times in PLOS ONE | www.plosone.org July 2013 | Volume 8 | Issue 7 | e67721 6 Financial Press and Markets in Times of Crisis Table 4. Pairwise and joint causality test p-values. Descriptive Statistics Sij,t~ 1{l 1{lt X t{1 k~0 lk(Xi,t{k{ Xi,t)(Xj,t{k{ Xj,t) ð4Þ ð4Þ Rij,t~ Sij,t ffiffiffiffiffiffiffi Si,t p ffiffiffiffiffiffiffi Sj,t p ð5Þ ð5Þ Secondly, it is worth noting that after the dramatic events of September/October 2008, the financial press in the UK was strongly criticized for boosterism and excessive embeddedness. [60] explained that, in the autumn of 2008, a turning point was achieved in the relationship between press and markets, epitomized especially by the Financial Times. This was called the ‘‘media’s moral compass’’ to mean that the relationship between the press and the market shifted from a ‘‘cozy co- dependence’’ to a more critical stance. This would explain why the Financial Times suggested a pessimistic interpretation of economic events. for i,j~US,UK,IT where Xi,t was the empirical average over Xi,t{tz1, . . . ,Xi,t, lw0 was a smoothing factor and tw0 was a forgetting factor. g g Fig. 2 shows the results of our non-parametric estimation procedure. The first row shows the log-returns of the FTSE, DowJones and MIB market indexes at a daily base for closing values. The graphs in the second row show the level of log- volatility (i.e. log Si,t) for each index. Although we did not report data before 1st September 2008, it is worth noting that market volatility significantly increased after September 2008. This is evident when looking at the beginning of our sample (see the graphs in the second row of Fig. 2). It is worth clarifying that this was not due to a lack of data in the estimates at the beginning of the sample. Indeed, the first estimate of the volatility was calculated starting from a window of 60 initial observations, which were not represented in the first row of Fig. 2. This means that our sample fully reflected a period of significant market turmoil. Results showed that market dynamics were similar. More specifically, while the level of volatility was similar at the beginning, at the end of the sample the Italian market showed higher log-volatility than the UK and U.S. stock markets. Furthermore, the level of correlation between the three market indexes increased after September 2008 to quickly reach 0.6 for DowJones and FTSE, 0.7 for MIB and DowJones and 0.9 for MIB and FTSE. Secondly, our results showed that the correlation between these three markets was positive. Descriptive Statistics If we look at values before and after the beginning of the period under observation, it is worth noting that the UK and Italian markets were more highly dependent than the U.S. and UK and U.S. and Italy respectively. Th l fi i i i i l fi di h The next step was to calculate the correlation between newspapers (see Tab. 1). Results showed that newspaper pessimism was significantly positively correlated. The more positive correlations were between the Wall Street Journal and Financial Times, and between the Wall Street Journal and Il Sole24ore. If we consider the difference between periods of high and low market volatility, it is worth noting that the higher correlation was in high volatility periods between Wall Street Journal and Financial Times, whereas correlations were not- significant or negative in periods of low volatility. This meant that in periods of higher market volatility, differences between Wall Street Journal and Financial Times drastically diminished. Indeed, in this period, these two leading newspapers basically conformed both in terms of timing and intensity of pessimism. On the other hand, significant differences persisted for other periods, where market volatility was less pronounced. Market Volatility and Correlation y Let Xi,t indicate the log-return at time t for FTSE (i~UK), DowJones (i~US) and MIB (i~IT), the three stock market financial indexes to which the three newspapers refer more frequently. Let us calculate their volatility and correlation dynamics by means of a generalized autoregressive conditional These results confirm certain previous empirical findings on the higher correlation between markets during a financial crisis (e.g., [65]; [66]), especially in periods of higher volatility of the U.S. July 2013 | Volume 8 | Issue 7 | e67721 PLOS ONE | www.plosone.org 7 Financial Press and Markets in Times of Crisis Table 5. Left: the effect of the bad news indexes at the first lag on market correlations; Right: the effect of market correlations at the first lag on the bad new indexes. Impact on correlations h ^W12,1 t-stat p-val US-UK w12,1 US{UK,W 20.0023 22.3541 0.0193 * w12,1 US{UK,F 0.0004 0.4882 0.6263 w12,1 US{UK,S 0.0016 0.7422 0.4589 US-IT w12,1 US{IT,W 0.0005 0.2482 0.8044 w12,1 US{IT,F 20.0003 20.2901 0.7718 w12,1 US{IT,S 20.0029 22.7522 0.0064 * UK-IT w12,1 UK{IT,W 0.0001 0.1411 0.8881 w12,1 UK{IT,F 0.0004 1.1182 0.2646 w12,1 UK{IT,S 20.0005 21.1302 0.2598 Impact on newspaper h ^W21,1 t-stat p-val W w21,1 W,US{UK 24.6537 23.5851 0.0004 * w21,1 W,US{IT 3.4669 2.8091 0.0053 * w21,1 W,UK{IT 5.9194 4.0342 0.0000 * F w21,1 F,US{UK 22.3772 20.7242 0.4700 w21,1 F,US{IT 1.6567 0.5301 0.5962 w21,1 F,UK{IT 7.4877 2.0162 0.0449 * S w21,1 S,US{UK 28.0680 22.9283 0.0037 * w21,1 S,US{IT 5.1940 1.9836 0.0485 * w21,1 S,UK{IT 12.8542 4.1272 0.0000 * Columns: the parameter h (first), estimates ^h (second), value of the t-statistics (third), p-value of the t-statistics (fourth) and ‘‘*’’ indicates the significance of the parameter at the 5% significance level (last). doi:10.1371/journal.pone.0067721.t005 ð6Þ Mi,t~nizyi,FBF,tzyi,WBW,tzyi,SBS,tzei,t ð6Þ with i~1, . . . ,6, and ei,t*N (0,j2 i ) i.i.d. Vt,i. with i~1, . . . ,6, and ei,t*N (0,j2 i ) i.i.d. Vt,i. with i~1, . . . ,6, and ei,t*N (0,j2 i ) i.i.d. Vt,i. Table 2 shows that all bad news coefficients were positive. This means the any increase of pessimism by newspapers had a positive impact on the volatility of markets. Obviously, the impact was not the same for each newspaper or market. The Wall Street Journal had a strong impact on all markets. Market Volatility and Correlation The bad news of Wall Street Journal and Financial Times had a significant impact (at 5% significance) on the simultaneous level of log-volatility in all markets (see the left panel in Tab. 2). Finally, Il Sole24Ore bad news influenced the volatility of the UK and Italian stock markets. y As regards to market correlation, it is worth noting that Wall Street Journal’s pessimism had a significant impact on all correlations. Vice-versa, the Financial Times and Il Sole24Ore had an impact only on their respective markets (see the right panel of Tab. 2). This would confirm the leadership of Wall Street Journal in influencing the stock market and its worldwide impact. On the other hand, it is interesting to note that any increase of pessimism by the Wall Street Journal had a negative impact on the correlation between UK and Italian markets. This could be explained by the fact that the Wall Street Journal mainly focused on domestic affairs and negative news on the U.S. stock market could have lead investors to move their investments towards other markets or in general to explore a variety of investment strategies. This could have contributed to generate heterogeneity in stock market behaviour globally. Statistical Causal Relations To look at the causes of statistical relationships in more detail, we performed a Granger-causality test that examined the lagged dependence structure between bad news and market correlation and volatility. This allowed us to verify whether bad news had any predictive value for market volatility and correlation. We considered each possible dependence between markets and information, by setting Bt~(BF,t,BW,t,BS,t) and considering VAR models. Volatility A joint test on the statistical causal relationships of volatility and pessimism was based on the following VAR model of dimension 6 and order p: Vt~Y10z X p j~1 Y11,jVt{jzY12,jBt{j   ze1,t ð7Þ Bt~Y20z X p j~1 Y21,jVt{jzY22,jBt{j   ze2,t ð8Þ Vt~Y10z X p j~1 Y11,jVt{jzY12,jBt{j   ze1,t ð7Þ Columns: the parameter h (first), estimates ^h (second), value of the t-statistics (third), p-value of the t-statistics (fourth) and ‘‘*’’ indicates the significance of the parameter at the 5% significance level (last). doi:10.1371/journal.pone.0067721.t005 Columns: the parameter h (first), estimates ^h (second), value of the t-statistics (third), p-value of the t-statistics (fourth) and ‘‘*’’ indicates the significance of the parameter at the 5% significance level (last). doi:10.1371/journal.pone.0067721.t005 ð7Þ stock markets ([67]). Secondly, they would confirm recent findings on the increasing interdependence of European stock markets ([68]). ð8Þ with (e1,t,e2,t)*N 6(O6,J) i.i.d. Vt, Y10 0~(yUS,yUK,yIT) and Y20 0~(yF,yW,yS) were the intercept and the 3-dimensional square matrices Y11,j, Y12,j, Y21,j, Y22,j, were the autoregressive coefficients of the VAR6(p) model. Analysis Let Vt~( log SUS,t, log SUK,t, log SIT,t)’ be the vector of log- volatilities and Zt~(q(SUS,UK,t), q(SUS,IT,t), q(SUK,IT,t))’, with q(x)~ log (1{x){ log (1zx), the vector of logistic-transformed correlations. Let us define Mt~(Vt0,Zt0)’. We examined the relationship between bad news and the variance and correlation of the three financial indexes. We considered static models as follows: In order to disentangle the relation between volatility and the press, we first looked at the statistical significance of the relationship between volatility at time t and news at time t{k, which depended on the matrices Y12,k, k~1, . . . ,p with elements y12,k ij , i~US,UK,IT and j~F,W,S. Then, we looked at the relation between bad news at time t and volatility at time t{k, PLOS ONE | www.plosone.org July 2013 | Volume 8 | Issue 7 | e67721 8 Financial Press and Markets in Times of Crisis Table 6. Pairwise and joint causality test p-values. H0 : C B H0 : B C US-IT UK-IT US-UK All US-IT UK-IT US-UK All W 0.1017 0.0295* 0.0595 0.0001* 0.0121* 0.6951 0.0451* 0.0221* F 0.6570 0.0100* 0.0108* 0.0223* 0.4739 0.3627 0.3627 0.6034 S 0.1026 0.0029* 0.0029* 0.0001* 0.9723 0.8733 0.8733 0.4141 All 0.1455 0.0026* 0.1780 0.0066* 0.0219* 0.4631 0.0208* 0.0423* The null hypotheses (H0) were as follows: correlation (C) did not cause (in the Granger sense) financial press pessimism (B) (C B, left panel), financial press did not cause correlation (B C, right panel). ‘‘All’’ indicates all variables are included in the test and ‘‘*’’ indicates the null is rejected at the 5% significance level. doi:10.1371/journal.pone.0067721.t006 The null hypotheses (H0) were as follows: correlation (C) did not cause (in the Granger sense) financial press pessimism (B) (C B, left panel), financial press did not cause correlation (B C, right panel). ‘‘All’’ indicates all variables are included in the test and ‘‘*’’ indicates the null is rejected at the 5% significance level. doi:10.1371/journal.pone.0067721.t006 which depended on elements y12,k ij , i~F,W,S and j~US,UK,IT of the matrices Y21,k, k~1, . . . ,p. For our purpose and for the sake of simplification, we have included only a subset of the VAR coefficients, i.e., the elements of Y12,1 and Y21,1 only for the first lag (see Table 3). Analysis Our results (see Table 5, left column) showed that Wall Street Journal bad news (one lag) had a negative impact on the correlation between U.S. and UK stock markets. Indeed, bad news in the Wall Street Journal reduced the co-movement of these markets. This could be explained in terms of outflow of capital from the U.S. stock market and inflow into the UK market. On the other hand, Financial Times bad news (one lag) had no significant impact on market correlations. Il Sole24ore bad news decreased the correlation between the U.S. and Italian stock markets. The same explanation is a possible reason. Tab. 4 shows the results of these joint and pairwise tests. First, the p-value of the joint test in the last column and last row, in the left panel, indicates that newspaper bad news was fully caused by market turmoil. Therefore, stock market behaviour was the essential source of bad news and newspapers did not have unrealistically pessimistic stances. Secondly, if we look at the p- values of almost all the pairwise causality tests (see the left panel), this statistical causality direction from markets to newspapers was true for all log-volatility and bad news indexes. On the other hand, if we look at the p-values in the last column, last row in the right panel, we should conclude that, in general, the financial press did not determine market volatility. More specifically, results showed that Wall Street Journal bad news alone had a strong statistical, causal impact on market volatility (see the first row in the right panel). Generally speaking, we could not predict market volatility only by looking at financial press bad news. In addition, results (see Table 6, right column) showed that Wall Street Journal bad news reflected all one lag correlations. An increase in the correlation between the U.S. and UK stock markets decreased the journal’s pessimism, whereas an increase in the U.S.-IT and UK-IT correlations increased it. Furthermore, higher (one lag) correlation between the UK and Italian stock markets increased the pessimism of the Financial Times. Finally, all correlations had a significant impact on Il Sole24Ore, similar to the Wall Street Journal. We performed a joint and pairwise Granger causality test to rigorously asses the presence of causal relationships, as we did for log-volatility. The last column, last row in the left panel of tab. Analysis Ct~W10z X q j~1 W11,jCt{jzW12,jBt{j   zg1,t ð9Þ Bt~W20z X q j~1 W21,jCt{jzW22,jBt{j   zg2,t ð10Þ Ct~W10z X q j~1 W11,jCt{jzW12,jBt{j   zg1,t ð9Þ ð9Þ Results showed that Wall Street Journal bad news (lagged by one period) significantly increased the volatility of the three market indexes in the subsequent period (see the left panel in Tab. 3). This is further confirmation of the leadership of this newspaper and its worldwide impact. The bad news of other newspapers did not have any significant impact on market volatility, with the exception of the lagged relation between Il Sole24Ore bad news and the volatility of the U.S. market. Considering the effect of market volatility on bad news, it is worth noting that the FTSE volatility had a significant and positive impact on the bad news of each newspaper (see the right panel in Tab. 3). This would confirm the recent worldwide importance of the London stock market. Finally, it is worth noting that the volatility of the Dow Jones index had a significant and negative impact on Il Sole24ore bad news. Bt~W20z X q j~1 W21,jCt{jzW22,jBt{j   zg2,t ð10Þ ð10Þ with (g1,t,g2,t)*N 6(O6,S) i.i.d. Vt, where W10~(wUS,wUK,wIT) and were the intercept and Wij,k, i,j~1,2 k~1, . . . ,q, were the autoregressive coefficients of the VAR6(1) model. We examined the statistical significance of the relationship between correlation at time t and bad news at time t{1, which was given by the matrix W12,k with elements w12,k ij , i~US{UK,US{IT,UK{IT and j~F,W,S. Then, we also looked at the relationship between bad news at time t and correlation at time t{1, which was given by the elements w21,k ij , i~F,W,S and j~US{UK,US{IT,UK{IT of the matrix W21,k. Note that, for the shortage of space, we included only the autoregressive coefficients at the first lag, not all the estimated coefficients of the VAR models (see Table 5). We tested the hypothesis that volatility did not jointly statistically cause, in a Granger sense, the bad-news indexes. To look at the causal relationship between each market-specific volatility and the three newspapers, we also tested separately the hypothesis that neither each one of the three bad-news indexes, nor all three indexes jointly considered statistically caused, in a Granger sense, the market-specific volatility. To look at the relationship between each newspaper and the three markets, we did the same for newspaper bad news. Analysis 6 shows that all correlations had a Granger causal effect on all bad news indexes. More specifically, while U.S.-IT stock market correlation did not statistically cause the bad news index, UK-IT Correlations The VAR model of order q for bad-news indexes and correlations was as follows: PLOS ONE | www.plosone.org July 2013 | Volume 8 | Issue 7 | e67721 9 Financial Press and Markets in Times of Crisis Table 7. Robustness analysis of the dynamic analysis for close, high and low returns. Table 7. Robustness analysis of the dynamic analysis for close, high and low returns. Impact on correlations h ^h Close High Low US-UK 20.0023 * 20.0047 * 20.0056 * 0.0004 20.0009 0.0006 0.0016 0.001 0.0005 US-IT 0.0005 20.0061 * 20.0072 * 20.0003 20.0003 0.0007 20.0029 * 20.0003 0.0003 UK-IT 0.0001 0.0121 * 0.0118 * 0.0004 0.0006 0.0008 20.0005 20.0018 20.0024 Impact on newspaper h ^h Close High Low W 24.6537 * 22.26757 * 22.7215 * 3.4669 * 1.9398 * 1.7358 * 5.9194 * 3.5517 * 4.0098 * F 22.3772 24.7657 * 26.2373 * 1.6567 6.3302 * 5.6573 * 7.4877 * 9.2883 * 11.4475 * S 28.068 * 22.9451 23.527 * 5.194 * 2.2545 2.3755 12.8542 * 7.0939 * 8.2052 * doi:10.1371/journal.pone.0067721.t007 Impact on correlations h ^h Close High Low US-UK 20.0023 * 20.0047 * 20.0056 * 0.0004 20.0009 0.0006 0.0016 0.001 0.0005 US-IT 0.0005 20.0061 * 20.0072 * 20.0003 20.0003 0.0007 20.0029 * 20.0003 0.0003 UK-IT 0.0001 0.0121 * 0.0118 * 0.0004 0.0006 0.0008 20.0005 20.0018 20.0024 Impact on newspaper h ^h Close High Low W 24.6537 * 22.26757 * 22.7215 * 3.4669 * 1.9398 * 1.7358 * 5.9194 * 3.5517 * 4.0098 * F 22.3772 24.7657 * 26.2373 * 1.6567 6.3302 * 5.6573 * 7.4877 * 9.2883 * 11.4475 * S 28.068 * 22.9451 23.527 * 5.194 * 2.2545 2.3755 12.8542 * 7.0939 * 8.2052 * doi:10.1371/journal.pone.0067721.t007 doi:10.1371/journal.pone.0067721.t007 and U.S.-UK correlations had a Granger causal effect on the bad news indexes of Financial Times and Il Sole24ore. Secondly, looking at the last column, last row of the right panel, we can see that the joint test did not reject the null hypothesis of the absence of Granger causality between all bad news and correlation indexes. Finally, results showed that Wall Street Journal bad news determined U.S.-IT and U.S.-UK market correlation but not that of UK-IT. contributed to market volatility. At the same time, our findings also corroborated the second hypothesis (H2), which argued that bad news could even influence the correlation of markets. July 2013 | Volume 8 | Issue 7 | e67721 Correlations More specifically, we found that the Wall Street Journal had a significant impact on market correlation, although different for different markets involved and directions. H3 was not fully confirmed as it claimed that, in period of financial turmoil, the press and markets influenced each other, possibly contributing to a cascade of pessimism. We found that market correlation and newspapers exerted mutual influence only in specific cases. In particular, our results showed that the Wall Street Journal strongly predicted market correlation and volatility. Therefore, any bad news in the Wall Street Journal predicted correlation between the U.S. and the other stock markets. Following certain peculiarities of Wall Street Journal as discussed in Sect 3 (see Table 2), such as its worldwide recognized leadership and strong focus on domestic affairs, this meant that investors considered any bad news published in this influential newspaper as a good prediction of market behaviour and promptly reacted by drastically modifying their global investment strategies. Our findings confirm the sociological argument of the reflexive nature of stock markets. We found that media and markets are so systematically related to extend this reflexivity also to qualitative, subjective, broad picture information sources, whose true economic value should be seriously questioned from a mainstream, ‘efficient market hypothesis’ approach. On the other hand, especially if we look at the stronger influence of Wall Street Journal, we can argue against the common sense belief that To sum up, we can say that results corroborated the first hypothesis (H1), i.e., bad news published in the newspapers’ banner headlines had a significant influence on market volatility. More specifically, we found that Wall Street Journal alone PLOS ONE | www.plosone.org July 2013 | Volume 8 | Issue 7 | e67721 July 2013 | Volume 8 | Issue 7 | e67721 10 Financial Press and Markets in Times of Crisis Table 8. Sensitivity analysis of the impact of different choices of the smoothing factor on volatility and correlation estimations. Correlations Impact on correlations h ^h l~0:99 l~0:95 l~0:90 US-UK 20.0023 * 20.015 * 20.0242 * 0.0004 0.0013 0.0032 0.0016 20.0003 20.0022 US-IT 0.0005 20.0175 * 20.0256 * 20.0003 0.001 0.0019 20.0029 * 20.0021 20.0039 UK-IT 0.0001 20.0003 0.0001 0.0004 0.0026 * 0.0047 * 20.0005 0.0017 0.0034 Impact on newspaper h ^h l~0:99 l~0:95 l~0:90 W 24.6537 * 22.9682 * 1.7003 * 3.4669 * 0.1451 20.5796 5.9194 * 1.8159 * 1.1167 * F 22.3772 21.9593 20.2972 1.6567 1.1568 20.0351 7.4877 * 6.1841 * 2.9076 * S 28.068 * 20.7873 0.4785 5.194 * 21.3902 * 21.6124 * 12.8542 * 3.63475 * 1.9182 * doi:10.1371/journal.pone.0067721.t008 Figure 3. Financial Times bad news index per group of journalists from 1st September 2008 to 1st September 2009 on a daily base: central journalists are in black solid lines, non- central journalists are in blue dashed lines. Peaks of bad news are indicated with vertical dashed lines. doi:10.1371/journal.pone.0067721.g003 Figure 3. Financial Times bad news index per group of journalists from 1st September 2008 to 1st September 2009 on a daily base: central journalists are in black solid lines, non- central journalists are in blue dashed lines. Peaks of bad news are indicated with vertical dashed lines. doi:10.1371/journal.pone.0067721.g003 columns), so confirming previous findings. Furthermore, results showed that the Wall Street Journal not only had a significant effect on the U.S.-UK market correlation, but also on other correlations. On the other hand, the effect of market correlation on the Wall Street Journal bad news was more significant for closing returns than for low and high returns. This meant that the higher the correlation between markets, especially between the U.S. and UK markets, the lower the bad news index of the Wall Street Journal was. The situation with the Financial Times was different. In this case, the effect of correlation for low and high returns were statistically stronger than that of closing returns. Il Sole24ore showed weak correlation effects in these downturn and upturn cases. It is important to note that the FTSE-MIB correlation had a strong effect on the Il Sole24ore bad news independent of the type of market phases. We then examined the robustness of the results on the choice of the smoothing l factor in the estimation of the volatility and correlation. Lower values of l corresponded to higher weights for the most recent observations in the window considered. Correlations This leads to volatility and correlation that were more sensitive to large variations in returns. Thus, we verified whether these results survived to the inclusion of a higher level of noise in the estimation of variance and correlation. Table 8 shows that Wall Street Journal bad news index was significant for the DJ-FTSE correlation as in previous analysis, but that it was also significant for DJ-MIB correlation. It is worth noting that, in this case, the Financial Times became statistically significant for FTSE-MIB correlation. doi:10.1371/journal.pone.0067721.t008 newspapers would have over-exaggerated the dramatic events of 2008/2009 by imposing a critical stance which contributed to a cascade and contagion of pessimism. We also investigated the case of the Financial Times better, trying to understand especially whether the relationship between its bad news and stock market was influenced by a small number of critical journalists. First, we distinguished two bad news indexes, the first, BFC,t, for the group of central journalists and the second, BFNC,t, for the residual group (non-central journalists). We assumed that a journalist was central when he/she wrote more than nine commentaries in periods of high newspaper’s pessimism. We defined a period of high pessimism whenever the Financial times bad news index was higher than two. These criteria gave us nine central Financial Times journalists, such as Krishna Guha, Francesco Guerrera (now at the Wall Street Journal), and Ralph Atkins among others. Robustness and Sensitivity Analysis In order to corroborate our findings better, we performed further statistical tests to verify if certain specificities of market behaviour, such as downturn or upturn phases, could have had a consequence on the predictive power of the bad news index on market volatility and correlation. To do so, we re-estimated the VAR models for volatility and correlation described in the previous section by using alternatively the lowest and the highest returns of each trading day in the calculation of volatility and correlation. This was to estimate the impact of bad news on more point-to-point market behaviour, where it is less likely that stock markets fully reverberated all potential effects of a bad news. Table 7 shows that Wall Street Journal bad news had a significant impact on extreme returns (see High and Low Fig. 3 shows that the two indexes for the two groups had different dynamics and often moved in opposite directions leading July 2013 | Volume 8 | Issue 7 | e67721 PLOS ONE | www.plosone.org July 2013 | Volume 8 | Issue 7 | e67721 11 Financial Press and Markets in Times of Crisis Table 9. Left: the effect of the Financial Times central and non-central journalists (parameter yi,FC, i~1, . . . ,3) on volatility; Right: the effect of the Financial Times central and non-central journalists (parameter yi,FC, i~4, . . . ,6) on correlations. Robustness and Sensitivity Analysis Impact on volatilities h ^h t-stat p-val US n1 27.1128 2169.9311 0.0000 * y1,W 0.1284 4.7021 0.0001 * y1,FC 0.0266 3.1034 0.0021 * y1,FNC 0.0266 1.4692 0.1431 y1,S 0.0354 5.3513 0.000 UK n2 27.1553 2182.3652 0.0000 * y2,W 0.1127 4.4023 0.0000 * y2,FC 0.0356 3.1587 0.0018 * y2,FNC 0.0367 1.5176 0.1306 y2,S 0.0678 5.7961 0.0000 * IT n3 26.8698 2236.6942 0.0000 * y3,W 0.0654 2.9420 0.0036 * y3,FC 0.0273 2.7861 0.0057 * y3,FNC 0.0298 1.4161 0.1515 y3,S 0.0509 5.0062 0.0000 * Impact on correlations h ^h t-stat p-val US-IT n4 0.6908 42.9045 0.0000 * y4,W 20.0029 20.2801 0.6871 y4,FC 20.0018 0.3921 0.6954 y4,FNC 20.0055 20.5602 0.5763 y4,S 20.0073 21.5322 0.1276 UK-IT n5 0.8347 77.1971 0.0000 y5,W 0.0148 2.1081 0.0104 * y5,FC 0.0063 2.0330 0.0398 * y5,FNC 0.0007 0.1091 0.0398 y5,S 0.0031 0.9532 0.0225 UK-US n6 0.7198 38.0473 0.0000 * y6,W 20.0208 22.0301 0.0211 * y6,FC 0.0028 0.5631 0.5742 y6,FNC 0.0110 1.0191 0.3091 y6,S 20.0063 21.2051 0.2297 Columns: the parameter h (first), estimates ^h (second), value of the t-statistics (third), p-value of the t-statistics (fourth) and ‘‘*’’ indicates significance of the parameter at the 5% significance level (last). doi:10.1371/journal.pone.0067721.t009 to a negative correlation 20.2305 (statistically significant at the 5% level). We then considered Vt~( log SUS,t, log SUK,t, log SIT,t)’ and Zt~(q(SUS,UK,t), q(SUS,IT,t), q(SUK,IT,t))’, with q(x)~ log (1{x){ log (1zx) and Mt~(Vt0,Zt0)’ as in the previous sections and estimated. to a negative correlation 20.2305 (statistically significant at the 5% level). We then considered Vt~( log SUS,t, log SUK,t, log SIT,t)’ and Zt~(q(SUS,UK,t), q(SUS,IT,t), q(SUK,IT,t))’, with q(x)~ log (1{x){ log (1zx) and Mt~(Vt0,Zt0)’ as in the previous sections and estimated. Mi,t~nizyi,FCBFC,tzyi,FNCBFNC,t zyi,WBW,tzyi,SBS,tzei,t ð11Þ ð11Þ with i~1, . . . ,6, and ei,t*N (0,j2 i ) i.i.d. Vt,i. with i~1, . . . ,6, and ei,t*N (0,j2 i ) i.i.d. Vt,i. Table 9 shows that the bad news index of the group of central journalists had a significant impact (at the 5% significance level) on the contemporaneous level of log-volatility in all markets (see the left panel). On the contrary, there was no relationship between the pessimism of non-central journalists and market volatility and correlation. This could indicate that the opinion of certain influential commentators was more considered by the market and revealed a specific strategy of the newspaper, i.e., to assign commentaries to more critical journalists in specific phases of the crisis. References Koutmos G (1995) Asymmetric volatility transmission in international stock markets. Journal of International Money and Finance 14(6): 747–762. 29. Barber B, Loeffler D (1993) The ‘‘dartboard’’ column: Second-hand information and price pressure. Journal of Financial and Quantitative Analysis 28(2): 273– 284. 10. Koutmos G (1998) Asymmetries in the conditional mean and the conditional variance: Evidence from nine stock markets. Journal of Economics and Business 50(3): 277–290. 30. Preis T, Reith D, Stanley HG (2010) Complex dynamics of our economic life on different scales: insights from search engine query data. Philosophical Transactions of the Royal Society A 368: 5707–5719. 11. 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Description S1 Text file with a description of the dataset. Description S1 Text file with a description of the dataset. Acknowledgments We would like to gratefully acknowledge help by Serena Cascio and Stefano Bacchiocchi for the collection of data and comments by Gaetano Carmeci, Alya Guseva, Francesco Ravazzolo, Akos Rona-Tas and Matthias Thiemann on a preliminary version of the paper. We would like to thank three anonymous referees for useful suggestions. Moreover, it is also important to note that press pessimism and market behaviour could also be conditioned by other media, such as the new social media and the Internet (e.g., [31]). Comparing behaviour and impact of various media would be essential to provide a more precise analysis of the 2008/2009 crisis and draw more general remarks of the complex ways through which market sentiment tends to form today (e.g., see [30]). The same holds for References 1. Reinhart CM, Rogoff KS (2009) This Time is Different. Eight Centuries of Financial Follies. Princeton, NJ: Princeton University Press. 21. Oberlechner T, Hocking S (2004) Information sources, news, and rumors in financial markets: Insights into the foreing exchange market. Journal of Economic Psychology 25(3): 407–424. 2. Tickell A (1995) Making a melodrama out of a crisis: reinterpreting the collapse of barings bank. Environment and Planning D: Society and Space 14(1): 5–33. y gy ( ) 22. Shiller R (2005) Irrational Exuberance. Princeton, NJ: Princeton University Press. of barings bank. Environment and Planning D: Society and Space 14(1): 5–33. 3. Thrift NJ (2001) It’s the romance, not the finance, that makes the business worth i Di l i k l E d S i 30 412 432 of barings bank. Environment and Planning D: Society and Space 14(1): 5 33. 3. Thrift NJ (2001) It’s the romance, not the finance, that makes the business worth pursuing: Disclosing a new market culture. Economy and Society 30: 412–432. 23. Knorr Cetina K, Preda A (2005) The Sociology of Financial Markets. Oxford: Oxford University Press. pursuing: Disclosing a new market culture. Economy and Society 30: 412–432. 4. Clark GL, Thrift N, Tickell A (2004) Performing finance: The industry, the media and its image. Review of International Political Economy 11(2): 289–310. 24. Antweiler W, Frank MZ (2004) Is all that talk just noise? the information content of internet stock message boards. Journal of Finance 49(3): 1259–1269. 5. Suttles GD, Jacobs MD (2010) Front Page Economics. Chicago: University of Chicago. of internet stock message boards. Journal of Finance 49(3): 1259– 25. Tetlock PC (2007) Giving content to investor sentiment: The role of media in the stock market. Journal of Finance 62: 1139–1168. 6. Pixley J (2002) Finance organizations, decisions and emotions. British Journal of Sociology 53: 41–65. stock market. Journal of Finance 62: 1139–1168. 26. Engelberg JE, Parsons CA (2011) The causal impact of media in financial markets. Journal of Empirical Finance LXVI (1): 67–97. 7. Shiller R (2002) Bubbles, human judgement, and expert opinion. Financial Analysts Journal 58(3): 18–23. 27. Preda A (2007) The sociological approach to financial markets. Journal of Economic Surveys 21(3): 506–533. 8. Thaler RH (1993) Advances in Behavioral Finance. New York: Russell Sage Foundation. y ( ) 28. Schindler M (2007) Rumors in Financial Markets. Insights into Behavioral Finance. Chichester: John Wiley & Sons. 9. Discussion Secondly, our findings showed that the increasing globalization of financial markets and their PLOS ONE | www.plosone.org PLOS ONE | www.plosone.org July 2013 | Volume 8 | Issue 7 | e67721 July 2013 | Volume 8 | Issue 7 | e67721 12 Financial Press and Markets in Times of Crisis the idea of including other newspapers and markets, which may help us to have a more complete picture of the recent crisis and its global dimension. correlation in times of crisis require the press to truly cover the international dimension of business and be less parochial. This challenges the quality of the press coverage of global market dynamics and indicates the need for improving the public understanding of the intricate mechanisms of stock markets. Author Contributions Conceived and designed the experiments: FS. Performed the experiments: RC. Analyzed the data: RC. Contributed reagents/materials/analysis tools: RC. Wrote the paper: FS. Conceived and designed the experiments: FS. Performed the experiments: RC. Analyzed the data: RC. Contributed reagents/materials/analysis tools: RC. Wrote the paper: FS. Supporting Information Finally, certain limitations of our work should be considered. First, we did not study the influence of the financial press on stock markets but only that of bad news. This gave us a narrow view of the link between the press and markets. Secondly, we studied the relationship of the financial press and stock markets in an ‘‘abnormal’’ market phase, where market behaviour was strongly subjected to irrational expectation and social mood. We inten- tionally selected this period as we expected that, in these situations, the pessimism of the financial press and its cross-sectoral dynamics could help us to understand the crisis better. While our results do not contribute to a general theory of the link of press and markets, they can provide important insights to understand the ‘social construction’ of pessimism between press and markets. Dataset S1 Excel file reporting the newspaper data used for the construction of the bad news indexes. (XLSX) Discussion In an interesting personal account on 2008/2009 events, Peter S. Goodman, now executive business editor of the Huffington Post, past national correspondent for the New Work Times, reported that: ‘‘Inside our newsroom in midtown Manhattan, we understood that were were not merely passive chroniclers of external events. The sportswriter can describe what is happening on the field from a dispassionate distance, without imagining that the words he types may somehow influence the events he is witnessing. Not so for those of us writing about the financial crisis: were were effectively on the field while the game was still under way. Investors and markets and ordinary people would move their money in reaction to what we and other major media were reporting, and this would in turn affect the policy climate, the perception of need for emergency measures, the politics of the debate over those measures, and the public mood, which then reverberated back on everything else’’ ([63], p. 110). This personal view testifies to the reflexive nature of markets and the limitation of the mantra of the market efficiency hypothesis. Our findings showed that the idea of reflexivity may contemplate that, in periods of crisis and market unpredictability, even distant information, subjective opinion and economically irrelevant facts may influence market behaviour. Probably, this is due to the fact that today economic, information, technological, and social systems are more strongly and globally coupled than in the past. This means that investors cannot easily predict future outcomes and tend to extend their social reflexivity towards non- operational, non strictly market related events and processes, e.g., by considering that general events, board picture information and the opinion of influential newspapers might have an informative value for markets. This findings has interesting implications for the financial press. Indeed, following Goodman’s argument, a competent, reliable and responsible information is crucial for markets to work well, especially in periods of financial turmoil. This means that it is important to carry out more serious investigation on the ethics and responsibility of the financial press to establish new standards of conduct and better incentives and sanctions to support reliable information (e.g., [61]; [14]). 1. Reinhart CM, Rogoff KS (2009) This Time is Different. Eight Centuries of Financial Follies. Princeton, NJ: Princeton University Press. 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The Art of Everyday Objects: A Non-Invasive In Situ Investigation of Materials and Techniques of Italian Pop Art Paintings on Aluminium

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 Citation: Longoni, M.; Cicala, N.; Guglielmi, V.; Poldi, G.; Bruni, S. The Art of Everyday Objects: A Non- Invasive In Situ Investigation of Materials and Techniques of Italian Pop Art Paintings on Aluminium. Heritage 2022, 5, 42–60. https:// doi.org/10.3390/heritage5010003 Keywords: contemporary art; synthetic organic pigments; binders; non-invasive analyses; Raman spectroscopy; Fourier-transform infrared spectroscopy; spectrofluorimetry; visible reflectance spec- troscopy; IR reflectography; false colour IR; UV induced fluorescence; portable microscopy Heritage 2022, 5, 42–60. https:// doi.org/10.3390/heritage5010003 The Art of Everyday Objects: A Non-Invasive In Situ Investigation of Materials and Techniques of Italian Pop Art Paintings on Aluminium Margherita Longoni 1 , Norma Cicala 1, Vittoria Guglielmi 1, Gianluca Poldi 2,* and Silvia Br Norma Cicala 1, Vittoria Guglielmi 1, Gianluca Poldi 2,* and Silvia Bruni 1,* 1 Dipartimento di Chimica, Università degli Studi di Milano, Via C. Golgi, 19, 20133 Milan, Italy; [email protected] (M.L.); [email protected] (N.C.); [email protected] (V.G.) 2 Dipartimento di Lettere, Filosofia e Comunicazione, Università degli Studi di Bergamo, Via Pignolo, 76, 24121 Bergamo, Italy g y * Correspondence: [email protected] or [email protected] (G.P.); [email protected] (S.B.) Abstract: Two paintings, made on aluminium support by Silvio Pasotti (among the major exponents of 1960s Italian pop art) were investigated in a totally non-invasive manner to identify the materials used by the artist. Raman spectroscopy, Fourier-transform infrared spectroscopy (FTIR), visible reflectance spectroscopy, and spectrofluorimetry with visible excitation were exploited as molecular analysis techniques, which are particularly suitable to recognise also synthetic organic materials, such as pigments and binders. The effectiveness of this multi-analytical approach was demonstrated, leading to the identification of several synthetic organic pigments, both conventional and “special effect” ones, introduced during the first half of the 20th century, as well as some well-established inorganic ones. Combining FTIR results both in the medium and near IR ranges, considerations regarding the binders employed by the artist could also be made, suggesting the use of both nitrocellulose and acrylic paints. Imaging techniques, such as IR reflectography, false colour IR, UV induced fluorescence, and portable microscopy, were also used to achieve a better knowledge of the painting practice. heritage heritage 1. Introduction Academic Editors: Valeria Di Tullioand and Brenda Doherty The need for a scientific non-invasive approach to the identification of materials and techniques is of course well established for paintings of any period in the history of art. Nevertheless, when artworks dating to the 1950s and 1960s are considered, this task is even more challenging due to the fast evolution of synthetic materials in that period and in the immediately previous decades of the 20th century. Received: 2 November 2021 Accepted: 15 December 2021 Published: 23 December 2021 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affil- iations. Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affil- iations. As regards synthetic binders, acrylic resins had been finally developed, while nitro- cellulose paints were still used by important artists such as Jackson Pollock in the early 1950s and Richard Hamilton in the second half of the same decade and in the 1960s [1]. On the other hand, as for synthetic organic pigments, it was in that period that new molecular classes such as benzimidazolones, isoindolinone, and others were introduced to obtain vivid colours [2]. In the artistic panorama of those years, the Pop Art movement was particularly innovative for the choice of themes, the use of objects (or their parts) taken from everyday life, the mixing of expressive techniques and, of course, the exploitation of newly available or unconventional painting materials. As an example, the well-known use of fluorescent pigments by the leader of the Pop Art Andy Warhol could be cited among others. 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/). For choosing the most suitable investigation techniques, the large prevalence of or- ganic materials in contemporary paintings needs to be considered. In fact, it limits the effectiveness of non-invasive methods of elemental analysis routinely used for more ancient https://www.mdpi.com/journal/heritage Heritage 2022, 5, 42–60. https://doi.org/10.3390/heritage5010003 Heritage 2022, 5 43 artworks, such as X-ray fluorescence, in favour of molecular-specific analytical techniques. The use of Fourier-transform infrared (FTIR) spectroscopy is well established for the iden- tification of binders [1], but its application in reflection mode to in situ analyses without sampling has somewhat more limited examples in the literature [3]. 1. Introduction The spectral response in the near-IR (NIR) range is also of particular interest being related to a greater depth in the painting stratigraphy in comparison with the mid-IR region [3]. Inorganic pigments that are still in use can also be recognized employing FTIR or Raman spectroscopy. At the same time, the Raman technique is particularly suitable for the identification of synthetic organic pigments [4], even if its use can be partially hindered by the fluorescence emission due to aged binders or to pigments themselves [5]. For this reason, in a recent paper, the possibility of identifying synthetic organic pigments through their emission spectra was also considered [6]. Some of the advantages and limitations of reflectance spectrometry (vis-RS) applied to 20th-century paintings have been established, as well as the need to integrate this methodology with others [7]; it was used due to its ability to recognize certain classes of modern pigments, especially green, blue, and violet. Imaging techniques including IR reflectography, false colour IR, and UV induced fluo- rescence [8] were also used to verify the presence of underdrawings and changes, retouches or repainting, fluorescent pigments, and to choose the measurement areas. Portable optical microscopy helped to better focus some technical issues, such as the use of spray paint or brush, the mixing of different pigments, and the presence of overlapping layers of different colours (including any ground layer). In the present work, molecular spectroscopic methods were tested for a complete characterization of materials in two paintings on aluminium of the Italian Pop artist Silvio Pasotti, namely the right panel of the frieze “The Daily Neo-Mythology” (1966−1967, ab. 15 m2 each, Property of Comune di Segrate; Figure 1) and “The Monster” (1966, 150 × 100 cm2, oval, Collection of the painter; Figure 2a) [9,10]. The latter painting was examined during the exhibition dedicated to the artist, that took place in 2018 in Segrate (Milano, Italy). The first painting, also investigated by means of elemental analysis ED-XRF, is permanently on show in the Auditorium of the “Giuseppe Verdi” Cultural Centre in Segrate (Figure 1, top). The paintings were chosen for their wide chromatic range, for their palette, and their relative simplicity of technique: Pasotti generally avoids impasto and overlapping of pictorial layers, except for a few areas, and the priming is uniform, always the same, allowing one to reduce the complexity of integrated analyses. 1. Introduction So, they were preferred to other works due to the possibility of enriching the personal database, verifying the limits of detection of the techniques used, interviewing the author, and as well for their unusual metal support. Silvio Pasotti (Bergamo, 1933), who attended the Carrara Academy of Bergamo, es- tablished his studio in Milan in 1958, the city where he still resides today, except for a long period in Paris between 1969 and 1976. He participated in numerous exhibitions in Italy and abroad and is considered one of the most representative Italian pop artists. In fact, in the 1960s, after having met American pop art, he was the author of works that often combined metal and painting, sometimes even of monumental proportions, in which images of everyday objects (such as cars or washing machines) were used to develop reflections on social issues. He also worked with mixed media and different techniques, including ceramic sculpture. Heritage 2022, 5 Heritage 2022, 5, ritage 2022, 5, FOR 443 Figure 1. Silvio Pasotti, “The Daily Neo-Mythology”: (top) the frieze in its context in the Auditorium of the “Giuseppe Verdi” Cultural Centre in Segrate and (centre and bottom) the right and left panels. Figure 1. Silvio Pasotti, “The Daily Neo-Mythology”: (top) the frieze in its context in the Auditorium of the “Giuseppe Verdi” Cultural Centre in Segrate and (centre and bottom) the right and left panels. Figure 1. Silvio Pasotti, “The Daily Neo-Mythology”: (top) the frieze in its context in the Auditorium of the “Giuseppe Verdi” Cultural Centre in Segrate and (centre and bottom) the right and left panels. Figure 1. Silvio Pasotti, “The Daily Neo-Mythology”: (top) the frieze in its context in the Auditorium of the “Giuseppe Verdi” Cultural Centre in Segrate and (centre and bottom) the right and left panels. Figure 1. Silvio Pasotti, “The Daily Neo-Mythology”: (top) the frieze in its context in the Auditorium of the “Giuseppe Verdi” Cultural Centre in Segrate and (centre and bottom) the right and left panels. igure 1. Silvio Pasotti, “The Daily Neo-Mythology”: (top) the frieze in its context in the Auditorium f the “Giuseppe Verdi” Cultural Centre in Segrate and (centre and bottom) the right and left panels. Figure 1. Silvio Pasotti, “The Daily Neo-Mythology”: (top) the frieze in its context in the Auditorium of the “Giuseppe Verdi” Cultural Centre in Segrate and (centre and bottom) the right and left panels. Figure 1. 2. Materials and Methods The measurement areas in the two paintings are shown in Figure 2 and will be indicated with the prefix M when belonging to “The Monster” and the prefix NQ when referring to “The Daily Neo-Mythology”. The in situ Raman analyses were performed in the range of 200–1900 cm−1 using a Jasco RPM-100 microprobe, equipped with a notch filter and an Olympus 50× objec- tive and interfaced via optical fibres to the laser source and to a Lot-Oriel MS125 spec- trometer (1200 lines/mm grid). The latter was equipped with an Andor CCD detector (1024 × 128 pixels), cooled by a Peltier device. A diode laser emitting at 785 nm was used as the excitation source, maintaining an incident laser power of a few milliwatts. Raman spectra were obtained by collecting 30 scans, with an exposure time of 2 s. For the acquisi- tion of the spectra on the paintings in their exhibition venue, the microprobe was mounted on a tripod equipped with a xyz stage. Reflection FTIR measurements were performed directly on Pasotti’s paintings using an Alpha Bruker FTIR spectrometer. This instrument is equipped with a deuterated triglycine sulfate (DTGS) detector operating at room temperature, and a specular reflection module allowed for non-contact measurements. The FTIR spectra were collected in the spectral range of 7500–400 cm−1 as a sum of 200 scans with a resolution of 4 cm−1. An integrated video camera allows one to select exactly the area of the samples to be analysed, having a diameter of 0.6 cm. In the mid-IR range the reflectance spectra were processed by the Kramers-Kronig transform, while in the near-IR (NIR) range the same spectra were converted into pseudo-absorbance log(1/R). p g A portable microprobe was used for the fluorescence analyses. The microprobe is equipped with an Olympus 20× objective and is connected via optical fibres to a halogen source (maximum power 150 W) and to the same Lot Oriel MS125 spectrometer already described above, here equipped with a 400 lines/mm grating. The wavelength scale was calibrated using the emission spectrum of a neon lamp. In the microprobe, the radiation from the source is sent along a direction perpendicular to the objective of the microscope. The microprobe is equipped with an interference filter to select the excitation wavelength and a dichroic filter to eliminate the component due to the exciting radiation from the spectrum. 1. Introduction Silvio Pasotti, “The Daily Neo-Mythology”: (top) the frieze in its context in the Auditorium of the “Giuseppe Verdi” Cultural Centre in Segrate and (centre and bottom) the right and left panels. ure 1. Silvio Pasotti, “The Daily Neo-Mythology”: (top) the frieze in its context in the Auditorium he “Giuseppe Verdi” Cultural Centre in Segrate and (centre and bottom) the right and left panels. Figure 2. Measurement areas in the two paintings by S. Pasotti: (a) “The monster” (M); (b) “The Daily Neo-Mythology”, right panel, detail (NQ). gure 2. Measurement areas in the two paintings by S. Pasotti: (a) “The monster” (M); (b) “The aily Neo-Mythology”, right panel, detail (NQ). Figure 2. Measurement areas in the two paintings by S. Pasotti: (a) “The monster” (M); (b) “The Daily Neo-Mythology”, right panel, detail (NQ). Figure 2. Measurement areas in the two paintings by S. Pasotti: (a) “The monster” (M); (b) “The Daily Neo-Mythology”, right panel, detail (NQ). gure 2. Measurement areas in the two paintings by S. Pasotti: (a) “The monster” (M); (b) “The ily Neo-Mythology”, right panel, detail (NQ). Figure 2. Measurement areas in the two paintings by S. Pasotti: (a) “The monster” (M); (b) “The Daily Neo-Mythology”, right panel, detail (NQ). Heritage 2022, 5 45 2. Materials and Methods In particular, the interference filter was centred at 435 nm, while the dichroic one had a transmission range between 458 and 680 nm. The fluorescence spectra were collected as a sum of 30 scans with an exposure time of 2 s and the analyses were preceded by the acquisition of a background spectrum in the absence of incident radiation. The emission spectra collected on the paintings were compared with the laboratory database of fluorescence spectra of synthetic organic pigments in mock-up paint samples [6], also performing principal component analysis (PCA) using the Minitab software version 14. Diffuse reflectance spectrometry in the spectral range of 360–740 nm (vis-RS) was performed with a handheld spectrophotometer Minolta CM-2600d equipped with an inner integrating sphere (10 nm acquisition step, d/8 geometry, UV included, 3 mm diameter spot, measurement time 1 s, specular component included and excluded). Colorimetric data in the CIELAB colour space were also acquired. A personal database, together with literature data, was used to compare spectra. X-ray fluorescence (ED-XRF) was carried out on some chosen areas of “The Daily Neo-Mythology” to verify some results obtained by molecular spectroscopy and to acquire information on the support and primer, the same used in “The Monster”. An Oxford Instrument X-Met 8000 energy dispersive handheld spectrometer (X-Flash SDD detector, 6 mm diameter spot, Rh target X-ray tube operating both at 8 kV, 50 µA and 40 kV, 8 µA). Spectra were collected with a measurement time of 100 s: 74 s at 8 kV, to detect light elements, and 26 s at 40 kV, for heavier ones. Data were processed using Artax proper software (version 4.9). Spectroscopic analyses were generally carried out on the same chosen points, but, due to the shorter measurement time vis-RS was applied to a greater number of areas. Heritage 2022, 5 46 Regarding imaging methods, IR reflectography (IRR) and false colour IR (IRFC) were performed with a 20 Mpx Samsung digital camera (filtered to operate in the IR range 0.85–1 µm) and a 1000 W halogen source. For UV induced fluorescence (UVF) the same camera, operating in the visible range, and a Labino bulb UV beam lamp were employed (MPX technology, emission peak at 365 nm). In situ optical digital microscopy (DM) was carried out with a Dino-Lite USB digital microscope (CMOS 5 Megapixel sensor, equipped with polarizer, anti-reflection, and IR cut-filter >650 nm, interface USB 2.0), directly connected to PC. 2. Materials and Methods Images were acquired at 50× and 230× magnifications. 3. Results and Discussion As for the pictorial technique, both works examined are painted on aluminium panels. The Segrate frieze, in particular, consists of separate panels placed side by side and hooked on the back, each made as a sandwich structure, where two aluminium sheets about 1 mm thick are separated by an internal wooden frame. As observed and also confirmed by Pasotti himself during a series of meetings that preceded a recent exhibition dedicated to him [9], the aluminium surface of the support was brushed in most of the areas to be painted so as to become rough and ensure better adhesion of the paint. On the contrary, it remains smooth in unpainted areas. Then, a light brown paint base was applied as a primer. The subsequent painting was applied by spray, generally without overlapping between the different colours. Despite the probable use of cardboard masks, useful to avoid dirtying the adjacent backgrounds, at high magnifications (50–230×) small scattered drops of colour can be observed that encroach on the neighbouring areas, without significantly changing the final tone (Figure 3, M5 and M7). In some cases, on the contrary, the spraying of one colour on the other is desired by the painter to obtain a blurred effect in terms of colour (Figure 3, image “close to M9”). Table 1. Pigments identified in the paintings “The Monster” and “The Daily Neo-Mythology” by Silvio Pasotti, with Raman, FTIR and fluorescence data supporting the identification. In case of mixtures, the band assignments are reported in parentheses. In vis-RS column, −indicates the minimum, + the maximum, “s” the shoulder, “e” the edge (increasing slope) and “de” the decreasing edge (slope) of the spectrum. Colour Area Raman Bands (cm−1) 1 IR Bands (cm−1) 1,2 vis-RS Bands (nm) Emission Maximum (nm) 1 Pigment The Monster (M) Red M1 985 (BaSO4) - 510e, 560–570e, 630s 610 cadmium red PR108? M12, M13 - - 410s (TiO2), ab. 3. Results and Discussion 490e, 550−, 630s 604 rhodamine 6G M14 793, 982, 1120, 1183, 1214, 1252, 1317, 1332, 1395, 1445, 1496, 1555, 1622 - 570e, 640s 630 β-naphthol PR3 Orange M10 445, 604 (TiO2), 963, 1044, 1365, 1426, 1493, 1555, 1582 (PR10) - 410s (TiO2), 460e, 480+ (weak), 490e, 540−560s 525 (530, 560 and 582 shoulders) (PY109) naphthol AS PR10 (or PO38) + isoindole PY109 Yellow M11, M20 445, 604 (TiO2) - 460e, 480+ (weak), 490e, 530s 525 (530, 560 and 582 shoulders) (PY109) isoindole PY109 Heritage 2022, 5 47 Table 1. Cont. Colour Area Raman Bands (cm−1) 1 IR Bands (cm−1) 1,2 vis-RS Bands (nm) Emission Maximum (nm) 1 Pigment The Monster (M) Green M4, M6 667, 747, 770, 1212, 1278, 1336, 1539 - 410s (TiO2), ab. 460s, 510–520+, 620de, 650−, 710+, 720− - phthalocyanine PG36 M16 684, 742, 775, 819. 960, 977, 1002, 1083, 1214, 1289, 1343, 1541 - 410s (TiO2), ab. 460s, 510–520+, 620de, 650−, 710+, 730− - phthalocyanine PG7 Blue M7 440, 605 (TiO2), 677, 745, 950, 1140, 1182, 1338, 1450, 1528 (PB15) 2093 (PB27) 410s (TiO2), 460+, ab. 710− - phthalocyanine PB15(:1) + Prussian blue PB27 M9 443, 602 (TiO2), 677, 745, 951, 1140, 1338, 1450, 1528 (PB15) - 420s (TiO2), 480+, 580de, 610−, 660−670+, 690−700− - phthalocyanine PB15(:1) Violet M15 440, 602 (TiO2), 1262, 1400 (PV5) - 420+, 540de, 560+ (weak), 590− 640 anthraquinone PV5 White M2 440, 602 - 380e, 420s (TiO2) - titanium white PW6 The Daily Neo-Mythology (NQ) Red NQ1 963, 1044, 1365, 1426, 1493, 1555, 1582 - ab. 560e, 620s 600 naphthol AS PR10 (or PO38) NQ12 (red- purple) 795, 1125, 1179, 1323, 1393, 1444 (PR3) - 580e, 640s, 690− (weak) 630 (610 shoulder, PV19?) β-naphthol PR3 + violet pigment (PV19?) NQ9 (fuchsia) - - not examined 612 PV19? Orange NQ8 789, 945, 997,1135, 1252, 1311, 1484, 1623 (PY1) 1669, 1602, 1559, 1509, 1450, 1386, 1293, 1271, 1175, 1137, 1120, 1084, 950, 912, 802, 770 (PY1) ab. 480e, 530e, 600s 590 (unidentified orange pigment) arylide PY1 + unidentified orange pigment Yellow NQ7 950, 1000, 1137, 1214, 1254, 1312, 1389, 1488, 1536, 1565, 1626 not available 480-490e, ab. 550s; 620−(weak), 700+ (weak) 540 arylide PY1 Table 1. Cont. Heritage 2022, 5 48 Table 1. Cont. 3. Results and Discussion Colour Area Raman Bands (cm−1) 1 IR Bands (cm−1) 1,2 vis-RS Bands (nm) Emission Maximum (nm) 1 Pigment The Daily Neo-Mythology (NQ) Green NQ5 (light green) 682, 741, 1285, 1340, 1542 (PG7), 1145 (PY3?) - 410s/(+) (TiO2), ab. 470s, 530+, 620de, 650−, 710+, 730− 524 phthalocyanine PG7 + arylide PY3 NQ6 684, 742, 775, 819, 977, 1002, 1083, 1214, 1289, 1343, 1541 - not examined - phthalocyanine PG7 NQ10 (dark green) 680, 740, 774, 816, 1290, 1539 (PG7), 956, 1145, 1342, 1451, 1530 (PB15) - 410s/(+) (TiO2), ab. 460s, 500+, 580de, 610−, 660+, 690− - phthalocyanine PG7 + PB15 Blue NQ2 see text 2100 410s (TiO2), 460+, 670−710− - Prussian blue PB27 NQ4 677, 746, 1001, 1143, 1343, 1534 670 (TiO2) 420s (TiO2), 490+, 580de, 610−, 670+, 700− - phthalocyanine PB15(:1) NQ11 548 1015, 450 450+, 590−, ab. 650e - ultramarine blue PB29 Violet NQ3 1262 - 410s (TiO2), 430+, 550−, 560+ (weak), 580− 640 anthraquinone PV5 1 A dash indicates that no bands attributable to the pigment were detected in the spectrum. 2 FTIR spectra allowed also the identification of the binders used in the different areas, that are nitrocellulose for the examined details of “The Monster” and acrylic for most examined areas of “The Daily Neo-Mythology” with the exception of area NQ2 where again nitrocellulose was instead detected, as detailed in Section 3.4. Table 1. Cont. 1 A dash indicates that no bands attributable to the pigment were detected in the spectrum. 2 FTIR spectra allowed also the identification of the binders used in the different areas, that are nitrocellulose for the examined details of “The Monster” and acrylic for most examined areas of “The Daily Neo-Mythology” with the exception of area NQ2 where again nitrocellulose was instead detected, as detailed in Section 3.4. 1 A dash indicates that no bands attributable to the pigment were detected in the spectrum. 2 FTIR spectra allowed also the identification of the binders used in the different areas, that are nitrocellulose for the examined details of “The Monster” and acrylic for most examined areas of “The Daily Neo-Mythology” with the exception of area NQ2 where again nitrocellulose was instead detected, as detailed in Section 3.4. For this series of works, the painter remembers the use of nitrocellulose paints, but also acrylics. 3. Results and Discussion Interventions with brushes or felt-tip pens are found in some zones to, respectively, define small coloured areas and some details (such as the edges of some backgrounds, for example). Multispectral images—IRR, IRFC and UVF—allowed us to check the preservation state and to choose the measurement points, avoiding retouching due to restorations. The only small damages (small losses), mainly integrated during the last restoration a few years ago, were found in the frieze, while “The Monster” appears to be very well preserved. IR reflectograms (IRR) showed only a few lines of underdrawing; probably a fairly transparent drawing medium or thin drawing lines largely covered by paint were used. The outline drawing is precise, very accurate, with small corrections made with the paint. The most interesting IRR result regards “The Monster”, where a previous version can be seen (Figure 4). The figure was originally bigger and its profile less rotated, its larger eye placed below the visible one and its beak extending to the left. Heritage 2022, 5 Heritage 2022, 49 Figure 3. Digital microscopy details (230×) of the measurement areas indicated (see Table 1 for descriptions). The lower images refer to the brushed aluminium-alloy support and to the light brown primer painted on it. T bl 1 Pi id ifi d i h i i “Th M ” d “Th D il N M h l ” b Figure 3. Digital microscopy details (230×) of the measurement areas indicated (see Table 1 for descriptions). The lower images refer to the brushed aluminium-alloy support and to the light brown primer painted on it. EW 9 nitrocellulose for the examined details of “The Monster” and acrylic for most examined areas of “The Daily Neo-Mythology” with the exception of area NQ2 where again nitrocellulose was instead detected, as detailed in Section 3.4. Multispectral images—IRR, IRFC and UVF—allowed us to check the preservation state and to choose the measurement points, avoiding retouching due to restorations. The only small damages (small losses), mainly integrated during the last restoration a few years ago, were found in the frieze, while “The Monster” appears to be very well preserved. IR reflectograms (IRR) showed only a few lines of underdrawing; probably a fairly transparent drawing medium or thin drawing lines largely covered by paint were used. The outline drawing is precise, very accurate, with small corrections made with the paint. 3. Results and Discussion The most interesting IRR result regards “The Monster”, where a previous version can be seen (Figure 4). The figure was originally bigger and its profile less rotated, its larger eye placed below the visible one and its beak extending to the left. Figure 3. Digital microscopy details (230×) of the measurement areas indicated (see Table 1 for descriptions). The lower images refer to the brushed aluminium-alloy support and to the light brown primer painted on it. Figure 3. Digital microscopy details (230×) of the measurement areas indicated (see Table 1 for descriptions). The lower images refer to the brushed aluminium-alloy support and to the light brown primer painted on it. transparent drawing medium or thin drawing lines largely covered by paint were used. The outline drawing is precise, very accurate, with small corrections made with the paint. The most interesting IRR result regards “The Monster”, where a previous version can be seen (Figure 4). The figure was originally bigger and its profile less rotated, its larger eye placed below the visible one and its beak extending to the left. g p g y y gy y Silvio Pasotti, with Raman, FTIR and fluorescence data supporting the identification. In case of mixtures, the band assignments are reported in parentheses. In vis-RS column, −indicates the minimum, + the maximum, “s” the shoulder, “e” the edge (increasing slope) and “de” the decreasing edge (slope) of the spectrum. Colour Area Raman bands (cm−1) 1 IR bands (cm−1) 1,2 vis-RS Bands (nm) Emission maximum (nm) 1 Pigment The Monster (M) Red M1 985 (BaSO4) ‒ 510e, 560– 570e, 630s 610 cadmium red PR108? M12, M13 ‒ ‒ 410s (TiO2), ab. 490e, 550−, 630s 604 rhodamine 6G Figure 4. “The Monster”: the same detail under (left) visible diffused light ad (right) IRR. The first version of the chicken, a larger one, can be seen in the IRR. IRFC and IRR were also useful to discriminate layers made with different pigments Figure 4. “The Monster”: the same detail under (left) visible diffused light ad (right) IRR. The first version of the chicken, a larger one, can be seen in the IRR. Figure 4. “The Monster”: the same detail under (left) visible diffused light ad (right) IRR. The first version of the chicken, a larger one, can be seen in the IRR. IRFC and IRR were also useful to discriminate layers made with different pigments Figure 4. 3. Results and Discussion “The Monster”: the same detail under (left) visible diffused light ad (right) IRR. The first version of the chicken, a larger one, can be seen in the IRR. Heritage 2022, 5 50 IRFC and IRR were also useful to discriminate layers made with different pigments but with the same colour, as in the very dark area in the centre of Figure 5. The different IR transparency allows to distinguish the distribution of black and dark blue pigment, i.e., ultramarine blue (as explained later, see NQ11 in Table 1), showing its characteristic red tint in IRFC image (Figure 5c). W 10 10 IRFC and IRR were also useful to discriminate layers made with different pigments but with the same colour, as in the very dark area in the centre of Figure 5. The different IR transparency allows to distinguish the distribution of black and dark blue pigment, i.e., ultramarine blue (as explained later, see NQ11 in Table 1), showing its characteristic red tint in IRFC image (Figure 5c). W 10 10 Figure 5. “The Daily Neo-Mythology”, right panel: the same detail seen in (a) visible diffused light, (b) IRR, and (c) IRFC. Figure 5. “The Daily Neo-Mythology”, right panel: the same detail seen in (a) visible diffused light, (b) IRR, and (c) IRFC. Figure 5. “The Daily Neo-Mythology”, right panel: the same detail seen in (a) visible diffused light, (b) IRR, and (c) IRFC. Figure 5. “The Daily Neo-Mythology”, right panel: the same detail seen in (a) visible diffused light, (b) IRR, and (c) IRFC. Figure 5. “The Daily Neo-Mythology”, right panel: the same detail seen in (a) visible diffused light, (b) IRR, and (c) IRFC. Figure 5. “The Daily Neo-Mythology”, right panel: the same detail seen in (a) visible diffused light, (b) IRR, and (c) IRFC. UV fluorescence showed the existence of highly fluorescent pigments, in some pink areas, which were then subjected to the spectroscopic investigation, while the UV fluorescence of the other pigments was generally low and the unpainted aluminium support returned a bluish colour due to the reflected UV component (see below). UV fluorescence showed the existence of highly fluorescent pigments, in some pink areas, which were then subjected to the spectroscopic investigation, while the UV fluo- rescence of the other pigments was generally low and the unpainted aluminium support returned a bluish colour due to the reflected UV component (see below). 3. Results and Discussion UV fluorescence showed the existence of highly fluorescent pigments, in some pink areas, which were then subjected to the spectroscopic investigation, while the UV fluorescence of the other pigments was generally low and the unpainted aluminium support returned a bluish colour due to the reflected UV component (see below). h l ll h d b d b Al % The aluminium alloy was characterized by XRF, constituted by Al (99.4 ± 0.1%) with very small amounts of Mn, Fe, Cu, Zn, Ga, and Pb (see the black spectrum in Figure 6). Its quantitative composition, close to “1060 Al-alloy”, was derived by calibration standards and proper software. The aluminium alloy was characterized by XRF, constituted by Al (99.4 ± 0.1%) with very small amounts of Mn, Fe, Cu, Zn, Ga, and Pb (see the black spectrum in Figure 6). Its quantitative composition, close to “1060 Al-alloy”, was derived by calibration standards and proper software. The aluminium alloy was characterized by XRF, constituted by Al (99.4 ± 0.1%) with very small amounts of Mn, Fe, Cu, Zn, Ga, and Pb (see the black spectrum in Figure 6). Its quantitative composition, close to “1060 Al-alloy”, was derived by calibration standards and proper software. Figure 6. XRF spectra (acquired at 40 kV) of the “The Daily Neo-Mythology”, a comparison among the metal support, the primer (close to NQ9) and a green area (NQ10). Figure 6. XRF spectra (acquired at 40 kV) of the “The Daily Neo-Mythology”, a comparison among the metal support, the primer (close to NQ9) and a green area (NQ10). Figure 6. XRF spectra (acquired at 40 kV) of the “The Daily Neo-Mythology”, a comparison among the metal support, the primer (close to NQ9) and a green area (NQ10). Figure 6. XRF spectra (acquired at 40 kV) of the “The Daily Neo-Mythology”, a comparison among the metal support the primer (close to NQ9) and a green area (NQ10) Figure 6. XRF spectra (acquired at 40 kV) of the “The Daily Neo-Mythology”, a comparison among he metal support, the primer (close to NQ9) and a green area (NQ10). Figure 6. XRF spectra (acquired at 40 kV) of the “The Daily Neo-Mythology”, a comparison among the metal support, the primer (close to NQ9) and a green area (NQ10). 3.1.1. Blue and Green For the blue and green hues, phthalocyanine pigments were abundantly used, even mixed with other organic or inorganic pigments, and could be identified through their Raman spectra. For blue, phthalocyanine blue PB15 was recognised in areas M9, M7 and NQ4 (Figure 7e, Figures S1c and S2c, Supplementary Materials) and supposed to be in the form PB15:1 due to the presence of a very weak band at around 1182 cm−1 [11]. As will be shown below, further spectroscopic data demonstrated that it was mixed with Prussian blue in the M7 area. PB15 was also detected by means of vis-RS, thanks to its characteristic bands (i.e., the broad 580–610 nm absorption band and the narrow 690–700 nm one) the relative maximum between these at 660–670 nm, and the reflectance peak at 460–480 nm [12]. However, on the basis of vis-RS it appears to be difficult to distinguish between PB15 and PB15:1. For green, both fully chlorinated PG7 and partially brominated PG36 phthalocyanine could be detected. In particular, PG7 was recognised in both paintings, in areas M16 and NQ6, again thanks to the Raman spectra (Figure 7f) [13–15]. In the dark green area NQ10, the same pigment was used in mixture with PB15, as indicated by the coexistence of Raman bands at 680, 740, 774, 816, and 1290 cm−1, due to PG7, and at 956, 1145, 1342, 1451 cm−1, due to PB15. Furthermore, the signal at 1530–1540 cm−1 contains both one component at about 1530 cm−1 for PB15 and another at approximately 1539 cm−1 for PG7 (Figure S2e, Supplementary Materials). For this area, vis-RS measurements could only distinguish the presence of PB15, although some traces of phthalocyanine were found in the yellow paint of NQ7, showing the sensitivity of this technique to this class of pigments. For the bright green NQ5 area of the same painting, the Raman spectrum suggests that PG7 was likely mixed with PY3 (Figure S2c, Supplementary Materials), as detailed below. The presence of a yellow pigment in some green mixtures such as NQ5 can be observed by microscopy (Figure 3, NQ5). Also, vis-RS allows the detection of PG7 due to the absorption band at 620–650 nm and the narrow one at 730 nm, with a relative maximum at 710 nm, in accordance with the literature [12]. 3.1. Synthetic Organic Pigments In the paintings analysed, a rather wide choice of synthetic organic pigments was detected. Throughout the paper, the pigments are named according to the conventional Colour Index (CI) nomenclature system, which requires each colourant to be designed by a CI name, referring to its colour, and a CI number, giving some information about the chemical constitution of the pigment (e.g., Pigment Red 1, or PR1). Specifically, for names, the matches are R for red, Y for yellow, O for orange, B for blue, G for green, V for violet and W for white. The chemical class to which each pigment belongs is reported in Table 1. 3. Results and Discussion Heritage 2022, 5 51 The light brown primer that can be sometimes seen at the edges of some coloured areas and under the paint where small lacunas can be found by magnification (Figure 3, bottom right), contains ochre/iron-oxides according to vis-RS and XRF. In particular, X- ray fluorescence (Figure 6, yellow spectrum vs. black one) evidenced the presence of Fe (abundant), Ca, Sr, Ti, Ba, Zn, S, Pb (traces), where Ca, Sr, and S (better read with the lower elements excitation 8 kV) can be due to calcium sulphate with celestite impurities, while Ti and Zn are linked to white pigments, titanium and zinc oxides mixed in the primer. p g p The materials identified in the two paintings will be discussed in detail below and are summarised in Table 1. In parallel, the performances of the different techniques adopted in the present study will be examined for each class of materials. 3.1.1. Blue and Green When PY3 is mixed, as in the NQ5 area, it produces the shift of the maximum from about 500–510 nm to 530 nm, and of the first edge from ab. 450 nm to 470–480 nm. Heritage 2022, 5 52 PG36 was identified on the basis of the Raman spectra in M4 and M6 areas of “The monster” (Figure 7g and Figure S1b, Supplementary Materials) [13]. It is interesting to note that this pigment was produced starting from 1959 [16], therefore only a few years before the painting under investigation. Phthalocyanine PG36 was not identified by vis-RS: probably its bands are too close to PG7 ones. 12 probably its bands are too close to PG7 ones. Figure 7. Raman spectra (λexc = 785 nm) of areas: (a) orange M10; (b) red M14; (c) violet M15; (d) yellow NQ7; (e) blue M9; (f) green NQ6; (g) green M4. The inserted box shows an enlargement of spectra (a,c) to highlight some weaker bands. Figure 7. Raman spectra (λexc = 785 nm) of areas: (a) orange M10; (b) red M14; (c) violet M15; (d) yellow NQ7; (e) blue M9; (f) green NQ6; (g) green M4. The inserted box shows an enlargement of spectra (a,c) to highlight some weaker bands. Figure 7. Raman spectra (λexc = 785 nm) of areas: (a) orange M10; (b) yellow NQ7; (e) blue M9; (f) green NQ6; (g) green M4. The inserted b spectra (a,c) to highlight some weaker bands. Figure 7. Raman spectra (λexc = 785 nm) of areas: (a) orange M10; (d) yellow NQ7; (e) blue M9; (f) green NQ6; (g) green M4. The inserted b spectra (a,c) to highlight some weaker bands. igure 7. Raman spectra (λexc = 785 nm) of areas: (a) orange M10; (b) red M14; (c) violet M15; (d) ellow NQ7; (e) blue M9; (f) green NQ6; (g) green M4. The inserted box shows an enlargement of pectra (a,c) to highlight some weaker bands. Figure 7. Raman spectra (λexc = 785 nm) of areas: (a) orange M10; (b) red M14; (c) violet M15; (d) yellow NQ7; (e) blue M9; (f) green NQ6; (g) green M4. The inserted box shows an enlargement of spectra (a,c) to highlight some weaker bands. Heritage 2022, 5 53 Of course, none of the above pigments gave rise to fluorescence emission upon visible excitation, most likely due to the quenching effect of the paramagnetic Cu2+ ion [17]. 3.1.2. Red For red colours, two different synthetic organic pigments could be identified. The first one is the β-naphthol PR3, positively indicated by its characteristic Raman bands [4] in the red area M14 of “The Monster” (Figure 7b), but also in the red-purple area NQ12 of “The Daily Neo-Mythology” (Figure S2f, Supplementary Materials). An interesting fluorescence response was obtained on the same areas upon excitation at 435 nm. As can be seen in Figure S3a (Supplementary Materials), an emission maximum around 630 nm is observed, in accordance with what previously reported for this pigment [6]. Furthermore, an excellent correspondence was obtained in the PCA for the emission spectrum of the M14 area in comparison with those of mock-up samples painted on canvas with several red organic pigments [6] (Figure 8). OR PEER REVIEW Figure 8. Score plot of the first two principal components of the emission spectra (λ the red areas M14 and M1 (pink circles with arrows) in comparison with those of pigments in painting mock-up samples with oil or acrylic binder on canvas [6]. Figure 8. Score plot of the first two principal components of the emission spectra (λexc = 435 nm) of the red areas M14 and M1 (pink circles with arrows) in comparison with those of red and orange pigments in painting mock-up samples with oil or acrylic binder on canvas [6]. Figure 8. Score plot of the first two principal components of the emission spectra (λe the red areas M14 and M1 (pink circles with arrows) in comparison with those of r pigments in painting mock-up samples with oil or acrylic binder on canvas [6]. Figure 8. Score plot of the first two principal components of the emission spectra (λexc = 435 nm) of the red areas M14 and M1 (pink circles with arrows) in comparison with those of red and orange pigments in painting mock-up samples with oil or acrylic binder on canvas [6]. The emission maximum of NQ12 is also found around 630 nm (Figure mentary Materials), in agreement with the same pigment PR3 detected by troscopy, but a broadening of the band towards 610 nm is also evident. Ind purple hue of that measurement area suggests a second component, possibl such as the β-form of PV19 (Figure S3a, Supplementary Materials). In fact, shows a fluorescence maximum around 605 nm [6]. 3.1.1. Blue and Green y q g p g ED-XRF could detect very small amounts of Cu, which also belong to the Al alloy, but at least in one point, NQ10, where the green is darker (i.e., more concentrated) a higher intensity of Cu was measured (Figure 6, green spectrum). 3.1.2. Red Correctly, it does not match any spectrum of our database, which actually does not contain those pigments. Materials), corresponding to those of PR10 [4], but also of the orange pigment PO38 [15] which belongs to the same structural class. The emission maximum observed for the same area upon visible excitation is around 600 nm (Figure 9a). Correctly, it does not match any spectrum of our database, which actually does not contain those pigments. Figure 9. Emission spectra (λexc = 435 nm) of areas: (a) red NQ1; (b) violet M15; (d) red M comparison, the emission spectrum (c) of a red fluorescent paint containing rhodamine 6G ( Fluo colour by Lefranc and Bourgeois) is also reported. Note: the vertical scale is not the sa the four spectra; bands in spectra (c,d), due to “special effect” fluorescent pigments, are abo order of magnitude more intense than those in spectra (a,b). Figure 9. Emission spectra (λexc = 435 nm) of areas: (a) red NQ1; (b) violet M15; (d) red M12. For comparison, the emission spectrum (c) of a red fluorescent paint containing rhodamine 6G (Flashe Fluo colour by Lefranc and Bourgeois) is also reported. Note: the vertical scale is not the same for the four spectra; bands in spectra (c,d), due to “special effect” fluorescent pigments, are about one order of magnitude more intense than those in spectra (a,b). Figure 9. Emission spectra (λexc = 435 nm) of areas: (a) red NQ1; (b) violet M15; (d) red M comparison, the emission spectrum (c) of a red fluorescent paint containing rhodamine 6G Fluo colour by Lefranc and Bourgeois) is also reported. Note: the vertical scale is not the sa the four spectra; bands in spectra (c,d), due to “special effect” fluorescent pigments, are abo order of magnitude more intense than those in spectra (a,b). Figure 9. Emission spectra (λexc = 435 nm) of areas: (a) red NQ1; (b) violet M15; (d) red M12. For comparison, the emission spectrum (c) of a red fluorescent paint containing rhodamine 6G (Flashe Fluo colour by Lefranc and Bourgeois) is also reported. Note: the vertical scale is not the same for the four spectra; bands in spectra (c,d), due to “special effect” fluorescent pigments, are about one order of magnitude more intense than those in spectra (a,b). 3.1.2. Red The same Raman bands were recorded for the orange area M10 of “The Mo (Figure 7a), where also a yellow pigment, not detectable by the Raman spectrum The same Raman bands were recorded for the orange area M10 of “The Monster” (Figure 7a), where also a yellow pigment, not detectable by the Raman spectrum, was identified by spectrofluorimetry as will be detailed below. identified by spectrofluorimetry as will be detailed below. As hypothesized and already verified [7], vis-RS is not effective in distingui As hypothesized and already verified [7], vis-RS is not effective in distinguishing among modern red pigments, as well as yellow ones, if not in special cases. 3.1.2. Red Furthermore, it has a w response in comparison with PR3, and this may explain why its bands are in the Raman spectrum of NQ12. It is interesting to note that a fuchsia deta painting, NQ9, shows an emission maximum always around 610 nm (Figure mentary Materials), with an excellent correspondence with the fluorescence PV 19 β (Figure S3a Supplementary Materials) also with regard to the band The emission maximum of NQ12 is also found around 630 nm (Figure S3c, Supplemen- tary Materials), in agreement with the same pigment PR3 detected by Raman spectroscopy, but a broadening of the band towards 610 nm is also evident. Indeed, the red-purple hue of that measurement area suggests a second component, possibly a violet one such as the β-form of PV19 (Figure S3a, Supplementary Materials). In fact, this pigment shows a fluorescence maximum around 605 nm [6]. Furthermore, it has a weaker Raman response in comparison with PR3, and this may explain why its bands are not observed in the Raman spectrum of NQ12. It is interesting to note that a fuchsia detail of the same painting, NQ9, shows an emission maximum always around 610 nm (Figure S3b, Supplementary Materials), with an excellent correspondence with the fluorescence spectrum of PV 19 β (Figure S3a, Supplementary Materials) also with regard to the band shape. PV 19 β (Figure S3a, Supplementary Materials) also with regard to the band The second red organic pigment is a naphthol AS (or naphthol red) identified in the NQ1 area on the basis of its Raman bands (Figure S2a Su g pp y g p The second red organic pigment is a naphthol AS (or naphthol red) one and was identified in the NQ1 area on the basis of its Raman bands (Figure S2a, Supplementary Heritage 2022, 5 54 Materials), corresponding to those of PR10 [4], but also of the orange pigment PO38 [15] which belongs to the same structural class. The emission maximum observed for the same area upon visible excitation is around 600 nm (Figure 9a). Correctly, it does not match any spectrum of our database, which actually does not contain those pigments. Materials), corresponding to those of PR10 [4], but also of the orange pigment PO38 [15] which belongs to the same structural class. The emission maximum observed for the same area upon visible excitation is around 600 nm (Figure 9a). among mode 3.1.3. Yellow 3.1.3. Yellow No Raman bands were observed for the yellow pigment in the M11 and M20 of “The monster”, but a characteristic emission spectrum was recorded for both with a maximum at 525 nm and shoulders at about 537, 560, and 582 nm (Figur Supplementary Materials). This pattern has a good correspondence with the fluores spectrum previously reported for the isoindole yellow PY109 [6] and was also obs for the orange area M10 (Figure S4b, Supplementary Materials), where the red pig PR10 (or PO38) was identified by Raman spectroscopy. The PCA of the emission s obtained on “The Monster” compared with those of yellow synthetic organic pigme painting mock up samples [6] shows nevertheless a certain distance in the space No Raman bands were observed for the yellow pigment in the M11 and M20 areas of “The monster”, but a characteristic emission spectrum was recorded for both areas, with a maximum at 525 nm and shoulders at about 537, 560, and 582 nm (Figure S4a, Supplementary Materials). This pattern has a good correspondence with the fluorescence spectrum previously reported for the isoindole yellow PY109 [6] and was also observed for the orange area M10 (Figure S4b, Supplementary Materials), where the red pigment PR10 (or PO38) was identified by Raman spectroscopy. The PCA of the emission spectra obtained on “The Monster” compared with those of yellow synthetic organic pigments in painting mock-up samples [6] shows nevertheless a certain distance in the space of the first two principal components between the spectra from the painting and those of the reference PY109 paints (Figure 10), which however do not in themselves form a very close Heritage 2022, 5 55 group. Furthermore, a minor contribution to the emission spectra due to the additional red pigment recognised for M10 cannot be excluded. W 16 group. Furthermore, a minor contribution to the emission spectra due to the additional red pigment recognised for M10 cannot be excluded. W 16 Figure 10. Score plot of the first two principal components of the emission spectra (λexc = 435 nm) of the yellow areas M10 and M20 in “The Monster” (red squares with arrows) and NQ5 and NQ7 in “The Daily Neo-Mythology” (green diamonds with arrows) in comparison with those of red and orange pigments in painting mock-up samples with oil or acrylic binder on canvas [6]. Figure 10. among mode 3.1.3. Yellow Score plot of the first two principal components of the emission spectra (λexc = 435 nm) of the yellow areas M10 and M20 in “The Monster” (red squares with arrows) and NQ5 and NQ7 in “The Daily Neo-Mythology” (green diamonds with arrows) in comparison with those of red and orange pigments in painting mock-up samples with oil or acrylic binder on canvas [6]. Figure 10. Score plot of the first two principal components of the emission spectra (λexc = 435 nm) of the yellow areas M10 and M20 in “The Monster” (red squares with arrows) and NQ5 and NQ7 in “The Daily Neo-Mythology” (green diamonds with arrows) in comparison with those of red and orange pigments in painting mock-up samples with oil or acrylic binder on canvas [6]. Figure 10. Score plot of the first two principal components of the emission spectra (λexc = 435 nm) of the yellow areas M10 and M20 in “The Monster” (red squares with arrows) and NQ5 and NQ7 in “The Daily Neo-Mythology” (green diamonds with arrows) in comparison with those of red and orange pigments in painting mock-up samples with oil or acrylic binder on canvas [6]. The possible identification of the pigment PY109 is of interest, as it was introduced in the same years when Pasotti painted “The Monster”. Indeed, this type of pigment was first described in 1964 [18]. The vis-RS characteristics of this material are listed in Table 1 (M10, M11, M20). The possible identification of the pigment PY109 is of interest, as it was introduced in the same years when Pasotti painted “The Monster”. Indeed, this type of pigment was first described in 1964 [18]. The vis-RS characteristics of this material are listed in Table 1 (M10, M11, M20). ( ) For the yellow area NQ7 and the green one NQ5 in “The Daily Neo-Mythology” two different yellow pigments were identified. For the yellow area NQ7 and the green one NQ5 in “The Daily Neo-Mythology” two different yellow pigments were identified. y p g In the first area the arylide yellow (or Hansa yellow) PY1 could be recognised on the basis of its Raman spectrum (Figure 7d) [4]. To further verify the performance of spectro- fluorimetry in the identification of such pigments, the emission spectrum of the NQ7 area was also recorded (Figure S4d, Supplementary Materials) and a remarkable correspond- ence was obtained with the spectra of arylide yellows. among mode 3.1.3. Yellow In fact, as expected [6] the emission maximum was located around 540 nm and the attribution was confirmed by PCA (Figure 10). PY1 was also detected by Raman (Figure S2d, Supplementary Materials) and by FTIR spectroscopy (Figure 11b) [19] in the orange area NQ8 of “The Daily Neo-Mythology”. The obvious presence of a second red or orange component in such area is confirmed by the fluorescence band with a maximum at 590 nm, that prevails on the emission of the yellow dye but unfortunately does not correspond to the spectrum of any pigment present in our database [6] In the first area the arylide yellow (or Hansa yellow) PY1 could be recognised on the basis of its Raman spectrum (Figure 7d) [4]. To further verify the performance of spectrofluorimetry in the identification of such pigments, the emission spectrum of the NQ7 area was also recorded (Figure S4d, Supplementary Materials) and a remarkable correspondence was obtained with the spectra of arylide yellows. In fact, as expected [6] the emission maximum was located around 540 nm and the attribution was confirmed by PCA (Figure 10). PY1 was also detected by Raman (Figure S2d, Supplementary Materials) and by FTIR spectroscopy (Figure 11b) [19] in the orange area NQ8 of “The Daily Neo- Mythology”. The obvious presence of a second red or orange component in such area is confirmed by the fluorescence band with a maximum at 590 nm, that prevails on the emission of the yellow dye but unfortunately does not correspond to the spectrum of any pigment present in our database [6]. in our database [6]. In the green area NQ5, where the Raman spectrum of phthalocyanine green PG7 was obtained (see above), the visible-excited emission spectrum (Figure S4c, Supplementary Materials), with a maximum at 524 nm, should be attributed to a yellow component. A reasonable assignment, also on the basis of PCA (Figure 10), could be the arylide yellow PY3 [6]. A very weak Raman band observed at approximately 1140 cm−1 (Figure S2c, Supplementary Materials) could further support this hypothesis [4]. Heritage 2022, 5 Heritage 2022, 5, FO 56 17 Figure 11. Supplementary Materials), sh in the literature for the anthra 3.2. “Special Effect” Pigments q p g [ ] p tabase, which does not include the aforementioned pigment, it should be noted that a characteristic emission at 640 nm (Figure 9b) was observed upon excitation at 435 nm. Vis-RS can detect two bands around 550 and 590 nm, and a small increase in the reflectance factor at 560 nm, while above 590-600 nm the curve begins to rise. 3.2.“. Special Effect” Pigments The so-called “special effect” pigments include among others UV- and daylight- fluorescent pigments [20], that were adopted by pop artists, first Andy Warhol in the 1960s, for their exceptional brightness. Fluorescent pigments are typically composed of a 5% fluorescent dye diluted by a transparent resin. Usually, several dyes are combined to obtain a wider range of different hues. Among them, xanthene dyes such as rhodamine B or 6G are often employed [21]. 3.2. . Special Effect Pigments The so-called “special effect” pigments include among others UV- and daylight-flu- orescent pigments [20], that were adopted by pop artists, first Andy Warhol in the 1960s, for their exceptional brightness. Fluorescent pigments are typically composed of a 5% flu- orescent dye diluted by a transparent resin. Usually, several dyes are combined to obtain a wider range of different hues Among them xanthene dyes such as rhodamine B or 6G Emission spectra were acquired for areas M12 and M13 of “The Monster”, showing a peculiarly high intensity. Their shape and wavelength of the fluorescence maximum (604 nm) resulted to be characteristic of rhodamine 6G. This is evidenced in Figure 9d by comparison with the fluorescence spectrum of a commercial fluorescent paint whose formulation was already demonstrated to contain such dye [22]. a wider range of different hues. Among them, xanthene dyes such as rhodamine B or 6G are often employed [21]. Emission spectra were acquired for areas M12 and M13 of “The Monster”, showing a peculiarly high intensity. Their shape and wavelength of the fluorescence maximum (604 nm) resulted to be characteristic of rhodamine 6G. among mode 3.1.3. Yellow (right) MIR reflection spectra (after Kramers-Kronig transform) of: (a) blue area NQ2 ( band due to Prussian blue PB27); (b) orange area NQ8 ( bands due to PY1); (c) blue area NQ11 ( bands due to ultramarine blue PB29); (d) green area M6; (left) NIR reflection spectra of: (e) blue area NQ11; (f) yellow area M11. Figure 11. (right) MIR reflection spectra (after Kramers-Kronig transform) of: (a) blue area NQ2 (• band due to Prussian blue PB27); (b) orange area NQ8 (♦bands due to PY1); (c) blue area NQ11 (■bands due to ultramarine blue PB29); (d) green area M6; (left) NIR reflection spectra of: (e) blue area NQ11; (f) yellow area M11. Figure 11. (right) MIR reflection spectra (after Kramers-Kronig transform) of: (a) blue area NQ2 ( band due to Prussian blue PB27); (b) orange area NQ8 ( bands due to PY1); (c) blue area NQ11 ( bands due to ultramarine blue PB29); (d) green area M6; (left) NIR reflection spectra of: (e) blue area NQ11; (f) yellow area M11. Figure 11. (right) MIR reflection spectra (after Kramers-Kronig transform) of: (a) blue area NQ2 (• band due to Prussian blue PB27); (b) orange area NQ8 (♦bands due to PY1); (c) blue area NQ11 (■bands due to ultramarine blue PB29); (d) green area M6; (left) NIR reflection spectra of: (e) blue area NQ11; (f) yellow area M11. In the gre 3.1.4. Violet obtained (see above), the visible-excited emission spectrum (Figure S4c, Supplementary Materials), with a maximum at 524 nm, should be attributed to a yellow component. A reasonable assignment, also on the basis of PCA (Figure 10), could be the arylide yellow PY3 [6]. A very weak Raman band observed at approximately 1140 cm−1 (Figure S2c, Sup- plementary Materials) could further support this hypothesis [4]. 3.1.4. Violet The violet tint is present in both paintings, respectively, in the M15 and NQ3 areas. The same Raman spectral pattern was obtained for the two areas (Figure 7c and Figure S2b, Supplementary Materials), showing a good correspondence with the spectrum reported in the literature for the anthraquinone pigment PV5 [4]. To complement the available database, which does not include the aforementioned pigment, it should be noted that a characteristic emission at 640 nm (Figure 9b) was observed upon excitation at 435 nm. The violet tint is present in both paintings, respectively, in the M15 and NQ3 areas. The same Raman spectral pattern was obtained for the two areas (Figures 7c and S2b, ( g ) p Vis-RS can detect two bands around 550 and 590 nm, and a small increase in the reflectance factor at 560 nm, while above 590–600 nm the curve begins to rise. 3.3. Inorganic Pigments 3.3. Inorganic Pigments It was identified thanks to vis-RS typical spectrum with 590 nm ab- sorption band, as well as to the characteristic Raman signal at 548 cm−1 [23] (Figure S2f, Supplementary Materials) and, in the IR spectrum, to the strong and wide band around 1015 cm−1 and the medium one at 450 cm−1 [24] (Figure 11c). p y ) , p , g cm−1 and the medium one at 450 cm−1 [24] (Figure 11c). As anticipated above, Prussian blue was recognized in the area NQ2 of “The Daily Neo-Mythology” and in the area M7 of “The Monster”, where it is admixed with phthal- ocyanine blue PB15. In both cases, it could not be identified on the basis of the Raman spectrum, simply since its strongest Raman band was located outside the spectral range accessible with a single acquisition by our equipment On the contrary it was clearly in- As anticipated above, Prussian blue was recognized in the area NQ2 of “The Daily Neo- Mythology” and in the area M7 of “The Monster”, where it is admixed with phthalocyanine blue PB15. In both cases, it could not be identified on the basis of the Raman spectrum, simply since its strongest Raman band was located outside the spectral range accessible with a single acquisition by our equipment. On the contrary, it was clearly individuated by the intense IR signal around 2090 cm−1 (Figure 11a). a e i e i a i g e a qui i io y ou equip e O e o a y, i a ea y i dividuated by the intense IR signal around 2090 cm−1 (Figure 11a). Finally, an interesting case is represented by the red pigment employed in the area M1 of “The Monster”. The most significant Raman band are located at 985 cm−1 and 1085 cm−1 and can be assigned respectively to BaSO4 and CaCO3 as calcite [23], which were most probably extenders of the paint used by the artist. It is interesting to note that in the Raman spectrum of cadmium red reported in ref. 16 the band at 985 cm−1 due to the filler barium sulphate is indeed the strongest one. In parallel, in another work [25] the Raman spectrum Finally, an interesting case is represented by the red pigment employed in the area M1 of “The Monster”. 3.3. Inorganic Pigments 3.3. Inorganic Pigments Traditional inorganic pigments are also represented in the paintings under investi- ti Traditional inorganic pigments are also represented in the paintings under investigation. gation. Titanium white TiO2 as rutile is ubiquitous and its typical Raman bands around 445 and 605 cm−1 [23] are observed in the spectra of white details such as M2 (data not shown) and also of several examined areas having different colours (see for example Figure 7a,b,e). The Ti signal was measured by ED-XRF in all the examined points, partly due to the primer, partly to the outer coloured layer (cf. spectra NQ9 and NQ10 in Figure 6), and the rutile 410 nm shoulder (sometimes shifted to 420 nm) was clearly detected by vis-RS Titanium white TiO2 as rutile is ubiquitous and its typical Raman bands around 445 and 605 cm−1 [23] are observed in the spectra of white details such as M2 (data not shown) and also of several examined areas having different colours (see for example Figure 7a,b,e). The Ti signal was measured by ED-XRF in all the examined points, partly due to the primer, partly to the outer coloured layer (cf. spectra NQ9 and NQ10 in Figure 6), and the rutile 410 nm shoulder (sometimes shifted to 420 nm) was clearly detected by vis-RS in many spectra (see Figure 12, MQ27 line). in many spectra (see Figure 12, MQ27 line). Two inorganic blue pigments were also recognised (i.e., ultramarine blue PB29 and Prussian blue PB27) thanks to the vibrational spectra and, very easily, through the reflec- Two inorganic blue pigments were also recognised (i.e., ultramarine blue PB29 and Prussian blue PB27) thanks to the vibrational spectra and, very easily, through the re- flectance spectra in the visible range. p y y g tance spectra in the visible range. Ultramarine blue could be detected in a dark blue detail (NQ11) of the “The Daily Neo-Mythology”. It was identified thanks to vis-RS typical spectrum with 590 nm absorp- tion band, as well as to the characteristic Raman signal at 548 cm−1 [23] (Figure S2f, Sup- plementary Materials) and, in the IR spectrum, to the strong and wide band around 1015 Ultramarine blue could be detected in a dark blue detail (NQ11) of the “The Daily Neo-Mythology”. Supplementary Materials), sh in the literature for the anthra 3.2. “Special Effect” Pigments This is evidenced in Figure 9d by comparison with the fluorescence spectrum of a commercial fluorescent paint whose for- mulation was already demonstrated to contain such dye [22] The higher fluorescence was detected by UVF images and vis-RS spectra in some pink and red-pink areas, as the shoes of the pair of female legs painted on the left side of “The Daily Neo-Mythology” right frieze, and a peculiar red hue of “The Monster”. The same vis-RS spectrum was obtained in all cases, with a relative maximum at 480 nm, a very intense fluorescence peak at 600 nm, and absorption bands at 530 nm (stronger), 630, and 690 nm (Figure 12). Heritage 2022, 5 57 630 Figure 12. (top left) visible light detail and (bottom left) UVF detail of “The Daily Neo-Mythology”; (right) comparison of vis-RS spectra of fluorescent pigments (rhodamine 6G) in different red or pink areas of the two examined paintings. 420 nm intense shoulder in the fuchsia line (MQ27, regarding the shoe illustrated) due to titanium white in the mixture. Figure 12. (top left) visible light detail and (bottom left) UVF detail of “The Daily Neo-Mythology”; (right) comparison of vis-RS spectra of fluorescent pigments (rhodamine 6G) in different red or pink areas of the two examined paintings. 420 nm intense shoulder in the fuchsia line (MQ27, regarding the shoe illustrated) due to titanium white in the mixture. Figure 12. (top left) visible light detail and (bottom left) UVF detail of “The Daily Neo-Mythology”; (right) comparison of vis-RS spectra of fluorescent pigments (rhodamine 6G) in different red or pink areas of the two examined paintings. 420 nm intense shoulder in the fuchsia line (MQ27, regarding the shoe illustrated) due to titanium white in the mixture. Figure 12. (top left) visible light detail and (bottom left) UVF detail of “The Daily Neo-Mythology”; (right) comparison of vis-RS spectra of fluorescent pigments (rhodamine 6G) in different red or pink areas of the two examined paintings. 420 nm intense shoulder in the fuchsia line (MQ27, regarding the shoe illustrated) due to titanium white in the mixture. 3.4. Binders The investigation of binders was entirely based on the use of reflection FTIR spec- troscopy, both in the MIR and in the NIR region. The experimental results pointed to the prevailing use of nitrocellulose paint for “The Monster” and of acrylic paint, at least for the most part, in the case of “The Daily Neo-Mythology”. Nitrocellulose could be identified as a binder in “The Monster” thanks to bands at 1650, 1282, 1074, and 845 cm−1, observed in the transformed reflection spectrum in the MIR range [27–29] (Figure 11d). In “The Daily Neo-Mythology”, the acrylic binder could be recognised in the MIR spectrum based on signals at 1736, 1446, 1382, and 1160 cm−1 [3,27] (Figure 11b,c). Among the examined areas, just in the blue detail NQ2 nitrocellulose could instead be detected (Figure 11a). g Unlike what was observed in the MIR region, the NIR spectra obtained for the two paintings are quite similar and show bands at 4680, 4430, and 4350 cm−1, that can be assigned to an acrylic binder (Figure 11e,f). In particular, in the NIR spectra of details of “The Monster” it is not possible to observe the bands characteristic of the NO2 group of nitrocellulose at 4835 and 5262 cm−1 [28]. Considering the greater penetration depth of NIR compared to MIR radiation [3], it can be supposed that an acrylic primer was used by the artist on the aluminium surface before applying the nitrocellulose paint. 3.3. Inorganic Pigments 3.3. Inorganic Pigments The most significant Raman band are located at 985 cm−1 and 1085 cm−1 and can be assigned respectively to BaSO4 and CaCO3 as calcite [23], which were most probably extenders of the paint used by the artist. It is interesting to note that in the Raman spectrum of cadmium red reported in ref. [16] the band at 985 cm−1 due to the filler barium sulphate is indeed the strongest one. In parallel, in another work [25] the Raman spectrum of the same red pigment could not be obtained upon excitation at 785 nm, the wavelength used in the present study for the in situ measurements. These two facts, and the lack of other significant signals, led us to hypothesize that our Raman data suggested indirectly the use of cadmium red as pigment in the examined area. As reported in ref. [6], such pigment Heritage 2022, 5 58 can be identified by its characteristic NIR emission upon visible excitation. However, in the present work, just the fluorescence response in the visible range was investigated and a weak emission around 610 nm was observed (Figure S3c, Supplementary Materials). The position of the band is coincident with that of the absorption edge of the same area (data not shown), and this fact seems to corroborate the above hypothesis. Indeed, as reported in the literature [26], semiconductor cadmium pigments can also exhibit a weak band edge luminescence at a wavelength close to the transition edge of the reflectance spectrum, besides the stronger one due to the deep trap emission. g p p XRF was not carried out on “The Monster”, but Cd and Se signals were found in a red of “The Daily Neo-Mythology” not listed in Table 1. 4. Conclusions During the 1960s, Silvio Pasotti began to temporarily abandon the canvas support experimenting with Al-alloy panels (Al above 99%), chosen for the possibility to work on a flat and durable surface, as well as for its aesthetic appearance, with differently reflective zones left unpainted. He used both brush and spray, usually avoiding mixing pigments, except for a few cases, to achieve peculiar effects. p p The non-invasive analytical approach adopted in this investigation allowed an in- depth study of the painting technique, including the changes that occurred during painting, and a comprehensive identification of the pigments and binders employed by the Italian pop artist, highlighting a wide choice of materials and some specific limitations of the analytical techniques used. The materials also included recently introduced pigments at the time of making the paintings and special effect coloring substances, such as rhodamine- based fluorescent pigments. Both acrylic and nitrocellulose binders were also recognised. Finally, from the methodological point of view, the synergic and complementary use of large area multispectral imaging techniques and molecular spectroscopic techniques, including the optical fluorescence, although so far little exploited, has proved to be an effective approach to the complex identification of materials in contemporary art, especially as regards the vast range of synthetic organic pigments. Heritage 2022, 5 59 Supplementary Materials: The following are available online at https://www.mdpi.com/article/10 .3390/heritage5010003/s1. Figure S1: Supplementary Raman spectra from areas of “The Monster”; Figure S2: Supplementary Raman spectra from areas of “The Daily Neo-Mythology”; Figure S3: Emission spectra from red areas of “The Monster” and “The Daily Neo-Mythology”; Figure S4: Emission spectra from yellow and green areas of “The Monster” and “The Daily Neo-Mythology”. Author Contributions: Conceptualization, G.P. and S.B.; methodology, S.B. and G.P.; formal analysis, S.B. and M.L.; investigation, S.B., N.C., V.G. and G.P.; resources, S.B. and G.P.; data curation, S.B., M.L., N.C. and G.P.; writing—original draft preparation, S.B., M.L. and G.P.; writing—review and editing, S.B.; visualization, M.L. and G.P.; supervision, S.B. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. Funding: This research received no external funding. Funding: This research received no external funding. Institutional Review Board Statement: Not applicable. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: Not applicable. Data Availability Statement: Not applicable. Conflicts of Interest: The authors declare no conflict of interest. Conflicts of Interest: The authors declare no conflict of interest. References 1. Learner, T. Modern paints: Uncovering the choices. In Modern Paints Uncovered; Learner, T.J.S., Smithen, P., Krueger, J.W., Schilling, M.R., Eds.; The Getty Conservation Institute: Los Angeles, CA, USA, 2007; pp. 3–16. g y g pp 2. Lomax, S.; Learner, T. A review of the classes, structures, and methods of analysis of synthetic organic pigments. J. Am. Inst. Conserv. 2007, 45, 107–125. [CrossRef] Conserv. 2007, 45, 107–125. [CrossRef] 3. Rosi, F.; Daveri, A.; Moretti, P.; Brunetti, B.G.; Miliani, C. Interpretation of mid and near-infrared reflection properties of synthetic polymer paints for the non-invasive assessment of binding media in twentieth-century pictorial artworks. Microchem. J. 2016, 124, 898–908. [CrossRef] 4. Scherrer, N.C.; Zumbuehl, S.; Delavy, F.; Fritsch, A.; Kuehnen, R. 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In Science and Art: The Contemporary Painted Surface; Sgamellotti, A., Brunetti, B.G., Miliani, C., Eds.; The Royal Society of Chemistry: London, UK, 2020; pp. 67–94. y pp 8. Pinna, D.; Galeotti, M.; Mazzeo, R. (Eds.) Scientific Examination for the Investigation of Paintings: A Handbook for Conservators-Restorers; Centro Di: Firenze, Italy, 2009. y Fiorucci, L.; Nicoletti, L.P. (Eds.) Pop ’60 Silvio Pasotti. Intorno al Fregio del Municipio di Segrate; Editoriale Umb 8. (In Italian) 9. Poldi, G.; Fiorucci, L.; Nicoletti, L.P. (Eds.) Pop ’60 Silvio Pasotti. Intorno al Fregio del Municipio di Segra Italy, 2018. (In Italian) y 10. Cavallo, L. Silvio Pasotti. Catalogo Ragionato Della Pittura; Skira: Milano, Italy, 2008. (In Italian) 10. Cavallo, L. Silvio Pasotti. Catalogo Ragionato Della Pittura; Skira: Milano, Italy, 2008. (In Italian) y 11. Defeyt, C.; Vandenabeele, P.; Gilbert, B.; Van Pevenage, J.; Clootse, R.; Strivaya, D. 4. Conclusions Acknowledgments: The Municipality of Segrate (Milano, Italy) and the artist Silvio Pasotti are acknowledged for granting permission to analyse the paintings. Maria Letizia Amadori is thanked for the use of XRF equipment. References Contribution to the identification of α-, β- and ε-copper phthalocyanine blue pigments in modern artists’ paints by X-ray powder diffraction, attenuated total reflectance micro-Fourier transform infrared spectroscopy and micro-Raman spectroscopy. J. Raman Spectrosc. 2012, 43, 1772–1780. [CrossRef] 12. Poldi, G.; Caglio, S. Phthalocyanine identification in paintings by reflectance spectroscopy. A laboratory and in situ study. Opt. Spectrosc. 2013, 114, 1018–1025. [CrossRef] 11. Defeyt, C.; Vandenabeele, P.; Gilbert, B.; Van Pevenage, J.; Clootse, R.; Strivaya, D. 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Coccato, A.; Bersani, D.; Coudray, A.; Sanyova, J.; Moens, L.; Vandenabeele, P. Raman spectroscopy of green minerals and reaction products with an application in Cultural Heritage research. J. Raman Spectrosc. 2016, 47, 1429–1443. [CrossRef] 14 Zieba Palus J ; Was Gubała J An investigation into the use of micro Raman spectroscopy for the analysis of car paints and single p pp g p 14. Zieba-Palus, J.; Was-Gubała, J. An investigation into the use of micro-Raman spectroscopy for the analysis of car paints and single textile fibres. J. Mol. Struct. 2011, 993, 127–133. [CrossRef] 15. Fremout, W.; Saverwyns, S. Identification of synthetic organic pigments: The role of a comprehensive digital Raman spectral library. J. Raman Spectrosc. 2012, 43, 1536–1544. [CrossRef] Heritage 2022, 5 60 16. Lenoir, J. Organic Pigments. In The Chemistry of Synthetic Dyes; Venkataraman, K., Ed.; Academic Press: New York, NY, USA, 1971; Volume 5, p 390 , p 17. Skoog, D.A.; Holler, F.J.; Crouch, S.R. 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Linguagens em múltiplas faces: uma agenda de estudos teóricos e aplicados

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L755 Linguagens em múltiplas faces: uma agenda de estudos teóricos e aplicados / Organizadores Héliton Diego Lau, André Luiz Souza-Silva, Zuleica Aparecida Michalkiewicz. – São Paulo: Pimenta Cultural, 2023. CONSELHO EDITORIAL CIENTÍFICO Doutores e Doutoras Doutores e Doutoras Ary Albuquerque Cavalcanti Junior Universidade Federal de Mato Grosso, Brasil Asterlindo Bandeira de Oliveira Júnior Universidade Federal da Bahia, Brasil Bárbara Amaral da Silva Universidade Federal de Minas Gerais, Brasil Bernadétte Beber Universidade Federal de Santa Catarina, Brasil Bruna Carolina de Lima Siqueira dos Santos Universidade do Vale do Itajaí, Brasil Bruno Rafael Silva Nogueira Barbosa Universidade Federal da Paraíba, Brasil Caio Cesar Portella Santos Instituto Municipal de Ensino Superior de São Manuel, Brasil Carla Wanessa do Amaral Caffagni Universidade de São Paulo, Brasil Carlos Adriano Martins Universidade Cruzeiro do Sul, Brasil Carlos Jordan Lapa Alves Universidade Estadual do Norte Fluminense Darcy Ribeiro, Brasil Caroline Chioquetta Lorenset Universidade Federal de Santa Catarina, Brasil Cássio Michel dos Santos Camargo Universidade Federal do Rio Grande do Sul-Faced, Brasil Christiano Martino Otero Avila Universidade Federal de Pelotas, Brasil Cláudia Samuel Kessler Universidade Federal do Rio Grande do Sul, Brasil Cristiana Barcelos da Silva. Universidade do Estado de Minas Gerais, Brasil Cristiane Silva Fontes Universidade Federal de Minas Gerais, Brasil Daniela Susana Segre Guertzenstein Universidade de São Paulo, Brasil Daniele Cristine Rodrigues Universidade de São Paulo, Brasil Dayse Centurion da Silva Universidade Anhanguera, Brasil Ary Albuquerque Cavalcanti Junior Universidade Federal de Mato Grosso, Brasil Asterlindo Bandeira de Oliveira Júnior Universidade Federal da Bahia, Brasil Bárbara Amaral da Silva Universidade Federal de Minas Gerais, Brasil Bernadétte Beber Universidade Federal de Santa Catarina, Brasil Bruna Carolina de Lima Siqueira dos Santos Universidade do Vale do Itajaí, Brasil Bruno Rafael Silva Nogueira Barbosa Universidade Federal da Paraíba, Brasil Caio Cesar Portella Santos Instituto Municipal de Ensino Superior de São Manuel, Brasil Carla Wanessa do Amaral Caffagni Universidade de São Paulo, Brasil Carlos Adriano Martins Universidade Cruzeiro do Sul, Brasil Carlos Jordan Lapa Alves Universidade Estadual do Norte Fluminense Darcy Ribeiro, Brasil Caroline Chioquetta Lorenset Universidade Federal de Santa Catarina, Brasil Cássio Michel dos Santos Camargo Universidade Federal do Rio Grande do Sul-Faced, Brasil Christiano Martino Otero Avila Universidade Federal de Pelotas, Brasil Cláudia Samuel Kessler Universidade Federal do Rio Grande do Sul, Brasil Cristiana Barcelos da Silva. Doutores e Doutoras Universidade do Estado de Minas Gerais, Brasil Cristiane Silva Fontes Universidade Federal de Minas Gerais, Brasil Daniela Susana Segre Guertzenstein Universidade de São Paulo, Brasil Daniele Cristine Rodrigues Universidade de São Paulo, Brasil Dayse Centurion da Silva Universidade Anhanguera, Brasil Adilson Cristiano Habowski Universidade La Salle, Brasil Adriana Flávia Neu Universidade Federal de Santa Maria, Brasil Adriana Regina Vettorazzi Schmitt Instituto Federal de Santa Catarina, Brasil Aguimario Pimentel Silva Instituto Federal de Alagoas, Brasil Alaim Passos Bispo Universidade Federal do Rio Grande do Norte, Brasil Alaim Souza Neto universidade Federal de Santa Catarina, Brasil Alessandra Knoll Universidade Federal de Santa Catarina, Brasil Alessandra Regina Müller Germani Universidade Federal de Santa Maria, Brasil Aline Corso Universidade do Vale do Rio dos Sinos, Brasil Aline Wendpap Nunes de Siqueira Universidade Federal de Mato Grosso, Brasil Ana Rosangela Colares Lavand Universidade Federal do Pará, Brasil André Gobbo Universidade Federal da Paraíba, Brasil Andressa Wiebusch Universidade Federal de Santa Maria, Brasil Andreza Regina Lopes da Silva Universidade Federal de Santa Catarina, Brasil Angela Maria Farah Universidade de São Paulo, Brasil Anísio Batista Pereira Universidade Federal de Uberlândia, Brasil Antonio Edson Alves da Silva Universidade Estadual do Ceará, Brasil Antonio Henrique Coutelo de Moraes Universidade Federal de Rondonópolis, Brasil Arthur Vianna Ferreira UniversidadedoEstadodoRiodeJaneiro Brasil Adilson Cristiano Habowski Universidade La Salle, Brasil Adriana Flávia Neu Universidade Federal de Santa Maria, Brasil Adriana Regina Vettorazzi Schmitt Instituto Federal de Santa Catarina, Brasil Aguimario Pimentel Silva Instituto Federal de Alagoas, Brasil Alaim Passos Bispo Universidade Federal do Rio Grande do Norte, Brasil Alaim Souza Neto universidade Federal de Santa Catarina, Brasil Alessandra Knoll Universidade Federal de Santa Catarina, Brasil Alessandra Regina Müller Germani Universidade Federal de Santa Maria, Brasil Aline Corso Universidade do Vale do Rio dos Sinos, Brasil Aline Wendpap Nunes de Siqueira Universidade Federal de Mato Grosso, Brasil Ana Rosangela Colares Lavand Universidade Federal do Pará, Brasil André Gobbo Universidade Federal da Paraíba, Brasil Andressa Wiebusch Universidade Federal de Santa Maria, Brasil Andreza Regina Lopes da Silva Universidade Federal de Santa Catarina, Brasil Angela Maria Farah Universidade de São Paulo, Brasil Anísio Batista Pereira Universidade Federal de Uberlândia, Brasil Antonio Edson Alves da Silva Universidade Estadual do Ceará, Brasil Antonio Henrique Coutelo de Moraes Universidade Federal de Rondonópolis, Brasil Arthur Vianna Ferreira Universidade do Estado do Rio de Janeiro, Brasil Humberto Costa Universidade Federal do Paraná, Brasil Igor Alexandre Barcelos Graciano Borges Universidade de Brasília, Brasil Inara Antunes Vieira Willerding Universidade Federal de Santa Catarina, Brasil Ivan Farias Barreto Universidade Federal do Rio Grande do Norte, Brasil Jaziel Vasconcelos Dorneles Universidade de Coimbra, Portugal Jean Carlos Gonçalves Universidade Federal do Paraná, Brasil Jocimara Rodrigues de Sousa Universidade de São Paulo, Brasil Joelson Alves Onofre Universidade Estadual de Santa Cruz, Brasil Jónata Ferreira de Moura Universidade São Francisco, Brasil Jorge Eschriqui Vieira Pinto Universidade Estadual Paulista Júlio de Mesquita Filho, Brasil Jorge Luís de Oliveira Pinto Filho Universidade Federal do Rio Grande do Norte, Brasil Juliana de Oliveira Vicentini Universidade de São Paulo, Brasil Julierme Sebastião Morais Souza Universidade Federal de Uberlândia, Brasil Junior César Ferreira de Castro Universidade de Brasília, Brasil Katia Bruginski Mulik Universidade de São Paulo, Brasil Laionel Vieira da Silva Universidade Federal da Paraíba, Brasil Leonardo Pinheiro Mozdzenski Universidade Federal de Pernambuco, Brasil Lucila Romano Tragtenberg Pontifícia Universidade Católica de São Paulo, Brasil Lucimara Rett Universidade Metodista de São Paulo, Brasil Manoel Augusto Polastreli Barbosa Universidade Federal do Espírito Santo, Brasil Marcelo Nicomedes dos Reis Silva Filho Universidade Estadual do Oeste do Paraná, Brasil Marcio Bernardino Sirino Universidade Federal do Estado do Rio de Janeiro, Brasil Dayse Sampaio Lopes Borges Universidade Estadual do Norte Fluminense Darcy Ribeiro, Brasil Diego Pizarro Instituto Federal de Brasília, Brasil Dorama de Miranda Carvalho Escola Superior de Propaganda e Marketing, Brasil Edson da Silva Universidade Federal dos Vales do Jequitinhonha e Mucuri, Brasil Elena Maria Mallmann Universidade Federal de Santa Maria, Brasil Eleonora das Neves Simões Universidade Federal do Rio Grande do Sul, Brasil Eliane Silva Souza Universidade do Estado da Bahia, Brasil Elvira Rodrigues de Santana Universidade Federal da Bahia, Brasil Éverly Pegoraro Universidade Federal do Rio de Janeiro, Brasil Fábio Santos de Andrade Universidade Federal de Mato Grosso, Brasil Fabrícia Lopes Pinheiro Universidade Federal do Estado do Rio de Janeiro, Brasil Felipe Henrique Monteiro Oliveira Universidade Federal da Bahia, Brasil Fernando Vieira da Cruz Universidade Estadual de Campinas, Brasil Gabriella Eldereti Machado Universidade Federal de Santa Maria, Brasil Germano Ehlert Pollnow Universidade Federal de Pelotas, Brasil Geymeesson Brito da Silva Universidade Federal de Pernambuco, Brasil Giovanna Ofretorio de Oliveira Martin Franchi Universidade Federal de Santa Catarina, Brasil Handherson Leyltton Costa Damasceno Universidade Federal da Bahia, Brasil Hebert Elias Lobo Sosa Universidad de Los Andes, Venezuela Helciclever Barros da Silva Sales Instituto Nacional de Estudos e Pesquisas Educacionais Anísio Teixeira, Brasil Helena Azevedo Paulo de Almeida Universidade Federal de Ouro Preto, Brasil Hendy Barbosa Santos Faculdade de Artes do Paraná, Brasil Jocimara Rodrigues de Sousa Universidade de São Paulo, Brasil Joelson Alves Onofre Universidade Estadual de Santa Cruz, Brasil Jónata Ferreira de Moura Universidade São Francisco, Brasil Jorge Eschriqui Vieira Pinto Universidade Estadual Paulista Júlio de Mesquita Filho, Brasil Marcos Pereira dos Santos Universidad Internacional Iberoamericana del Mexico, México Marcos Uzel Pereira da Silva Universidade Federal da Bahia, Brasil Maria Aparecida da Silva Santandel Universidade Federal de Mato Grosso do Sul, Brasil Maria Cristina Giorgi Centro Federal de Educação Tecnológica Celso Suckow da Fonseca, Brasil Maria Edith Maroca de Avelar Universidade Federal de Ouro Preto, Brasil Marina Bezerra da Silva Instituto Federal do Piauí, Brasil Michele Marcelo Silva Bortolai Universidade de São Paulo, Brasil Mônica Tavares Orsini Universidade Federal do Rio de Janeiro, Brasil Nara Oliveira Salles Universidade do Estado do Rio de Janeiro, Brasil Neli Maria Mengalli Pontifícia Universidade Católica de São Paulo, Brasil Patricia Bieging Universidade de São Paulo, Brasil Patricia Flavia Mota Universidade do Estado do Rio de Janeiro, Brasil Raul Inácio Busarello Universidade Federal de Santa Catarina, Brasil Raymundo Carlos Machado Ferreira Filho Universidade Federal do Rio Grande do Sul, Brasil Roberta Rodrigues Ponciano Universidade Federal de Uberlândia, Brasil Robson Teles Gomes Universidade Federal da Paraíba, Brasil Rodiney Marcelo Braga dos Santos Universidade Federal de Roraima, Brasil Rodrigo Amancio de Assis Universidade Federal de Mato Grosso, Brasil Rodrigo Sarruge Molina Universidade Federal do Espírito Santo, Brasil Rogério Rauber Universidade Estadual Paulista Júlio de Mesquita Filho, Brasil Rosane de Fatima Antunes Obregon Universidade Federal do Maranhão, Brasil Samuel André Pompeo Universidade Estadual Paulista Júlio de Mesquita Filho, Brasil Sebastião Silva Soares Universidade Federal do Tocantins, Brasil Silmar José Spinardi Franchi Universidade Federal de Santa Catarina, Brasil Simone Alves de Carvalho Universidade de São Paulo, Brasil Simoni Urnau Bonfiglio Universidade Federal da Paraíba, Brasil Stela Maris Vaucher Farias Universidade Federal do Rio Grande do Sul, Brasil Tadeu João Ribeiro Baptista Universidade Federal do Rio Grande do Norte Taiane Aparecida Ribeiro Nepomoceno Universidade Estadual do Oeste do Paraná, Brasil Taíza da Silva Gama Universidade de São Paulo, Brasil Tania Micheline Miorando Universidade Federal de Santa Maria, Brasil Tarcísio Vanzin Universidade Federal de Santa Catarina, Brasil Tascieli Feltrin Universidade Federal de Santa Maria, Brasil Tayson Ribeiro Teles Universidade Federal do Acre, Brasil Thiago Barbosa Soares Universidade Federal do Tocantins, Brasil Thiago Camargo Iwamoto Pontifícia Universidade Católica de Goiás, Brasil Thiago Medeiros Barros Universidade Federal do Rio Grande do Norte, Brasil Tiago Mendes de Oliveira Centro Federal de Educação Tecnológica de Minas Gerais, Brasil Vanessa Elisabete Raue Rodrigues Universidade Estadual de Ponta Grossa, Brasil Vania Ribas Ulbricht Universidade Federal de Santa Catarina, Brasil Wellington Furtado Ramos Universidade Federal de Mato Grosso do Sul, Brasil Wellton da Silva de Fatima Instituto Federal de Alagoas, Brasil Yan Masetto Nicolai Universidade Federal de São Carlos, Brasil Marina Bezerra da Silva Instituto Federal do Piauí, Brasil Michele Marcelo Silva Bortolai Universidade de São Paulo, Brasil Mônica Tavares Orsini Universidade Federal do Rio de Janeiro, Brasil Nara Oliveira Salles Universidade do Estado do Rio de Janeiro, Brasil Neli Maria Mengalli Pontifícia Universidade Católica de São Paulo, Brasil Patricia Bieging Universidade de São Paulo, Brasil Patricia Flavia Mota Universidade do Estado do Rio de Janeiro, Brasil Raul Inácio Busarello Universidade Federal de Santa Catarina, Brasil Avaliadores e avaliadoras Ad-Hoc Alessandra Figueiró Thornton Universidade Luterana do Brasil, Brasil Alexandre João Appio Universidade do Vale do Rio dos Sinos, Brasil Bianka de Abreu Severo Universidade Federal de Santa Maria, Brasil Carlos Eduardo Damian Leite Universidade de São Paulo, Brasil Catarina Prestes de Carvalho Instituto Federal Sul-Rio-Grandense, Brasil Elisiene Borges Leal Universidade Federal do Piauí, Brasil Elizabete de Paula Pacheco Universidade Federal de Uberlândia, Brasil Elton Simomukay Universidade Estadual de Ponta Grossa, Brasil Francisco Geová Goveia Silva Júnior Universidade Potiguar, Brasil Indiamaris Pereira Universidade do Vale do Itajaí, Brasil Jacqueline de Castro Rimá Universidade Federal da Paraíba, Brasil Lucimar Romeu Fernandes Instituto Politécnico de Bragança, Brasil Marcos de Souza Machado Universidade Federal da Bahia, Brasil Michele de Oliveira Sampaio Universidade Federal do Espírito Santo, Brasil Pedro Augusto Paula do Carmo Universidade Paulista, Brasil Samara Castro da Silva Universidade de Caxias do Sul, Brasil Thais Karina Souza do Nascimento Instituto de Ciências das Artes, Brasil Viviane Gil da Silva Oliveira Universidade Federal do Amazonas, Brasil Weyber Rodrigues de Souza Pontifícia Universidade Católica de Goiás, Brasil William Roslindo Paranhos Universidade Federal de Santa Catarina, Brasil Jacqueline de Castro Rimá Universidade Federal da Paraíba, Brasil Lucimar Romeu Fernandes Instituto Politécnico de Bragança, Brasil Marcos de Souza Machado Universidade Federal da Bahia, Brasil Michele de Oliveira Sampaio Universidade Federal do Espírito Santo, Brasil Pedro Augusto Paula do Carmo Universidade Paulista, Brasil Samara Castro da Silva Universidade de Caxias do Sul, Brasil Thais Karina Souza do Nascimento Instituto de Ciências das Artes, Brasil Viviane Gil da Silva Oliveira Universidade Federal do Amazonas, Brasil Weyber Rodrigues de Souza Pontifícia Universidade Católica de Goiás, Brasil William Roslindo Paranhos Universidade Federal de Santa Catarina, Brasil Alessandra Figueiró Thornton Universidade Luterana do Brasil, Brasil Alexandre João Appio Universidade do Vale do Rio dos Sinos, Brasil Bianka de Abreu Severo Universidade Federal de Santa Maria, Brasil Carlos Eduardo Damian Leite Universidade de São Paulo, Brasil Catarina Prestes de Carvalho Instituto Federal Sul-Rio-Grandense, Brasil Elisiene Borges Leal Universidade Federal do Piauí, Brasil Elizabete de Paula Pacheco Universidade Federal de Uberlândia, Brasil Elton Simomukay Universidade Estadual de Ponta Grossa, Brasil Francisco Geová Goveia Silva Júnior Universidade Potiguar, Brasil Indiamaris Pereira Universidade do Vale do Itajaí, Brasil Jacqueline de Castro Rimá Universidade Federal da Paraíba, Brasil Parecer e revisão por pares Parecer e revisão por pares Parecer e revisão por pares Os textos que compõem esta obra foram submetidos para avaliação do Conselho Editorial da Pimenta Cultural, bem como revisados por pares, sendo indicados para a publicação. Todas as questões que envolvem o uso da língua não são apenas questões linguísticas; são tam- bém questões políticas, históricas, sociais e culturais. Irandé Antunes SUMÁRIO Prefácio........................................................................................................14 Uma introdução não basta diante das mistas faces da língua(gem)............................................17 CAPÍTULO 1 Héliton Diego Lau Bichês como língua(gem) identitária da comunidade LGBTQIA+: uma análise orientada pela Linguística Aplicada....................................................28 CAPÍTULO 2 André Luiz Souza-Silva Por uma Sociolinguística militante na escola: contribuições para uma prática linguística anti-hegemônica...................................46 CAPÍTULO 3 Zuleica A. Michalkiewicz Retomando a questão da língua portuguesa no ensino: um debate infindável..........................................................................................70 CAPÍTULO 4 Venan Alencar Mábia Camargo Linguística Queer: sutilezas, complexidades, monstruosidades...........................................................94 CAPÍTULO 5 Tamires Tolomeotti Pereira Jamil Cabral Sierra Atos de fala em Austin e Butler: o performativo nos discursos de ódio neoconservadores sobre gênero e sexualidade.....................................................112 Prefácio........................................................................................................14 .14 Uma introdução não basta diante das mistas faces da língua(gem)............................................17 CAPÍTULO 1 Héliton Diego Lau Bichês como língua(gem) identitária da comunidade LGBTQIA+: uma análise orientada pela Linguística Aplicada....................................................28 CAPÍTULO 2 André Luiz Souza-Silva Por uma Sociolinguística militante na escola: contribuições para uma prática linguística anti-hegemônica...................................46 CAPÍTULO 3 Zuleica A. Michalkiewicz Retomando a questão da língua portuguesa no ensino: um debate infindável..........................................................................................70 Uma introdução não basta diante das mistas faces da língua(gem)............................................17 CAPÍTULO 1 Héliton Diego Lau Bichês como língua(gem) identitária da comunidade LGBTQIA+: uma análise orientada pela Linguística Aplicada....................................................28 CAPÍTULO 2 André Luiz Souza-Silva Por uma Sociolinguística militante na escola: contribuições para uma prática linguística anti-hegemônica...................................46 CAPÍTULO 3 Zuleica A. Michalkiewicz Retomando a questão da língua portuguesa no ensino: um debate infindável..........................................................................................70 CAPÍTULO 4 Venan Alencar Mábia Camargo Linguística Queer: sutilezas, complexidades, monstruosidades...........................................................94 CAPÍTULO 5 Tamires Tolomeotti Pereira Jamil Cabral Sierra Atos de fala em Austin e Butler: o performativo nos discursos de ódio neoconservadores sobre gênero e sexualidade....................................................112 .70 .94 CAPÍTULO 5 CAPÍTULO 6 Lucas Possatti Atitudes linguísticas e acomodação dialetal de falantes cariocas em João Pessoa........................... 132 CAPÍTULO 6 Lucas Possatti Atitudes linguísticas e acomodação dialetal de falantes cariocas em João Pessoa........................... 132 CAPÍTULO 6 Lucas Possatti Atitudes linguísticas e acomodação dialetal de falantes cariocas em João Pessoa........................... 132 CAPÍTULO 7 Ronna Freitas de Oliveira Silvely Brandes Quando Freire e a educação de línguas dialogam: algumas aproximações entre ensino de línguas e perspectivas freireanas............... 152 CAPÍTULO 8 Jaqueline Ângelo dos Santos Denardin Caroline Soder Keila Gentil Neves de Lima Susana de Freitas Vaz A importância das práticas de ensino desenvolvidas pelo intérprete de Libras e da compreensão do surdo em relação a aprendizagem da Língua Portuguesa......................................165 CAPÍTULO 9 Denise Gabriel de Oliveira Eliziane Manosso Streiechen Cristiane Malinoski Pianaro Angelo O processo de aprendizagem da Libras como L2 na perspectiva de professores em formação......................................................... 179 CAPÍTULO 10 Joyce da Silva Cruz de Mendonça Abrindo horizontes inclusivos: o ensino da língua brasileira de sinais como L2 para crianças ouvintes de uma escola pública do interior da Paraíba............................ 202 Jaqueline Ângelo dos Santos Denardin Caroline Soder Keila Gentil Neves de Lima Susana de Freitas Vaz CAPÍTULO 9 CAPÍTULO 11 Danielle dos Santos Mendes Coppi A argumentação para além da redação do ENEM: uma breve reflexão sobre a produção do gênero cartaz no ensino fundamental........................................................... 222 CAPÍTULO 12 Juliana Ferreira Benke Karina Pacheco dos Santos Vander Broock Caroline Kretzmann Sequência didática para leitura e produção de texto dissertativo-argumentativo.................... 239 CAPÍTULO 13 Gabriel Fernandes de Oliveira A escrita significativa num projeto de letramento em contexto de ensino remoto.......................... 262 CAPÍTULO 14 Merylin Ricieli dos Santos Pré-livros enquanto vetores para a construção de uma sociedade antirracista.............................281 CAPÍTULO 15 Maísa Cardoso Mas afinal, o que é ler? Algumas concepções e provocações sobre a prática da leitura e a formação do leitor..................................... 305 CAPÍTULO 16 José Cristovão Maia Lucena Marreiro Leonardo Carvalho de Oliveira Histórias em quadrinhos como incentivo à leitura: uma revisão bibliográfica................................................................................. 326 CAPÍTULO 11 CAPÍTULO 11 Danielle dos Santos Mendes Coppi . 222 CAPÍTULO 12 Leonardo Kominek Barrentin Héliton Diego Lau Resistência trans em uma perspectiva discursiva: Resistência trans em uma perspectiva discursiva: a contrarreligião de Urias em Diaba................................................................... 342 CAPÍTULO 18 Larissa da Silva Duarte Letícia Petel de Sousa Héliton Diego Lau O silênci(ament)o em “Apesar de Você”, de Chico Buarque: da censura à esperança................................................................................... 358 CAPÍTULO 19 Samuel Barbosa Silva O discurso sobre a educação moral do movimento escola sem partido e a oposição às discussões de gênero/sexualidades.................................................................377 CAPÍTULO 20 Maria Vanessa Monteiro das Chagas A leitura literária em sala de aula de língua portuguesa: reflexões sobre o espaço da autoria feminina no livro didático do ensino fundamental............................................................ 406 CAPÍTULO 21 Samuel Filipe Guedes do Nascimento Manassés Morais Xavier José Walbérico da Silva WCosta Construções sintáticas adjetivas como enunciados concretos no gênero artigo de opinião: uma proposta de análise.................................................................................. 429 Residência pós-doutoral no Programa de Pós-Graduação em Direito da Universidade Federal de Minas Gerais, na linha “História, Poder e Liberdade” (2023-). Doutorado em Sociologia pelo Pro- grama de Pós-Graduação em Sociologia da Universidade Federal da Paraíba (2020). Mestrado em Ciências Jurídicas pela Universidade Federal da Paraíba (2014). Líder do Grupo de Pesquisa e Estudos sobre Desvio e Controle Social (GEDECON). Integra a “Road Initiative” - Rede Internacional de Pesquisas e Observatório acerca de Desaparecimento Forçado. Pesquisador das áreas de Crim- inologia, Estudos de Gênero e Sexualidade e Direito Antidiscriminatório. PREFÁCIO S U M Á R I O CAPÍTULO 21 429 CAPÍTULO 22 André Luiz Souza-Silva Klecio de Assis Raimundo “Monstros à solta” na prática de análise linguística: proposta de ensino à luz de fenômenos de gramaticalização................................ 449 CAPÍTULO 23 Natássia Thais do Nascimento Ribeiro Variação e mudança linguísticas em gramáticas escolares de língua portuguesa da década de 2000....................................................475 CAPÍTULO 24 Emily Gonçalves de Medeiros Ferreira As Regras da lingua portugueza, espelho da lingua latina, de Jerónimo Contador de Argote (1725), e a gramatização da colocação pronominal: apontamentos historiográficos.......................................................................... 494 CAPÍTULO 25 Willian Ferreira Furtado de Lacerda A realização do fonema lateral /l/ em inglês como L2 por falantes brasileiros......................................515 CAPÍTULO 26 Marina Chiara Legroski A mentira como ato de fala: uma proposta de análise dentro da teoria de Searle............................................. 536 Sobre a organização............................................................................. 560 Sobre as autoras e os autores........................................................... 562 CAPÍTULO 22 André Luiz Souza-Silva Klecio de Assis Raimundo “Monstros à solta” na prática de análise linguística: proposta de ensino à luz de fenômenos de gramaticalização................................ 449 CAPÍTULO 23 Natássia Thais do Nascimento Ribeiro Variação e mudança linguísticas em gramáticas escolares de língua portuguesa da década de 2000....................................................475 CAPÍTULO 24 Emily Gonçalves de Medeiros Ferreira As Regras da lingua portugueza, espelho da lingua latina, de Jerónimo Contador de Argote (1725), e a gramatização da colocação pronominal: apontamentos historiográficos.......................................................................... 494 CAPÍTULO 25 Willian Ferreira Furtado de Lacerda A realização do fonema lateral /l/ em inglês como L2 por falantes brasileiros......................................515 CAPÍTULO 26 Marina Chiara Legroski A mentira como ato de fala: uma proposta de análise dentro da teoria de Searle............................................. 536 Sobre a organização............................................................................. 560 Sobre as autoras e os autores........................................................... 562 Índice remissivo......................................................................................572 449 Emerson Erivan de Araújo Ramos1 Quando recebi o convite generoso de Héliton Diego Lau, André Souza-Silva e Zuleica Michalkiewicz para escrever o prefácio deste livro, pensei que estivessem superestimando minha capaci- dade de transitar entre as áreas das ciências humanas. Malgrado eu perceba que os limites entre os campos científicos estejam cada vez mais porosos (permitindo o rico intercruzamento de ferramentas analíticas), não tenho produções diretamente ligadas à Linguística. Afirmo isso porque, como Pierre Bourdieu (1996, 1998) deci- frou de maneira densa, um campo do saber (como subproduto do campo intelectual) não é formado exclusivamente pelo patrimônio das formulações mentais que aparecem em livros, artigos científicos, conferências, orientações acadêmicas e outras atividades intelectu- ais mais ou menos cotidianas. Mais que isso, um campo do saber é um espaço social específico que possui também uma dimensão prá- tica de legitimação do saber, envolvendo uma complexa agonística entre os agentes do campo e regras internas a serem vencidas para que um saber seja legitimado. De forma mais clara, qualquer campo do saber (o que inclui a Linguística) é formado por um conjunto finito de informações e produções que é localizado como pertencente a um campo específico do saber não só devido a suas propriedades ínsitas, mas a partir também de decisões políticas de agentes do 14 S U M Á R I O campo que decidem sobre quais produções podem ou não ser con- sideradas como pertencentes a uma disciplina. Essas decisões são tomadas em práticas como as ementas das disciplinas, as decisões editoriais de revistas conceituadas, os financiamentos públicos e pri- vados de pesquisa, as seleções nos programas de pós-graduação etc.; os quais, em conjunto e mediante reiteração, definem os limites e os conteúdos de uma determinada área do saber. Esse caráter prático da formação das agendas de estudo de um campo do saber normalmente é apagado, de modo que são pri- vilegiadas as discussões em torno da propriedade (e qualidade) das formulações mentais desse campo, em detrimento da autorreflexão sobre os critérios político-sociais de legitimação e classificação de um saber. Cito um exemplo: faz parte das leituras incontornáveis dos estudantes de Ciência Política os textos de Max Weber, a fim de discutir conceitos essenciais para a formação na área, como os conceito de “burocratização” e “dominação”. Emerson Erivan de Araújo Ramos1 É relativamente raro, contudo, que esses mesmos estudantes sejam estimulados a realiza- rem leituras sobre formas específicas de dominação (que igualmente envolvem teorizações sobre o poder), como produções feministas sobre o patriarcado, textos de teoria crítica da raça ou ainda obras importantes sobre cisheterossexismo. O que faz com que as produ- ções de Max Weber sobre dominação sejam tidas como um objeto por excelência da Ciência Política, mas não sejam assim também compreendidos os estudos de capacidades, raça, gênero e sexuali- dade? A resposta pode ser encontrada no espaço social que estabe- lece as regras próprias desse campo do saber. Quando fui generosamente convidado pelos coorganizado- res para realizar o prefácio de Linguagens em múltiplas faces: uma agenda de estudos teóricos e aplicados, aceitei porque o convite veio acompanhado de uma postura epistemológica a qual me afilio: a defesa da inter ou transdisciplinaridade, que é tanto uma forma de produção do saber quanto uma estratégia política de combate à violência simbólica deveras corriqueira nas regras de legitimação 15 internas ao campo intelectual. Desenrijecer as fronteiras disciplina- res é uma atitude democrática que diversifica as agendas de deba- tes, o que pode ser encontrado nesta obra. S U M Á R I O Trata-se de uma coletânea em que é latente o esforço con- junto (presente já desde o título) de diversificar a agenda de deba- tes da Linguística, transitando desde um “núcleo duro” dos estudos sobre a linguagem até produções que entendem a língua como objeto privilegiado das lutas por reconhecimento. O efeito direto desse esforço é a presença maciça de textos que problematizam a língua como expressão e parte constitutiva das estratégias de domi- nação, tomada nesta obra como elemento de disputa política no seio de uma sociedade assimétrica, motivo pelo qual são aqui especial- mente abordados temas como gênero, sexualidade, raça, capacida- des, colonialidade etc. Por fim, acredito que a presente obra contribui para atu- alizar e expandir a agenda de debates da Linguística, fornecendo um panorama contemporâneo e democratizante dos estudos sobre linguagem – útil também para pensar os problemas próprios de outros campos do saber. REFERÊNCIAS BOURDIEU, Pierre. As regras da arte: Gênese e estrutura do campo literário. São Paulo: Companhia das Letras, 1996. BOURDIEU, Pierre. O poder simbólico. Rio de Janeiro: Bertrand Brasil, 1998. BOURDIEU, Pierre. O poder simbólico. Rio de Janeiro: Bertrand Brasil, 1998. 16 UMA INTRODUÇÃO NÃO BASTA DIANTE DAS MISTAS FACES DA LÍNGUA(GEM) A respeito do fazer científico na área de linguagem, Borges Neto (2006), em uma entrevista, na qual lhe questionam sobre os desafios para a linguística do século 21, ele afirma ser o desafio da tolerância crítica. Para além disso, declara o seguinte: Não tem ninguém burro fazendo linguística, não tem nenhum idiota envolvido com funcionalismo ou com for- malismo, e se alguém acha interessante trabalhar com determinado assunto, a gente tem que respeitar o quanto possível, ler e entender, compreender o ponto de vista do outro (BORGES NETO, 2006, p. 50). Nesse sentido, não se trata apenas de adjetivar se o que estamos apresentando aqui é de caráter formalista e/ou funciona- lista, mas de pensar em sua relevância para a compreensão sobre a linguagem, não só porque algum fenômeno nos interessa particular- mente, mas porque agrega conhecimento para nós e para todos que buscarem ler os textos que compõem esta coletânea de trabalhos acadêmicos. É preciso sempre lembrarmos que a linguagem é um fenômeno de mistas faces e não precisamos entrar no ringue das hiperteorias que consolidem ou ascendam a linguística como ciên- cia, pois a linguagem não cabe nas gavetas, não se limita às poltro- nas de escritório ou é manipulável por meio de tubos em laboratório, porque a linguagem está fora disso tudo e ao mesmo tempo ema- ranhada em tudo isso. Portanto, faz-se importante não se ter uma visão, exclusivamente, utilitarista e/ou material da ciência. 17 Entendendo os estudos da linguagem como um campo inter/transdisciplinar, temos uma agenda bastante ampla acerca de estudos teóricos e aplicados no campo do que podemos chamar de Linguísticas, no intento de indicar como temos um horizonte amplo e múltiplo nessa produção de conhecimento. Nesse sentido, da Lin- guística Teórica à Linguística Aplicada, o objeto de estudo é sempre a língua(gem) em que se transgride fronteiras disciplinares conven- cionais, objetivando desenvolver novas agendas de pesquisa enca- beçadas por uma ampla variedade de disciplinas que nada têm de subalternas, mas de poderosos caminhos para a ação. Estas estão em célebres discussões subsidiando uma a outra para estabelecer diálogos profícuos nos diversos campos de investigação, trazendo consigo o caminho intercambiável em diferentes áreas do conheci- mento, de soluções para problemas linguísticos socialmente relevan- tes e de construções conceituais conjuntas. Nessa direção, apresentamos o livro Linguagem em múltiplas faces: uma agenda de estudos teóricos e aplicados. UMA INTRODUÇÃO NÃO BASTA DIANTE DAS MISTAS FACES DA LÍNGUA(GEM) De início, temos o capítulo de Héliton Diego Lau, no qual se discute, à luz da Linguística Aplicada, como sujeitos LGBTQIA+ fazem uso da língua(gem) em um movimento identitário. Para tanto, o autor desenvolve a análise de um episódio do podcast “Não me Critica”, no qual homossexuais masculinos opinam sobre vocábulos encontra- dos num dicionário, bem como sobre o uso social da língua, variações de prestígio, grau de escolaridade, etc. Por fim, Lau argumenta sobre a língua(gem) da referida comunidade ser evitada e somente falada em tom de riso, por exemplo, com a intenção de “imitar”, de forma “debochada” e estereotipada, o modo como gays se expressam. André Luiz Souza-Silva discute em seu capítulo sobre as contribuições da Sociolinguística para o ensino de língua no intento de uma conduta militante na escola. Para tal, ressalta o papel polí- tico do ensino de língua(gem) mediante documentos oficiais. Seu corpus se constitui a partir do recorte de uma análise de entrevista 18 S U M Á R I O semiestruturada acerca das atitudes linguísticas de sujeitos da comunidade LGBTQIA+ sobre o seu modo de falar, bem como pro- blematiza um fenômeno linguístico em que seu locus consiste nas identidades sexuais e de gênero para análise em sala de aula. Para o autor, a Sociolinguística defendida por ele engloba ideias variacionis- tas, interacionais, discursivas e educacionais. A seguir, temos o capítulo de Zuleica A. Michalkiewicz contex- tualizando políticas linguísticas. Nesse sentido, a autora apresenta um panorama breve das políticas educacionais que direcionam as políticas de línguas no Brasil. Para isso, Michalkiewicz discute, a par- tir da reforma pombalina e da política nacionalista da Era Vargas, sobre a compreensão de que a percepção acerca da língua se modi- fica entre os temas sociais e linguísticos. Logo, as ideologias “socio- linguísticas” atuam de maneira dialógica e relacionam os aspectos sociais às hierarquias de poder. Venan Alencar e Mábia Camargo realizam um percurso teó- rico sobre a Linguística Queer, levantando questões sobre identi- dade, subversão, performance e assujeitamento. Discorrem ainda a respeito das sutilezas, complexidades e monstruosidades que podem ser compreendidas na Teoria Queer devido à ação e reprodu- ção considerada normal nas pesquisas acadêmicas.Diante disso, os autores questionam como praticar pesquisas no presente e no futuro e suas implicações políticas por meio de mobilizações reacionárias. . UMA INTRODUÇÃO NÃO BASTA DIANTE DAS MISTAS FACES DA LÍNGUA(GEM) Tamires Tolomeotti Pereira e Jamil Cabral Sierra discutem acerca da performatividade nos discursos de ódio neoconserva- dores sobre gênero e sexualidade. Apoiadas em Austin e Butler, as autoras entendem os discursos de ódio como performativos e assim não é possível se ater apenas ao caráter supostamente descritivo, representativo e essencialista da linguagem. Dessa forma, defendem que os discursos são atribuições de caráter social e reiterativo para além das enunciações linguísticas e das ações individuais, logo são modos corpóreos e plurais de exercício da política. 19 Adiante, há a produção de Lucas Possatti sobre atitudes lin- guísticas e acomodação dialetal de falantes cariocas da cidade de João Pessoa, na Paraíba, isso com o objetivo de dialogar sobre as atitudes linguísticas e seu papel na acomodação dialetal, também chamando a atenção para a importância de uma leitura detalhada a partir de uma análise qualitativa que busque observar os dados de modo individual e comparativamente. A partir desses objetivos, Possatti apresenta como as atitudes, empoçadas em crenças, este- reótipos e preconceitos, auxiliam no julgamento dos diversos falares, interferindo na acomodação dialetal. Ronna Freitas de Oliveira e Silvely Brandes apresentam a aproximação entre os estudos de Paulo Freire e o ensino de línguas. As autoras destacam que o colonialismo que opera nas universidade afastou o teórico dos estudos no Brasil, logo os estudos decoloniais requerem o suleamento da pesquisa, sobretudo sobre as teorizações de Paulo Freire. A partir da Pedagogia do oprimido, Oliveira e Bran- des destacam que o sonho do oprimido é se tornar opressor e, nesse pensamento, Freire traz a pedagogia da libertação a fim de superar essa contradição. Além disso, o pensamento de Freire coaduna com os estudos do círculo de Bakhtin que, embora a linguagem seja uma arena de disputa de poder é também por meio dela que se efetiva a prática reflexiva, a ressignificação da visão de mundo, o olhar para o outro e para si trazendo novos sentidos para agir na atuação polí- tica em sala de aula. Jaqueline Ângelo dos Santos Denardin, Caroline Soder, Keila Gentil Neves de Lima e Susana de Freitas Vaz trazem uma importante reflexão acerca do trabalho do intérprete de língua de sinais para aprender a Língua Portuguesa. Para este capítulo, as autoras obje- tivam compreender como os intérpretes têm desenvolvido práticas educativas que facilitem a compreensão de alunos surdos no desen- volvimento e aprendizagem da Língua Portuguesa. UMA INTRODUÇÃO NÃO BASTA DIANTE DAS MISTAS FACES DA LÍNGUA(GEM) 20 A seguir, temos uma investigação sobre a aquisição/apren- dizagem da Libras como L2 para o adulto, considerando o contexto nos cursos de licenciatura em História, Pedagogia e Letras (Portu- guês, Inglês e Espanhol). Denise Gabriel de Oliveira, Eliziane Manosso Streiechen e Cristiane Malinoski Pianaro Angelo, neste capítulo, bus- cam conhecer as experiências dos acadêmicos dos cursos mencio- nados sobre a aquisição/aprendizagem da Libras como L2. As auto- ras versam o incentivo dos ouvintes a aprender a Libras para reduzir as barreiras de comunicação que os surdos enfrentam diariamente. A seguir, Joyce da Silva Cruz de Mendonça discute o ensino da Libras como L2 para crianças ouvintes em uma escola pública do interior da Paraíba. A autora lança um projeto que visa discutir a inclusão do sujeito surdo, sua cultura e linguagem em sala de aula por meio da pesquisa-ação. A motivação que a levou a investigar tal estudo surgiu de inquietações vivenciadas pela pesquisadora em contextos escolares para criar um projeto de educação de cons- cientização e inclusão. A respeito da argumentação, Danielle dos Santos Mendes Coppi trata deste assunto em seu capítulo. A partir de estudos da argumentação da Linguística Textual, a autora apresenta algumas reflexões acerca da produção do gênero cartaz, no Ensino Funda- mental, com foco na argumentação para além da redação do Enem. Nessa direção, seu trabalho sinaliza, como o professor de língua por- tuguesa, ao trabalhar com os mais diversos gêneros textuais/discur- sivos, sob uma perspectiva processual de escolarização, pode con- tribuir para que os estudantes produzam, coerentemente, um gênero textual de grande impacto social, como é o caso da redação do Enem. O capítulo de Juliana Ferreira Benke, Karina Pacheco dos San- tos Vander Broock e Caroline Kretzmann discute a categorização da estrutura do gênero dissertativo-argumentativo com base nos con- ceitos de argumentação e categorias argumentativas da Nova Retó- rica. O objetivo do trabalho é sugerir uma melhoria na sequência 21 S U M Á R I O didática para o trabalho com leitura e interpretação do texto disserta- tivo-argumentativo para os estudantes do programa de nivelamento de língua portuguesa. Gabriel Fernandes de Oliveira apresenta uma discussão sobre como o uso dos gêneros textuais/discursivos pode mediar uma conscientização do valor social da escrita nas aulas de língua portuguesa. UMA INTRODUÇÃO NÃO BASTA DIANTE DAS MISTAS FACES DA LÍNGUA(GEM) O autor analisa uma sequência de produções de car- tazes conscientizadores, elaborados como parte de um projeto de letramento desenvolvido por via remota com o objetivo de conscien- tizar sobre os impactos da pandemia no cotidiano das pessoas. Merylin Ricieli dos Santos traz uma discussão inovadora sobre a sobre o Letramento Racial Crítico nos pré-livros infantis nos primeiros anos das crianças. Ainda que possa parecer complexa a relação entre pré-livros e a teoria antirracista, a autora enfatiza que o racismo é uma construção social, logo quanto mais cedo se reco- nhecer as diferenças étnicas e raciais, o enfrentamento desta ideo- logia pode trazer resultados mais relevantes para a valorização de culturas minoritárias. Diante da questão: “o que é ler?”, Maísa Cardoso alinha con- siderações sobre concepções de leitura e direciona algumas provo- cações sobre a prática da leitura e a formação de leitores. Na direção dessas ideias, Cardoso discorre sobre o fenômeno da linguagem no que diz respeito à prática leitora, assim, a autora delineia como a ação de ler pressupõe se posicionar, se arriscar, inferir, relacionar conheci- mentos e produzir novos, também sendo importante formar leitores mais agentes no contexto da educação. Para isso, é necessário que a família e outras instituições sociais compartilhem esse ideal. As HQs são consideradas exemplos de textos multimodais que combinam palavras e imagens para representar uma narra- tiva. Sobre esta temática encontra-se o trabalho de José Cristóvão Maia Lucena Marreiro e Leonardo Carvalho de Oliveira. Segundo 22 S U M Á R I O os autores, o objetivo deste capítulo é discutir como as HQs podem ser utilizadas na sala de aula como uma estratégia pedagógica que pode contribuir para a formação de leitores competentes e críticos. Este artigo é de grande importância para educadores, pais e pes- quisadores que buscam compreender as potencialidades das HQs como uma ferramenta no processo de ensino e aprendizado escolar. A contribuição seguinte é a de Leonardo Kominek Barrentin e Héliton Diego Lau que, a partir de Análise do Discurso de linha francesa, analisam  trechos de uma canção de Urias - Diaba - com a finalidade de identificar o embate religioso promovido pela discur- sividade da canção da cantora transexual, alinhando uma interpre- tação numa espécie de contrarreligião, a partir da contradição e da ressignificação de elementos depreciativos, considerando os efeitos de sentido do material de análise. UMA INTRODUÇÃO NÃO BASTA DIANTE DAS MISTAS FACES DA LÍNGUA(GEM) Feito isso, os autores apontam para desidentificação frente ao discurso religioso e como a produção de linguagem em questão acolhe, protege e marca existência/resis- tência de modo claro e, se preciso for, violento. No capítulo posterior, Larissa da Silva Duarte, Letícia Petel de Sousa e Héliton Diego Lau versam uma análise sobre o silên- ci(ament)o na canção “Apesar de Você”, de Chico Buarque, consi- derando um movimento que vai da censura à esperança, isso com base em Análise do Discurso de linha francesa. A partir da análise da canção, os autores indicam como existem “ecos” na sociedade por força das memórias discursivas da formação ideológica apre- sentadas na canção do referido intérprete, haja vista a repetição discursiva desses temas na atualidade, assim, a memória se faz nas condições de produção. Nos últimos anos, tem havido um aumento de discursos que acusam professores de disseminar imoralidade aos alunos e, como resultado, um movimento chamado “Escola Sem Partido” surgiu no Brasil com o objetivo de combater a suposta doutrinação ideológica nas escolas. O foco principal do capítulo de Samuel Barbosa Silva é o 23 S U M Á R I O artigo 4 do anteprojeto de Lei Federal do movimento e a seção “Edu- cação Moral” em seu site. A partir dos estudos da Análise de Discurso de linha francesa de Michel Pêcheux, o objetivo de seu trabalho é investigar a posição ideológica do movimento “Escola Sem Partido” em relação ao discurso da Educação Moral, analisando quais senti- dos devem ser mantidos e quais devem ser punidos por lei. Já o capítulo de Maria Vanessa Monteiro das Chagas trata da leitura literária em sala de aula de língua portuguesa, tecendo refle- xões sobre o espaço da autoria feminina no livro didático do ensino fundamental. Para tal empreitada, Chagas se atém aos aspectos interseccionais que compõem o perfil-autor presente em livros didá- ticos dirigidos ao 9º ano do Ensino Fundamental, com base na Aná- lise Crítica do Discurso. Ao fim, a autora constata que o perfil-autor predominante é o do homem branco sudestino, logo, há baixa repre- sentatividade da autoria feminina nos livros analisados, uma vez que não apenas há poucos textos escritos por mulheres, mas também o perfil das autoras é mais restrito. Samuel Filipe Guedes do Nascimento, Manassés Morais Xavier e José Walbérico da Silva WCosta apresentam uma proposta de análise linguística das Construções sintáticas adjetivas no artigo de opinião. UMA INTRODUÇÃO NÃO BASTA DIANTE DAS MISTAS FACES DA LÍNGUA(GEM) Os autores fazem um recorte de um artigo de opinião que trata da CPI da COVID-19 e analisam as valorações presentes nos enunciados, destacando-os como estratégias do discurso em marcar e demarcar pontos de vista. Por meio da teoria dialógica da linguagem, Nascimento, Xavier e WCosta defendem que a posição social na qual os sujeitos da enunciação se encontram, sendo favo- ráveis ou desfavoráveis diante das situações que demandam posi- cionamentos demandam a atividade interativa daqueles que usam a linguagem, evidenciando que há uma relação direta entre o sujeito e a filiação ideológica a qual ele pertence. André Luiz Souza-Silva e Klecio de Assis Raimundo analisam usos linguísticos que diferem daqueles prescritos pela gramática 24 S U M Á R I O tradicional como “corretos”. Para isso, será utilizado um corpus inédito composto por transcrições de entrevistas realizadas com falantes do interior da Paraíba, coletadas por estudantes da Educação Básica. A escolha desse corpus segue a perspectiva da Linguística Funcional, que analisa dados reais de uso da língua retirados de situações con- cretas de comunicação. As contribuições defendidas têm um caráter pedagógico e fundamentadas nos estudos funcionais da língua, com foco na gramaticalização, a fim de analisar a relação entre as estru- turas gramaticais e os contextos efetivos de comunicação. O capítulo de Natássia Thais do Nascimento Ribeiro mate- rializa discussões do campo da Historiografia Linguística. A autora desenvolve uma análise sobre a variação e a mudança linguísticas em gramáticas escolares de língua portuguesa da década de 2000. Para isso, Ribeiro objetiva analisar os textos de apresentação de gramáticas escolares do período selecionado, para perceber se as gramáticas analisadas trazem, por meio dos conceitos de variação e mudança linguísticas, uma retórica que demonstre intenção de rom- per com os preceitos dogmáticos da tradição gramatical, alinhando- -se a uma virada sociodiscursiva. Por fim, percebe-se como as gramá- ticas escolares analisadas se desvinculam dos dogmas da gramática tradicional, mas que ainda coexistem, à época, manuais propondo um trabalho exclusivamente amparado na tradição gramatical. Emily Gonçalves de Medeiros Ferreira apresenta uma discus- são acerca da abordagem da colocação pronominal na obra “Regras da lingua portugueza, espelho da lingua latina”, de Jerónimo Conta- dor de Argote (1725), e de suas motivações e implicações no pro- cesso de gramatização da língua portuguesa no século 18. A autora reflete sobre o papel das políticas linguísticas na constituição da nação. UMA INTRODUÇÃO NÃO BASTA DIANTE DAS MISTAS FACES DA LÍNGUA(GEM) Para tanto, considera que a “cultura, os princípios, os valo- res, tudo aquilo que molda a nação como tal é expresso e apreen- dido a partir de sua língua”. Nessa direção, a gramática serve como instrumento que define regras e referências linguísticas, ainda que de um lugar idealizado. Assim, a autora discorre, numa perspectiva 25 S U M Á R I O historiográfica, sobre o lugar da obra de Contador de Argote na pro- dução gramatical portuguesa dos setecentos, com ênfase na ques- tão do posicionamento dos pronomes átonos. Willian Ferreira Furtado de Lacerda discorre sobre  a varia- ção no nível fonético-fonológico destacando que esta possui fun- ções diferentes no português brasileiro. Assim, quando o /l/ ocupa a posição de coda silábica no PB, ele pode não acarretar mudança de significado da palavra, no entanto no inglês poderá ser determinante para o entendimento da mensagem que se deseja transmitir. Embora o objetivo da pesquisa fosse encontrar pontos de convergência, os resultados apontam que  as variáveis foram julgadas de formas distintas, afirmando que  o fator social interfere no comportamento linguístico do falante. Por último, o capítulo de Marina Chiara Legroski se debruça sobre a mentira como um ato de fala, apresentando uma proposta de análise contextualizada à teoria de Searle. Nesse sentido, Legroski caracteriza a mentira a partir de algumas características, buscando compreendê-la no escopo da linguística, pensando em suas condi- ções de realização, quais regras a definem e nos atos locucionário, ilocucionário e perlocucionário envolvidos em sua realização. Feito isso, a autora mapeia traços de ato de fala e descreve que tipo de ação ressoa no mundo a partir da mentira, pois, na contemporaneidade, esse tipo de discussão é relevante por implicações práticas e éticas. Diante do exposto, é válido enfatizar como essa agenda de estudos da linguagem - teóricos e/ou práticos - é um acordo da plu- ralidade social que apresenta diferentes atividades mediadas pela língua(gem), seja em sua imanência ou em sua aplicabilidade. Desse modo, concordamos com o que afirma Carvalho (2014, p. 59):  “A sociedade se apresenta cada vez mais complexa, exigindo dos indi- víduos um arsenal razoável de conhecimento para poder entendê-la, para poder reagir aos problemas que surgem num turbilhão, para poder participar da discussão e resolução desses problemas”. UMA INTRODUÇÃO NÃO BASTA DIANTE DAS MISTAS FACES DA LÍNGUA(GEM) 26 Nesta apresentação, entendemos como o fazer acadêmico na área das linguagens é realmente múltiplo e em decorrência de diferentes questões que envolvem o fazer científico, tanto de ordem teórica quanto metodológica, bem como suas possibilidades peda- gógicas e aplicáveis à vida cotidiana. Enfim, isso posto, desejamos aos leitores um olhar crítico-reflexivo frente a todas as discussões aqui presentes e um convite aos pares para interagirmos sobre o que se propõe, analisa e discute nesta coletânea. A organização REFERÊNCIAS BORGES NETO, José. Entrevista com José B. Neto. In: XAVIER, Antonio Carlos; CORTEZ, Suzana (Org.). Conversa com linguistas: virtudes e controvérsias da linguística. São Paulo: Parábola Editorial, 2006, p. 37-50. CARVALHO, Eneida Dornellas de. Percursos acadêmicos da transdisciplinaridade em linguística. In: LINS, Juarez Nogueira; LINS, Cleuma Regina Ribeiro da Rocha (Orgs.). Diálogos interdisciplinares: linguística, literatura e ensino. Recife: Editora UFPE, 2014, p. 59-70. CARVALHO, Eneida Dornellas de. Percursos acadêmicos da transdisciplinaridade em linguística. In: LINS, Juarez Nogueira; LINS, Cleuma Regina Ribeiro da Rocha (Orgs.). Diálogos interdisciplinares: linguística, literatura e ensino. Recife: Editora UFPE, 2014, p. 59-70. 27 INTRODUÇÃO S U M Á R I O A comunidade de lésbicas, gays, bissexuais, travestis, trans- gêneros, transexuais, queers, intersexo, assexuais e mais (LGBQ- TIA+) está cada vez mais sendo discutida e ganhando visibilidade em novelas, séries de TV, discursos políticos etc., trazendo à tona o respeito que ela tanto quer. O ano de 2014 foi aquele no qual a comu- nidade LGBTQIA+ ganhou um maior espaço em todos os tipos de mídia, tanto positiva quanto negativamente. Graças a essa visibilidade, pôde-se notar a diversidade das pessoas, quer seja na forma de se vestir, de se comportar, até a forma de seus relacionamentos. Além disso, houve um rompimento do binarismo, ou seja, a (des/re)construção do que nos fora imposto através de uma visão cis-heteronormativa e, muitas vezes, machista. Neste capítulo, é dado foco à comunidade LGBTQIA+, mos- trando a questão da língua(gem) em que associamos somente a eles, classificada como bajubá, que possui matrizes africanas, popu- larmente conhecida como “Bichês”. O corpus consiste em excer- tos retirados de um programa chamado Não Me Critica, via mídia podcast. Nele, três rapazes que se identificam como homossexuais comentam/criticam a respeito de alguns vocábulos encontrados num dicionário, ou como diríamos em “Bichês”, numa “dicionária”, já que o gênero “neutro”, se assim podemos chamar, é o feminino. Também são comentadas/criticadas questões como de uso social, variação de prestígio, nível de escolaridade, entre outros. 29 S U M Á R I O A LINGUÍSTICA APLICADA E SUAS CONTRIBUIÇÕES A Linguística Aplicada (LA) é um campo transdisciplinar (KLEIMAN & GRANDE, 2015) e indisciplinar (MOITA LOPES, 2006) de estudo que investiga, identifica e oferece soluções para proble- mas relacionados com a linguagem da vida real. Por sua interdisci- plinaridade, alguns campos acadêmicos como educação, linguística, psicologia, antropologia e sociologia. Seu teor provém da Linguís- tica, digamos “teórica”. A grosso modo, não é uma tarefa fácil conceituar exatamente o que é LA devido à sua interdisciplinaridade, sua mestiçagem e suas diferentes formas de trabalhar, mas podemos observar e notar que os trabalhos relacionados à LA tratam de sujeitos que são/estão à margem da sociedade, como os pobres, negros, a comunidade LGB- TQIA+, entre outros. Portanto, “o foco principal tem sido o sujeito ins- crito na produção do conhecimento ou a sua redescrição em outras bases” (MOITA LOPES, 2011, p. 87). Ou seja, através dos estudos com a LA podemos perceber que a forma como esses sujeitos falam/ escrevem, pensam sobre a fala/escrita também é importante e deve ser estudada e refletida. [...] o conhecimento tem de ser novo não simplesmente porque o mundo está diferente, mas porque tais mudan- ças requerem processos de construção de conhecimento que devem, necessariamente, envolver implicações de mudança na vida social. As mudanças têm sido nos dois sentidos: uma resposta à vida social que implica ques- tões de natureza epistemológica. Em um sentido, a opção tem a ver com conhecimentos que refletem as mudanças radicais da vida contemporânea e, em outro, na direção de um projeto epistemológico com implicações sobre a vida social (MOITA LOPES, 2011, p. 91). 30 É importante e necessário que se reconheça a visão do Outro (HALL, 2006), como nesse caso, linguisticamente. Por exemplo, é importante aprendermos inglês, mas qual fora a visão estipulada pelos professores de língua inglesa no século XX? Achávamos que aprendíamos ou o “inglês americano” ou “inglês britânico” e nem pensávamos no inglês falado no Canadá, por exemplo, e em outros países em que o inglês é considerado língua materna. Atualmente, os professores de língua inglesa e pesquisadores chamam o ensino de língua estrangeira moderna, no caso, o inglês, de Inglês como Língua Franca (CANAGARAJAH, 2006; JORDÃO, 2014; MARSON & JORDÃO, 2022), mostrando uma nova forma de pensarmos o ensino de língua inglesa no século XXI. A LINGUÍSTICA APLICADA E SUAS CONTRIBUIÇÕES Também devemos considerar que, no presente século, as mudanças que ocorrem na língua(gem) dos falantes é muito rápida e mais visível, principalmente em meios midiáticos, como as redes sociais (LAU, 2014), por exemplo. Foi a tecnoinformação do meio de avanços tecnológicos [...] que possibilitou um mundo mais veloz, de discursos que atravessavam o globo em um piscar de olhos no chamado tempo real, que mudam a economia na tela do computador, que nos aproximam de forma surpreendente, que nos possibilitam ser e ver outros virtualmente como também “conversar” com pessoas que nunca vamos ver, que nos assustam como alteridades nunca imaginadas – provocando a construção de discursos fundamentalistas, que podem abrir nossos olhos para outras formas polí- ticas de viver tanto a vida íntima e pública que, aliás, se confundem cada vez mais [...], questionando verdades naturalizadas em todos os sentidos, embora possam tam- bém confirma-las, já que, como sabemos, são muitos os discursos que nos chegam (MOITA LOPES, 2011, p. 91-92). As redes sociais que conhecemos e utilizamos agora, como Twitter e Instagram, apresentam uma gama imensa de informações em um tempo muito mais rápido, diferente do que temos na televi- são, por exemplo. Os assuntos que repercutem nas mídias sociais, 31 alguns no caso, servem para (des)construir um conceito que tínha- mos sobre determinado assunto, como a linguagem não-binária (LAU, 2019a, 2019b), por exemplo. Outro exemplo é a questão dos direitos igualitários para a comunidade LGBTQIA+ e casamento civil igualitário repercutiu e ainda repercute nas redes sociais, inclusive petições que servem para criminalizar a homolesbobitransfobia, con- quistada em 2019. Ou seja, os discursos que nos eram impostos sobre questão de gênero e sexualidade, em que família é somente homem e mulher, estão enfraquecendo e abrindo portas para a diversidade das famílias, sexualidade e gênero. S U M Á R I O A chamada ciência moderna já foi amplamente criticada por se basear em um sujeito homogêneo e essencializado como branco, homem, heterossexual de classe média que as teorias feministas, queer, antirraciais, pós-coloniais e pós-modernistas se encarregam de desconstruir. Essa questão está relacionada à redescrição de um mundo constituído por uma ciência de significado objetivo, i.e., “as últimas verdades”, que nos fez acreditar em uma única explicação para os fatos sociais, que não era de modo alguns entendidos como atravessados pelo exercício do poder, despolitizando e tornando autônomo o conheci- mento (MOITA LOPES, 2011, p. 101-102). UMA CRÍTICA A RESPEITO DA CRÍTICA O programa Não Me Critica é um programa de rádio online gravado em podcasts, desde 12 de setembro de 2012, o qual surgiu com quatro apresentadores que se identificam como homossexuais: Elmer Dias (ED), Francisco Carbone (FC), Thiago Arzakom (TA) e Vinícius Ribeiro. O programa apresenta diversos temas discutidos semanalmente às quartas-feiras, alguns voltados mais ao público LGBTQIA+, outros mais gerais. Após um ano, teve a saída de Viní- cius Ribeiro, idealizador do projeto, mas os demais continuaram. 32 Os temas são, em alguns casos, sugeridos por ouvintes, em outros, são escolhidos pelos próprios apresentadores, que afirmam que o programa se trata de conversas gravadas do grupo a respeito do tema. Os temas são, em alguns casos, sugeridos por ouvintes, em outros, são escolhidos pelos próprios apresentadores, que afirmam que o programa se trata de conversas gravadas do grupo a respeito do tema. Nos programas gravados, os rapazes criticam a respeito do tema em questão, muitas vezes sendo o título do podcast, em que eles colocam suas opiniões, críticas, comentários e sugestões de uma forma, em alguns casos, cômica e divertida. De todos os programas hospedados, o selecionado para fazer parte do corpus deste trabalho foi o podcast número 83, pos- tado no dia 24 de junho de 2014, com duração de 49 minutos e 49 segundos, sob o título de “Bichês”, no qual os três apresentadores comentam/criticam a língua(gem) utilizada por alguns membros da comunidade LGBTQIA+. As transcrições foram feitas de forma para facilitar a leitura dela para melhor compreensão da análise, omitindo risadas e algumas falar inaudíveis devido, em alguns momentos, todos falarem ao mesmo tempo. O programa em si trata das gírias/vocábulos tidas como uti- lizadas pela comunidade LGBTQIA+. Os apresentadores, no início, ficam em dúvida se “a dicionária” existe fisicamente, e de fato, sim, sendo de autoria de Fred Lib e Vitor Angelo2. Recorte 1: ED: Vocês usam esses dialetos no dia a dia? TA: Não. ED: Como assim não usa? Você não fala que vai “desaquendar”? TA: Eu não falo “corar o fax”. ED: Como “corar o fax”? Recorte 1: ED: Vocês usam esses dialetos no dia a dia? ED: Vocês usam esses dialetos no dia a dia? TA: Não. TA: Não. ED: Como assim não usa? Você não fala que vai “desaquendar”? ED: Como assim não usa? Você não fala que vai “desaquendar”? TA: Eu não falo “corar o fax”. UMA CRÍTICA A RESPEITO DA CRÍTICA ED: Como “corar o fax”? A respeito da dicionária, recomendo a leitura de Lau (2015) e Souza-Silva (2021). A respeito da dicionária, recomendo a leitura de Lau (2015) e Souza-Silva (2021). 2 33 33 FC: É uma expressão antiga, né, gente? Porque fax já não existe. TA: Que é prestar atenção. ED: “Corar o fax”? Gente, mas a pessoa tava criativa, né? FC: Lê a explicação científica de “chuca”. TA: Lavagem intestinal. Elmer, qual é a cor do seu “chicotol” aí? ED: É nude, é nude. “Chicotol” é cabelo? FC: Cabelo é “picumã”. TA: Cueca. [...] Então, gente, baseado nesse pequeno, como fala: ED: Dialeto? TA: Não, nessa pequena demonstração, nessa desgusta- ção... Comumente eu não uso isso. Você usa algum? ED: Você fala sim, Thiago! TA: “Adoro”, só. ED: “Adoro”; “ai, que cu”. FC: Como assim, “ai que cu” é uma palavra? TA: Esse eu uso raramente. ED: “Arrasou”, você não usa “arrasou”? TA: “Arrasou” e “adoro”, que nem tá aí, porque já caiu no uso comum. ED: Várias coisas: “caminhoneira”, você usa; “colar vel- cro”, você usa. “Bicha-pão-com-ovo”, “poc-poc”, essas coisas você usa. FC: Tipo, eu realmente não tenho, tipo, não falo 24 horas por dia nesse dialeto, né, mas conheço muita gente que é FC: É uma expressão antiga, né, gente? Porque fax já não existe. ED: “Corar o fax”? Gente, mas a pessoa tava criativa, né? FC: Lê a explicação científica de “chuca”. ED: “Corar o fax”? Gente, mas a pessoa tava criativa, né? FC: Lê a explicação científica de “chuca”. TA: Lavagem intestinal. Elmer, qual é a cor do seu “chicotol” aí? ED: Dialeto? TA: Esse eu uso raramente. TA: Esse eu uso raramente. ED: “Arrasou”, você não usa “arrasou”? TA: “Arrasou” e “adoro”, que nem tá aí, porque já caiu no uso comum. ED: Várias coisas: “caminhoneira”, você usa; “colar vel- cro”, você usa. “Bicha-pão-com-ovo”, “poc-poc”, essas coisas você usa. FC: Tipo, eu realmente não tenho, tipo, não falo 24 horas por dia nesse dialeto, né, mas conheço muita gente que é bem integrada, que sabe mais do que eu. 34 ED: Mas geralmente esse dialeto não tá associado a um homossexual com trejeitos e essas coisas? FC: Ih, gente... ED: Não estou falando que todo mundo é e que seja uma coisa ruim, só estou tentando dar assunto ao tema [...]. Através das pesquisas que eles fizeram a respeito do Bichês, podemos notar quando ED pergunta se os demais utilizam e eles ficam em dúvida se utilizam, em destaque TA, que, podemos pressu- por, por questão da língua portuguesa “padrão” ter um certo “prestígio”, diferente da linguagem do Bichês. Na perspectiva da LA, devemos [...] repensar a questão da legitimidade da língua em uso em função da lógica democrática da controvérsia, da ruptura e do dissenso como vetores de produção e não de perturbação, perda ou degradação linguística. “Legi- timidade dos usos linguísticos” é aqui compreendida em termos socioculturais e políticos e não puramente linguís- ticos (SIGNORINI, 2011, p. 169-170). Palavras como “adoro” e “arrasou”, que ele afirmou que nem estava no site em que estava olhando a respeito dos vocábulos do Bichês serem consideradas por ele “comuns”, a palavra “arrasou”, na dicionária, pode possuir significados diferentes ao que comu- mente associamos a esta palavra, ou seja, esta possui diversos sig- nificados (LAU, 2015). Também podemos observar que, sabendo que todos os apre- sentadores se identificam como homossexuais e a língua(gem) está caricata ao público LGBTQIA+, geralmente pensamos que: se faz parte, responde a todos os estereótipos criados, portanto, deve-se saber falar/usar o Bichês (LAU, 2016). Nesse caso, podemos notar que TA até pode utilizar alguns vocábulos, mas ele que se identificar como o “homossexual que fala Bichês”, por isso a “negação” da uti- lização dessa língua(gem), além do mais, ser identificado como um homossexual afeminado. 35 Podemos continuar analisando durante o programa os dis- cursos formados a respeito dessa linguagem foneticamente: Recorte 2: FC: E aí... ED e TA: Nhaí! ED: Dialeto? S U M Á R I O FC: Na dicionária tá “e aí”: expressão de cumprimento, talvez a mais usada no meio homossexual. ED: E aí, tá boa, querida? TA: Eu acho que o “e aí” é pra quem é hétero; agora o “nhaí”, sim. Aquele vídeo do “Comédia MTV”, mesmo sendo uma comédia, uma coisa engraçada, explica que não é essas palavras simplesmente lidas, como fonetica- mente elas [...]. Tem um contexto, né? Essa questão do contexto, juntamente com o vídeo que eles assistiram do Comédia MTV, que é de uma sala de aula em que o pro- fessor está dando uma aula de Bichês para os alunos, desde vocá- bulos até frases, possíveis traduções do português para o Bichês e a parte fonética, como foi o caso de os apresentadores terem citado. Juntamente a isso, além de ter o contexto, no caso, pressuposta- mente, em que os falantes dessa língua(gem) utilizariam com outros falantes, além da diferença do “sotaque” entre homossexuais e a comunidade LGBTQIA+, que falam mais anasalado. Também é interessante discutir a respeito do que TA apon- tou: a questão da diferença da fala entre o heterossexual e o homos- sexual. Através dessa diferença por causa da orientação sexual, podemos pressupor uma hierarquia, em que a língua(gem) do hete- rossexual (“e aí”) possa receber um prestígio maior, por ser conside- rada uma variação informal, mas, de uma certa forma, “padrão”, afinal, grande parte utiliza essa expressão, do que a de um homossexual (“nhaí”), por estar ligado ao cômico, ao fato de não ser alfabetizado, entre outros fatores negativos. 36 A igualdade entre falantes, independentemente das posi- ções numa dada ordem sociolinguística, é primeira e fun- damental para que se coloque a questão da legitimidade da língua em uso [...], pois é anterior a toda hierarquização. [...] sua condição de falante – ou seja, sua capacidade de interagir verbalmente no coletivo, seu estatuto de inter- locutor autorizado naquele coletivo – é o que legitima os usos que ele faz dessa mesma língua. Não são determi- nados usos que ele faz da língua, ou determinadas formas que utiliza, que os legitimam como falante “competente” daquela língua (SIGNORINI, 2011, p. 171). S U M Á R I O Ou seja, a fala de uma determinada classe que ainda é consi- derada “padrão” pela sociedade cis-heterossexual, torna um falante “competente”, excluindo as demais falas, obrigando um membro da comunidade LGBTQIA+ a sucumbir-se à língua(gem) heterossexual, privando de uma outra forma de fala, sem ser menosprezado. ED: E aí, tá boa, querida? Recorte 3: ED: Uma coisa que acontece é que esses dialetos têm levas, safras, temporadas. Até pouco tempo, a gente tava usando “isso é choque, querida, arrasou!”. FC: Tem a ver com um evento, um meme, personali- dade da web. Inês Brasil, tem super a ver. A própria dos “bons drink” virou uma. ED: Que nunca falou “me disseram que eu estava na pior, porrãn!”? TA: Isso que o ED falou no início, que é das bichas mais afetadas, “quaquá”, “pão-com-ovo”... Mas é, gente, princi- palmente culturalmente falando. Onde existe uma cultura voltada para a... talvez a escassez de recursos de conhe- cimento, de estudo, eu acho... FC: Imagina bichas riquíssimas: “você já encontrou aquela ‘bicha pão-com-ovo’ hoje?” TA: Jamais! TA: Jamais! 37 Percebemos, que por mais que possamos pensar que são grupos “deslocados da língua(gem) da maioria”, há semelhanças, seja em casos de gírias que mudam, ou como o próprio ED comen- tou, terem safras, temporadas. Acontece muito com a língua(gem) cis-heterossexual, se é assim que podemos/devemos classificar. Em termos do comportamento e desenvolvimento huma- nos, as ideias clássicas sobre nossas ações e seus sig- nificados adquiridos com base em suas funções no sis- tema social foram substituídas pela visão de que o que fazemos tem um papel primordial em moldar os lugares onde vivemos, e, longe de sermos compreendidos como socializados com base nas normas de um grupo social cujo monitoramento subsequentemente nos mantém moralmente alinhados, há agora a compreensão de que nos “agrupamos” com base em uma grande quantidade de opções mutáveis, decidindo o que é correto ou errado para nós mesmos (RAMPTON, 2011, p. 113). S U M Á R I O Por mais que as variações linguísticas sejam diversas, em cada região há um significado diferente para a língua(gem), para as gírias e dialetos é a mesma coisa. Bourdieu (2008, p. 25) fala da lín- gua(gem) como “desvio individual em relação à norma linguística”, ou seja, junto da questão identitária do sujeito, não utilizamos uma forma de língua(gem) para nos comunicar. Rampton (2011) comenta a respeito da “comunidade de fala”, cujo termo foi questionado e estudado pela Sociolinguística, agora, tomado por duas direções: a primeira, “onde há uma análise em close da interação face a face em vários contextos e relações sociais muito bem estabelecidos tais como oficinas de trabalho, salas de aula e grupos profissionais de um tipo ou de outro”; e a segunda, “quando ela é analisada como uma representação semiótica nos discursos ideológicos que constroem e naturalizam agrupamentos muito gran- des [...]” (RAMPTON, 2011, p. 115-116). Calvet (2002, p. 115) também discute sobre isso, mas ele denomina como “comunidade linguística”: 38 “[...] onde se encontra a pertinência dessas diversas variações, atra- vés do tempo, do espaço ou dos estratos sociais”. Recorte 4: TA: Você conhece muitos “PAMs”, ED? ED: Pães? Adoro pão! FC: Pan é aquilo de pansexual, né? TA: Não, “PAM” é a sigla pra “Passiva Até a Morte”. ED: Ai, sim, eu conheço algumas. FC: Tem também os ativos até a morte. Recorte 4: Recorte 4: Recorte 4: TA: Você conhece muitos “PAMs”, ED? ED: Pães? Adoro pão! TA: Jamais! FC: Pan é aquilo de pansexual, né? TA: Não, “PAM” é a sigla pra “Passiva Até a Morte”. ED: Ai, sim, eu conheço algumas. FC: Tem também os ativos até a morte. Recorte 4: TA: Você conhece muitos “PAMs”, ED? ED: Pães? Adoro pão! FC: Pan é aquilo de pansexual, né? TA: Não, “PAM” é a sigla pra “Passiva Até a Morte”. ED: Ai, sim, eu conheço algumas. FC: Tem também os ativos até a morte. S U M Á R I O Essa questão que eles comentam durante o programa, é a questão das siglas utilizadas em determinados vocábulos do Bichês, como no caso: “PAM” e “pan”. Ao comentarem sobre essas duas siglas, houve um certo estranhamento, já que elas eram desconhe- cidas pelo grupo. Numa questão de gênero, a respeito da primeira sigla mencionada, podemos pressupor que para a comunidade LGB- TQIA+, no caso em que o sujeito homossexual em que este recebe a classificação de ativo, há um binarismo nisso, pois se associa o sujeito passivo a mulher, alguém “submisso”, esta questão ideológica sendo vista desde a Bíblia e utilizada para propagar o machismo. Butler (2003) comenta a respeito do binarismo, sobre a questão da heteronormatividade: pois quando vem à nossa mente a palavra “casal”, imaginamos um homem e uma mulher, geralmente. Há asso- ciações errôneas de que um casal homossexual seja chamado de “par”, pois ambos são do mesmo gênero, iguais, como um “par de brincos”, por exemplo. Supondo por um momento a estabilidade do sexo binário, não decorre daí que a construção de “homens” aplique- -se exclusivamente a corpos masculinos, ou que o termo “mulheres” interprete somente corpos femininos. Além disso, mesmo que os sexos pareçam não problemati- camente binários em sua morfologia e constituição [...], 39 não há razão para supor que os gêneros também devam permanecer em número de dois (BUTLER, 2003, p. 24). não há razão para supor que os gêneros também devam permanecer em número de dois (BUTLER, 2003, p. 24). Outro fato ligado ao homossexual é essa questão de “quem é o homem/mulher da relação?”, sabendo que a relação homoafetiva é diferente da heteroafetiva. Na relação homossexual pode haver: ativo, passivo, versátil e gouine, sendo este último uma relação que não envolve penetração. Algo semelhante e que estava sendo bas- tante comentado e criticado eram os g0ys, que se identificam como homens heterossexuais, entretanto, podem fazer sexo oral e mas- turbação em outro homem heterossexual. O que não se é permitido é penetração e ter um relacionamento afetivo com outro homem (LAU & FÁTIMA, 2018, 2020). O termo “passivo” remete à figura do homossexual com a “visibilidade do estigma”, ou seja, daquele que apresenta atitudes que identificam sua preferência sexual. [...] o homossexual ativo tende a ganhar status de mais macho, chegando ao ponto de, em raras exceções, os machos que “comem bichas” não serem classificados de maneira dife- rente dos “homens verdadeiros” devido ao seu desem- penho do papel ativo. Inclusive, muitos homens que têm relações homossexuais não se consideram homossexu- ais, desde que não pratiquem sexo anal ou que exerçam o papel “ativo” na relação sexual [...] (ALMEIDA, 2011, p. 9). Ou seja, infelizmente, alguns membros da comunidade LGB- TQIA+ que não se identificam como gays, porém fazem sexo com outros homens, desde que sejam somente ativos, não se consideram gays pela rotulação de que ser gay é ser passivo, “ser mulher”, por ser penetrado. Independentemente do tipo com quem cada parceiro se relaciona, seja ativo, passivo, versátil ou se ambos são gouines, a prática em si é considerada homossexual da mesma forma. Recorte 5: TA: Engraçado, né, gente, o “a”, que é um artigo feminino, no mundo gay, fica “A Pedro”, “a Mário” ... TA: Engraçado, né, gente, o “a”, que é um artigo feminino, no mundo gay, fica “A Pedro”, “a Mário” ... 40 ED: Não, rola um feminino: “A Thiaga”. TA: Eu não falo: “a fota”, “a relógia” e “a dicionária”. FC: “A prédia”, gente. Como assim? Além de Bichês, é burra. TA: Não foi bem alfabetizada. FC: Não, nada bem alfabetizada. TA: Por isso que eu digo... FC: Olha, TA voltando na polêmica... TA: Calma, gente! Deixa eu falar! Eu acho que muito gente ... acho que essas expressões vieram dos homossexuais da periferia, que tem uma condição financeira mais baixa, que não sabe falar o português direito, e que surgem essas gírias. não há razão para supor que os gêneros também devam permanecer em número de dois (BUTLER, 2003, p. 24). Você vê também muitas travestis usando essas gírias, esses dialetos. Então acabou isso aí. Não necessariamente todos os homossexuais, aliás, a grande maioria dos homossexuais não utiliza esse dialeto. Um assunto para o qual os apresentadores chama- ram atenção nessa parte foi a questão da alfabetização, tendo a crença de quem possui escolaridade fala “direito”, aprendeu o por- tuguês “corretamente”, excluindo outras formas de aprendizado que envolvem a língua. [...] a sobrevivência na escola é comumente vista como sinônimo da aquisição dos bens culturais de prestígio – ser “estudado” é ser “educado”, “mais elevado” – e, ao mesmo tempo, como sinônimo de aquisição dos recursos necessários ao sucesso na ação social de base discursiva, independentemente dos contextos situacionais em jogo – ser “estudado” é saber falar “direito”, é raciocinar/agir/ avaliar “certo”. [...] tanto o não-acesso à escola quanto o fracasso escolar são vistos como sinônimos de déficit desses mesmos bens culturais – não ser “estudado” é ser ignorante, é “não saber das coisas” – e, ao mesmo tempo, como sinônimos de déficit de recursos necessários à ação social de base discursiva – não ser “estudado” é não [...] a sobrevivência na escola é comumente vista como sinônimo da aquisição dos bens culturais de prestígio – ser “estudado” é ser “educado”, “mais elevado” – e, ao mesmo tempo, como sinônimo de aquisição dos recursos necessários ao sucesso na ação social de base discursiva, independentemente dos contextos situacionais em jogo 41 falar “direito”, é estar sempre vulnerável diante da multipli- cidade de situações e interlocutores do cotidiano e, con- sequentemente, estar sempre sujeito ao fracasso na con- secução de objetivos próprios (SIGNORINI, 1995, p. 162). S U M Á R I O Esse fato pode ser feito uma ligação do que já foi comen- tado quanto a variação de prestígio, a língua(gem) heterossexual, pois um dos apresentadores afirma que a língua(gem) surgiu dos homossexuais da periferia, associando a quem mora nesse lugar um nível baixo de alfabetização. Também as travestis, que, devido a sua identidade de gênero, abandonam/abandonaram os estudos, foram expulsas de casa. Isso volta novamente a questão da normatividade da língua, desconsiderando esse tipo de língua(gem) algo “correto”, entretanto, algumas expressões da língua(gem) homossexual podem ser repensadas e aceitas pelos demais. CONSIDERAÇÕES FINAIS Toda língua apresenta inúmeras variações em decorrên- cia das diferenças entre os falantes dela, quer seja de classe social, profissão, grau de escolaridade etc. Uma variação que geralmente é menosprezada, ou observada como menos importante, é aquela relativa às identidades de gênero e sexual. Embora possam ser de comunidades de fala semelhantes, dois falantes podem ter dificul- dades em se comunicar por um deles ser homossexual e o outro ser heterossexual, por exemplo. Alguns membros da comunidade LGBTQIA+, pela necessi- dade de se comunicar de forma mais velada, sem que outros per- cebam do que se trata, ou mesmo para falar de forma “engraçada”, como reforçando de forma caricata o estereótipo que lhes é reser- vado, utilizando para isso o Bichês. 42 O estereótipo pode não ser completamente falso, mas fre- quentemente exagera alguns traços da realidade e omite outros. O estereótipo pode ser mais ou menos tosco, mais ou menos violento. Entretanto, necessariamente, lhe fal- tam nuanças, uma vez que o mesmo modelo é aplicado a situações culturais que diferem consideravelmente umas das outras (BURKE, 2004, p. 156). Por se tratar de uma língua(gem) informal, diferente do “por- tuguês padrão”, falada por pessoas de classes sociais inferiores, é vista como uma língua(gem) a ser evitada e somente falada com teor humorístico, até mesmo com intenção de “imitar” de maneira “debochada” a forma de se expressar de gays que correspondem ao estereótipo, chamados de “poc-poc” ou “pão-com-ovo”, termos do próprio Bichês para se referir de forma negativas a esses sujeitos. A comunidade LGBTQIA+ que utiliza o Bichês em seu coti- diano, ou seja, com a intenção primária de transmitir a mensagem sem teor humorístico ou com intenção de menosprezar, são vistos como de classe social inferior, menor escolaridade e até mesmo de menos inteligência. Além disso, são muitas vezes classificados como “mais gays”, isto é, pessoas que não correspondem ao padrão cis-he- teronormativo da sociedade. REFERÊNCIAS ALMEIDA, Daniel Mazzaro Vilar de. “Sou gay, porém totalmente discreto”: os estereótipos e a criação do ethos em um site de relacionamento gay. ReVeLe, Minas Gerais, n. 3, p. 1-23, ago. 2011. ALMEIDA, Daniel Mazzaro Vilar de. “Sou gay, porém totalmente discreto”: os ALMEIDA, Daniel Mazzaro Vilar de. “Sou gay, porém totalmente discreto”: os estereótipos e a criação do ethos em um site de relacionamento gay. ReVeLe, Minas Gerais, n. 3, p. 1-23, ago. 2011. ARZAKOM, Thiago; DIAS, Elmer; CARBONE, Francisco. NMC #083: Bichês. Disponível em: <http://www.naomecritica.com.br/nmc-083/>. Acesso em: 10 dez. 2014. BOURDIEU, Pierre. A economia das trocas linguísticas: o que falar quer dizer. 2 ed. São Paulo: EDUSP, 2008. 43 BURKE, Peter. Testemunha ocular: história e imagem. Bauru, SP: EDUSC, 2004. BUTLER, Judith. Problemas de gênero: feminismo e subversão da identidade. Rio de Janeiro: Civilização Brasileira, 2003. CALVET, Louis-Jean. Sociolinguística: uma introdução crítica. 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Na pretensão do novo, a presença do velho: a prática discursiva e a identificação g0y em questão. Travessias, Cascavel, v. 12, n. 4, ed. Esp., p. 69-85, dez. 2018. LAU, Héliton Diego; FÁTIMA, Wellton da Silva de. Na pretensão do novo, a presença do velho: a prática discursiva e a identificação g0y em questão. Travessias, Cascavel, v. 12, n. 4, ed. Esp., p. 69-85, dez. 2018. LAU, Héliton Diego; FÁTIMA, Wellton da Silva de. Questões de identificação sexual: a posição-sujeito g0y. In: LAU, Héliton Diego; FÁTIMA, Wellton da Silva de (Orgs.). Raça, gênero e sexualidade em perspectivas discursivas: efeitos e práticas da/na violência, v. 2. São Paulo: Pimenta Cultural, 2020, p. 286-305. LAU, Héliton Diego. A (des)informação do bajubá: fatores da linguagem da comunidade LGBT para a sociedade. Temática, Paraíba, v. 11, n. 2, p. 90-101, fev. 2015. LAU, Héliton Diego. A linguagem de universitários nas redes sociais através do ponto de vista sociolinguístico. 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MARSON, Isabel Cristina Vollet; JORDÃO, Clarissa Menezes. Multiliteracies, English as a Lingua Franca and Translingual Practices: opening Pandora’s box. Muitas Vozes, Ponta Grossa, v. 11, p. 1-19, 2022. MOITA LOPES, Luiz Paulo da. Linguística Aplicada e a vida contemporânea: problematização dos construtos que têm orientado a pesquisa. Em diferentes investigações, com a finalidade de representar uma maior pluralidade de identi- dades sexuais e de gênero, é comum identificarmos diversos formatos de siglas. Aqui, assumo o uso de LGBTQIA+, considerando o sinal “+” como representativo das demais formas de viver as sexualidades e identidades de gênero. Dito isso, espero que todos da comunidade possam, de alguma forma, sentir-se aqui representados. Luiz Paulo da (Org.). Por uma Linguística Aplicada Indisciplinar. 2 ed. São Paulo: Parábola, 2011, p. 85-107. MOITA LOPES, Luiz Paulo da. Ideologia linguística: como construir discursivamente o português no século XXI. 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Sociodialeto, Mato Grosso do Sul, v. 12, n. 34, p. 13-27, jul. 2021. 45 INTRODUÇÃO Este texto é potencializado por meus contextos de formação acadêmica, científica, docente e pessoal. Então, a partir de estudos anteriores, especialmente do que desenvolvi durante o mestrado (cf. SOUZA-SILVA, 2022), pretendo fornecer material teórico para uma agenda pedagógica anti-hegemônica e aberta para a diversi- dade linguística. Logo, justifico o interesse por essas ideias a fim de contribuir para o fortalecimento e o desenvolvimento da linguística, especificamente, da vertente da Sociolinguística Educacional (cf. BORTONI-RICARDO, 2017). As questões a serem levantadas aqui contribuem social- mente, haja vista a LGBTfobia ser uma realidade nacional (GASTALDI et al, 2021) e seu combate também ser possível de compreensão via aspectos da linguagem, uma vez que entendemos a linguagem como aquilo que o homem tem de mais íntimo e que representa sua sub- jetividade, não sendo exagero dizer que, quando críticas são feitas à linguagem de alguém, isso funciona como uma arma que fere tanto quanto qualquer outra atitude violenta, conforme indica Leite (2008). Nessa direção, o caráter pedagógico transparece, uma vez que os estudos sociolinguísticos colocam em evidência o papel social da língua(gem). Assim, destaco as possibilidades de reflexão dos/as docentes em formação inicial ou continuada considerando que as variedades linguísticas estigmatizadas também devem ser objetos de reflexão e análise linguística, como apontam documen- tos oficiais. Desta feita, é valioso realinhar uma educação sociolin- guística que não inflija, por exemplo, pessoas LGBTQIA+ (lésbica, gay, bissexual, transexual, travesti, queer, intersexo, assexual e mais)3 3 47 S U M Á R I O ao lugar da irracionalidade, da ignorância, do misticismo, da promis- cuidade e/ou da monstruosidade (SILVA, 2016), bem como outros sujeitos socialmente marcados. Ademais, sou motivado pessoalmente, tendo em vista meu compromisso com o combate a toda e qualquer discriminação, con- siderando minha própria realidade, história de vida e experiências humanas e acadêmicas, uma vez que tenho me interessado pelas discussões sobre linguagem, gênero e sexualidade desde a gradua- ção, tendo em vista algumas produções já realizadas a exemplo de Souza-Silva & Coppi (2020), Souza-Silva & Silva Jr (2021) e Souza- -Silva, Dias & Bezerra (2021). Por fim, este capítulo se organiza em seções, após este introito: 1) apresento considerações sobre o expoente campo da Sociolinguística e o que defendo ser uma conduta militante; logo depois 2) indico o papel político do ensino de língua(gem) mediante documentos oficiais; e finalizo com 3) reflexões teórico-analíticas para o contexto de formação docente e discente. INTRODUÇÃO Ao fim, teço consi- derações finais tocante ao tema geral. SOCIOLINGUÍSTICA(S): PARA ALÉM DE TEORIA(S), UMA CONDUTA! No século XX, os estudos linguísticos são legitimados com postulações teóricas e rigor metodológico a partir dos fundamentos de Ferdinand de Saussure – linguista suíço conhecido, na contem- poraneidade, como o pai da linguística moderna. A abordagem saus- suriana ficou conhecida como estruturalista, por evidenciar a face interna do signo linguístico e investigar a linguagem verbal numa sis- temática homogênea, desconsiderando, para os fins daquela época, questões extralinguísticas. Para tanto, trabalhou com as conhecidas 48 S U M Á R I O dicotomias, formadas por pares que se complementam e direcio- nam o olhar investigativo diante da língua. Paralelamente, fomen- tada e estabelecida a linguística junto ao seu objeto de investigação, outras vertentes de estudos da língua emergem no horizonte por analisar o sistema linguístico à luz do uso, a exemplo dos estudos da Sociolinguística. Para a Sociolinguística, devem ser feitas considerações internas e externas a respeito do signo linguístico. Desse modo, a investigação, nessa subárea, considerará não só a sistemática das línguas, mas suas relações pragmáticas, haja vista seu interesse não somente no uso, mas nos falantes e em como (re)configuram a língua, desempenhando sua função primordial, enquanto falantes, que é a de manterem a língua viva e funcional. Os estudos sociolinguísticos são muitos e a Sociolinguística é consagrada em âmbito nacional, tendo diversos estudos seja em micro ou em macro comunidades linguísticas brasileiras. Os estudos que consideram o linguístico e social como indissociáveis trabalham sob o “guarda-chuva” Sociolin- guística (cf. HORA, 2021): Dialetologia, Geolinguística, Sociolinguís- tica Interacional, Sociolinguística Educacional e a Sociolinguística Variacionista – esta última passa a englobar estudos que podem ser de primeira, segunda ou terceira onda (ECKERT, 2005; VELOSO, 2014; SOUZA-SILVA & LUCENA, 2021), bem como estudos de Teo- ria da Acomodação (GILES et al, 1991) e de Atitudes Linguísticas (cf. LUCENA, 2022). Nesse expoente, por exemplo, há a vertente Interacional (GUMPERZS, 1998), a qual também considera os aspectos sociais, investigando as variações que ocorrem na linguagem a partir de determinados contextos comunicativos a fim de analisar as diversi- dades linguística e cultural; bem como sua abordagem mais conhe- cida e difundida: a Variacionista, a qual investiga as influências extralinguísticas em direção à estrutura linguística, considerando inúmeros fatores para estabelecer processos de variação e a con- solidação de mudanças das línguas, uma vez que compreende que 49 “[...] pressões internas, estruturais, e as pressões sociolinguísticas agem em alternância sistemática [...]” (LABOV, 2008, p. 214). SOCIOLINGUÍSTICA(S): PARA ALÉM DE TEORIA(S), UMA CONDUTA! De modo mais emblemático para o que se pretende discutir aqui, temos o paradigma educacional da Sociolinguística, o qual é proposto por Bortoni-Ricardo (2017) e que se faz pelos esforços da aplicação de estudos sociolinguísticos quantitativos e qualitativos na solução de questões linguísticas no contexto da sala de aula e na proposição de trabalho pedagógico efetivo e funcional, paradigma este que dialoga com áreas afins e de diferentes abordagens da lin- guística contemporânea. Desse modo, concebo que reflexões, resul- tados e proposições da Análise do Discurso, da Linguística Aplicada, da Linguística Queer, da Linguística Funcional, etc. são relevantes para as interpretações no interior da comunidade educacional. Dentro dessa vertente proposta por Bortoni-Ricardo (2017), a autora elenca princípios que direcionam o fazer prático dos estudos dessa vertente sociolinguística e um deles é o de se conscientizar de modo crítico professores/as e alunos/as quanto à variação linguís- tica e à desigualdade social que ela reflete. Diante das sociedades pós-modernas, é pertinente pensar em abordagens sociolinguísti- cas que se façam em um continuum, no qual a linguagem interliga os eixos linguístico e sociocultural em um movimento encaixado tal qual de engrenagens. Desse movimento decorrem tanto questões estritamente linguísticas quanto amplamente sociais, possibilitando uma aborda- gem sociolinguística que vá de um extremo ao outro e com zona intermediária ampla. Nessa direção, podemos estabelecer, conforme Calvet (2002), que há estudos macrossociolinguísticos que estu- dam o que os/as falantes perpetram com suas línguas e variedades, considerando atitudes, ideologias e posições de poder; e estudos microssociolinguísticos que investigam em que medida a estrutura social influencia o uso e a produção das variedades linguísticas pelos falantes. Então, a primeira perspectiva está mais interessada em 50 S U M Á R I O compreender como a sociedade é configurada pela língua e a segunda se interessa por como a língua é configurada pela sociedade. Nesse sentido, é preciso compreender que é possível falar em Sociolinguísticas, de forma plural, não nos limitando a um quadro teórico único, logo, “[...] nos fechar na língua ou nos fechar na socie- dade nos condena à impotência” (CALVET, 2002, p. 126-127). Dito isso, para desenvolver uma pesquisa no campo da linguística, não se pode deixar de adotar uma perspectiva que guiará toda e qualquer discussão, crenças e ações frente ao objeto a ser analisado, inclusive na sala de aula. Portanto, compartilho com Bagno (2004, p. SOCIOLINGUÍSTICA(S): PARA ALÉM DE TEORIA(S), UMA CONDUTA! 10) uma abordagem que tem a seguinte premissa: “[...] o compromisso polí- tico de converter a sociolinguística num instrumento de luta contra toda forma de discriminação e de exclusão social pela linguagem. Porque não basta descrever e analisar as relações entre língua e sociedade – é preciso, também, transformá-las”. É nessa empreitada que considero uma Sociolinguística mili- tante, pois, conforme Bagno (2017), a língua também é uma bandeira por meio da qual grupos específicos se congregam para defender e/ ou reivindicar direitos, também sendo uma bandeira que o Estado e suas diferentes agências hasteiam com a finalidade de desempenhar sua política de controle social. A partir disso, além de adotar uma postura investigativa, deve-se assumir um conceito de língua. Para tanto, entendo o seguinte: A língua é sistema, ela é um conjunto de elementos inter- -relacionados em vários níveis, no nível morfológico, fonológico-morfológico, sintático. Mas ela só se realiza enquanto prática social, quer dizer, os seres humanos nas suas práticas sociais usam a língua e a língua só se configura nessas práticas e é constituída nessas práticas (KOCH, 2006, p. 124, grifos meus). A língua é sistema, ela é um conjunto de elementos inter- -relacionados em vários níveis, no nível morfológico, fonológico-morfológico, sintático. Mas ela só se realiza enquanto prática social, quer dizer, os seres humanos nas suas práticas sociais usam a língua e a língua só se configura nessas práticas e é constituída nessas práticas (KOCH, 2006, p. 124, grifos meus). Ao enaltecer a prática social, se coloca em ênfase que o interesse na prática linguística se manifesta nas variadas práticas sociais, não desconsidero a necessidade de um sistema linguístico, 51 mas me interesso pela língua no seio das práticas sociais. Assim, a Sociolinguística que defendo tem caráter transgressivo e se faz pelo foco em questões sociopolíticas que favorecem a existência ou não de determinados códigos linguísticos, bem como de determinadas práticas linguísticas e seus agentes. Ao abordar a questão sociopolítica centrada em práticas lin- guísticas, pode-se direcionar a comunidades específicas, como é o caso da comunidade LGBTQIA+. Dessa forma, é possível e per- tinente estar aberto para discussões sobre conceitos como iden- tidade, gênero, sexualidade e performatividade4, uma vez que a Sociolinguística, como a que defendo, se interessa pelas práticas lin- guísticas protagonizadas por sujeitos marginalizados e socialmente marcados. Este conceito abarcará, conforme delineamentos de Borba (2020), a compreensão tanto do corpo que fala quanto daquilo que se fala sobre o corpo, lançando um olhar para contextos de inter- ação particulares e os sentidos sócio-históricos-culturais que, conforme o autor, possibilitam e constrangem ações normativas que, simultaneamente, auxiliam em sua transformação. Assim, é possível ter uma visão dos usos linguísticos como oriundos de atos de fala performáticos, sendo fenômenos linguístico-corporais localmente inseridos e constituídos por ações sociológicas e dis- cursivas amplas. SOCIOLINGUÍSTICA(S): PARA ALÉM DE TEORIA(S), UMA CONDUTA! Dito isso, a relação entre língua e sociedade é muito mais extensa, íntima e complexa do que apenas influência ou reflexo, con- forme afirma Bagno (2012). Conforme Bagno & Rangel (2005 apud BAGNO, 2017), diz respeito à possibilidade de adquirir, desenvolver e ampliar o conhecimento linguístico e sobre outros sistemas semióticos a partir de fatores socioculturais, também favorecendo a ampliação de saberes acerca de crenças e repre- sentações que compõem a sociedade e seus grupos, o que os autores dizem compor o imaginário linguístico em nossa sociedade. O PAPEL POLÍTICO DO ENSINO DE LÍNGUA(S) Não tive a pretensão de lançar uma proposta pedagógica em minha dissertação, mas acredito na contribuição que minhas ideias e dados podem dar para as práticas de ensino de língua(s), uma vez que acredito em um ensino de língua que possibilite a construção de um “novo senso comum” sobre o uso da(s) língua(s), favorecendo a 52 tolerância com e pela linguagem. Desse modo, caso as discussões e dados sejam “didatizados” para a análise e reflexão linguísticas, opor- tuniza-se uma postura sociopolítica na sala de aula frente ao que foi coletado, haja vista os estereótipos que cercam as performances dos sujeitos com os quais trabalhei. E, conforme argumenta Rajagopalan (2013), por muito tempo, estudos foram conduzidos numa ideia pos- sível e legítima de se abordar a linguagem sem lançar olhares para as questões políticas que envolvem os fenômenos focalizados. Junto a essas ideias, documentos oficiais reconhecem a legi- timidade do trabalho com a variação linguística, demonstrando a importância dos estudos sociolinguísticos e suas contribuições para o ensino de língua(s). Desse modo, é importante que as práticas de combate ao preconceito linguístico e o ensino de língua(s) possam ser democráticas e que ocorram em vias de respeito e comprometi- mento com valores éticos, assim, a democratização do ensino tam- bém valoriza uma educação linguística5. Os Parâmetros Curriculares Nacionais de Língua Portuguesa (PCN), há mais de 20 anos, já apontaram para a necessidade de se trabalhar o preconceito linguístico como resultado de avaliações sub- jetivas dos grupos sociais, que deve ser combatido vigorosamente e que “a discriminação de algumas variedades lingüísticas (sic), trata- das de modo preconceituoso e anticientífico, expressa os próprios conflitos existentes no interior da sociedade” (BRASIL, 1998, p. 82). Já a Proposta Curricular de Língua Portuguesa para a Educa- ção de Jovens e Adultos (PCEJA), na proposição do que venha ser a prática de análise linguística, enfatiza que tal atividade tem cará- ter reflexivo voltado à língua o que auxilia no desenvolvimento de 5 53 habilidades intelectuais e na compreensão de aspectos do universo social (BRASIL, 2002), logo, oportuniza a análise de fatores sociais no que tange ao uso da língua. Este conceito é movediço nos estudos da linguagem, mas, aqui, concebo-o como a habilidade de ser consciente sobre as condições sociais, culturais e políticas que atravessam as práticas sociolinguísticas, nas quais os sujeitos estão inseridos, proporcionando combate às opressões mediadas pela linguagem (cf. BAGNO, 2017). O PAPEL POLÍTICO DO ENSINO DE LÍNGUA(S) Além dessa proposta, o documento Orientações Curriculares de Língua Portuguesa para o Ensino Médio (OCEM) diz ser, nas inte- rações em diferentes instituições sociais, que o sujeito aprenderá e apreenderá as formas de funcionamento da língua e seus diversos modos de manifestação pela linguagem, o que lhe possibilitará “[...] conhecimentos relativos aos usos da língua e da linguagem em dife- rentes situações” (BRASIL, 2006, p. 24). Por último, há a Base Nacional Comum Curricular (BNCC), documento que frisa a importância de se compreender, na educação básica, como a língua é fenômeno (geo)político, histórico, cultural, social, variável, heterogêneo e sensível aos contextos de uso. Essa compreensão possibilitará o reconhecimento de “[...] suas varieda- des e vivenciando-as como formas de expressões identitárias, pes- soais e coletivas, bem como agindo no enfrentamento de preconcei- tos de qualquer natureza” (BRASIL, 2018, p. 490). Apresentados esses excertos, identifico como o encontro das questões linguísticas e sociais faz parte da sistemática curricular, sendo legítimas e pertinentes não só para uma formação escolar, mas para o letramento6 dos/as discentes que são cidadãos/cidadãs e também serão atuantes em âmbitos profissionais diversos. Por- tanto, faz-se necessária uma prática pedagógica para o ensino de língua(s) que valorize todo e qualquer aspecto cultural como legí- timo, isso em relação a diferentes grupos. Desse modo, concordo com Leite (2011, p. 23): 6 54 O reconhecimento e aceitação da diversidade cultural significam o reconhecimento e aceitação de que, embora as culturas sejam diferentes umas das outras, são todas igualmente estruturadas, coerentes e complexas. Este pensamento derruba as teorias de que uma cultura é mais importante do que a outra, ou que um grupo social é, por natureza, moral, intelectual e culturalmente mais privilegiado do que outros. Fazer qualquer tipo de clas- sificação por hierarquia das culturas das populações é cientificamente incorreto. Assim, o ensino de língua(s) que se abre para diferentes aspectos da cultura dos/as próprios/as discentes, de grupos margi- nalizados, de povos originários, pessoas com deficiência, etc., possi- bilita um espaço de múltiplas identidades e colabora para suas mani- festações plenas. Portanto, devemos ser professores/as engajados/ as numa prática não só da variação linguística, mas que é contra a homogeneidade numa visão de perfeição, encanto e desejo. O PAPEL POLÍTICO DO ENSINO DE LÍNGUA(S) Então, destaco algo já defendido por mim: [...] não há justificativas para a inferiorização de gays, lésbicas, transexuais, travestis, bissexuais, ou quaisquer outras manifestações plurissignificativas da sexualidade e das identidades de gênero, uma vez que a aceitação da superioridade de um subjaz na inferiorização do outro. Assim, o que é aceitável é a adoção de práticas anti-he- gemônicas, as quais rejeitam a perpetuação do precon- ceito linguístico, evidenciando a heterogeneidade como espaço da pluralidade social (SOUZA-SILVA, 2020, p. 50). Essa perspectiva favorece a compreensão não só dos dife- rentes comportamentos linguísticos, mas também das diferentes manifestações identitárias, dentro e fora de nossas salas de aula. E, não podemos nos esquecer de que assumir posturas teóricas e metodológicas implica assumir também, conforme Oliveira & Wilson (2017), valores e crenças a elas vinculadas. Por fim, reforço a importância de um ensino de língua(s) ancorado nos estudos da linguagem com a predileção de instru- mentalizar os/as alunos/as para a leitura coerente, respeitosa e 55 S U M Á R I O científica daquilo que abrange os usos da língua, a exemplo da polê- mica que envolveu a questão do pajubá no ENEM (Exame Nacional do Ensino Médio) em 2018, e a respeito disso já defendi que não se trata do ensino da “linguagem de travesti” ou de persuadir os/as discentes a terem contato com práticas linguísticas desnecessárias, mas trata-se da aceitação de toda e qualquer variedade da língua como código válido para a prática de análise e reflexão linguística (cf. SOUZA-SILVA, 2020). REFLEXÕES PEDAGÓGICAS E PROPOSIÇÕES ANALÍTICAS Nesta seção, irei realizar duas ações: a) analisar, a partir de entrevista semiestruturada, a atitude de pessoas LGBTQIA+ – 1 mulher trans-heterossexual, 1 mulher cis-lésbica, 2 mulheres cis-bis- sexuais e 1 homem cis-gay – frente ao trabalho com a linguagem de seu grupo restrito em contexto de sala de aula e b) apresentar um fenômeno linguístico para análise em sala de aula, considerando as identidades sexuais e de gênero como variáveis a serem problema- tizadas. Desse modo, selecionei os enunciados dos/as participantes LGBTQIA+ quando questionados/as sobre a linguagem LGBTQIA+ ser um assunto para se refletir na sala de aula junto a estudantes, no bloco sobre consciência e avaliação sobre a diversidade linguística. Então, de modo objetivo, a atitude linguística é uma mani- festação da atitude social dos/as falantes no que tange, especifica- mente, à língua e ao uso que se faz dela no seio da sociedade. Posto isso, Baker (1992 apud HORA, 2011) indica que há alguns tópicos de interesse dos estudos de atitudes linguísticas, a exemplo da atitude em relação a grupos de línguas, comunidades e minorias, bem como em relação às lições de língua. Nessa direção, é oportuno indicar o 56 S U M Á R I O que pessoas LGBTQIA+ (indicadas como participantes a partir da letra “P”) valoram sobre a questão supracitada: Quadro 1 – O que pessoas LGBTQIA+ dizem sobre o estudo de sua linguagem de grupo em contexto escolar? P1 = Acredito que sim, pois o modo que as pessoas aprendessem sobre a linguagem LGBTQIA+ poderia evitar violências como transfobia e homofobia, que são casos graves (mulher transexual). P2 = Claro! É uma maneira de inclusão, acredito (mulher bissexual). P3 = Com certeza! Bom, é um dos grupos sociais que... estão em ascensão, né... de reconhecimento... político, social, enfim. Então, eu acho que a sala de aula é um local pra gente lidar com essas... é... novas expressões de liberdade, seja quanto à população LGBT, tudo que cerca essa população, ou quanto questões de gênero quando a gente fala da violência contra mulheres, contra idosos, enfim... eu acho que a sala de aula é o local pra gente lidar com isso [...] (mulher bissexual). P4 = Deveria ser trazido, sim... e eu acho por como questão de respeitar, sabe? REFLEXÕES PEDAGÓGICAS E PROPOSIÇÕES ANALÍTICAS Pra galera entender: olha, é o jeito que essa galera, que essa comunidade se expressa [...] (mulher lésbica). P5 = Sem dúvida alguma! Isso vai fazer com que as crianças e adolescentes que cada vez mais empoderadas nascem, tão nascendo aí, chegando é... à adolescência cada vez mais empoderadas, tenham cada vez menos dúvidas do local ao qual elas pertencem, da identidade que elas têm que vivenciar e de quem elas precisam ser, seelasquiseremsereoqueelasqueremfazercomsuasprópriasvidasecomseuspróprioscorpos(homemgay) Quadro 1 – O que pessoas LGBTQIA+ dizem sobre o estudo de sua linguagem de grupo em contexto escolar? P4 = Deveria ser trazido, sim... e eu acho por como questão de respeitar, sabe? E não pra virar chacota, porque como eu falei anteriormente, na maioria das vezes, uma palavra específica, essa questão do dialeto, ela é vista como pra gerar entretenimento, como pra gerar palhaçada, e eu acho que tem que ser além disso, entende? Então eu acho que poderia sim ser trazido em sala de aula tanto pra questão de conhecimento quanto pra romper essas barreiras do preconceito, né? Pra galera entender: olha, é o jeito que essa galera, que essa comunidade se expressa [...] (mulher lésbica). P5 = Sem dúvida alguma! Isso vai fazer com que as crianças e adolescentes que cada vez mais empoderadas nascem, tão nascendo aí, chegando é... à adolescência cada vez mais empoderadas, tenham cada vez menos dúvidas do local ao qual elas pertencem, da identidade que elas têm que vivenciar e de quem elas precisam ser, se elas quiserem ser e o que elas querem fazer com suas próprias vidas e com seus próprios corpos (homem gay). Fonte: o autor REFLEXÕES PEDAGÓGICAS E PROPOSIÇÕES ANALÍTICAS E não pra virar chacota, porque como eu falei anteriormente, na maioria das vezes, uma palavra específica, essa questão do dialeto, ela é vista como pra gerar entretenimento, como pra gerar palhaçada, e eu acho que tem que ser além disso, entende? Então eu acho que poderia sim ser trazido em sala de aula tanto pra questão de conhecimento quanto pra romper essas barreiras do preconceito, né? Pra galera entender: olha, é o jeito que essa galera, que essa comunidade se expressa [...] (mulher lésbica). P5 = Sem dúvida alguma! Isso vai fazer com que as crianças e adolescentes que cada vez mais empoderadas nascem, tão nascendo aí, chegando é... à adolescência cada vez mais empoderadas, tenham cada vez menos dúvidas do local ao qual elas pertencem, da identidade que elas têm que vivenciar e de quem elas precisam ser, se elas quiserem ser e o que elas querem fazer com suas próprias vidas e com seus próprios corpos (homem gay). Fonte: o autor Quadro 1 – O que pessoas LGBTQIA+ dizem sobre o estudo de sua linguagem de grupo em contexto escolar? P1 = Acredito que sim, pois o modo que as pessoas aprendessem sobre a linguagem LGBTQIA+ poderia evitar violências como transfobia e homofobia, que são casos graves (mulher transexual). P2 = Claro! É uma maneira de inclusão, acredito (mulher bissexual). P3 = Com certeza! Bom, é um dos grupos sociais que... estão em ascensão, né... de reconhecimento... político, social, enfim. Então, eu acho que a sala de aula é um local pra gente lidar com essas... é... novas expressões de liberdade, seja quanto à população LGBT, tudo que cerca essa população, ou quanto questões de gênero quando a gente fala da violência contra mulheres, contra idosos, enfim... eu acho que a sala de aula é o local pra gente lidar com isso [...] (mulher bissexual). P4 = Deveria ser trazido, sim... e eu acho por como questão de respeitar, sabe? E não pra virar chacota, porque como eu falei anteriormente, na maioria das vezes, uma palavra específica, essa questão do dialeto, ela é vista como pra gerar entretenimento, como pra gerar palhaçada, e eu acho que tem que ser além disso, entende? Então eu acho que poderia sim ser trazido em sala de aula tanto pra questão de conhecimento quanto pra romper essas barreiras do preconceito, né? Fonte: o autor. A mulher trans-heterossexual – P1 – acredita que o contato com a linguagem da comunidade LGBTQIA+, no contexto escolar, evitaria violências de cunho homo e transfóbico. Nessa direção, a compreensão dos fatores que envolvem a criação da linguagem de grupo é relevante porque representa a possibilidade de adqui- rir conhecimentos relacionados a saber a língua e a saber sobre a língua, conforme indica Martins (2013). Logo, os fatos linguísticos podem receber um olhar crítico frente às discriminações pela lingua- gem e à exclusão social. 57 A mulher cis-bissexual – P2 – também defende o mesmo argumento, afirmando ser uma forma de inclusão, reconhecendo que a escola possa ser um espaço para o debate sobre a heterogenei- dade. Desse modo, ao indicar que tal ação é algo inclusivo, a parti- cipante oportuniza pensarmos na construção da cidadania dos/as discentes. Então, figura-se um dos objetivos centrais da escola que, de acordo com Antunes (2009), favorece a participação consciente, crítica e relevante dos/as discentes na construção de uma sociedade mais justa onde todos/as têm vez e voz. Também há P3 – mulher cis-bissexual. Essa participante defende que a ascensão da comunidade LGBTQIA+ no que tange à representatividade, crescente social e politicamente, favorece o contexto da sala de aula como oportuno para o trato das diferen- tes “expressões de liberdade”, como ela chama, e tudo que cerca a população dessa comunidade, logo, não se limita ao espaço da sala de aula de língua(gem). Assim, como indica P3, a sala de aula é um lugar para lidarmos com isso, uma vez que “tudo que é contrário, pois, ou diferente do padrão estabelecido pela escola como legítimo, é deficiente e deve ser modificado pelo ensino” (LEITE, 2011, p. 22), posição que rejeito, haja vista hierarquias culturais, sociais e linguís- ticas serem anticientíficas. O enunciado de P4 – mulher cis-lésbica – corrobora o de P1, favorecendo um ensino de língua e sobre a língua, beneficiando a compreensão das demandas discursivas que envolvem a linguagem, uma vez que P4 defende um ensino que possa conscientizar os/as discentes sobre sujeitos LGBTQIA+ não serem tratados/as em tom jocoso, abandonando posturas estereotipadas sobre o que possa ser gay, lésbica, travesti, bissexual, etc., não servindo unicamente ao riso e à piada. Desse modo, promover a conscientização do “[...] grande significado da linguagem para a construção do sentido de todas as coisas” (ANTUNES, 2009, p. 43, grifos meus). Fonte: o autor. 58 Por último, temos as posições de P5 (homem cis-gay), as quais favorecem a possibilidade de os/as discentes terem espaço e liberdade para vivenciarem, assim como outros/as cidadãos/ãs que experienciam suas identidades sexuais e de gênero não dominantes, com respeito e segurança. Logo, a abertura da sala de aula para a o trato da linguagem LGBTQIA+ favorece esse entendimento, “se elas quiserem ser e o que elas querem fazer com suas próprias vidas e com seus próprios corpos”, algo que não é simples para nosso sis- tema de ensino, haja vista a escola ser produtora de diferenças, dis- tinções e desigualdades, como aponta Louro (2014). Além disso, a autora nos afirma que a sistêmica escolar na qual vivemos foi ela- borada pela sociedade ocidental moderna que começou separando adultos de crianças, depois católicos de protestantes e “[...] também se fez diferente para os ricos e para os pobres e imediatamente sepa- rou meninos de meninas” (LOURO, 2014, p. 61). Essas ideias servem para conscientizar os/as professores/ as sobre o que pessoas LGBTQIA+ podem argumentar sobre suas identidades sexuais e de gênero quando lançam olhares para o papel da linguagem na manutenção dessas identidades e de suas perfor- mances, favorecendo a reflexão sobre o papel da escola e das aulas de língua(gem) diante disso. Na direção dessas reflexões, o/a profes- sor/a pode refletir com os/as alunos/as sobre o seguinte fenômeno: o uso do vocativo. E, de acordo com a investigação de Nogueira (2019) – em uma comunidade gay de Serra Talhada-PE – os termos bicha, mulher [mulhé, mulé], viado e amiga são os que mais fun- cionam como vocativos. De forma geral, a investigação de Nogueira (2019) indicou que 52% dos vocativos têm maior frequência de uso na conversa entre amigos do que no contexto do trabalho e da entrevista reali- zada por ela. Um dado interessante da pesquisa é que os sujeitos participantes realizam uma dupla marcação de vocativo, uso não reconhecido pela GT (Gramática Tradicional), fugindo dos padrões do que se considera falar e escrever bem. Vejamos alguns exemplos 59 S U M Á R I O selecionados do corpus de Nogueira (2019): a) Larissa, eu amo essa casa, bicha; b) Mulé, a senhora tava apressando, mulé; e c) Mulhé, deixe de ser baixo, viado. Fonte: o autor. É possível identificar em (a) o uso do termo ‘bicha’ tendo como referente ‘Larissa’; em (b) a variante ‘mulé’ é repetida para reforçar o direcionamento da mensagem ao interlocutor e, em (c), o uso de ‘mulhé’ e ‘viado’ como marcadores do mesmo receptor, o qual só podemos presumir que seja do sexo masculino por causa do uso do adjetivo “baixo”, em gênero masculino na oração. Afinal, é comum a prática de tratamento por “mulher” entre homossexuais masculinos. Esses usos permitiram à pesquisadora estabelecer que “[...] o uso do duplo vocativo serve ao propósito comunicativo como recurso enfático, dado o seu exagero de marcação do destinatário na conversa” (NOGUEIRA, 2019, p. 69). Então, mediante a coleta de Nogueira (2019), selecionei os vocativos e solicitei que os/as 20 participantes (divididos em Grupo 1: LGBTQIA+ e Grupo 2: Cis/Héteros) indicassem sua preferência de uso em relação a esse recurso linguístico, sendo também uma ação investigativa do contexto das atitudes linguísticas. Diante disso, os resultados obtidos com os 20 participantes indicam o seguinte: Gráfico 1 – Preferência de vocativos associados às práticas de LGBTQIA+ Fonte: o autor. Gráfico 1 – Preferência de vocativos associados às práticas de LGBTQIA+ Gráfico 1 – Preferência de vocativos associados às práticas de LGBTQIA+ Fonte: o autor. 60 60 Ao observar o Gráfico 1, identifica-se que os LGBTQIA+ lideram o uso dos itens. Os vocativos bicha, mulher, amiga e gata estão entre os mais frequentes na pesquisa de Nogueira (2019), sendo indicados por nossos/as participantes LGBTQIA+ como de uso corrente em suas comunicações cotidianas, mas bicha guarda maior marcação LGBTQIA+ do que os demais, corroborando a análise da autora, junto ao correlato viado, de acordo com a qual esses vocativos reafirmam as identidades desviantes no contexto da comunidade em questão. E, um informante do gênero masculino e outra do feminino ambos do grupo Cis/Hétero indicaram o uso do item bicha, acentu- ando nossa interpretação de que não devemos ser categóricos em análises estilísticas, uma vez que, conforme Freitag (2015), generali- zações são perigosas, inclusive aquelas generalizações feitas a partir de resultados de sexo/gênero em estudos sociolinguísticos nacionais. Vale a interpretação sobre o uso do termo bicha, o qual é tão comum e corriqueiro para LGBTQIA+, mas que por percepção comum é tão utilizado como insulto entre homens, seja numa espécie de “brincadeira” ou com intenção de atacar, verdadeiramente, suas masculinidades. Uma investigação de Souza-Silva, Dias & Bezerra (2021), sobre o uso de termos de chamamento em contexto escolar e seus níveis de agressividade nesse mesmo contexto, indicou bicha como o mais frequente e entre os três tidos como mais agressivos. Logo, devemos estar conscientes que no interior das comunidades, especialmente as de prática, o termo bicha funciona com diferentes intenções, mas sempre associado e tendo como referência o sujeito homossexual, particularmente, afeminado. De acordo com Green (2019), sobre esse termo não se sabe a origem exata, há a hipótese de que tenha aparecido no começo do século 20, bem como se acredita que seja uma adaptação do vocábulo francês “biche”, que significa corsa, feminino de veado. O termo “biche” também era usado para se referir a uma jovem mulher 61 S U M Á R I O francesa. Já no contexto brasileiro, o item “biche”, que viraria “bicha”, seria usado no início do século 20 para designar prostitutas, espe- cialmente, prostitutos afeminados. Dito isso, o termo está associado aos domínios do feminino, remetendo à fragilidade e passividade, características que são socialmente inferiorizadas e colocadas como opostas ao que é masculino. A partir disso, fica em desataque que, num movimento de “inversão performativa da injúria” (BUTLER, 1997 apud BORBA, 2020), LGBTQIA+ ressignificam seus chamamentos em símbolo de batalha e indignação social, como ocorre também com viado, sapa- tão e travesti. Nesse sentido, temos um mecanismo que indica a materialização linguística do efeito espada/escudo (SOUZA-SILVA, 2022), bem como ocorreu com o termo queer no contexto estaduni- dense. Sobre essa ressignificação: “[...] uma estratégia de ressignifi- cação que vira a ofensa do avesso, dobra-a sobre si e se apropria de seu poder político para produzir lugares de identificação e aliança. Essas reapropriações de sentido são uma das principais estratégias de contestação queer [...]” (BORBA, 2020, p. 10). Nessa direção, acrescento que para além de se reapropriar do termo, faz-se necessário que no interior da comunidade LGBT- QIA+, especialmente entre homens gays, que o uso de bicha seja valorado para além de parâmetros de feminilidade, construindo entre cis-gays, que se identificam como “másculos”, uma cultura de compreensão sobre a importância em não estereotipar o que seja ser bicha, pois não há hierarquia sexual entre ser bicha, viado, mona, gay, homossexual, etc., uma vez que esses termos foram cria- dos para rotular uma questão geral: homens que sentem atração sexual por outros homens. Outrossim, compreendo que, como indica Trevisan (2018), por força da Aids – e a doença ter sido rotulada como “peste gay” – muitos queriam se distanciar da figura da bicha feminina, tão recor- rente entre os anos 60 e 70. Entretanto, a diferença que existe entre 62 S U M Á R I O esses rótulos é atributo social, resultado de diversas violências por força de diferentes questões, como a socioeconômica. Portanto, a ideia de que “existe o gay e existe a bicha” deve ser fortemente com- batida no interior da comunidade, para que possamos dar um passo à frente no combate à discriminação. Para além da preferência desses itens, é possível refletir sobre a posição junto às orações. No estudo de Nogueira (2019), verifica-se maior ocorrência dos vocativos na posição final (44%), à direita; seguida pela posição inicial (19%), à esquerda; e dupla mar- cação (vocativo + oração + vocativo) como a menos usual (8%). No andamento das análises e comparações, a autora indica que não há uma especificidade em relação ao posicionamento do vocativo na “fala gay”. Entretanto, pareceu oportuno observar essa preferência em meio aos grupos LGBTQIA+ e Cis/Hétero da pesquisa que rea- lizei. Para tanto, essas posições foram apresentadas aos/às partici- pantes e obtive os seguintes dados: Gráfico 2 – Preferência de localização do vocativo em sentença Fonte: o autor. Como se identifica no Gráfico 2, há LGBTQIA+ que indicam preferência nas três posições. Entretanto, 50% preferem a ordem em posição final junto à oração, pouco maior que os Cis/Héteros (40%); já em relação à ordem inversa, Cis/Héteros saem na frente, regis- trando 60% e LGBTQIA+ 30%. Logo, em relação às posições padrão Gráfico 2 – Preferência de localização do vocativo em sentença Fonte: o autor. Gráfico 2 – Preferência de localização do vocativo em sentença Gráfico 2 – Preferência de localização do vocativo em sentença Gráfico 2 Preferência de localização do vocativo em sentença Fonte: o autor. Como se identifica no Gráfico 2, há LGBTQIA+ que indicam preferência nas três posições. Entretanto, 50% preferem a ordem em posição final junto à oração, pouco maior que os Cis/Héteros (40%); já em relação à ordem inversa, Cis/Héteros saem na frente, regis- trando 60% e LGBTQIA+ 30%. Logo, em relação às posições padrão 63 S U M Á R I O e reconhecidas formalmente pela GT, os dois grupos guardam maior preferência por esses usos. Entretanto, 20% do grupo LGBTQIA+ (um gay e uma bissexual) indicou preferência pela dupla marcação, posição não reconhecida pela GT, afinal, para os preceitos desse antiguíssimo compêndio, marcar um item duas vezes seria algo desnecessário, mas “[...] o uso do duplo vocativo serve ao propósito comunicativo como recurso enfático [...]” (NOGUEIRA, 2019, p. 69). Portanto, a depender da intenção enunciativa do/a falante/ escrevente, a ordem dos itens que compõem um enunciado pode ser modificada. Assim, caso o/a falante/escrevente tenha interesse em colocar em evidência mais o interlocutor do que o acontecimento, pode trazer o vocativo para a posição inicial junto à oração para que ganhe maior destaque do que o acontecimento, também podendo ocorrer ao contrário: para evidenciar mais o acontecimento do que o interlocutor, o/a falante/escrevente leva o vocativo para a posição final junto à oração, colocando a ação em destaque, por exemplo. Portanto, é no jogo das atividades linguísticas que se pode mapear essas intenções discursivas, considerando variáveis diver- sas, como tópico da conversa, interlocutores, seus papéis sociais, etc. Todavia, de modo mais geral, podemos indicar que há LGBT- QIA+ que optam por marcar como usual aqueles itens mais estigma- tizados, consequentemente, tornando-os mais marcados. Diante da noção de que a escola é nossa principal agência de letramento formal e responsável, por vezes, pela perpetuação de estigmas sociais e valoração da GT como modelo ideal de língua, mas tal espaço é relevante para a desmistificação, compreensão e reflexão de estigmas. Logo, a Sociolinguística Educacional pode favorecer o trato dessas questões nas aulas de língua(gem). Assim, as amostras de análises aqui desenvolvidas favorecem o estudo da variação linguística, a compreensão da normatividade, o impacto da identidade social no uso da língua e a leitura crítica de fenômenos linguísticos estilísticos. 64 Nesse cenário, pode o/a professor/a de língua portuguesa, antes de analisar tais fenômenos com os/as alunos/as, solicitar que eles/as respondam quais vocativos usariam, qual a posição de voca- tivo que preferem, outros vocativos comuns nas comunicações do contexto familiar e da escola, o que acham sobre o uso desses voca- tivos que marcam identidades sexuais e de gênero, possibilitando um trabalho sociolinguístico na sala de aula, mas sempre ciente de que não é só adentrar em sala com um almanaque de variantes linguísti- cas, pois a militância está na postura frente aos discursos intoleran- tes, logo, o professor de Português deve abandonar o  título de caça- -erros e passar a ser agente combativo da exclusão pela linguagem. CLOSES FINAIS A Sociolinguística na qual acredito não se preocupa ape- nas com questões teóricas e metodológicas, trata-se, também, de uma postura frente aos diferentes falares e falantes marginalizados/ as que têm suas identidades marcadas socialmente por diferentes fatores: raça, etnia, classe social, origem, gênero e sexualidade. Essa postura é combativa, é militante, resultando numa Sociolinguística de caráter transdisciplinar, pois a leitura das práticas linguísticas de grupos mais herméticos lança mão do que nomeio como arsenal da Sociolinguística. Tal arsenal conglomera ideais variacionistas, pois parte das variantes e variáveis linguísticas, buscando compreender em que medida os processos são independentes e quais são condicionados; interacionais, porque considera a influência dos contextos comuni- cativos no que tange ao uso das variantes linguísticas; discursivos, já que mapeia os efeitos de sentido, as intenções e atitudes que envol- vem os usos linguísticos, bem como dialoga com a questões educa- cionais, para evidenciar práticas pedagógicas anti-hegêmonicas que 65 S U M Á R I O viabilizem a reflexão sobre todo e qualquer código linguístico e seus condicionadores sociolinguísticos. Essa conduta possibilita o empoderamento linguístico, o qual deve ser compreendido no propósito de que os/as falantes não que- rem apenas representar a língua, mas também se sentirem repre- sentados/as por ela. Desse modo, entendo que o preconceito lin- guístico é poderoso por sua “invisibilidade”, sendo concretizado na discriminação pela linguagem, assim, o empoderamento linguístico é a “visibilidade”, uma vez que a performance pela linguagem poderá fornecer proatividade aos/às falantes, concretizando elevação de poder, ampliação política e mudanças na qualidade de vida, desde que falantes estejam conscientes da dialogicidade desse fenômeno. Por fim, acredito que os desdobramentos das ideias aqui defendidas favorecem a Sociolinguística contemporânea, tendo valia por evidenciar um grupo minoritário, seus usos linguísticos e performances, oportunizando direcionamentos pedagógicos, pois revela usos marginais à GT e evidencia a influência de identidades no momento de uso da linguagem. REFERÊNCIAS ANTUNES, Irandé. Língua, texto e ensino: outra escola possível. São Paulo: Parábola Editorial, 2009. BAGNO, Marcos. Dicionário crítico de Sociolinguística. São Paulo: Parábola Editorial, 2017. 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Devassos no Paraíso: a homossexualidade no Brasil, da colônia à atualidade. 4. Ed. Rio de Janeiro: Objetiva, 2018. VELOSO, Rafaela. As três ondas da Sociolinguística e um estudo em comunidades de práticas. In: XVII Congreso Internacional Asociación de Lingüística y Filología de América Latina. João Pessoa: Ideia, 2014, p. 1740-1749. 69 S U M Á R I O INTRODUÇÃO Desde a Brasil colônia até os dias atuais, assistimos a um cenário político educacional enredado por políticas linguísticas implí- citas, ou seja, ações que interferem na dinâmica linguística de uma língua, mas não nominadas como política linguística, porque não há uma legislação explícita sobre ela. Entretanto, as políticas públicas e linguísticas – sejam elas explícitas ou implícitas, nortearam ações que, em certa medida, delinearam os usos linguísticos e o ensino de língua portuguesa. Diante disso, neste capítulo faço um recorte da pesquisa inti- tulada “Políticas e ideologias linguísticas: uma análise dialógica das avaliações do Enade”, no qual apresento um breve panorama das políticas educacionais que orientam as políticas de línguas no Brasil (MICHALKIEWICZ, 2020). A grosso modo, como destaca Bagno (2020), no Brasil não temos políticas linguísticas definidas e as poucas que tivemos foram impositivas e não democráticas. A primeira foi a política repressora do Diretório dos Índios, quando Pombal decretou o uso da Língua Portuguesa em todo território nacional, suplantando a língua geral e todas as línguas indígenas. A segunda diz respeito à Era Vargas, que decretou o fim das línguas imigrantes, sobretudo o italiano e o alemão. O pesquisador faz essa afirmação em contraste com as políticas linguísticas e investimentos de outros países da Europa e da América Latina. Todavia, não se pode deixar de destacar que embora as polí- ticas tenham inicialmente o cunho impositivo, atualmente temos a Lei nº 10.436, de 24 de abril de 2002, que dispõe sobre a Língua Brasileira de Sinais – Libras, as Leis de cooficialização de línguas indígenas e línguas de imigração em alguns estados. Ainda que não sejam legislações federais, as cooficializações são políticas explícitas 71 e tem seu valor. Embora Bagno (2020) destaque que as leis estaduais ficam à mercê de mudanças políticas locais que votam pela retirada delas, por isso são legislações mais frágeis na visão do pesquisador. COMPREENDENDO AS POLÍTICAS EDUCACIONAIS QUE ORIENTAM AS POLÍTICAS LINGUÍSTICAS NO BRASIL No Brasil, a instituição escolar surgiu em 1849, quando Padre Manuel da Nóbrega e a Companhia de Jesus organizaram as casas de Be-a-bá, em Salvador, na Bahia. Entretanto, apesar de a escola fazer parte da história do país desde o Brasil Colônia, a educação brasileira sempre foi destinada a poucos. As casas de Be-a-bá serviam para catequizar as crianças ameríndias por meio dos catecismos bilíngues em Tupi-Português. Mais tarde, as escolas da Companhia de Jesus serviram para educar os filhos dos proprietários de terras e negros escravizados. Contudo, a hegemonia da Companhia de Jesus teve fim em 1759, quando aconteceram as reformas pombalinas, as quais instituí- ram as aulas régias de língua portuguesa, do latim e do grego, além de retórica e filosofia, proibindo o uso da língua geral e todas as línguas indígenas. Vale destacar que os padres da companhia exerceram o controle da educação no Brasil durante 210 anos, por isso a história da educação está entrelaçada com a educação europeia via igreja. Ferreira Jr (2010, p. 19), em contraste, afirma que “os traços da nossa sociedade são alicerçados na escravidão, latifúndio e na monocultura e, devido a essa sociedade, poucos avanços ocorriam na educação, já que ela era praticamente inexistente”. 72 A catequização da Companhia de Jesus começou com os adultos, porém, devido à resistência dos ameríndios em relação à cultura, os padres se voltaram para os curumins. Evidencia-se a ide- ologia que as crianças eram neutras e não contaminadas com os elementos culturais do seu povo, assim seria mais fácil incorporarem a cultura da civilização em detrimento da barbárie indígena. O primeiro passo foi gramaticar a língua em um catecismo bilíngue em português e tupi, entendida aqui como um documento de política linguística no país, ainda que implícita, já que alterava os usos da língua indígena e determinava o ensino de língua na escola. Assim sendo, os catecismos jesuíticos do século XVI se constituíram num instrumento de duplo significado: de um lado, possibilitavam o aprendizado das primeiras Letras tanto no português quanto no tupi, isto é, transforma- ram-se em “cartilhas” que eram utilizadas como material didático do processo pedagógico desenvolvido no âmbito das casas de bê-á-bá, embriões dos futuros colégios da Companhia de Jesus e, do outro, veiculavam a concepção de mundo da chamada “civilização ocidental cristã” por meio da violência simbólica contra os elementos estrutu- rais da cultura ameríndia (FERREIRA JR, 2010, p. 21). COMPREENDENDO AS POLÍTICAS EDUCACIONAIS QUE ORIENTAM AS POLÍTICAS LINGUÍSTICAS NO BRASIL Além da língua geral gramaticizada pelos padres, havia o nheengatu, que quer dizer “língua boa”. Convém ressaltar que o nheengatu era também uma língua geral – a língua geral da Amazô- nia, assim como havia a língua geral paulista. A língua nheengatu foi levada durante a conquista portuguesa da Amazônia a regiões onde não era nativa. Ainda hoje o nheengatu é uma língua cooficial de São Gabriel da Cachoeira. Ela foi também a língua geral da catequese do norte e nordeste do Brasil. O método de ensino jesuítico – o Ratio Studiorium – era por meio da exposição de regras concisas, num ensino mnemônico. O método foi elaborado no final do século XVI, centrado nos princí- pios herdados da Universidade Medieval, era utilizado em todas 73 S U M Á R I O as regiões do Novo Mundo em fase de ocupação. Um método de ensino que garantia a padronização, a formação uniforme de todos os que viessem a frequentar os colégios da Companhia de Jesus em qualquer lugar do mundo. Compreende-se então que foi o primeiro material didático, na época entendido como um manual prático e sistematizado, já que apresentava ao professor a metodologia de ensino a ser utilizada em suas aulas. Mariani (2003) e Severo (2018) elucidam que nesse contexto complexo de catequização e colonização do novo território, as polí- ticas linguísticas jesuíticas estavam numa “arena conflito” (SEVERO, 2018). De um lado, a Igreja objetivava catequizar o maior número de não cristãos e, para que houvesse a compreensão dessa relação sócio-discursiva, a companhia de Jesus não se furtava a utilizar a língua geral que mediava o processo de comunicação entre os mis- sionários e os gentis. No entanto, a igreja tinha um acordo com a coroa para a expansão das terras da metrópole, que tem a língua portuguesa como língua nacional gramatizada e escrita, a qual, por sua vez, representa a civilização. Nesse cenário sociolinguístico em que residem línguas indí- genas, língua geral e língua portuguesa, se a igreja alfabetizar em língua geral já há o apagamento das demais línguas indígenas, mas também ocorre o silenciamento da língua portuguesa. Dessa forma, é preciso haver um consenso para atender tanto a realeza quanto a igreja num projeto político-linguístico, pois “Legitimam-se, em uma concepção linguística, uma teoria religiosa e uma outra de natureza político-jurídica, ambas servindo como justificativa para expansão das terras da metrópole” (MARIANI, 2003, p. 75). COMPREENDENDO AS POLÍTICAS EDUCACIONAIS QUE ORIENTAM AS POLÍTICAS LINGUÍSTICAS NO BRASIL Mariani (2003, p. 75) ainda destaca que a heterogeneidade linguística, em que os sentidos precisam ser construídos, “são deter- minados em situações enunciativas singulares, situações histórica e paulatinamente engendradas que vão dando lugar ao surgimento de uma língua e de sujeitos nacionais”. A pesquisadora conclui que 74 a ideologia de uma língua está aglutinada numa junção linguísti- co-jurídica em que a língua portuguesa é uma instituição nacional. Dessa forma, em um cenário linguístico complexo, a língua portu- guesa passa a ser ensinada com base no latim. 1. Controle disciplinar rígido das normas pedagógicas estabelecidas; 2. Repetição (leitura por meio da memori- zação/aprendizagem mnemônica); 3. Disputas (emulação entre os grupos de alunos da mesma turma tendo como conteúdo as obras lidas, ou seja, exercícios coletivos de fixação dos conhecimentos por meio de perguntas e res- postas); 4. Composição (redação de textos tendo como referência os temas de estudo); 5. Interrogações (ques- tões formuladas sobre as obras clássicas latinas estuda- das); 6. Declamação (exposição oral dos conhecimentos aprendidos por meio da retórica); 7. Prática sistemática de exercícios espirituais (FERREIRA JR, 2010, p. 29). S U M Á R I O Com essa perspectiva de ensino e controle disciplinar rígido, os colégios jesuítas eram considerados os melhores do mundo, porém eram fortes na formação humanística e intelectual e fracos nas ciências naturais. Enquanto as escolas das primeiras letras nas- ceram destinadas a catequizar os indígenas, os colégios eram para ensinar os filhos dos proprietários de terras. Os escravizados, indí- genas adultos, mestiços e brancos pobres ficavam de fora da escola porque eram destinados ao trabalho braçal. Em 1700, o reino de Portugal passava por uma crise econô- mica e a política do padroado existente entre o estado e a igreja per- mitiu à igreja poder paralelo ao governo português, tanto do ponto de vista econômico quanto político. Durante os 210 anos de atuação da Companhia de Jesus no Brasil, o poder político e econômico dos padres era maior que do próprio rei. Nesse contexto político, Marquês de Pombal, então primei- ro-ministro da coroa, percebeu o atraso lusitano em relação à trans- formação econômica da Europa Ocidental e promoveu mudanças 75 dos aparelhos estatais que administravam o império português pelo mundo. COMPREENDENDO AS POLÍTICAS EDUCACIONAIS QUE ORIENTAM AS POLÍTICAS LINGUÍSTICAS NO BRASIL Diante disso, Pombal assinou o tratado de Madrid, ampliando as fronteiras do norte e do sul, e acusou os jesuítas de conspiração devido ao poderio econômico, expulsou-os de Portugal e seus domínios em 1759. [...] o Marquês de Pombal expulsou a Companhia de Jesus (1759) dos domínios portugueses e, em seguida, promo- veu uma reforma educacional que extinguiu o sistema de ensino jesuítico, pois, para ele, o atraso lusitano em relação à modernidade gerada pelo mundo burguês era causado pela ação jesuítica na gestão dos negócios do Estado português (FERREIRA JR, 2010, p. 30). S U M Á R I O A partir da expulsão, as missões passaram a ser controla- das por funcionários do governo. As capelas foram transformadas em paróquias e os vigários que as assumiram eram nomeados pelo rei. Os indígenas tiveram que deixar de usar seus nomes originais, passando a ter nomes portugueses. Os caciques foram nomeados capitães e juízes e as lideranças passaram a ser vereadores muni- cipais. A política repressiva determinou que todos os indígenas, a partir daquele momento, eram cidadãos portugueses. Não é neces- sário explicitar o grande prejuízo dessa política para as comunida- des indígenas. A educação, que antes era exclusividade dos jesuítas, sofreu um grande recuo porque não havia professores suficientes para atender o público. Talvez uma das mudanças mais agressivas foi em relação ao idioma. O Marquês de Pombal tornou obrigatório o uso da língua portuguesa no Brasil, por meio de reformas no ensino de Portugal e suas colônias. O português foi incluído no currículo nacional e o uso de outras línguas totalmente proibido, especialmente a língua geral de base tupi, falada tanto por indígenas quanto pelos coloniza- dores e seus filhos. Essa política impositiva de Pombal dialoga com o domínio de terras e da nação, introduzindo uma nova língua que impõe a 76 desculturalização dos povos nativos. Processo esse que definia tal imposição como civilização do Bárbaro. Institucionaliza-se, na colônia, A língua portuguesa com SUA memória de filiação ao latim. O Diretório busca colo- car em silêncio a língua geral e seus falantes, caracteri- zando-a como uma “invenção diabólica”. Não se fala em um português-brasileiro. Ele ou não existe aos olhos da metrópole, ou, se existe, precisa ser corrigido, melhorado, reformatado de acordo com os moldes gramaticais por- tugueses. Aos olhos da metrópole precisa ser a continui- dade da imaginária homogeneidade que confere o caráter nacional a Portugal. COMPREENDENDO AS POLÍTICAS EDUCACIONAIS QUE ORIENTAM AS POLÍTICAS LINGUÍSTICAS NO BRASIL Mas os processos históricos, como se sabe, são continuidade e mudança sempre (MARIANI, 2003, p. 78, grifos da autora). Institucionaliza-se, na colônia, A língua portuguesa com SUA memória de filiação ao latim. O Diretório busca colo- car em silêncio a língua geral e seus falantes, caracteri- zando-a como uma “invenção diabólica”. Não se fala em um português-brasileiro. Ele ou não existe aos olhos da metrópole, ou, se existe, precisa ser corrigido, melhorado, reformatado de acordo com os moldes gramaticais por- tugueses. Aos olhos da metrópole precisa ser a continui- dade da imaginária homogeneidade que confere o caráter nacional a Portugal. Mas os processos históricos, como se sabe, são continuidade e mudança sempre (MARIANI, 2003, p. 78, grifos da autora). S U M Á R I O As mudanças preconizadas por Pombal eram desarticuladas e precisavam de unidade, o literário e verbalista da companhia de Jesus foi comprometido pelas ações de Pombal e causaram a falta de recursos para a educação, vinte anos após a expulsão, em toda a Bahia não havia mais que dois professores. Várias escolas foram fechadas e as bibliotecas dos conventos foram abandonadas ou destruídas. As aulas régias só passaram a ser ministradas depois de 1722, quando a coroa portuguesa instituiu o subsídio literário. Ade- mais, os projetos de Pombal estavam em concordância com a evolu- ção do mundo urbano burguês, porque ele queria manter os marcos de um império colonial, o que resultou na estagnação e demora para a implantação de universidades no país. É pertinente lembrar que com a vinda da família real para o Rio de Janeiro, foi criada a imprensa no Brasil, o que serviu para difundir a Língua Portuguesa. Em face disso, em 1826, iniciou-se uma discussão no parlamento brasileiro sobre a questão da língua nacional do Brasil. Em 1827, começam a ocorrer intensas discussões sobre o que deveria ser objeto de ensino de língua. Uns achavam que a escola deveria ensinar a ler e escrever utilizando a gramática da língua nacional. Nesse contexto, a língua portuguesa tornou-se 77 S U M Á R I O a língua da nação brasileira. Isso porque a determinante de ensino era o Ratio Studiorum da Companhia de Jesus, que não permitia o ensino do vernáculo. As crianças eram alfabetizadas em língua por- tuguesa e, em seguida, passavam a aprender em Latim. Nos cur- sos superiores se ensinava a gramática latina e a retórica. COMPREENDENDO AS POLÍTICAS EDUCACIONAIS QUE ORIENTAM AS POLÍTICAS LINGUÍSTICAS NO BRASIL 3º Elementos da geografia A instrução primária consta de dois graus: no primeiro se ensina a ler e a escrever, a prática das quatro operações e princípios religiosos e, no segundo, a ler e escrever, Aritmética até proporções, A GRAMÁTICA DA LÍNGUA NACIONAL, noções gerais dos deveres religiosos e morais. A instrução primária compreende a leitura e a escrita, a prática e a teoria de Aritmética até proporções, inclusive as noções mais gerais de geometria prática, A GRAMÁTICA DA LÍNGUA NACIONAL, princípios da Moral Cristã e a doutrina da Religião do Estado. Fonte: Castanha (2007). Quadro 1 – Currículos das escolas da corte COMPREENDENDO AS POLÍTICAS EDUCACIONAIS QUE ORIENTAM AS POLÍTICAS LINGUÍSTICAS NO BRASIL O quadro abaixo deixa evidente que a língua portuguesa, delineada nos inte- resses políticos, é a língua da nação e, como definido nos currículos abaixo, subentende-se que ensinar língua é ensinar gramática. Quadro 1 – Currículos das escolas da corte CORTE (1827) RIO DE JANEIRO (1837) MATO GROSSO (1837) PARANÁ (1846) Os professores ensinarão a ler e escrever, as quatro operações de aritmética, práticas de quadrado, decimais e proporções, as noções mais gerais de geometria prática, A GRAMÁTICA DA LÍNGUA NACIONAL, e os princípios da moral cristã e da doutrina da religião Católica, Apostólica e Romana, proporcionados à compreensão dos meninos, preferindo para as leituras a constituição do Império e a História do Brasil As Escolas Públicas de Instrução Primária compreendem as três classes de ensino: 1ª leitura e escrita, as quatro operações de aritmética sobre números inteiros, frações ordinárias, decimais e proporções, princípios da Moral Cristã e a Religião do Estado, A GRAMÁTICA DA LÍNGUA NACIONAL. 2º Noções gerais de geometria teórica e prática. 3º Elementos da geografia A instrução primária consta de dois graus: no primeiro se ensina a ler e a escrever, a prática das quatro operações e princípios religiosos e, no segundo, a ler e escrever, Aritmética até proporções, A GRAMÁTICA DA LÍNGUA NACIONAL, noções gerais dos deveres religiosos e morais. A instrução primária compreende a leitura e a escrita, a prática e a teoria de Aritmética até proporções, inclusive as noções mais gerais de geometria prática, A GRAMÁTICA DA LÍNGUA NACIONAL, princípios da Moral Cristã e a doutrina da Religião do Estado. Fonte: Castanha (2007). Quadro 1 – Currículos das escolas da corte CORTE (1827) RIO DE JANEIRO (1837) MATO GROSSO (1837) PARANÁ (1846) Os professores ensinarão a ler e escrever, as quatro operações de aritmética, práticas de quadrado, decimais e proporções, as noções mais gerais de geometria prática, A GRAMÁTICA DA LÍNGUA NACIONAL, e os princípios da moral cristã e da doutrina da religião Católica, Apostólica e Romana, proporcionados à compreensão dos meninos, preferindo para as leituras a constituição do Império e a História do Brasil As Escolas Públicas de Instrução Primária compreendem as três classes de ensino: 1ª leitura e escrita, as quatro operações de aritmética sobre números inteiros, frações ordinárias, decimais e proporções, princípios da Moral Cristã e a Religião do Estado, A GRAMÁTICA DA LÍNGUA NACIONAL. 2º Noções gerais de geometria teórica e prática. CORTE (1827) Os professores ensinarão a ler e escrever, as quatro operações de aritmética, práticas de quadrado, decimais e proporções, as noções mais gerais de geometria prática, A GRAMÁTICA DA LÍNGUA NACIONAL, e os princípios da moral cristã e da doutrina da religião Católica, Apostólica e Romana, proporcionados à compreensão dos meninos, preferindo para as leituras a constituição do Império e a História do Brasil Fonte: Castanha (2007). Fonte: Castanha (2007). Como destacado no quadro, é possível observar que o ensino da gramática da língua nacional se constitui como uma polí- tica linguística. Na esteira política da época, fica evidente que saber gramática é sinônimo de bem falar e bem ler, o que garante a boa 78 retórica para exercer os cargos públicos. Porém, essa ideologia de saber gramática para bem falar e bem ler perdurou por muitos anos no ensino (MOURA NEVES, 2004)7. Em primeiro lugar, os poucos que se escolarizavam (século XVI ao XVIII) pertenciam a camadas privilegiadas, cujo interesse e objetivo era seguir o modelo educacional da época, que se fundava na aprendizagem do latim e através do latim, fugindo à tradição dos sistemas pedagógicos de então atribuir às línguas nacionais estatuto de disciplina curricular; em segundo lugar, o português não era a lín- gua dominante no intercâmbio social, não havendo, por isso, motivação para instituí-lo em disciplina curricular; e em terceiro lugar [...], o português ainda não se constituíra em área de conhecimento em condições de gerar uma disciplina curricular (SOARES, 2002, p. 158-159). S U M Á R I O Como a autora pontua, o português não se constituía em área de conhecimento que pudesse ter status de disciplina curricu- lar. Somente em 1871 que o cargo de professor de português surgiu por meio de decreto imperial. O perfil desse docente – que lecionava as disciplinas de Retórica e Gramática – era o de um intelectual inte- grante das elites da época, uma vez que não havia cursos de forma- ção para professores. Mais tarde, no governo Varguista, Getúlio Vargas criou uma série de medidas nas quais objetivou limitar o uso de línguas estran- geiras no Brasil. Um exemplo dessas medidas foi a proibição de alfa- betizar em qualquer outra língua que não fosse o português. A obra da respectiva pesquisadora explica que durante muito tempo o ensino de gramática era determinante para o bem falar e o bem escrever. CORTE (1827) Essa proibição objetivava que as comunidades de imigrantes que falavam suas línguas maternas ou dialetos passassem primeiramente a ser bilíngues e depois deixassem de lado a língua trazida pela primeira geração de imigrantes. No entanto, de acordo com os trabalhos de Margotti (2004) sobre comunidades italianas no Rio Grande do Sul, 7 79 Fritzen (2008) sobre os migrantes alemães em Blumenau, Jacu- masso (2009) acerca de comunidades ucranianas em Irati, entre muitos outros que discutem acerca das práticas linguísticas nas comunidades migrantes, é possível observar que a comunidade de migrantes no Brasil assumiram a brasilidade, mas não negam sua diversidade. Tanto que os estudos mostram que em várias comuni- dades de migrantes ainda hoje há práticas bi/multilingues. Os problemas políticos e sociais que nasceram no Brasil-co- lônia refletem e refratam a baixa qualidade de ensino, pois há falta de políticas educacionais mais efetivas. A ausência de projetos de educação que não sejam apenas promessas de governo e itens de reformas supérfluas, que em nada colaboram para a evolução e cres- cimento do sistema educacional reiteram continuamente a condição social e educacional do país, arrastando século após século a marca do elitismo e exclusão de classes minorizadas. DELINEANDO AS POLÍTICAS LINGUÍSTICAS PARA A LÍNGUA PORTUGUESA Face ao exposto até aqui, no Brasil, as políticas linguísticas, no que tange ao ensino da língua, não são definidas explicitamente. As políticas públicas para a educação mostram um panorama amplo que abrange os métodos de ensino, deliberações para o funciona- mento das escolas e cursos. Sendo assim, procuro clarificar a ques- tão do ensino da língua portuguesa e da formação docente. Há muitos trabalhos que são de extrema relevância nesse cenário após a Lei de diretrizes e Base de 1996, nos primeiros quinze anos de 2000, surgem muitos trabalhos sobre o currículo, livro didá- tico e os documentos parametrizadores para o ensino de língua por- tuguesa, centralizados na esfera Federal, foram publicados, como: 80 Bunzen (2009) – na tese “Dinâmicas discursivas na aula de Portu- guês: os usos do livro didático e projetos didáticos autorais”, Angelo (2005) – na tese “Revisitando o ensino tradicional de língua portu- guesa”, Faccina (2000) no trabalho “Políticas linguísticas e ensino de língua portuguesa: da República Velha à Constituição de 1934.”, Lauria (2004), na tese “Livro didático de português: entre as concep- ções de ensino, trilhos da lei e as sendas do texto.”; De Pietri (2003), “A constituição do discurso da mudança no ensino de língua portu- guesa.”; Razzini (2000), “O espelho da nação: a Antologia Nacional e o ensino de português e de literatura.”, entre outros. Nesses trabalhos, retoma-se a história da educação, como apresentado até aqui, e num processo de escrutínio da história da educação linguística, esquadrinha-se o ensino de língua portuguesa no Brasil. Sendo assim, destaco duas políticas linguísticas explícitas que afetam o ensino de língua portuguesa e as políticas para a edu- cação. A saber, a reforma pombalina, que propôs o ensino da gramá- tica portuguesa ao lado da gramática latina, e a política nacionalista da Era Vargas, com a proposta de uma política linguística da língua nacional, contrapondo-se às línguas trazidas pelos imigrantes. Cronologicamente, de 1532 até 1600, assiste-se à constitui- ção da língua nacional, no período Brasil-colônia, com a chegada dos portugueses. Nesse momento, três línguas eram usadas no país: a língua portuguesa, a língua geral e o latim. O sistema de ensino estava sob domínio dos jesuítas, que usavam a língua geral e as línguas indígenas, sendo que a língua portuguesa não constava no currículo escolar. DELINEANDO AS POLÍTICAS LINGUÍSTICAS PARA A LÍNGUA PORTUGUESA De 1600 até 1800, temos a expulsão dos jesuítas, a chegada de africanos escravizados e o decreto de Pombal, que exige a obriga- toriedade do ensino de língua portuguesa tanto na escola como nos demais usos, suplantando as demais línguas e criando-se a ideolo- gia do país monolíngue. 81 Com a vinda da Família Real em 1808 e mais portugueses que chegam juntamente com a realeza, há uma mudança cultural no Rio de Janeiro e a relação com as línguas ali faladas (ANGELO, 2005). A língua portuguesa passa a ser veículo de circulação com a criação, nessa época, da imprensa no Brasil. Com a independência em 1822 e o estabelecimento do Estado brasileiro, a questão da lín- gua se evidencia. De acordo com Orlandi (2009), há a instrumenta- lização da língua por meio da escrita de gramáticas e dicionários e o florescimento da literatura brasileira, contribuindo para a constru- ção da língua nacional. Importante pontuar, conforme Orlandi (2013, p. 231), a maneira como se constroem as gramáticas são indicações de como se deve praticar o ensino da língua, ainda mais porque os mesmos intelec- tuais que faziam as gramáticas eram os que ensinavam a língua. Foi o ano de 1826 a data fixada para o término desse período, quando o Parlamento brasileiro - leia-se poder constituído - formulou a ques- tão da língua nacional do Brasil (LORENSET, 2016, p. 197). Em sentido semelhante a Lorenset (2016), Bunzen (2011) esclarece que, após a Independência do Brasil, construiu-se uma política linguística de construção e afirmação da língua nacional: Do ponto de vista de uma política linguística pós-inde- pendência, uma decisão política no parlamento deter- minou que o ensino da língua deveria se dar através do uso da gramática da “língua nacional”, utilizando-se essa categorização “como uma forma de não nomear a língua da nova Nação pelo nome do antigo colonizador” (GUI- MARÃES, 2005, p. 15). Nesse contexto político, a “língua nacional” passou a ser um dos saberes escolares neces- sários para a formação dos setores burocráticos e intelec- tuais da nova “nação”. No ensino secundário, a língua por- tuguesa e sua literatura foram sendo lentamente incluídas no currículo oficial (BUNZEN, 2011, p. 893). 82 Com a decisão do parlamento, os professores deveriam ensi- nar a ler e escrever em língua portuguesa. DELINEANDO AS POLÍTICAS LINGUÍSTICAS PARA A LÍNGUA PORTUGUESA No entanto, entre 1808 a 1820 cresceu a heterogeneidade linguística com a chegada de imi- grantes vindos da Alemanha, Itália, Polônia, Ucrânia, entre outros e as línguas alemã, italiana, polonês, ucraniana passaram a conviver com as línguas indígenas e as línguas africanas. S U M Á R I O São esses decretos e decisões políticas que impõem a lín- gua do colonizador aos povos colonizados. Para que essa língua se fortaleça cada vez mais, destaca-se a criação do Colégio Pedro II (SOARES, 2002), sendo o mais antigo do Brasil. Nele, o estudo da língua portuguesa foi incluído como currículo escolar dentro das dis- ciplinas de gramática, retórica e poética. No ano seguinte à criação do Colégio, foi regulamentada a gramática nacional como objeto de estudo, tanto que em 1871, por decreto imperial, foi criado o cargo de professor de português. Entretanto, não havia formação de professores. Conforme Bunzen (2011), os professores de português eram os intelectuais oriundos das elites. Intelectuais esses que, para adentrarem os cur- sos superiores no Império, prestavam exame obrigatório de língua portuguesa e também sobre a gramática latina. De acordo com Lorenzset (2016, p. 197), o Colégio Pedro II passou a ser um dos espaços privilegiados de configuração das políticas linguísticas: [...] o Colégio Pedro II passa a ser o lugar de formação e elaboração de programas que configuram formas de cidadania. Quanto à língua, apresenta-se como lugar de conhecimento legítimo, da garantia da unidade linguís- tica nacional e de domínios da “boa” língua, “boa” retó- rica, da “boa” escrita. Assim, a forma histórica do sujeito social brasileiro pode ser depreendida no modo como a língua é ensinada, notadamente em grandes colégios como o Colégio Pedro II: “no ensino da língua estão ins- critos valores, metas e perfis de formação de quadros para gerir nossas instituições e nossos projetos políticos de nação” (Ibid., p. 202). 83 Nesse cenário, começaram a ser produzidas as gramáticas dirigidas aos alunos, as quais ratificavam a pertinência dos estudos da gramática. Além disso, o Colégio Pedro II, com as exigências da época, consagrou o padrão do ensino de língua no Brasil. (RAZZINI, 2000). DELINEANDO AS POLÍTICAS LINGUÍSTICAS PARA A LÍNGUA PORTUGUESA Na década de 1930, foi criado o Ministério da Educação e da Saúde Pública e a chegada de Vargas ao poder consolidou os pro- gramas oficiais e as disciplinas escolares, por meio, por exemplo, do Decreto 19.890 de 1931: S U M Á R I O Nesse sentido, programas oficiais e disciplinas específi- cas para cada série do secundário aparecem no decreto 19.890 de 1931, documento responsável também pela equiparação de todos os colégios secundários ao Colé- gio Pedro II, pela seriação e frequência obrigatória para ingresso nas faculdades (RAZZINI, 2000). Em junho de 1931, a disciplina passa a ser denominada de Português, com objetivos e conteúdos fixados pelas instruções meto- dológicas para cada disciplina (BUNZEN, 2011, p. 896). Em 1939, o decreto 1.190 determinou que professores no ensino secundário deveriam ter formação na Faculdade de Filosofia, que teria as seguintes finalidades: Art. 1º A Faculdade Nacional de Filosofia, Ciências e Letras, instituída pela Lei n. 452, de 5 de julho de 1937, passa a denominar-se Faculdade Nacional de Filosofia. Serão as seguintes as suas finalidades: a) preparar tra- balhadores intelectuais para o exercício das altas ativi- dades de ordem desinteressada ou técnica; b) preparar candidatos ao magistério do ensino secundário e normal; c) realizar pesquisas nos vários domínios da cultura, que constituam objeto de ensino (BRASIL, 1939). Art. 1º A Faculdade Nacional de Filosofia, Ciências e Letras, instituída pela Lei n. 452, de 5 de julho de 1937, passa a denominar-se Faculdade Nacional de Filosofia. Serão as seguintes as suas finalidades: a) preparar tra- balhadores intelectuais para o exercício das altas ativi- dades de ordem desinteressada ou técnica; b) preparar candidatos ao magistério do ensino secundário e normal; c) realizar pesquisas nos vários domínios da cultura, que constituam objeto de ensino (BRASIL, 1939). Para exercer o magistério, a partir do decreto, o professor de português deveria formar-se no curso de Letras clássicas e precisava também da formação em didática, somando mais um ano à forma- ção em Letras. No documento, consta em seu “Art. 49. Ao bacha- rel, diplomado nos termos do artigo anterior, que concluir regular- mente o curso de didática referido no art. 20 desta lei será conferido 84 S U M Á R I O o diploma de licenciado no grupo de disciplinas que formam o seu curso de bacharelado.” (BRASIL, 1939) Logo, a licenciatura não era uma formação única, sendo condicionada ao bacharelado. DELINEANDO AS POLÍTICAS LINGUÍSTICAS PARA A LÍNGUA PORTUGUESA Com a exigência da formação docente em nível superior e com objetivos e conteúdos fixados pelas instruções metodológicas para cada disciplina, o programa de ensino de língua portuguesa “priorizava exercícios práticos de gramática e textos literários, aten- dendo a dois objetivos: “a habilitação do aluno para falar e escrever corretamente” e “a missão do professor de despertar no aluno o amor pela língua pátria e o gosto literário.” (BUNZEN, 2011, p. 898-899). Com o Estado Novo e os decretos de Vargas, a política lin- guística atingia, sobretudo, os imigrantes. As escolas deveriam ministrar aulas apenas em Língua Portuguesa, tendo como mote a formação de uma consciência de brasilidade, reforçando o imagi- nário da língua nacional. Segundo Razzini (2000, p 202), a reforma Capanema impôs ao país programas oficiais em língua uniforme, em que se sobrepunha a visão “mistificadora” das instituições nacio- nais e culto às autoridades. Em nome da língua nacional, a ditadura getulista exerceu forte repressão linguística: o poder central legislou sobre língua e identidade, língua e Estado”. Diante desse contexto, a língua portuguesa culta foi forta- lecida, sendo então modelo de língua, sinônimo de correção lin- guística formal. No entanto, a partir de 1950, a escola passou a ser reivindicada pelas classes trabalhadoras, que exigiam escola para seus filhos. Essa mudança na paisagem escolar trouxe uma série de transformações para a educação e para o ensino de língua por- tuguesa em especial. Houve um aumento considerável no número de alunos. Por isso, foram necessários mais professores para atender essa demanda; entretanto, não havia tantos profissionais disponíveis. Automatica- mente, houve um relaxamento na seletividade desses profissionais. 85 Soares (2002) enfatiza que mesmo com alunos advindos de classes populares, os manuais didáticos (que eram escritos pelos profissio- nais da elite intelectual) não foram alterados, o que criou um quadro complexo no ensino da língua, com baixo aproveitamento, desistên- cia escolar, etc. Ademais, Soares (2002) ainda afirma que, com a for- mação docente ampliada para atender a demanda, os professores não eram mais provenientes da elite intelectual. Observou-se assim uma perda de prestígio na função docente, porque a clientela para a formação superior era oriunda de contextos menos letrados. De acordo com Bunzen (2011), citando Barreto Barros (2008), como os novos professores não pertenciam mais às classes intelectuais, o material didático precisou ser adaptado. DELINEANDO AS POLÍTICAS LINGUÍSTICAS PARA A LÍNGUA PORTUGUESA Soares (2002) enfatiza que mesmo com alunos advindos de classes populares, os manuais didáticos (que eram escritos pelos profissio- nais da elite intelectual) não foram alterados, o que criou um quadro complexo no ensino da língua, com baixo aproveitamento, desistên- cia escolar, etc. Ademais, Soares (2002) ainda afirma que, com a for- mação docente ampliada para atender a demanda, os professores não eram mais provenientes da elite intelectual. Observou-se assim uma perda de prestígio na função docente, porque a clientela para a formação superior era oriunda de contextos menos letrados. De acordo com Bunzen (2011), citando Barreto Barros (2008), como os novos professores não pertenciam mais às classes intelectuais, o material didático precisou ser adaptado. Diante disso, no ensino primário e secundário, continuava a normativa explicitada anteriormente, que priorizava exercícios práticos de gramática e leitura de textos literários (BUNZEN, 2011). Todavia, o ensino não era uma questão resolvida, porque cada pro- fessor seguia uma linha, empregavam-se nomenclaturas variadas a depender da afinidade com os manuais. Para tanto, o MEC delegou a um grupo de gramáticos a tarefa de compilar termos técnicos na esfera gramatical para serem usados de forma uniforme em todo o país. Esse compilado foi conhecido como a Nomenclatura Gramati- cal Brasileira - NGB (FACCINI, 2000), mais uma política linguística brasileira que está vigente até os dias atuais. A despeito da criação da NGB, os problemas com o ensino permaneceram, tendo em vista que não se restringiam à nomencla- tura utilizada pelos professores. A questão era mais complexa, uma vez que o ensino de língua era variável e deve privilegiar o ensino de leitura, escrita e oralidade em situações de uso, muito mais que definição de conceitos. Dessa maneira, na década seguinte, Bunzen (2011, p. 899) destaca que: 86 Os anos 60 são fortemente marcados pelo processo de descentralização das questões educacionais mais gerais, uma vez que a Lei de Diretrizes e Bases (LDB) de 1961 apostou no federalismo e na autonomia dos Estados na definição de sua política educacional. Do ponto de vista do currículo prescrito, assistiu-se a uma maior flexibi- lidade, pois, diferentemente dos anos anteriores, houve uma maior abertura para construção do currículo pelos Estados, pelos professores e pelos próprios autores--e- ditores de livros didáticos. A LDB-61 apontava, por exem- plo, algumas indicações para o ensino de cada disciplina escolar que poderia ser desenvolvida e ampliada pelos programas estaduais. DELINEANDO AS POLÍTICAS LINGUÍSTICAS PARA A LÍNGUA PORTUGUESA A LDB de 1961 foi instituída como tentativa para o aperfeiçoa- mento do ensino, já que historicamente havia uma crise do ensino. Na respectiva lei, ficou determinado que a educação é direito de todos, todavia, admite-se no documento que há insuficiência de escolas e o encerramento de matrícula na falta de vagas. Como tentativa de minimizar esse problema, foram criados os Conselhos Estaduais de Educação, incumbidos de melhorar a qualidade do ensino, considerando a variedade de cursos, a flexi- bilização de currículos e articulação dos diferentes graus, conforme consta no artigo 12 da LDB de 1961. Como se pode observar, o ensino não era centralizado em escala federal, havia uma descentralização que dependia de cada estado estabelecer suas diretrizes de ensino. Nesse cenário, não havia como estabelecer diretrizes para avaliações em larga escala, no entanto o ensino requeria qualidade. Na formação docente, a dis- ciplina de linguística foi instituída no currículo dos Cursos de Letras. Evidente que essa decisão trouxe sérias consequências, pois não havia professores formados em linguística (em número suficiente) para lecionar essa disciplina. De acordo com Geraldi (1996), com a linguística no currículo de Letras, há uma desestabilização no diálogo entre a gramática e o ensino. Assim, o discurso da homogeneidade 87 começa a ruir e cria-se um conflito entre as concepções descritivas e o caráter normativo da gramática tradicional. Num cenário cada vez mais complexo no que tange ao ensino de língua, a Lei de Diretrizes e Bases de 1971 foi sancionada e nela foi estabelecida a língua nacional como instrumento de comunicação e expressão da cultura brasileira. A partir daí, a disciplina de língua portuguesa passou a ser Comunicação e Expressão, no 1º grau (1ª à 4ª série); Comunicação e Expressão em Língua Portuguesa (5ª à 8ª série) e Língua Portuguesa e Literatura Brasileira no 2º grau. A língua era vista como normativa porque expressava a cultura brasi- leira e se configurava como instrumento de comunicação nacional, havendo, assim, a perspectiva de que deveria haver homogeneidade linguística no território nacional. O ensino de língua servia à domina- ção do poder político e militar. Nesse contexto, no entanto, o ensino de língua portuguesa estava em crise na escola, como mostra Bunzen (2011, p. 902): Se a escola pública não conseguia ensinar a ler e a escre- ver, o ensino de língua materna encontrava-se em crise. DELINEANDO AS POLÍTICAS LINGUÍSTICAS PARA A LÍNGUA PORTUGUESA Ao lado da “crise da leitura e da escrita” e da “luta” por uma maior democratização do ensino, emerge fortemente no Brasil o “discurso da mudança” (PIETRI, 2003) que constrói discursivamente o chamado “ensino tradicional de português” (ANGELO, 2005). Por tal razão, amplia- ram-se consideravelmente as pesquisas sobre o ensino – desde a alfabetização até as redações dos vestibulandos. Uma afirmação de Faraco (1975, p. 5), quando o pesquisador apresenta as sete pragas no ensino de língua portuguesa corrobora com a citação acima: “O ensino de português tem se mostrado inútil (os resultados negativos nos autorizam tal classificação). Recursos humanos e materiais têm sido criminosamente desperdiçados numa vazia de significado: onze anos de escola e o indivíduo está menos instrumentalizado linguisticamente que ao entrar na escola”. 88 Nessa direção, pesquisadores e professores universitários passaram a propor reflexões críticas das práticas escolares, do ensino de língua, da concepção de língua(gem). Houve um movimento dos pesquisadores acerca das práticas de uso da língua no dizer-ouvir- -ler-escrever, registrados nos trabalhos de Geraldi a partir de 1984. Em vista dessas pesquisas e da divulgação desses trabalhos, o Con- selho Federal de Educação retomou a designação de Português no ensino fundamental e médio. Geraldi (1996, p. 311-312) destaca que: S U M Á R I O A chegada dos anos 80 deflagaria um intenso processo de revisão e questionamento do ensino em vigor, voltado para a reconceitualização geral dos objetivos, pressupos- tos e procedimentos didáticos para a área. Tal processo resultava de pelo menos duas ordens de fatores: a já refe- rida crise do ensino, função do despreparo da instituição escolar para as transformações quantitativas e qualita- tivas que a ela se impunham e a possibilidade de utili- zação de paradigmas emergentes recolhidos das ciên- cias da linguagem e das teorias do conhecimento, que haviam iniciado seu “desembarque” no país em meados da década anterior, representadas especialmente pela lin- guística da Enunciação e pelas contribuições dos autores sócio-históricos no campo da filosofia da linguagem e da psicologia. (Bakhtin e Vygotsky, sobretudo). O autor destaca que os programas de formação para aper- feiçoamento dos professores eram desenvolvidos para conven- cer o professor da importância das novidades e que deveriam se adaptar às mudanças. Esses programas eram normalmente lan- çados de cima para baixo, reforçando a assimetria institucional e a dependência, numa perspectiva fragmentada e burocratizada, das práticas de ensino. DELINEANDO AS POLÍTICAS LINGUÍSTICAS PARA A LÍNGUA PORTUGUESA Em meio a essa conjuntura do ensino de língua na década de 1980, foi através das universidades, da formação docente, da pesquisa em linguística, da implantação dos livros didáticos e trei- namentos para professores que o ensino de língua portuguesa ini- ciou uma nova etapa. Num processo de revisão, os objetivos e os 89 S U M Á R I O métodos de ensino foram sendo questionados. Soares (2002) reitera que em cada momento histórico, a disciplina de língua portuguesa, por conseguinte, o ensino de língua e a formação de professores foi determinada por fatores externos e condições sociais, econômicas e culturais. Sem deixar de destacar os fatores internos sobre os conhe- cimentos sobre as línguas para a formação de professores que vão atuar na educação básica. As políticas públicas federais impactaram sobremaneira a educação e o ensino de língua materna com a centralização e norma- tização do ensino com o surgimento das propostas curriculares ofi- ciais, que buscavam explicitar as diretrizes mínimas para cada nível de ensino e o estabelecimento das avaliações em larga escala, emba- sados e legitimados por meio da Lei de Diretrizes e Bases de 1996. A história como apresentada acima nos conduz a refletir sobre as concepções de lingua(gem), assim como do ensino com vistas a observar as rupturas, afastamentos, retrocessos e desigual- dades que vem se reproduzindo no Brasil. Essa construção sobre o ensino de língua portuguesa tem impactos em como se forma o professor para a educação básica. Essas políticas para o ensino de línguas, a meu ver, refletem ideologias linguísticas construídas histo- ricamente e, ainda que haja pesquisas e discussões sobre o assunto, essas políticas reiteram as ideologias de uma língua que deve ser ensinada, com vistas a atender um mercado linguístico amalgamado em desigualdades sociais. CONSIDERAÇÕES FINAIS O terreno de estudo das linguagens situa-se num imbri- camento de discursos que se entretecem e mobilizam olhares para inúmeras direções teóricas. Respeitando as discussões sobre 90 S U M Á R I O língua e linguagem em suas várias vertentes, as línguas atuam como competição política, as variedades que são associadas ao ator social, neste caso o professor formado em Letras, ou indexadas a ele, são supervalorizadas e as ultrapassam como símbolos de identidade de um grupo. Na verdade, passam a ser emblemas políticos, sociais, intelectuais e moralizantes. Portanto, a percepção sobre a língua varia entre os temas sociais e linguísticos, deixando evidente que as ideologias, sejam sociais ou de língua, atuam de maneira dialógica e relacionam os níveis sociais e hierarquias de poder. Assim, por meio do círculo de Bakhtin, por exemplo, a lingua- gem é vista como um constante processo de interação mediado pelo diálogo e não como um sistema autônomo. “A língua materna, seu vocabulário e sua estrutura gramatical, não conhecemos por meio de dicionários ou manuais de gramática, mas graças aos enunciados concretos que ouvimos e reproduzimos na comunicação efetiva com as pessoas que nos rodeiam” (BAKHTIN, VOLOSHINOV, 2009, p. 93) De acordo com essa concepção, a língua só existe em função do uso que locutores e interlocutores fazem dela em situações de comunicação, isto é, não havendo uma separação entre língua e lin- guagem, mas língua(gem). Portanto, ensinar, aprender e fazer uso da linguagem passam essencialmente pelo sujeito, que é o agente das relações sociais e responsável pela composição e pelo estilo dos dis- cursos. Logo, o sujeito se vale do conhecimento de enunciados ante- riores para formular suas falas e redigir seus textos. Além disso, um enunciado sempre é modulado pelo falante para o contexto social, histórico, cultural e ideológico. BAKHTIN, Mikhail; VOLOCHÍNOV, Valentin. Marxismo e filosofia da linguagem. 13.ed. São Paulo: Hucitec, 2009 BAKHTIN, Mikhail; VOLOCHÍNOV, Valentin. Marxismo e filosofia da linguagem. 13.ed. São Paulo: Hucitec, 2009 BRASIL. Decreto-lei nº 1.190, de 4 de abril de 1939. Dá organização à Faculdade Nacional de Filosofia. Diário Oficial da União, seção 1, 6/4/1939, p. 7929. Disponível em: <http:// bit.ly/3iP4dcu>. Acesso em: 23 de jan. de 2021. BUNZEN, Clecio. Dinâmicas discursivas na aula de português: os usos do livro didático e projeto didáticos autorais. 2009. 227 f. Tese de Doutorado em Linguística Aplicada – Universidade Estadual de Campinas, Campinas, 2009. BUNZEN, Clecio. A fabricação da disciplina escolar Português. Revista Diálogo Educ., Curitiba, v. 11, n. 34, p. 885-911, 2011 CASTANHA, André Paulo. O Ato Adicional de 1834 e a instrução elementar no Império: descentralização ou centralização? 2007. 563 f. Tese de Doutorado em Educação – Programa de Pós-Graduação em Educação, Universidade Federal de São Carlos, São Carlos, 2007. DE PIETRI, Ederson. O discurso da mudança do ensino de língua materna no processo de constituição da linguística brasileira. 2003. 202 f. Tese de Doutorado em Linguística Aplicada – Universidade Estadual de Campinas, Instituto de Estudos da Linguagem: Campinas, 2003. FACCINA, Rosemeire. Políticas linguísticas e ensino de língua portuguesa: da República Velha à Constituição de 1934. 2000. Disponível em: <https://bit.ly/3XoWoJB>. Acesso em: 12 fev. 2021. FARACO, Carlos Alberto. As sete pragas do ensino de Português. Construtora, v. 3, n. 1, p. 5-12, 1975 FERREIRA JR, Amarilio. História da educação brasileira: da colônia ao século XX. São Carlos: EdUFSCAR. Coleção UAB-UFSCAR, 2010. REFERÊNCIAS ANGELO, Graziela Lucci de. Revisitando o ensino tradicional de língua portuguesa. 2005. 265 f. Tese de doutorado em Linguística Aplicada – Universidade de Campinas, Campinas, 2005. ANGELO, Graziela Lucci de. Revisitando o ensino tradicional de língua portuguesa. 2005. 265 f. Tese de doutorado em Linguística Aplicada – Universidade de Campinas, Campinas, 2005. 91 BAGNO, Marcos. Sete erros aos quatro ventos a variação linguística no ensino do português. São Paulo: Parábola, 2013. FERREIRA JR, Amarilio. História da educação brasileira: da colônia ao século XX. São Carlos: EdUFSCAR. Coleção UAB-UFSCAR, 2010. GERALDI, João Wanderley; SILVA, Lilian Lopes Martins; FIAD, Raquel Salek. Linguística, ensino de língua materna e formação de professores. D.E.L.T.A., v. 12, n. 2, p. 307-326, 1996. 92 92 LAURIA, Maria Paula. Livro didático de português: entre as concepções de ensino, trilhos da lei e as sendas do texto. 2004. 425 f. Tese de Doutorado em Educação – Faculdade de Educação da Universidade de São Paulo, São Paulo, 2004. LAURIA, Maria Paula. Livro didático de português: entre as concepções de ensino, trilhos da lei e as sendas do texto. 2004. 425 f. Tese de Doutorado em Educação – Faculdade de Educação da Universidade de São Paulo, São Paulo, 2004. LORENSET, Rossaly Beatriz Chioquetta. Mostrando a língua: políticas linguísticas e historicidade do ensino de língua portuguesa no Brasil. ReVEL, v. 14, n. 26, 2016. MARIANI, Bethania. Políticas de colonização linguística. Letras, n. 27, p. 73-82, 2003. MOURA NEVES, Maria Helena. Que gramática estudar na escola? Norma e uso da Língua portuguesa. 2 ed. São Paulo: Contexto, 2004. SEVERO, Cristine Gorski. Uma visão panorâmica das políticas linguísticas no Brasil: construindo diálogos. Revista da Academia Brasileira de Letras, n. 94, p. 11-22, 2018. RAZZINI, Marcia de Paula Gregorio. O espelho da nação: A Antologia Nacional e o ensino de português e de literatura. 2000. 247 f. Tese de Doutorado – Universidade Estadual de Campinas, Campinas, 2000. SOARES, Magda. Português na escola: história de uma disciplina curricular. In: BAGNO, Marcos. (Org.). Linguística da norma. São Paulo: Loyola, 2002, p. 140-168. 93 S U M Á R I O INTRODUÇÃO As pesquisas em Estudos da Linguagem, de forma geral, abrangem uma gama de temas, sujeitos e perspectivas. Nosso obje- tivo ao posicionar a Linguística Queer neste breve capítulo é mos- trar como alguns estudos tidos como clássicos na área abordam as questões “avessas”, “estranhas”, por vezes pouco exploradas pela academia, e desafiá-las na medida em que a pesquisa é, por si só, um modo de ação e de reprodução de sentidos sobre o que pode ser considerado normal, correto, verdadeiro etc. Ao demonstrar tudo isso, buscamos também questionar como podemos praticar nossas pesquisas no presente e no futuro, à medida em que percebemos alguns problemas nas abordagens de alguns autores e autoras que, assim como nós, estão mais ou menos limitados por suas condi- ções sócio-histórico-culturais. Como explica Borges (2022, p. 344), “absorver isso como parte inerente do processo de pesquisa é peça- -chave para entendermos a importância de um escrutínio de nos- sas próprias práticas investigativas”, por isso, é preciso explicitar que “nossos posicionamentos enquanto sujeitos sociais cujos limites de leitura de mundo são dados pelos contingenciamentos comuns a qualquer processo sociossemiótico de construção de significado. Nossas inspirações se apoiam nos escritos de autoras e autores como Judith Butler, Mary Bucholtz, Kira Hall e Paul Preciado, por exemplo. Igualmente inspiradores foram e são os encontros do grupo de pesquisa EdQueer – Estudos Discursivos na perspectiva Queer, da Universidade Federal de Uberlândia, do qual o autor faz parte desde sua fundação em 2021. Outro grupo que também se prova relevante para nossas discussões é o NUDES – Núcleo de Estudos em Discursos e Sociedade, da Universidade Federal do Rio de Janeiro, do qual participa a autora. Por fim, no Brasil, pesquisas empreendidas por Rodrigo Borba, Luiz Paulo Moita Lopes e Guacira Lopes Louro no Brasil, por exemplo, nos interessam por traduzirem 95 S U M Á R I O para nossas perspectivas culturais questões da linguagem que, mui- tas vezes, são inerentes às realidades do norte global. Dessa forma, nossas reflexões estão organizadas de modo que, em um primeiro momento, trazemos um panorama da Lin- guística Queer ou Cuir no Brasil e fora dele. Em seguida, buscamos esclarecer alguns conceitos-chave para a área, como o de gênero, sexualidade, identidade e formas de assujeitamento e subversão. INTRODUÇÃO Propomos, ao final, uma reflexão acerca da maneira como temos conduzido algumas pesquisas nos Estudos da Linguagem, tendo em vista a “virada performativa” e o papel da linguagem em práticas investigativas responsivas aos fenômenos sociais atuais e às vidas das pessoas que estão “fora” da academia. Em conclusão, questionamos quais são as implicações de nos posicionarmos enquanto sujeitos de pesquisa, considerando que nossas investigações refletem análises parciais dos fenômenos estudados. Afinal de contas, não estamos fora da vida que nos cerca. É nesse sentido que podemos fazer pesquisa, construindo signifi- cados com os outros, que já foram nossos “objetos” de pesquisa. Trata-se, portanto, de “coconstruir leituras de mundo complemen- tares” (BORGES, 2022, p. 344), que se somam a algo que ainda não podemos responder, imaginamos, mas sobretudo indagar e buscar, em nossas reflexões, modos de queerizar a própria escrita e nossos movimentos em direção ao outro. LINGUÍSTICA QUEER O momento sócio-histórico chamado de pós-modernidade ou modernidade recente (RAMPTON, 2006; MELO e MOITA LOPES, 2014), associado ao fenômeno da globalização, configura a ruptura com modelos universais de se produzir conhecimento. Ao romper 96 S U M Á R I O com o sujeito do iluminismo, coloca em queda o ideal originário da uma suposta natureza humana: o homem, branco, heterossexual, protótipo primeiro, disputa seu espaço junto às identidades híbridas, marcas de um mundo dinâmico e fluído. Sendo a pós-modernidade, então, uma nova abordagem teórica, são notórios os seus impactos em diversas ciências, como as humanas, as sociais, as artes, e prin- cipalmente a linguística. Nesse sentido, os estudos queer, aliados à crítica feminista ou ao pós-feminismo, conduzida por Butler (1990, 1993), Cameron & Kulick (2013), Preciado (2014) dentre outras feministas, influenciadas por autores como Deleuze, Derrida e Foucault, inauguram este novo campo de saber/fazer. Desvinculando-se de ideais universalizantes que restringiram o corpo durante séculos, a partir destes autores e autoras, o corpo, o desejo, e as sexualidades desviantes, passam a serem problemas de análise. A linguística queer afilia-se aos teóricos citados anterior- mente, sob uma perspectiva não essencialista de compreender os processos discursivos em que nos engajamos para construir nos- sas subjetividades de sexualidade (FABRÍCIO, 2017, BORBA, 2015). Para Leap (2015, p. 661), “a linguística queer explora como a lingua- gem ativa (e por vezes disfarça) as intersecções entre sexualidade, gênero, raça, classe social e outras formas de inequidades sociais”. Nesse sentido, ela abre espaço para se explorar de forma ampla outros estudos que tratem de grupos minorizados e suas relações com formas de poder e normatividades. Pensando as normatividades como condições modernas que coexistem em um fluxo contingencial intenso contemporâneo, à esteira da antinorma, a proposta queer contesta concepções moder- nistas sobre as identidades ao mostrar o que é desviante da noção de normalidade. No sentido de desestabilizar as normatividades impostas não apenas ao gênero, mas também à raça, à etnia, à sexu- alidade, à religiosidade, à nacionalidade, ao corpo, ao desejo etc., 97 S U M Á R I O na medida mesma em que essencialismos, transgressões e subver- sões são constitutivas das práticas identitárias. LINGUÍSTICA QUEER O olhar da linguística queer sobre os fenômenos sociais per- passa a crítica às normas que regulam as sexualidades não perdendo de vista que autoridade e hierarquia não são preceitos estáticos, mas dinâmicos, pois se encontram entrelaçados com contextos sociais e históricos específicos. Interessa à linguística queer questionar sob quais circunstâncias determinadas vozes também queer emergem e como o fazem, enquanto outras são reprimidas e silenciadas. Trata- -se, pois, de um empreendimento transdisciplinar, em que relações de poder, colonialismo, raça, etnia, classe social e faixa etária, por exemplo, cruzam as análises das amostras nas pesquisas. Uma abordagem interessante é feita por Borba (2020, p. 14) para o termo queer: “deriva do prefixo protoindo-europeu terkw- que, acredita-se, deu origem ao verbo latino torquere, ou seja, ‘torcer’ ou ‘girar’”. Nesse sentido, nossa proposta enquanto pesquisadores queer é de fazer torcer as normatividades, os binarismos, suspeitar das rela- ções sociais já naturalizadas e estabelecidas como normais quando vinculadas a determinadas práticas sexuais e identitárias fixas. Além disso, o termo em inglês queer pode ser utilizado em sua forma latina cuir seguindo o processo de descolonização proposto por Trujillo (2016, p.72), de modo que a mobilização deste conceito busca estar em diálogo com uma geografia engajada em “posicionar as produções de conhecimentos” com as realidades do Sul global, confrontando as hegemonias não só de identidades, mas também geopolíticas, uma vez que há uma produção disciplinar de aportes queer que derivam do Norte. Portanto, os Estudos Cuir constituem um campo de saber fora do espectro identitário, pois correspondem às demandas de articulação política das subjetividades não norma- tivas e de práticas de desidentificação (a ideia de torcer, girar, nova- mente se mostra interessante) de forma cuirificada (TRUJILLO, 2016). 98 S U M Á R I O Esse termo também possui um grau de “elasticidade de uso” (DARING et al., 2012, p. 11), podendo ser mobilizado como um subs- tantivo, adjetivo ou como verbo. Para privilegiar um olhar não-nor- mativo e não-binário sobre as práticas sociais, o cuir deixou de ser substantivo (marcador identitário), passando pela qualificação dos sujeitos (adjetivo) ou posicionamento para o presente decolonial em que pode ser entendido como uma relação, um ato (verbo) de estranhar o que se constituiu sócio-historicamente como “normal”. Queerizar ou cuirizar significa, portanto, estranhar, torcer, colocar ao avesso alguma prática que está normalizada e normatizada. A noção de poder em Foucault ([1982] 1995, p. 243) envolve as relações entre os sujeitos. As relações de poder dependem de “dois elementos que lhes são indispensáveis”: “o outro”, aquele “sobre o qual o poder se exerce, deve ser inteiramente reconhecido e mantido como sujeito de ação”. E a abertura, diante da relação de poder, “de todo um campo de respostas, reações, efeitos e invenções possíveis”. Em outras palavras, só há relação de poder quando há alguma medida de liberdade. LINGUÍSTICA QUEER A análise dos fenômenos sociais pela linguística queer reco- nhece a necessidade de desaprender e despraticar a lógica capita- lista e colonialista engendrada nos processos sociais e culturais, e nos fluxos subjetivos nos quais o papel do discurso é central, sobre- tudo nas lutas de poder onde o capitalismo faz incidir todo o peso da sua repressão, instaurando e restaurando todos os tipos de territoria- lidades por meio de tecnologias de poder8 que tentam recodificar e controlar as pessoas (DELEUZE & GUATTARI, [1972], 2014). Neste sentido, a linguística passa a ser transgressora dos seus próprios referenciais constitutivos e indisciplinada em alguns círculos em relação aos campos de saber institucionalizados, disci- plinares e estanques ao focalizar dispositivos macro e micropolíticos de controle social em contextos vulnerabilizados, abrindo espaços para a produção de sentidos outros, dos saberes menores, mestiços e menos privilegiados (MOITA LOPES, 2006). 99 Igualmente relevante é a compreensão de dois termos que são caros à linguística queer: gênero e sexualidade. Ainda que, por algum tempo, tenham sido tomados como semelhantes ou pratica- mente idênticos, os estudos na área nos provam que é preciso por- menorizar as particularidades de cada um. GÊNERO, SEXUALIDADE E IDENTIDADE EM SUBVERSÃO Para darmos início a essa discussão, trazemos a explicação de Bucholtz & Hall (2004) sobre como os termos gênero e sexuali- dade tendiam (ou ainda tendem) a se mesclar em pesquisas na área, seguida de uma definição de sexualidade: [...] pesquisas anteriores em gênero haviam tendido a mesclar sexualidade no gênero, trabalhos mais recentes reconhecem que esses conceitos teóricos são separados, enquanto reconhecem que – assim como raça, classe e outras dimensões das relações sociopolíticas – eles não podem ser produtivamente discutidos de forma inde- pendente uma da outra. [...] Sexualidade: os sistemas de ideologias, práticas e identidades mutuamente constituí- das que dão significado sociopolítico ao corpo como um campo erotizado e/ou reprodutivo (BUCHOLTZ & HALL, 2004, p. 470, itálicos das autoras). As autoras desacreditam, então, que seja possível conduzir estudos sobre sexualidade que tenham como únicas premissas o sexo erótico ou o desejo. Tampouco são muito válidos estudos que se voltem a entender “a linguagem de homens gays” ou “a lingua- gem de mulheres lésbicas”, entre outros grupos, pois uma mesma comunidade não precisa compartilhar uma mesma linguagem, como foi demonstrado por Eckert & McConnell-Ginet (2010). Afinal, recur- sos linguísticos não são distribuídos uniformemente em um mesmo grupo, e os usuários da língua fazem associações convenientes entre 100 formas linguísticas e significados na sociedade para construírem suas subjetividades e as de outrem (BUCHOLTZ & HALL, 2004). O gênero, por sua vez, assim como outras categorias identi- tárias, são performatividades encenadas repetidamente por nós ao longo de nossas vidas. As identidades, quaisquer que sejam elas, emergem dos contextos socioculturais onde são inauguradas e só podem ser entendidas como efeitos de performances linguísticas localizadas em práticas discursivas situadas (BORBA, 2015). Tais prá- ticas discursivas precisam existir antes para que tenham sentido em determinada situação de comunicação: a produção disso que chamamos de identidade não está restrita ao uso explícito de categorias identitárias, mas ocorre por meio de diversos recursos linguísticos (sons, morfemas, estruturas sintáticas etc.). Ao serem usados em uma prática discursiva específica, esses signos retomam uma história que movimenta certos arcabouços interpre- tativos disponíveis socioculturalmente e lhes confere sen- tido no aqui e agora da enunciação (BORBA, 2020, p. 30). a produção disso que chamamos de identidade não está restrita ao uso explícito de categorias identitárias, mas ocorre por meio de diversos recursos linguísticos (sons, morfemas, estruturas sintáticas etc.). GÊNERO, SEXUALIDADE E IDENTIDADE EM SUBVERSÃO Ao serem usados em uma prática discursiva específica, esses signos retomam uma história que movimenta certos arcabouços interpre- tativos disponíveis socioculturalmente e lhes confere sen- tido no aqui e agora da enunciação (BORBA, 2020, p. 30). Práticas discursivas específicas requerem análises também específicas, pois as práticas são empreendidas por sujeitos que tam- bém devem ser levados em conta. Nas palavras de Cameron (2010, p. 133, itálicos da autora), “As pessoas desempenham gênero de modos diferentes em contextos diferentes”. A linguística queer não perde de vista esses sujeitos, tampouco as relações de poder que estão imbri- cadas em determinada situação de comunicação, pois elas dizem respeitos às normas que regulam o que pode e o que não pode ser dito, feito, encenado, performado. Ao compreender a linguagem a partir da performatividade (do gênero e da sexualidade), a linguística queer ultrapassa as noções de língua(gem) que se entendem como expressões da subjetividade por reconhecer que os significados não preexistem aos usos. Esta visão potencializa múltiplas possibilidades de forjar significados nas situa- ções comunicacionais, nos fluxos, na mobilidade, nas ficcionalidades, 101 S U M Á R I O nas corporeidades etc., revolvendo a tradição representacional no campo dos estudos da linguagem (MOITA LOPES e FABRÍCIO, 2018). A partir do exposto acima, o viés queer entende que a per- formatividade problematiza noções de poder e controle acerca de discursos, indicando como as subjetividades são discursiva e con- textualmente construídas. A normatividade, então, está relacionada à produção de significados ao indexar discursos, ideologias e valores que vão constrangendo e performativamente construindo categorias identitárias. É pela norma que noções de discurso e poder estão pre- sentes nas negociações linguísticas e são geralmente conformadas por uma disputa escalar entre discursos marginalizados e dominan- tes (HIRAMOTO, 2015). As identidades para a linguística queer possuem um caráter tático porque não é possível manter uma noção identitária estável ou essencializada sem regimentá-la. As subjetividades são construções situacionais e, ao serem mobilizadas na interação, invocam certas táticas de subjetivação. Estas táticas de intersubjetividade funcionam como um laborioso recurso linguístico e, por este motivo, funcionam como ferramentas teórico-analíticas que nos ajudam a entender os mecanismos discursivos e performativos através dos quais são cria- das as identidades (BUCHOLTZ e HALL, 2004). Abre-se, assim, campo para subversões, pois as vidas das pessoas socialmente minorizadas se encontram constrangidas por regulações constantes, sobretudo aquelas que dizem respeito à cis- -heteronorma9. A noção de cisgeneridade é problemática por carregar sentidos históricos de estabilidade e natu- ralidade em relação às binaridades de gêneros e às sexualidades heteronormais. Procuramos com essa definição produzir o debate sobre a possibilidade de existência de outros sujeitos, que assim como nós, procuram alternativas para os desdobramentos das categorias ‘homem’ e ‘mulher’ a fim de compreender as nossas próprias experiências em relação ao heteropatriarcado. As nossas motivações são políticas e com elas queremos “visibilizar e transversalizar as lutas feministas e cuir por meio de todas as alianças de corpos possíveis”, como propôs Trujillo (2016, p. 87). GÊNERO, SEXUALIDADE E IDENTIDADE EM SUBVERSÃO Nesse espaço de contestação, é importante enten- dermos conceitos como o de performance. 9 102 PERFORMANCE E ASSUJEITAMENTOS Para Butler (1990), o gênero é uma estilização repetida do corpo inserido a uma estrutura reguladora altamente rígida. O corpo é uma estilização da linguagem também, sendo a linguagem uma ação do corpo que o estiliza por meio dela. É na paródia que a sub- versão pode acontecer ao expor a natureza performativa das identi- dades, ao mesmo tempo, em que se constroem na paródia. Assim como o gênero, a raça também é uma estilização repetida do corpo em estrutura reguladora, do mesmo modo as cate- gorias identitárias nos conferem diretrizes de como devemos/pode- mos estilizar nossas práticas discursivas sobre a melanina de nossas peles. O uso de categorias limítrofes da vida, ainda se faz necessário como estratégia para demarcar um território discursivo de resistência não apenas à cis-heteronorma, como também ao racismo, à homo- fobia e ao preconceito. Os grupos chamados minoritários, ou de sexualidade dita desviante, que compreendem gays, lésbicas, bissexuais, travestis e transgêneros, pessoas não binárias e queer, inauguram performa- tividades alheias às configurações normativas vigentes. Do mesmo modo, criam inteligibilidade para os seus corpos desviantes e rei- vindicam o reconhecimento (oficial) de suas identidades, por ainda estarem excluídas da agenda dos direitos civis. São reféns da ordem cis-heteronormativa e estão alheias ao projeto, ainda que essencia- lizante, de identidade. O reconhecimento dessas vidas como inteligíveis é uma grande questão para os estudos de gênero, pois ao mesmo tempo que as regulações do Estado nos interpõem barreiras de acesso a direitos, precisamos dessa mesma instância para afirmamos nossa existência enquanto pessoas no mundo. Embora algum tipo de política identitária seja plausível em termos de direitos civis, 103 S U M Á R I O o ponto defendido é que diferentes posições de sujeitos agenciam interdições e silenciamentos de modo singular. Isso quer dizer que diferentes marcadores identitários apontam para discursos e siste- mas de opressões que interagem de modo relacional e interseccio- nal (CRENSHAW, 1991). A interseccionalidade colabora para estudar as assimetrias nas relações de poder em diferentes eixos de opressão e entende as nossas práticas identitárias imbricadas e constrangidas por uma matriz de dominação estrutural e disciplinar (BORGES, 2022). Nesse sentido, o viés queer orienta-se por uma visão de poder descentra- lizada, antiessencialista, anticolonialista, anticapitalista e mestiça, entende a identidade como performance, rejeita qualquer categoria de identificação em detrimento dá ênfase à diferença e às liberações desejantes (LEÓN, 2012). PERFORMANCE E ASSUJEITAMENTOS As experimentações queer tem o potencial de produzir socia- bilidades pautadas na diferença e em projetos sociais éticos, preocu- pados com equidade social. Enquanto isso não ocorre, determinadas pessoas se veem em um impasse de serem vistas como “monstros”, usando o termo de Preciado (2020, p. 25): “O monstro é aquele que vive em transição. Aquele cujo rosto, cujo corpo, cujas práticas e lin- guagens não podem ser ainda considerados como verdadeiros em um regime de saber e poder determinado”. O patriarcado e o colonialismo não são modelos já abando- nados, mas “infraestruturas cognitivas, regimes de representação, técnicas do corpo, tecnologias do poder, discursos e aparatos de verificação, narrativas e imagens que continuam operando no pre- sente” (PRECIADO, 2022, p. 31). Interessa à linguística queer investi- gar as formas possíveis de vida, de modo contracultural, para vencer esses agenciamentos que, muitas vezes, são naturalizados e repro- duzidos inclusive pelos próprios grupos minorizados. 104 O sujeito amparado de sua(s) identidade(s) olha diante de si mesmo e se vê sozinho, sendo capaz de se ver como um ser mais amplo além de si mesmo. Ao olhar para a sociedade, deseja se inte- grar nela, pertencer a ela. De acordo com Bakhtin, a existência do outro é o elemento chave de nossa existência, eu sou o que sou por- que existe o outro, o discurso de outrem me identifica e faz com que eu me torne o que eu sou (BAKHTIN, 1995). Desse modo, na pre- sença do não-eu, do diferente, do estranho, já não posso mais me considerar como sujeito permanente cuja essência centraliza toda ordem de ser e de estar no mundo. S U M Á R I O À esteira de Butler (1993), o sexo, assim como as identida- des, é social e discursivamente construído. A tentativa de caracte- rizar as pessoas por meios de suas práticas sexuais tem propiciado um debate intensivo na antropologia, nas ciências sociais, biológicas, nas políticas públicas dos estados-nação, nas instituições de ensino, na medicina. Cada vez mais nos deslumbramos com as inúmeras possibilidades de nos tornarmos seres inteligíveis por meio de nos- sas práticas sexuais, e mais curiosos, na medida em que, ampliamos as possibilidades do que podemos fazer com nossos corpos. Por isso, os discursos produzidos em torno das classificações identitárias, caracterizam-se como práticas que sistematicamente formam os objetos que elas nomeiam. PERFORMANCE E ASSUJEITAMENTOS Nomear, então, torna-se um instrumento de poder, a partir da sua efetividade de criar realidades. Preciado (2020) é crítico a essas realidades, pois como homossexuais, transexuais, trabalhadores do sexo, corpos racializados ou travestis temos tido do mesmo modo alterizados e animalizados, [...] o que a medicina e a psiquiatria têm desenvolvido nos últimos séculos é também um processo comparável à objetificação, à exotização, à normalização e, em último termo, à exter- minação institucional e políticas das minorias sexuais (PRECIADO, 2020, p. 46). 105 No cerne destas estruturas individualizadas, os gêneros são desenhados de acordo com binarismos e verdades estanques aos contextos dos sujeitos neles inseridos. Nesse sentido, a abordagem queer converge para a proposta foucaultiana de uma analítica da nor- malização propondo uma ruptura radical com a modernidade, dife- renciando-se em sua perspectiva de análise porque “foca nas subje- tividades dos sexualmente dissidentes buscando somar à crítica dos processos sociais normalizadores uma compreensão não-normativa da subjetividade daqueles que vivem em desacordo com as prescri- ções sexuais e de gênero hegemônicas” (MISKOLCI, 2009, p. 334). No queer há uma articulação da analítica do poder foucaul- tiana que pode ser aproveitada como um empreendimento político não-moralizador dos indivíduos, tendo em vista o paradoxo cis-hete- ronormativo e as políticas identitárias que privilegiam subjetivações normalizadas, estão sempre em disputa de um lado, reiterações, repetições, constrangimentos e assujeitamentos, do outro são pro- duzidos modos de vir a ser subversivos em fluxos transgressivos, criativos e inovadores. Não obstante, a institucionalização e o embranquecimento heteronormativo neoliberal caracterizam complexidades da prá- xis investigativa queer. Nesse sentido, Borges (2022, p. 353) alerta para o fato de que ao desconsiderar a branquitude, os processos excludentes de escolaridade, a heteronormartividade e o neolibe- ralismo como contingências sociais que “moldam as interações de pesquisa”, incorremos no equívoco de produzir leituras de mundo “enviesadas” e “ilegítimas”. Por isso, enquanto pesquisadoras e pesquisadores, sobre- tudo por praticarmos nossas pesquisas com uma visada crítica, pre- cisamos nos questionar até que ponto não estamos nos assujeitando também às sutilezas das normatividades, tanto do nosso corpo, da nossa agência, como das nossas escritas e do modo como condu- zimos nossos estudos. 106 REPENSANDO VIVÊNCIAS E PESQUISAS QUEER Segundo Moita Lopes e Fabrício (2018), o campo dos estu- dos da linguagem tem sido, por muito tempo, influenciado por uma forte tradição representacional, tradição que oferece as bases para uma linguística modernista rígida orientadora de um modelo de realidade autoritário e dicotômico sobre os sujeitos. Tal compreen- são, no entanto, perde sua potência na medida em que o cenário contemporâneo tem sido caracterizado em virtude de sua intensa mobilidade de recursos linguísticos e semióticos, e ainda, da comple- xidade dos processos da globalização potencializados pelos e nos ambientes digitais. Ao classificar as pessoas, produzimos dinâmicas de governa- mentalidade capazes de transformar sujeitos em categorias univer- sais, objetos ou abjetos. Categorizamos, ainda, quais normas funda- mentam e constroem suas práticas sociais, como propõe Butler (1990). Warner (1992) resume grande parte das discussões que trou- xemos até aqui sobre o ser queer da seguinte maneira: No terreno político diário, disputas sobre a sexualidade e suas regulações estão ligadas geralmente a visões de instituições sociais e normas dos tipos mais básicos. Toda pessoa que se entende como queer sabe, de um modo ou de outro, que sua estigmatização está conectada com o gênero, a família, as noções de liberdade individual, o Estado, o discurso público, consumo e desejo, natureza e cultura, amadurecimento, políticas de reprodução, fanta- sia racial e nacional, identidade de classe, verdade e con- fiança, censura, vida privada e pública, terror e violência, assistência de saúde e normas culturais profundas em relação ao corpo. Ser queer significa lutar contra essas questões todo o temo, de forma local e gradual, mas sem- pre com consequências (WARNER, 1992, p. xiii). 107 Se como pessoas queer vivemos constrangidos pelas inúme- ras maneiras como as normatividades se manifestam e se interpõem em nossas vivências, como bem descreve o autor acima, é preciso repensar os assujeitamentos causados por elas, de modo que não nos sujeitemos nem tanto às estigmatizações, tampouco pensemos que existe uma liberdade total para de tudo escaparmos. É possível, sim, fabricar possibilidade de vidas outras, conforme traz Preciado (2022), nesse mundo disfórico e “petrosexorracial”, caracterizado pela exploração de combustíveis fósseis contaminantes, a utilização de uma taxonomia excessiva para a classificação social dos indiví- duos por meio de tecnologias dos governos, a dominação de uns corpos sobre os outros no sistema binário e colonial. No campo de pesquisas, considerar relevante também bus- car cuirizar ou queerizar as maneiras como temos conduzido nossos estudos. REPENSANDO VIVÊNCIAS E PESQUISAS QUEER As “novas” abordagens que visionamos devem considerar relações menos verticais entre o sujeito que pesquisa e o sujeito sobre quem se pesquisa. Ainda que haja, de fato, uma relação desi- gual entre os sujeitos, pois, de um lado, sempre teremos um sujeito pesquisador assegurado e reconhecido publicamente por ação da instituição de ensino a que está vinculado, acreditamos nos cami- nhos possíveis e mais queer, cuirificados no que tange às nossas prá- ticas de investigação. Nosso poder enquanto pesquisadores queer é fazer uso de uma sorte de poder que é potência, criação e produção. Tal empreendimento pode ocorrer tanto em termos estrutu- rais, quando repensamos a organização da nossa escrita em artigos científicos, livros, capítulos, teses, dissertações e monografias, como em termos de condução de pesquisa e suas metodologias. BAKHTIN, Mikhail. Marxismo e filosofia da linguagem. São Paulo: Hucitec, 1995. BORBA, Rodrigo. Discursos transviados: por uma linguística queer.  1 Ed. São Paulo: Cortez, 2020. BORBA, Rodrigo. Discursos transviados: por uma linguística queer. 1 Ed. São Paulo: Cortez, 2020. BORGES, Thais Regina Santos. Branquitude e modos queer de pesquisar: por um olhar feminista, interseccional e decolonial. In: MELO, Glenda Cristina Valim de; JESUS, Dánie Marcelo (Orgs.). Linguística aplicada, raça e interseccionalidade na contemporaneidade. Rio de Janeiro: Mórula, 2022, p. 343-362. BUCHOLTZ, Mary.; HALL, Kira. Theorizing identity in language and sexuality research. Language in Society, 33, p. 469-515, 2004. BUTLER, Judith. Gender trouble. Feminism and the Subversion of Identity. New York; London: Routledge, 1990. BUTLER, Judith. Bodies that Matter: on the discursive limits of ‘sex’. Nova York: Routledge, 1993. REFERÊNCIAS BAKHTIN, Mikhail. Marxismo e filosofia da linguagem. São Paulo: Hucitec, 1995. BAKHTIN, Mikhail. Marxismo e filosofia da linguagem. São Paulo: Hucitec, 1995. 108 BORBA, Rodrigo. Linguística queer: por uma perspectiva pós-identitária para os estudos da linguagem. Entrelinhas, v. 09, n. 1, p. 91-107 jan./ jun., 2015. BORBA, Rodrigo. Linguística queer: por uma perspectiva pós-identitária para os estudos da linguagem. Entrelinhas, v. 09, n. 1, p. 91-107 jan./ jun., 2015. GUEDES, Lyra Brenda; COSTA, Silvia Almeida. Repensando a criança-consumidora: Novas práticas, novos paradigmas.  Comunicação, Mídia e Consumo. Ano 1, v. 1, n.1. Maio 2004. São Paulo: ESPM, 2012. GUEDES, Lyra Brenda; COSTA, Silvia Almeida. Repensando a criança-consumidora: Novas práticas, novos paradigmas. Comunicação, Mídia e Consumo. Ano 1, v. 1, n.1. Maio 2004. São Paulo: ESPM, 2012. HIRAMOTO, Mie. Who’s really normal? Language and sexuality in public spaces. National University of Singapore: Singapore, 2015. LEAP, William. Queer Linguistics as Critical Discourse Analysis. In: The Handbook of Discourse Analysis. 2nd edition. Chichester, UK: John Wiley & Sons, 2015, p. 661-681. LEÓN, Adriano de. Os labirintos do desejo: desenhando uma metodologia anarcoqueer. Revista de Ciências Sociais, n. 36, abril, 2012. MELO, Glenda Cristina Valim; MOITA LOPES, Luiz Paulo. Ordens de indexicalidade mobilizadas nas performances discursivas de um garoto de programa: ser negro e homoerótico. Língua(gem) em Discurso LemD, Tubarão, SC, v. 14, n. 3, p. 653-673, set./dez. 2014. MISKOLCI, Richard. Abjeção e desejo. Afinidades e tensões entre a teoria queer e a obra de Michel Foucault. In: RAGO, Margareth.; VEIGA-NETO, Alfredo (Orgs). Para uma vida não-fascista. Belo Horizonte: Autêntica, 2009. MOITA LOPES, Luiz Paulo da. Por uma linguística aplicada (in)disciplinar. São Paulo: Parábola, 2006/2016. BUTLER, Judith. Bodies that Matter: on the discursive limits of ‘sex’. Nova York: Routledge, 1993. CAMERON, Deborah.; KULICK, Don. Sexuality as identity: gay and lesbian language. In.: Cameron, Deborah; Kulick, Don (Orgs.). Language and sexuality. Cambridge: Cambridge University Press, p. 74-105, 2013. CAMERON, Deborah. Desempenhando identidade de gênero: conversa entre rapazes e construção da masculinidade heterossexual. In: Linguagem, Gênero, Sexualidade: clássicos traduzidos. São Paulo: Ed. Parábola, 2010, p. 129-150. CRENSHAW, Kimberlé Willians. Mapping the Margins: Intersectionality, Identity Politics, and Violence against Women of Color. Stanford Law Review, 43, 1991, 1241-1299. DARING, Channon. B.; ROGUE, Jen.; SHANNON, Deric; ABBEY VOLCANO. Queering Anarchism: Addressing and Undressing Power and Desire. AK Press: Oakland, 2012. DELEUZE, Gilles. GUATTARI, Félix. O anti-édipo: capitalismo e esquizofrenia I. São Paulo: Editora 34, [1972] 2014. ECKERT, Penelope; McConnell-Ginet, Sally. Comunidades de práticas: lugares onde co-habitam linguagem, gênero e poder. In: OSTERMANN, Ana Cristina; FONTANA, Beatriz (Orgs.). Linguagem, Gênero, Sexualidade: clássicos traduzidos. São Paulo: Parábola, 2010, p. 93-108. 109 S U M Á R I O FABRÍCIO, Branca. Falabella. Linguística aplicada e visão de linguagem: por uma INdisciplinaridade radical. RBLA, Belo Horizonte, v. 17, n. 4, p. 599-617, 2017. FOUCAULT, Michel. O sujeito e o poder. In: DREYFUS, Hubert; RABINOW, Paul. Foucault, Uma Trajetória Filosófica: Para além do estruturalismo e da hermenêutica. Trad. de Vera Porto Carrero e Antonio Carlos Maia. Rio de Janeiro: Forense Universitária, [1982] 1995. p. 231-249. MOITA LOPES, Luiz Paulo da. Por uma linguística aplicada (in)disciplinar. São Paulo: Parábola, 2006/2016. MOITA LOPES, Luiz Paulo; FABRÍCIO, Branca Falabella. Viagem textual pelo sul global: ideologias linguísticas queer e metapragmáticas translocais. Linguagem em (Dis) curso – LemD, Tubarão, SC, v. 18, n. 3, p. 759-784, set./dez. 2018. MOITA LOPES, Luiz Paulo; FABRÍCIO, Branca Falabella. Viagem textual pelo sul global: ideologias linguísticas queer e metapragmáticas translocais. Linguagem em (Dis) curso – LemD, Tubarão, SC, v. 18, n. 3, p. 759-784, set./dez. 2018. PRECIADO, Paul Beatriz. Manifesto Contrassexual. São Paulo: n-1 edições, 2014. PRECIADO, Paul Beatriz. Manifesto Contrassexual. São Paulo: n-1 edições, 2014. PRECIADO, Paul Beatriz. Yo soy el monstruo que os habla. (Nuevos Cuadernos Anagrama) (Spanish Edition). Barcelona: Editorial Anagrama, 2020. (Kindle Edition) PRECIADO, Paul Beatriz. Dysphoria mundi. Madrid: Anagrama, 2022. 110 Este texto apresenta, de forma revista, resultados da tese de doutorado intitulada “A performativ- idade política dos discursos de ódio neoconservadores na educação das dissidências sexuais e de gênero”, defendida em julho de 2022, no Programa de Pós-Graduação em Educação da Uni- versidade Federal do Paraná, que investiga como os discursos de ódio mobilizados contra as dissidências sexuais e de gênero têm figurado na atual governamentalidade neoconservadora, além de seus efeitos no campo da educação, considerando marcos políticos como o “kit gay”, a “ideologia de gênero” e a eleição de Bolsonaro. RAMPTON, Ben. Language in late modernity: interaction in an urban school. Cambridge University Press, 2006. TRUJILLO, Gracia. (Pre)ocupando al 15-M. Activismos feministas y queer/cuir em las protestas contra las políticas de austeridad. In: COLLING, Leandro. (Org.) Dissidências sexuais e de gênero. Salvador: EDUFBA, 2016, p. 69-90. S U M Á R I O WARNER, Michael. “Introduction” In: Fear of a Queer Planet: Queer Politics and Social Theory. Minneapolis: University of Minnesota Press, 1993, p. vii-xxxi. 111 INTRODUÇÃO S U M Á R I O Qualquer empreendimento crítico acerca da linguagem e de seus usos dificilmente escaparia às argumentações de seu caráter potencialmente destrutivo (e, porque não, produtivo, em termos fou- caultianos?) quando consideramos os enquadramentos neoconser- vadores gestados na ordem dos nossos dias. Uma breve reflexão sobre a emergência de ações centradas no combate ao reconhe- cimento de modos de vida mais dissidentes em relação à cishete- ronormatividade evidencia como os discursos de ódio gozam certa legitimidade e o quanto são requeridos nos traquejos da política con- temporânea brasileira. Polêmicas como o “kit gay” e a “ideologia de gênero”, que movimentam toda sorte de discursos e manifestações odiosas, apesar de soarem embotadas, constituem, afinal, mais do que uma espécie de pauta-comum em agendas eleitorais. Ao pro- mover “pânicos morais” (RUBIN, 2012; MISKOLCI, 2007; CÉSAR; DUARTE, 2017), investidas como essas arriscam perspectivas e ações antidiscriminatórias para além do campo educacional, afe- tando outras esferas da vida cotidiana, como o direito de aparecer nas ruas, de não sofrer violações, de acessar serviços públicos bási- cos ou mesmo ocupar postos de trabalho. Em nossas pesquisas10, ao discutir as ofensivas neoconserva- doras na educação dos gêneros e das sexualidades, tentamos articu- lar problemas suscitados quando pensamos a linguagem de ódio em contato com o que já não é (e talvez nunca tenha sido) apenas dis- cursivo. Especificamente, ao analisarmos discursividades produzidas 10 10 113 S U M Á R I O sobre as dissidências sexuais e de gênero na atual governamenta- lidade neoconservadora11, insistimos que os discursos de ódio têm mais a nos dizer do que as narrativas comuns usualmente supõem, isto é, não acreditamos que este tipo de discurso possa ser enten- dido como uma composição fixa do significado literal de suas partes: discurso e ódio. Desconfiamos, inclusive, das buscas por uma locali- zação pré-discursiva e mais “segura” do ódio, geralmente subsumida na soberania do sujeito ou dos signos. Sabemos que a procura por um sujeito que odeia (sempre patológico) ou mesmo por uma lista restrita de palavras consideradas ofensivas não são critérios muito úteis para rechaçar determinados enunciados e enunciações, pois para além da arbitrariedade dos signos, a linguagem também é aquilo que fazemos com as palavras (BUTLER, 1997). Que fazer diante disso? Não seria desonesto perguntar o que “sobra” da linguagem e, para além dela, quando saturamos do que nela é, contundentemente, violento. A governamentalidade neoconservadora pode ser entendida como uma racionalidade política “normativa e disciplinadora interiorizada pelos sujeitos contemporâneos, conformando-os ao ‘princípio universal da concorrência” (BIROLI; VAGGIONE; MACHADO, 2020, p. 26), que promove a regulação da moralidade sexual a partir da defesa da família heteronormativa, além da produção de cultura e subjetivação políticas fortemente atravessadas por paixões mortais como a LGBTIA+- fobia, o racismo, o classismo, o antifeminismo e o autoritarismo (BROWN, 2019). Uma discussão detalhada pode ser acessada em Pereira (2022). INTRODUÇÃO No entanto, se ela é uma das for- mas privilegiadas para o exercício do poder, falamos, talvez, de uma relação quiasmática entre vida e linguagem (BUTLER, 1997): que tipo de agência existiria fora da linguagem se dela dependemos para sig- nificar as coisas que “estão aí”, incluindo os nossos corpos, estiliza- ções e mobilizações políticas? Certamente, ao entender os discursos de ódio como performativos, abandonamos o caráter supostamente descritivo, representativo e essencialista da linguagem para pensá-la como atributiva em seu caráter social e reiterativo; adentramos um campo que não se encerra nas enunciações linguísticas, tampouco em ações individuais, mas se estende em modos corpóreos e plurais de exercício da política. Uma aposta que, nesse caso, parte da teoria 114 dos atos de fala de John Langshaw Austin (1990) para ser tensio- nada em perspectivas mais aliadas ao pós-estruturalismo, como as de Judith Butler (1997; 2003; 2018; 2019). Justamente, interessa-nos, aqui, arriscar uma leitura dos dis- cursos de ódio neoconservadores que possa contradizer o clamor ao uso meramente descritivo da linguagem sob a forma da opinião ou da liberdade de expressão e deslocar o foco da tríade sujeito agres- sor-ofensa-sujeito agredido. Nosso intento não é fazer uma revisão sistemática do performativo da linguagem, mas partir das perspecti- vas austinianas e butlerianas para analisar alguns enunciados, desde excertos de notas taquigráficas produzidas no âmbito da Câmara dos Deputados, além de tweets publicados em perfis reconhecidos publicamente por seu posicionamento odioso em relação às minorias sexuais e de gênero, pois entendemos que investigar esses discursos também nos permite arriscar um caminho outro em torno da análise da centralidade do ódio na linguagem política contemporânea. O PERFORMATIVO NOS DISCURSOS DE ÓDIO NEOCONSERVADORES Não pensamos a linguagem enquanto imagem do mundo, como um dado natural e transcendental, neutro, estável e destitu- ído de relações de poder. Ao contrário, entendemos que a relação inextricável entre linguagem e mundo não é denotacionista, mas atributiva; a linguagem é constitutiva do pensamento e do sentido que damos às palavras e às coisas (VEIGA-NETO, 2017); ou, ainda, performativa, isto é, capaz de produzir efeitos, estados de coisas, independentemente de uma forma linguística a priori (AUSTIN, 1990; OTTONI, 2002). Nesse sentido, importam as inquietações que Aus- tin propunha em sua disrupção à tradicional filosofia da linguagem, isto é, em seu deslocamento de temas em voga, como as formas 115 lógicas ou os elementos constitutivos da linguagem, para os proble- mas que tinham sido, até então, abandonados por essa mesma tradi- ção, como os usos ordinários da linguagem; é por este caminho que suas teorizações rompem lugares-comuns (OTTONI, 2002) e, conse- quentemente, por onde desejamos começar as nossas indagações. Em Quando dizer é fazer, Austin (1990) questiona a função supostamente constatativa de alguns proferimentos, sobretudo aqueles envolvidos em convenções sociais, circunscrevendo uma classe de proferimentos que realizariam alguma ação em decorrên- cia ou como efeito daquilo que é proferido. São esses proferimentos que, em um primeiro momento, serão chamados de “performativos”, por uma derivação do verbo inglês “to perform”, correlato do subs- tantivo “ação”. Como exemplo, dizer “aceito esta mulher como minha legítima esposa” em uma cerimônia de casamento faria mais do que apenas descrever a ação em curso ou declarar aquilo que se está fazendo, o proferimento “aceito” constituiria a ação de casar-se. Assim, para que os performativos realizem a ação que pres- supõem, ou para que sejam considerados “felizes”, algumas condi- ções devem ser cumpridas, entre elas: deve existir um procedimento convencionalmente aceito, que apresente um determinado efeito convencional e que inclua o proferimento de certas palavras, por certas pessoas, e em certas circunstâncias; e além disso, que as pessoas e circunstâncias particulares, em cada caso, devem ser adequadas ao procedimento específico invo- cado. O procedimento tem de ser executado, por todos os participantes, de modo correto e completo. O PERFORMATIVO NOS DISCURSOS DE ÓDIO NEOCONSERVADORES Nos casos em que, como ocorre com frequência, o procedimento visa às pessoas com seus pensamentos e sentimentos, ou visa à instauração de uma conduta correspondente por parte de alguns dos participantes, então aquele que participa do procedimento e o invoca deve de fato ter tais pensa- mentos ou sentimentos, e os participantes devem ter a intenção de se conduzirem de maneira adequada, e, além disso, devem realmente conduzir-se dessa maneira sub- sequente (AUSTIN, 1990, p. 31). 116 Se essas condições são infringidas, há um glossário austi- niano para dizer que o performativo será “infeliz”: malogrado, errado, fracassar, tropeço, desacerto, abusos, más execuções, más invoca- ções, más aplicações, nulos, desrespeito, dissimulações... E Austin (1990, p. 34) reconhece que “a infelicidade é um mal herdado por todos os atos cujo caráter geral é ser ritual ou cerimonial, ou seja, por todos os atos convencionais”. Não à toa o casamento é um exemplo recorrente em Quando dizer é fazer, já que supõe um rito de palavras e ações ou um jogo de condições entre autoridade e aquiescência; em suma, um evento cujo peso convencional satisfaria plenamente as condições para um performativo feliz, mas, que, paradoxalmente, é uma fonte inesgotável de modos como um performativo pode dar errado. Outros exemplos incluem o batismo de um navio, que neces- sita de uma pessoa escolhida para tal função e um nome de batismo adequado (embora não saibamos exatamente os critérios para isso), além do batizado de uma criança na Igreja Católica, que somente pode ser realizado por um padre a partir do proferimento de certas palavras em uma cerimônia específica. Seguindo os exemplos que Austin (1990) oferece, podemos concluir que os performativos estejam invariavelmente vinculados a convenções sociais, ou que a força de um performativo dependa, necessariamente, de um sujeito e um contexto estáveis. Como Sed- gwick (2018) percebe, o sujeito requerido para o performativo parece emergir na 1ª pessoa do singular do presente do indicativo, que fala e age naturalmente em situações que apelam à autoridade do Estado ou da Igreja, e que conta com uma rede de apoio de testemunhas solenes. Em outras palavras, é como se os performativos apenas se efetivassem quando pronunciados por sujeitos que ocupassem, de antemão, uma posição privilegiada para fazê-lo e, ao mesmo tempo, comandassem a cena enunciativa. O PERFORMATIVO NOS DISCURSOS DE ÓDIO NEOCONSERVADORES No entanto, essas assertivas deve- riam ser questões e não presunções até mesmo para o exemplo fun- dacional da teoria dos atos de fala, o casamento heterossexual, pois 117 o ‘eu’ que fala no ‘sim, quero’ é um ‘eu’ apenas na medida em que ele ou ela consente em formar parte de um ‘nós’ estabelecido e heterossexual, como tal, constituído na pre- sença de um ‘eles’, e sua capacidade de atuação e volição neste assunto depende inteiramente de um ritual confuso de hiperidentificação com os poderes (para os quais não há pronome válido) do Estado e, frequentemente, tam- bém da Igreja (SEDGWICK, 2018, p. 75, tradução livre). Ou seja, o performativo em uma cerimônia de casamento não acontece simplesmente via proferimento da autoridade represen- tada por um padre ou por um juiz ou uma juíza, ou pela deliberação dos sujeitos que dizem “sim”, mas principalmente pelo testemunho de outrem e pela repetição de uma convenção, já que essa cerimô- nia é um ato de expor e ritualizar o vínculo diádico, monogâmico e heteronormativo. O casamento é um bom exemplo da fragilidade das condições pressupostas por Austin (1990) para um performativo feliz porque, certamente, há maneiras incontáveis de fazer um casamento dar errado ou de torná-lo infeliz (para usar um termo austiniano) que não compreendem a clássica interrupção da cerimônia por uma ter- ceira parte interessada, ou a hipótese de um ator assumir o lugar do padre, ou, ainda, o estranho exemplo austiniano sobre a impossibili- dade de um padre casar dois ursos de pelúcia. O casamento entre pessoas de mesmo gênero poderia facil- mente ser um exemplo de perturbação das condições de um perfor- mativo feliz, não somente nos lugares em que permanece juridica- mente proibido, mas por ser considerado uma forma menos “moral” ou “natural” do matrimônio, mesmo onde é permitido. Ao contrário do casamento heterossexual, tem-se aí uma relação potencialmente menos segura ou complacente com a família, com as testemunhas (e não estamos falando somente das pessoas convidadas, mas do ato “público” de testemunhar), com a sanção religiosa e com o Estado que, inclusive, podem se tornar (e com frequência o são) vetores de ameaças e violências que tornam mais tênues o senso de direito e agência desses indivíduos, comumente alocadas em recusas ou 118 desvios da/pela lógica heterossexual (SEDGWICK, 2018). Em última instância, nesse caso, não estaríamos distantes daquilo que Austin (1990, p. O PERFORMATIVO NOS DISCURSOS DE ÓDIO NEOCONSERVADORES 36) chamou de “estiolamento da linguagem”, isto é, o enfra- quecimento que um ato de fala sofre ao ser utilizado de modo “para- sitário” em contextos não literais, como o teatro ou a ficção, ainda que o casamento heterossexual seja, por excelência, S U M Á R I O [...] uma espécie de quarta parede ou arco de proscênio invisível que circula no mundo (um casal heterossexual seguro de seu direito de dar as mãos na rua), que, em torno dela, reorienta continuamente as relações circunvi- zinhas de visibilidade e audiência, do tácito e do explícito, da possibilidade ou impossibilidade de uma determinada pessoa expressar uma determinada posição enunciativa (SEDGWICK, 2018, p. 75-76, tradução livre). Em um gesto parecido, Butler (2003) discute os processos de generificação que inauguram nossas vidas sociais a partir de um aparato discursivo médico-científico abreviado pelas célebres expressões “é um menino” ou “é uma menina”. Enunciados como esses não descrevem ou constatam materialidades pré-discursivas, embora seja convencionado, cisheteronormativamente, que sexo e gênero são conceitos depurados de historicidade; antes, esses enunciados estão atribuindo identidades de gênero e, de certo modo, identidades sexuais, uma vez que as normas de gênero operam pela incorporação violenta de ideais normativos de feminilidade e mascu- linidade que, frequentemente, são acompanhados pela idealização do vínculo heterossexual (DORLIN, 2009), o que torna esse ato “pri- mordial” um arranjo fundamental para a coerência entre sexo-gêne- ro-desejo ou, ainda, um briefing para o “eu aceito”/”sim” das cerimô- nias matrimoniais (BUTLER, 2019). Vale lembrar, inclusive, o kitsch heteronormativo que se reinventa, recentemente, na forma dos “chás de revelação do sexo”, que adiciona uma camada de “magia” a esse processo de nomeação (com bolo, balões e cores em tons de azul ou rosa, evidentemente), no qual o “revelar” funciona tanto como uma hipérbole dos dizeres clínicos e médicos quanto uma reiteração das normas sexuais e de gênero diante de testemunhas. 119 Objeções poderiam ser feitas nesses casos, já que uma criança não dispõe de condições para aceitar ou recusar esses rituais de generificação que acontecem antes mesmo do nascimento; aliás, que possibilidade de inteligibilidade existiria fora desses processos se as normas devem ser “citadas” a fim de nos constituirmos como sujeitos “viáveis”? Ainda assim, ao contrário do que poderiam supor as teorizações austinianas, esse performativo acontece, embora nem sempre funcione da maneira esperada: cisgênera e heterosse- xual. O PERFORMATIVO NOS DISCURSOS DE ÓDIO NEOCONSERVADORES Antes, é preciso que uma série de normas sejam reiteradas e negociadas ao longo de nossas vidas “em relação a um conjunto mais difuso e complicado de poderes discursivos e institucionais” (BUTLER, 2018, p. 36) para que a performatividade de gênero seja realizada, feliz ou infelizmente, nos termos de Austin. Em todo caso, essa tensão sobre as condições de felicidade de um performativo pode ser pensada junto ao contraditório esforço austiniano de delimitar os atos de fala performativos e, ao mesmo tempo, desmantelar a distinção constatativo/performativo. Para isso, Austin (1990) distingue os atos de fala locucionários, ilocucionários e perlocucionários, que, por serem atos, são performativos; o que os diferencia parece ser uma questão de força de efetividade. No primeiro ato, locucionário, é como se não houvesse uma força de ação, justamente porque esse tipo de ato, grosso modo, é simplesmente o ato de dizer algo. Ele compreende os atos fonéticos, ou a emissão de certos ruídos, os atos fáticos, ou o proferimento de palavras ou vocábulos organizados sintaticamente, e os atos réticos, ou operações semânticas de sentido e referência. Eles se relacio- nam à medida que sempre que realizamos um ato fático, realizamos um ato fonético (o contrário não é verdade) e, ao realizarmos um ato rético, realizamos um ato fático (aqui, o contrário também não é necessariamente verdade). Atos locucionários são, portanto, o ato de dizer algo ou de pro- duzir sentido, diferentemente dos atos ilocucionários, que consistem 120 S U M Á R I O na realização de um ato ao dizer algo, no momento mesmo do pro- ferimento, ou seja, implicitamente, existe uma redundância entre enunciado e ato, uma vez que aceitamos dizendo “aceito”, interro- gamos dizendo “posso?”, prometemos dizendo “juro que”, ou orde- namos dizendo “faça isso” (DELEUZE; GUATTARI, 2000). Em uma relação de forças, esses seriam os atos adequados para efetivar um performativo, mesmo porque, de modo mais direto, produzem con- sequências convencionais (AUSTIN, 1990), como as sentenças judi- ciais, os batismos, os casamentos e as inaugurações de propriedade que exercem um poder de conexão à ação realizada - uma sentença transforma o corpo acusado em corpo-prisioneiro, assim como um batismo transforma o corpo em corpo-cristão etc (BUTLER, 2019; DELEUZE; GUATTARI, 2000). Um ato ilocucionário está relacionado com a produção de efeitos se houver o “reconhecimento entre os interlocutores de que algo está assegurado” (OTTONI, 2002, p. 12 O proferimento foi publicado em 2011 no perfil de Feliciano e, posteriormente, apagado. No en- tanto, um print do tweet foi divulgado por alguns sites de notícia à época, como neste link: https://bityli.com/0TzX4. O PERFORMATIVO NOS DISCURSOS DE ÓDIO NEOCONSERVADORES 134) e se as ações desem- penhadas por esse tipo de ato forem as mesmas enunciadas, acon- tecendo no momento mesmo do proferimento. São esses aspectos que os diferenciam dos atos perlocucionários, já que estes concer- nem à produção de certos efeitos sobre quem a fala é dirigida, mas não produzem, necessariamente, nem o que é dito nem ao mesmo tempo que o proferimento acontece. Como exemplo, podemos citar um proferimento publicado no perfil do Twitter do pastor e deputado federal Marco Feliciano: “a podridão dos sentimentos dos homoafe- tivos levam [sic] ao ódio, ao crime, a [sic] rejeição”12. Este enunciado pode, sem dúvida, produzir reações de medo, indignação, raiva ou, quem sabe, não provocar reação alguma, bem como suscitar um júbilo LGBTIA+fóbico. É imprevisível. Se o que está sendo produ- zido pelo proferimento não é idêntico ao conteúdo do enunciado, isto é, se não há conexão e transformação no estado dos corpos no 12 121 S U M Á R I O momento do proferimento (não somente porque não é o caso de um discurso direto proferido em uma convenção ou cerimônia, mas porque se trata de uma falácia e de um discurso de ódio dirigido às minorias sexuais), há um espaço entre o ato de dizer e a con- cretização dessa ação, que não é realizada ao dizer algo, mas que se estende temporalmente tanto para o passado da situação de fala quanto para o futuro, abrindo outras possibilidades quanto a seus efeitos (BUTLER, 1997). Em síntese, sempre que realizamos um ato ilocucionário, temos um ato locucionário (o contrário não pode ser dito), embora não seja possível afirmar que esses atos possam ser definidos e até mesmo derivados de atos locucionários. Por outro lado, quando efe- tuamos os atos locucionários e ilocucionários podemos realizar um perlocucionário, mas que também não se deixa reduzir à soma dos atos anteriores (AUSTIN, 1990). Sem dúvidas, a distinção entre esses três tipos de atos é um pouco complexa, e talvez essa nem mesmo fosse uma questão séria para Austin, que trabalhava, ao que parece, contra as próprias idiossincrasias13. Bem por isso, convém esquecer, por um momento, a distinção propriamente dita para ampliar o pro- blema da força de um ato de fala ou da efetividade de um performativo. Como Sedgwick (2018) percebe a partir da leitura de Shoshana Felman, as conferências que compõem Quando dizer é fazer funcionam, analiticamente, como se fossem tentativas sucessivas de refutação dos exemplos e dos limites da própria teoria dos atos de fala que ali estava sendo construída: “[...] una de sus astutas características consiste en un repetido tropismo, una eviden- te fascinación ante un tipo particular de ejemplos de los enunciados performativos. Aparecen presentadas en primer lugar como performativas puras, originarias y definitorias del concepto y, finalmente, desdeñadas con um mero ‘caso marginal en el límite’, eso admitiendo que sea posible decir que los ejemplos o el concepto fueron capaces de ‘sobrevivir’ a la operación analítica que Austin llevó a cabo en el conjunto de sus conferencias” (SEDGWICK, 2018, p. 6). Os excertos a seguir são oriundos de notas taquigráficas produzidas na Câmara dos Deputados. O contexto do primeiro enunciado destacado envolve a rejeição ao PL nº 3.369/2015 que institui o Estatuto da Família do Século XXI; o segundo enunciado, para citar o conteúdo da própria nota taquigráfica, compõe um proferimento contra “o endeusamento da comunidade homossexual”; e o terceiro constitui um proferimento a respeito do filme “A tentação de Cristo”, da produtora de vídeos Porta dos Fundos. Ressaltamos que estes enunciados são parte de um conjunto mais amplo que constitui o material empírico produzido na tese de doutorado que deu origem a este texto e que pode ser acessado em Pereira (2022). O PERFORMATIVO NOS DISCURSOS DE ÓDIO NEOCONSERVADORES Austin (1990) propõe que localizemos o enunciado em uma “situação total de fala”, isto é, que delimitemos o ato de fala em seu contexto de proferimento. Para os ilocucionários, a questão seria saber se o sujeito que invoca a lei, a cerimônia ou o ritual conven- cional está autorizado a fazê-lo e se as circunstâncias para a rea- lização do ato estão corretas, ou seja, observar as condições para 13 122 S U M Á R I O um performativo feliz. Contudo, o próprio estatuto convencional sabota a possibilidade de uma determinação completa porque, explicitamente, pressupõe um caráter citacional persistente e instá- vel, ou seja, o poder, visto na figura de autoridade (padre, juiz, juíza etc), está sempre repetindo usos anteriores do ritual ou da lei a que refere, e é precisamente essa repetição que confere ao proferimento seu poder de nomear e/ou vincular (BUTLER, 2019). Como Butler (1997) nos diz, as ilocuções (como todos os enunciados) apresentam e instituem um plano de ação que não se restringe ao instante do proferimento. Assim, ainda que um ato de fala ilocucionário realize sua ação no momento mesmo em que se pronuncia o enunciado, à medida que o momento está ritualizado, nunca é simplesmente um momento único. O “momento” em um ritual é uma historicidade condensada: excede o passado e o futuro, é efeito de invocações prévias e futuras que, ao mesmo tempo, constituem e escapam à enunciação (BUTLER, 1997, p. 18-19). Uma ação performativa, seja como for, não cessa ao tér- mino de uma cerimônia ou de um ato convencional, mas persiste no tempo, ainda que isso possa ser percebido com maior facilidade a partir dos atos perlocucionários. Podemos tomar como exemplo alguns proferimentos realizados pelo deputado federal mais bem votado na Bahia nas eleições de 2018, Manoel Isidório de Santana Júnior (AVANTE-BA), também conhecido como “Pastor Sargento Isi- dório”, a respeito de algumas discussões que se deram no âmbito da Câmara dos Deputados14: 14 123 [...] tenho certeza de que este Parlamento não se aga- chará, que os Deputados e Deputadas desta Casa não se abaixarão para chamar de família um grupo sexual. Aí, entram no quarto pai e mãe, chamam os filhos e, daqui a pouco, chamam cavalo, cachorro e gato, daqui a pouco está a zoofilia envolvida com a família. Que tipo de famí- lia querem fazer no Brasil? O PERFORMATIVO NOS DISCURSOS DE ÓDIO NEOCONSERVADORES Daqui a pouco, esse mesmo grupo vai querer também incluir, na família, a zoofilia (BRASIL, 2019a, não paginado). Do jeito que a coisa está indo, daqui a pouco nós heteros- sexuais - a família de homem mais mulher é igual a filho - teremos que abaixar a cabeça para que eles passem. Nós queremos respeito (BRASIL, 2019b, não paginado). Noventa por cento dos brasileiros são agredidos na sua fé pelo comportamento desses malignos escarnecedores, profanos, inescrupulosos, torpes, imundos, infames, iní- quos, assassinos da fé, enganadores, injuriadores, deso- bedientes de pai e mãe, néscios, infiéis, endemoninhados, enfim, marginais, inimigos de Deus. Não à toa, a CNBB informa que o homossexualismo [sic] adoece nossa juventude (BRASIL, 2019c, não paginado). Para nos afastarmos mais uma vez de Austin, a efetividade ou poder de ferir desses enunciados certamente poderia ser pensado junto à história de perseguição e violência contra as minorias sexuais e de gênero, bem como de suas práticas discursivas, presentificadas, nestes excertos, por uma lista de adjetivos pejorativos e pela correla- ção entre as dissidências afetivas e/ou sexuais e a zoofilia, ou a uma doença, como bem denota o termo “homossexualismo”. No entanto, é válido lembrar que narrar o passado e o futuro dessas cenas (embora seja importante) soa como um trabalho de Sísifo, porque parte do que constitui uma situação total de fala é a sua própria impossibili- dade de apreensão espaço-temporal. Justamente, a historicidade do discurso não quer dizer simplesmente que os discursos estejam alo- cados em contextos históricos, mas implica a história como constitu- tiva das práticas discursivas, isto é, elas não existem descoladas de suas condições de possibilidade (BUTLER, 2019; FOUCAULT, 2014). 124 Não que se deva abandonar qualquer tentativa de delimitação contextual, mas ela sempre estará sujeita a uma (re)contextua- lização posterior, que não será dada, em todo caso, de forma uní- voca (BUTLER, 1997). Desvencilhando-nos desse embaraço, em um primeiro momento, estaríamos diante da possibilidade de pensar mais cri- ticamente o caráter performativo dos discursos, inclusive dos dis- cursos de ódio. O PERFORMATIVO NOS DISCURSOS DE ÓDIO NEOCONSERVADORES Aceitando o argumento de que a força performa- tiva desse tipo de discurso advém de uma historicidade dissimulada no momento do proferimento, como uma estratégia de exercício do próprio poder (BUTLER, 1997; FOUCAULT, 1988), não há um sujeito soberano em um proferimento odioso, porque as palavras pronun- ciadas sempre vêm citadas de algum lugar e estão sujeitas à reite- ração e à ampliação em novas invocações (BUTLER, 1997). Assim, o discurso LGBTIA+fóbico não se origina com o sujeito do proferi- mento, ainda que ele seja necessário para dar continuidade à cadeia citacional; o que se tem no momento do proferimento é uma espécie de conexão ou vínculo com uma comunidade de sujeitos LGBTIA+- fóbicos “historicamente transmitida” (BUTLER, 1997, p. 138) que nos revela o caráter genealógico dessas invocações, ou seja, não é pela intenção do sujeito do proferimento que um ato de fala LGBTIA+fó- bico tem êxito, mas porque esse ato “acumula a força da autoridade com a repetição ou a citação de um conjunto prévio de práticas auto- rizantes” (BUTLER, 2019, p. 375). Para ter êxito, um ato performativo precisa contar com o apoio e, ao mesmo tempo, dissimular as con- venções constitutivas pelas quais é mobilizado (BUTLER, 2019). Evidente que isso não implica a desresponsabilização do pro- ferimento de discursos de ódio. Nunca é demais lembrar que sobera- nia e responsabilidade são coisas distintas (BUTLER, 1997). A crítica à soberania que, aqui, toma lugar diz respeito ao descentramento do sujeito e uma politização de sua capacidade de agência; enquanto a responsabilidade tem a ver com assumir, deliberadamente, o ato de citar enunciados. Do mesmo modo como não estamos propondo o 125 S U M Á R I O “descarte” do sujeito para a enunciação, ou declarando uma espé- cie de agência sem corpo, mas apenas situando que “os efeitos da ação [...] têm sempre o poder de proliferar para além do controle do sujeito, para desafiar a transparência racional da intencionalidade desse sujeito” (BUTLER, 1998, p. 19). É por isso que os discursos estão sempre fora de controle de quem os profere, escreve ou sinaliza, ou que a “fala é excitável”, se quisermos usar o vocabulário de Butler (1997), isto é, o alcance da significação das expressões performativas persiste para além do sujeito (e, às vezes, contra suas próprias inten- ções) e do momento do proferimento (BUTLER, 2019). O PERFORMATIVO NOS DISCURSOS DE ÓDIO NEOCONSERVADORES Se, de algum modo, as próprias teorizações austinianas já nos mostravam que a vontade e a intenção do sujeito não são sufi- cientes para que um ato performativo se realize, ou mesmo para que possamos apreendê-lo (justamente pelos problemas que deixaram em aberto), à máxima “ao dizer algo, estamos fazendo algo” seria preciso acrescentar que o “fazer” não está alocado em um retorno ao poder soberano, centralizado, fonte única e originária do poder e do discurso, estendido ao sujeito. Isso é importante porque as ten- tativas de localizar a fonte do discurso ou de emanação do poder funcionam muito bem em prol de uma despolitização dos discur- sos de ódio. Quando dissemos, há pouco, que a historicidade de um enunciado era dissimulada no momento do proferimento, era senão porque a vontade ou a intenção de um sujeito soberano toma conta da cena enunciativa. A LGBTIA+fobia, por exemplo, teria como causa um sujeito LGBTIA+fóbico e não uma rede de saber e poder que produz, macro e micropoliticamente, o ódio e a violência, bem como formas de odiar e violentar as minorias sexuais e de gênero de modo estrutural. Assim, podemos interrogar ações como a decisão do Supremo Tribunal Federal em criminalizar a LGBTIA+fobia, no sentido de que talvez ela atenda majoritariamente à culpabiliza- ção do sujeito do proferimento; ou, que tipo de parâmetros serão criados para os limites do dizível, até porque as práticas religiosas 126 LGBTIA+fóbicas não parecem ser questionadas. E, não menos importante, se queremos legar tais configurações a um Estado que se recusa a promover condições de vida mais igualitárias para esses sujeitos, que negligencia sistematicamente demandas que são histo- ricamente pleiteadas, e que também é um agente ativo de violações como o assédio, a patologização e outras violências (MOMBAÇA, 2021). Perde-se aí um gesto fundamentalmente crítico a favor de uma tranquilidade fictícia, que pode ser deslocado, paradoxalmente, em direção às retóricas de “opinião” e “liberdade de expressão”, como nos mostra o proferimento do pastor Silas Malafaia (2015, não paginado): “opinião não é homofobia. Opinião não é crime. Os esquerdopatas, libertinos, ativistas gays, não suportam o contraditório. Estamos na democracia”. Do mesmo modo como essas retóricas podem resvalar na imunidade parlamentar, como podemos notar nos proferimentos do deputado federal Marco Feliciano e do presidente Jair Bolsonaro destacados a seguir: [...] Irão reconhecer o crime de ‘homofobia’ como crime de racismo. O PERFORMATIVO NOS DISCURSOS DE ÓDIO NEOCONSERVADORES Uma mordaça será colocada na boca dos pas- tores e padres. Pior do que mordaça na boca dos líderes, será a mensagem subliminar de tolhir [sic] o direito a fé, aos valores cristãos, ao uso da própria bíblia cristã. Pois o termo HOMOFOBIA é muito vago, não há como mensu- rar o que ele significa. Criará insegurança jurídica (FELI- CIANO, 2019, não paginado). [...] Eles não querem igualdade, eles querem privilégios. Eles querem é nos prender porque nós olhamos torto para eles, nos prender porque nós não levantamos de uma mesa para tirar nossos filhos ‘menor’ de idade [sic] de ver dois homens ou duas mulheres se beijando na nossa frente, como se no restaurante fosse um local para fazer isso. Eles querem é privilégios! Eles querem é se impor como uma classe à parte. [...] E eu tenho imunidade para falar que sou homofóbico, sim, com muito orgulho, se é para defender as crianças nas escolas (BOLSONARO, 2013, não paginado). 127 De fato, não precisamos conhecer a intencionalidade de um proferimento, tampouco de um sujeito empírico que enuncia, para dizer de sua capacidade performativa. Nem sempre o ódio LGB- TIA+fóbico encontra seus destinatários diretos. Não à toa pai e filho abraçados podem ser agredidos em praça pública quando a conduta é interpretada como homoafetiva, ou que livros com personagens e temática LGBTIA+ possam ser recolhidos em feiras literárias, ou que a performatividade de gênero possa ser tomada como sinônimo de homossexualidade ou transexualidade, por exemplo. Nesse exercício de sobreposição, a LGBTIA+fobia é menos direcionada às práticas sexuais propriamente ditas do que aos desvios da cisheteronormati- vidade naquilo que Michel Foucault chamou de “economia dos pra- zeres” (ERIBON, 2004; FOUCAULT, 2015), o que, decerto, ajuda a explicar outras questões, como a reatividade ao pensamento e às palavras nas perseguições em torno dos ativismos e produções aca- dêmicas feministas e LGBTIA+. CONSIDERAÇÕES FINAIS Ao longo do texto, compomos alguns argumentos sobre o caráter performativo dos discursos de ódio neoconservadores sobre gênero e sexualidade a partir de Austin (1990) e Butler (1997; 2003; 2018; 2019) uma vez que, no nosso entendimento, tais perspectivas sobre a performatividade da linguagem podem nos ajudar a pensar de um modo outro a centralidade do ódio no atual cenário político brasileiro, isto é, permitem trabalhar um pouco mais por fora dos ter- mos em que comumente a disseminação desse tipo de discurso é tomada: ora como uma emanação subjetiva de um sujeito que odeia, ora como uma questão puramente jurídica de localizar e punir o sujeito do proferimento. 128 Brevemente, afirmamos a insuficiência de supor as dissidên- cias sexuais e de gênero como alvos únicos e finais de um sentimento compartilhado por um grupo relativamente homogêneo que odeia e manifesta o ódio da mesma forma, tanto porque essas premissas se firmam em concepções personificadas e unilaterais quanto porque o ódio não deve ser apenas “um efeito” sobre determinados sujeitos, mas um conjunto de efeitos sobre cadeias mais amplas de afecções, que se projetam a partir de uma historicidade condensada e colo- cam em jogo uma futuridade imprevisível. Assim, também não nos parece que a delimitação de um contexto apropriado (com todas as condições austinianas de felicidade) seja imprescindível para consi- derar a efetividade de um performativo, não só porque o contexto de enunciação sempre escapa às tentativas de totalização, mas porque parte do trabalho dos discursos de ódio consiste em desfazer a partir da dor e da humilhação as posições de sujeito que ocupamos, ainda que essas posições sejam, por vezes, inabitáveis (AHMED, 2015). Para além do recorte que estabelecemos, aqui, caberia inves- tigar, portanto, não apenas o dano que a linguagem nos causa, mas como ela o faz, que não se deixa capturar pela soberania do sujeito, tampouco dos signos. Em última instância, significaria atentar aos limites do dizível que os discursos de ódio neoconservadores dispu- tam, que tem a ver, na verdade, com os modos pelos quais eles nos informam os próprios limites do inteligível. REFERÊNCIAS AHMED, Sara. La politica cultural de las emociones. 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POSSATTI, 2020), que revelam um pouco de como esse fator mostrou influen- ciar o processo de convergência dos falantes. Com isso, objetivo chamar atenção não apenas para o fator de atitudes linguísticas, mas também a importância de um olhar detalhado partindo de uma análise qualitativa que busca observar os dados de modo individual e comparativamente. O trabalho em questão envolve acomodação dialetal e se encaixa nos pressupostos teóricos da Teoria da Acomodação da Comunicação (GILES et al., 1991) e nos aportes teórico-metodoló- gicos da Teoria da Variação Linguística (LABOV, 1966, 2008 [1972]). O objetivo geral foi observar e analisar a acomodação dialetal de cariocas residentes na cidade de João Pessoa, na Paraíba, partindo da investigação do fenômeno da palatalização ou não palatalização da fricativa coronal /s/ em posição de coda final. Nessa posição de coda final, os dois dialetos possuem pronúncias diferentes da fricativa, ocorrendo, no dialeto pessoense, uma pronúncia alveolar ([s], [z]), e no dialeto carioca, uma pronúncia palatal ([ʃ], [Ʒ]). Essa diferença marca uma distinção bastante clara entre os dois diale- tos, marcando quando ocorre de fato a convergência e facilitando o processo de análise. Foram entrevistados 16 participantes e busquei verificar se e em que medida o processo de convergência (não-palatalização do /s/) ocorreu. Além disso, procurei identificar quais variáveis linguísti- cas e extralinguísticas exerceram alguma influência no processo de acomodação dialetal, e por fim, observei e descrevi as diferentes ati- tudes linguísticas dos falantes. 133 Os resultados revelaram forte influência das atitudes linguís- ticas e a identidade para o processo de acomodação linguística dos entrevistados. Para chegar a tal conclusão, foi necessária a realiza- ção de uma análise qualitativa atenta que buscou con, “‘trastar os dados de cada informante uns com os outros, assim como analisá- -los individualmente. BREVES NOTAS SOBRE ACOMODAÇÃO DIALETAL A acomodação dialetal é o processo de adaptar ou modificar a fala a fim de se adequar às necessidades e preferências linguísticas de indivíduos ou grupos que falam distintos dialetos ou variedades linguísticas. Isso pode incluir o uso de termos e expressões regionais ou locais apropriados, traços linguísticos específicos ao dialeto ou variedade em questão, ou até fatores como ritmo de fala. Um dos objetivos ou efeitos disso é proporcionar aproximação, garantindo uma comunicação amistosa e eficaz entre os falantes. Nem sempre, no entanto, deseja-se aproximar a maneira de falar a de um outro indivíduo ou grupo. Temos então algumas opções de ajuste da fala, com alguns conceitos simples: convergência, divergência e manutenção. Tais conceitos foram desenvolvidos por Howard Giles, professor de psicologia e comunicação, em 1973. Esses conceitos se referem à forma como as pessoas se adaptam às nor- mas linguísticas e sociais em diferentes situações de comunicação. De acordo com Giles (1973), a convergência, a divergência e a manutenção linguística são estratégias comportamentais que os falantes utilizam em situações de comunicação com seus interlocu- tores. Primeiro temos o conceito de convergência: este se refere a um movimento de aproximação da fala de um indivíduo A com um 134 S U M Á R I O indivíduo B, por exemplo. Ao aproximar a maneira de falar, o falante pode demonstrar solidariedade, conformidade e/ou o desejo de ser aceito. Em seguida temos o conceito de divergência, que por sua vez se refere ao movimento de se distanciar da forma de falar em relação a seu(s) interlocutor(es), ou seja, é o movimento inverso à conver- gência. A divergência pode ser resultado de atitudes negativas para com um determinado indivíduo ou grupo. Por fim, temos a manuten- ção, que visa manter a forma de falar, evitando alterações, a fim de se manter sua própria identidade linguística e social. A manutenção pode ser meramente por um desejo de se manter a identidade, mas pode também ser, assim como a divergência, uma ferramenta de dis- tanciamento do falante com seu(s) interlocutor(es). Giles (1973) argumenta que a escolha entre essas estraté- gias é influenciada por diversos fatores, incluindo a relação entre as pessoas, a situação de comunicação e a percepção de normas sociais e linguísticas. Um fator bastante influenciador é o das atitu- des linguísticas, como constatei em minha dissertação, sobre o qual trato mais a seguir. BREVES NOTAS SOBRE ATITUDES LINGUÍSTICAS Seja como for, 136 S U M Á R I O na tentativa de sanar quaisquer limitações para a observação das atitudes, faz-se necessário obter dados de diferentes naturezas, que revelem o máximo possível as crenças, valores e intenções dos indi- víduos, e que possam influenciar o fenômeno em estudo, para que então se faça uma análise qualitativa e comparativa desses dados. As atitudes que nos interessam são as atitudes linguísticas. Essas envolvem as opiniões, crenças, valores, predisposição e inten- ção que as pessoas têm em relação ao uso e às variedades da língua. As atitudes linguísticas podem influenciar como as pessoas usam a língua e como elas julgam o uso da língua por outras pessoas, podem ser positivas ou negativas e podem variar dependendo da situação e do contexto. Se um indivíduo tem uma atitude negativa em relação a uma determinada variedade de língua, ele pode evitar usá-la ou usá-la menos em situações sociais específicas. Se, por outro lado, um indivíduo tem uma atitude positiva para com uma determinada variedade de língua, ele pode ser mais propenso a usá-la e valorizá- -la. Isso foi corroborado pelos resultados de minha dissertação de mestrado, como viremos a discutir e exemplificar mais adiante. BREVES NOTAS SOBRE ATITUDES LINGUÍSTICAS De acordo com Ajzen (1988, p. 4), “uma atitude é uma dis- posição para responder favoravelmente ou desfavoravelmente a um objeto, pessoa, instituição ou evento”. As atitudes sofrem influência de fatores sociais e há a tendência de se ajustá-las “[...] para se ade- quarem àqueles que são predominantes nos grupos sociais a que se vinculam” (LASAGABASTER, 2004, p. 399), sendo elas mediadoras das respostas dos indivíduos. 135 Para Kaufmann (2011), a estrutura interna das atitudes se divide em três diferentes componentes: a componente cognitiva, que é o reflexo de convicções e crenças acerca do objeto da atitude (aquilo que é compreendido como sendo verdade e que julgamos e acreditamos saber); a componente afetiva, que considera a avaliação do objeto da atitude como algo positivo ou negativo (tudo aquilo que é tido como juízo de valor); e a componente conativa, na qual, como apontam Deprez & Persoons (1987, apud KAUFMANN, 2011, p.123), as crenças e valores são convertidos em intenções comportamentais (a transformação das componentes prévias em predisposição e inten- ções). Ajzen e Fishbein reduziram o conceito de atitude à compo- nente afetiva. Essa se chamaria de atitude, enquanto a componente cognitiva passa a ser a crença e a componente conativa passa a ser a intenção (KAUFMANN, 2011, p. 122-123). Contudo, as atitudes de um indivíduo nem sempre refletem em um comportamento específico. Como exemplificado por Kau- fmann (2011, p. 123), “mesmo não gostando de pessoas dos Estados Unidos em geral, pode-se querer dominar o inglês estadunidense fluentemente, porque isso pode trazer vantagens importantes no trabalho”. No exemplo dado pelo autor, apesar da pré-disposição do indivíduo, as vantagens que podem ser obtidas por realizar um com- portamento específico contrário a essa pré-disposição se sobres- saem. A atitude, de acordo com a visão mentalista, é um estado interno de prontidão, mas a resposta, nem sempre é representante das atitudes. Seguindo ainda a visão mentalista, poderíamos dedu- zir que os comportamentos de um indivíduo poderiam ser previs- tos a partir de suas atitudes, mas o esperado nem sempre vem a se concretizar. Infelizmente, apenas a resposta pode ser observada de maneira tangível e, portanto, temos um problema metodológico no que trata de se observar as atitudes. Já na visão behaviorista, as respostas dos indivíduos às situações sociais podem revelar as ati- tudes desses mesmos indivíduos, sendo assim possível investigar as atitudes a partir dos comportamentos observáveis. RELAÇÃO ENTRE ATITUDES LINGUÍSTICAS E IDENTIDADE As atitudes linguísticas estão estreitamente relacionadas com a identidade de um indivíduo, uma vez que a língua é um aspecto importante da identidade cultural, social e histórica. A identidade de um indivíduo, portanto, pode facilmente ser um fator influencia- dor para as atitudes linguísticas desse mesmo indivíduo. A exemplo disso, uma pessoa que tem uma forte identidade cultural pode ter atitudes positivas em relação à língua e às variedades da língua que são associadas a essa cultura, e pode se esforçar para preservar e 137 S U M Á R I O transmiti-las. Semelhantemente, o fato de uma pessoa ter uma iden- tidade social específica, como pertencer a um grupo étnico ou a uma classe social, pode influenciar suas atitudes para com as variedades da língua associadas a esse grupo ou classe, ou para com outros grupos ou classes. Ademais, o inverso pode acontecer, no qual a identidade de um indivíduo pode ser afetada por suas atitudes lin- guísticas. Se uma pessoa tem uma atitude negativa em relação à sua própria maneira de falar, isso pode afetar sua autoestima, que, por sua vez, afetaria sua identidade e sua autopercepção. Com tantos fatores sociais que exercem pressão na percep- ção e o uso da língua, as atitudes linguísticas de um indivíduo para com um determinado grupo, dialeto ou fenômeno da fala, por exem- plo, podem facilmente influenciar sua forma de falar quando em con- tato com esses. Esse indivíduo pode desejar ser considerado amigá- vel para com um determinado grupo e até mesmo como pertencente ao mesmo, ou desejar criar um distanciamento entre ele e o grupo. A depender do intuito desse indivíduo, que nem sempre é totalmente consciente, diferentes ajustes na fala podem ser realizados, também nem sempre conscientes. Pode ser desejável convergir sua fala, caso o indivíduo deseje receber apoio e/ou reconhecimento do grupo ao qual se dirige, e em outras situações, nas quais se deseje criar dis- tanciamento, a manutenção ou divergência podem ser desejáveis. Os traços da fala dos indivíduos são parte de sua cultura e identidade, portanto, é importante considerar que ouvintes podem vir a se sentir ofendidos se acharem que os traços de seu dialeto podem ser facilmente convergidos ou imitados. A imitação, em espe- cial, pode não soar natural, causando um estranhamento. RELAÇÃO ENTRE ATITUDES LINGUÍSTICAS E IDENTIDADE Para Giles (1980), haveria um nível de tolerância para a convergência, sendo ela aceitável e bem vista quando dentro desse limite, e negativa quando a convergência passasse desse nível. Além disso, a divergência também pode ser positiva, uma vez que a manutenção de traços divergentes, típicos de grupos ou 138 S U M Á R I O dialetos de maior prestígio, pode ser bem vista, como normalmente ocorre no Nordeste com as variedades do Sudeste, por exemplo. As atitudes dos indivíduos para com diferentes grupos ou dialetos são influenciadas pelo fator de prestígio que esses carregam. Diferentes fatores sociais podem atribuir prestígio, como status social do indi- víduo, grupo, ou dialeto associado, para listar alguns exemplos. Um grupo que obtém ascensão social será mais facilmente considerado como algo que se almeja. Diferentes dialetos possuem diferentes marcadores linguís- ticos, sendo estes específicos a suas comunidades de fala. Esses traços distintos podem ser mais ou menos salientes, ou seja, podem ser mais ou menos perceptíveis a depender do nível de contraste se comparado a outros dialetos. A saliência se caracteriza pela mudança linguística e estigmatização, resultando em um contraste e distanciamento fonético quando comparado a outros dialetos (TIM- BERLAKE, 1977; KERSWILL, 1985). São chamados de salientes os traços que são mais facilmente distinguidos, e esses são normal- mente marcadores linguísticos de uma comunidade de fala especí- fica, sendo mais facilmente percebidos por alguém não pertencente àquela comunidade de fala. Para exemplificar alguns marcadores linguísticos, temos o /t/ e /d/ nos dialetos nordestinos, que não são palatalizados antes da vogal /i/ e da semivogal /j/, como nas palavras [t]ia e [d]ia. Temos também, no mesmo dialeto, o /s/ e /z/ que são palatalizados antes de /t/ e /d/, como no caso das palavras fe[ʃ]ta e de[ʒ]de. Por serem característicos dos dialetos nordestinos, esses traços são marcado- res linguísticos desses dialetos. O que se espera é que os marcadores que são mais salien- tes sofram uma influência maior das atitudes linguísticas e a identi- dade dos falantes no processo de acomodação. Esses fatores viriam a favorecer ou inibir o processo de convergência desses falantes. RELAÇÃO ENTRE ATITUDES LINGUÍSTICAS E IDENTIDADE Na pesquisa que deu origem aos resultados que discutiremos neste 139 S U M Á R I O capítulo, temos o dialeto carioca em contato com o pessoense, e con- siderando que o dialeto pessoense foi tido como sendo de “menor prestígio” pelos informantes da pesquisa se comparado ao dialeto carioca, o resultado mais esperado já seria de que isso criaria uma resistência à convergência. Tais crenças foram expostas pelos pró- prios informantes entrevistados e é provável que em uma situação de contato dialetal inversa (pessoense morando no Rio de Janeiro), a convergência sofreria menos resistência no que trata das questões atitudinais e identitárias. Sendo o traço pertence a uma outra comunidade de fala e que se constitui como parte da identidade dessa comunidade, um aspecto a se levar em consideração é o de que, se esse traço for muito saliente, ele pode se tornar desconfortável para o falante aco- modar. Consequentemente, quando tais traços são adotados pelo falante de outra comunidade de fala, esse está, de certa forma, aban- donando sua própria identidade. Evidentemente, cada indivíduo terá trajetórias individuais na acomodação. Há ainda, o fato de que os traços não são assimilados todos simultaneamente, uma vez que uns são mais facilmente assimilados que outros, podendo haver, inclusive, aqueles que podem não fazer parte do inventário fonético de um indivíduo em situação de contato dialetal, o que causaria dificuldade de acomodação. Como já fora mencionado na introdução, o traço que é saliente no dialeto carioca e que foi escolhido como objeto de estudo de minha dissertação, é o /s/ palatal em posição de coda final, como ocorre em “carioca[ʃ]”. No dialeto pessoense, o /s/ nessa mesma posição possui uma pronúncia alveolar, como ocorre em “carioca[s]”. Por se tratar de um traço saliente, portanto, a diferença dele nos dois dialetos é evidente e facilmente observável. 140 A palavra sexo, aqui utilizada, “[...], significa a distinção biológica entre homens e mulheres, opon- do-se, portanto, à distinção gramatical de gênero entre o masculino e feminino” (COULTHARD, 1991, p. 76, apud FREITAG, 2015, p. 26). METODOLOGIA Uma análise qualitativa e comparativa dos dados de fala obtidos através de entrevistas foi importante porque me permitiu identificar as semelhanças e diferenças nas respostas dos partici- pantes entrevistados, o que por sua vez, possibilitou a identificação de tendências e padrões. Isso me possibilitou acessar a perspec- tiva dos entrevistados e resultou por revelar informações valiosas acerca das diferentes variáveis que exercem influência no processo de acomodação linguística, sobretudo no que trata dos fatores de ati- tudes linguísticas e a identidade desses indivíduos, por se tratarem de fatores subjetivos. Ao realizar comparações e se identificar diferenças e seme- lhanças entre as respostas de indivíduos pertencentes a diferentes grupos (com base em variáveis como sexo, idade, tempo de exposi- ção, etc.), foi possível aprofundar a compreensão de como as atitu- des linguísticas se relacionam com a identidade cultural e social das pessoas, e ainda, em que nível essas variáveis afetam o processo de acomodação desses falantes. A pesquisa foi composta por 16 participantes naturais da cidade de Rio de Janeiro que estavam morando na cidade de João Pessoa há pelo menos 1 ano e que tinham pelo menos 18 anos de idade. Todos foram estratificados de acordo com as variáveis sexo15, faixa etária e tempo de exposição. O meio utilizado para a coleta de dados foi a entrevista semiestruturada, que foi dividida em duas partes: a) a primeira com perguntas gerais acerca da vida do entre- vistado, para que este se sentisse à vontade e falasse mais natural- mente o possível. Nessa etapa o dado que interessava era o dado de 15 141 S U M Á R I O fala, então o intuito foi reduzir o efeito do paradoxo do observador; b) a segunda com perguntas específicas que buscavam averiguar infor- mações que revelassem alguma informação a respeito das atitudes linguísticas dos falantes e sua identidade. Para que fosse possível realizar essa coleta e a subsequente análise dos dados, foi utilizado um gravador digital. Foi feita uma análise quantitativa e os dados foram codificados e analisados através da utilização do programa estatístico Goldvarb X (SANKOFF, TAGLIAMONTE & SMITH, 2005). Esses dados quantitativos formaram uma base que permitiu que a subsequente análise qualitativa estivesse bem embasada. Dessa forma, a última etapa de análise buscou observar os informantes de forma individual e comparativa, utilizando como base os dados quantitativos obtidos previamente. RESULTADOS Em diferentes respostas dadas pelos informantes para dife- rentes perguntas da entrevista, ficou evidente a presença de suas atitudes. Algo que esteve bastante presente foram respostas que demonstravam atitudes favoráveis para com o dialeto carioca, ou seja, seu dialeto de origem. Por vezes, se evidenciou também atitu- des negativas ou neutras para com o falar pessoense. Foi através da identificação das atitudes linguísticas desses falantes, assim como das experiências vivenciadas por eles, que pude melhor compre- ender em que medida esses fatores influenciam seus processos de acomodação linguística. Atitudes positivas para com o dialeto carioca se mostra- ram ser inibidoras da acomodação, e, da mesma forma, as atitudes negativas para com o dialeto pessoense também inibiram a conver- gência a esse dialeto. 142 Vejamos a comparação entre respostas dadas pelas infor- mantes 1 e 2, a fim de melhor entender a influência dessas variá- veis. As duas são do sexo feminino e pertencem ao mesmo grupo de faixa etária. As diferenças imediatamente óbvias entre as duas são o tempo de exposição e a motivação da vinda a João Pessoa (A informante 1 teve a vinda obrigatória e a informante 2 teve a vinda espontânea). Ambas tiveram baixos índices de convergência, mas a informante 2, mesmo tendo menos tempo de exposição ao dia- leto pessoense, acomodou mais que a informante 1. O fator tempo de exposição, como demonstrado pelos dados quantitativos obtidos na pesquisa, mostrou-se favorecedor da acomodação. No entanto, nesse caso, outros fatores parecem ter exercido maior influência, se sobrepondo a essa variável. Considerando, então, a natureza da vinda das duas para João Pessoa, já era de se esperar uma maior aceitação do novo local e seu dialeto por parte da informante 2, que veio espontaneamente. Tudo isso em conjunto já sinaliza para uma potencial diferença atitudinal entre as duas. Vejamos alguns dados sobre as duas informantes e o que elas tiveram a dizer em resposta a algumas das perguntas realizadas: S U M Á R I O Tabela 1 – Informantes 1 e 2 INFORMANTE SEXO IDADE TEMPO DE EXPOSIÇÃO PERCENTUAL DE ACOMODAÇÃO 1 F 21 15 anos 3.6% 2 F 22 4 anos 9.4% Fonte: Possatti (2020). Vejamos uma das perguntas feitas às duas a seguir: Entrevistador (E) – Você considera que tem algum sotaque? Se sim, qual? Informante 1 (I1) – “Sim. Bastante (risos). [...]. RESULTADOS Todo mundo fica indignado: ‘Nossa, faz tempo que você mora aqui, você não é pra falar mais assim’, só que é uma coisa que 143 é de família mesmo, e como quase todo ano eu volto não tem como perder”. Informante 2 (I2) – “Do Rio. Apesar de estar mais ameni- zado um pouco, acho que predomina o de lá, do Rio”. As duas informantes consideram que têm o sotaque carioca e afirmam gostar de suas próprias maneiras de falar. Um fator que diferencia as duas é a frequência de contato que elas mantêm com pessoas de sua cidade natal. A informante 1 mantém mais contato com pessoas de sua família (que moram no Rio de Janeiro), quando comparada à informante 2. S U M Á R I O Com a próxima pergunta, busquei obter respostas para algu- mas das experiências vivenciadas por elas: E – Alguém já criticou, elogiou, riu ou comentou a respeito da sua forma de falar? I1 – “Ah, tudo isso! Então no começo, [...] logo quando eu vim pra cá, eu era pequena... e a aceitação, assim, no colé- gio, foi péssima. Porque ninguém queria ser meu amigo. Porque eu era de fora e aí, tipo, tinha uma rejeição grande. Até mudei de colégio por causa disso. Fui pra outro colé- gio, aconteceu a mesma coisa. E ninguém gostava, você tinha poucos amigos porque ‘Ah, era a menina que não é daqui’, ‘que não fala igual a gente’, ‘que fala estranho’. E passei por isso muito tempo, só que aí me acostumei. Não liguei e também não ia mudar... forçar uma coisa por causa das pessoas. Mas assim, quando cheguei na uni- versidade foi bem melhor. [...]. Porque assim, pelo menos a minha turma, a maioria não é daqui. É de Pernam- buco, é de Fortaleza. É bem diferente, então não tinha esse negócio assim”. I2 – “Criticar não, mas as pessoas brincam... levam muito na brincadeira às vezes é... o meu sotaque assim... fazendo gírias e tal [...]”. A partir de suas respostas, percebemos que a recepção e aceitação vivenciada pela informante 1 na escola, quando se mudou 144 S U M Á R I O para João Pessoa, deixou a desejar. Ela demonstrou ter dificuldade em fazer novas amizades por ser de fora e atribuiu sua forma de falar como uma característica que destacava essa diferença. RESULTADOS Esse tipo de pressão social normalmente influencia uma mudança de fala, favo- recendo a convergência e acomodação, mas outros fatores parecem ter se sobressaído, particularmente pelo fato de que ela expressa que não tinha intenções de mudar e “forçar uma coisa por causa das pessoas”. Não podemos, a partir disso, saber todos os exatos motivos dessa resistência à mudança, mas o fato é que resistiu à pressão de mudar, considerando desnecessário fazê-lo apenas para agradar as/ os colegas ou se encaixar. Isso é, sem dúvida, um reflexo de suas atitudes para com seu próprio dialeto e o novo dialeto local. Atitudes as quais estão ligadas a fatores identitários, que são, em conjunto, peças-chave para explicar o motivo do baixo índice de acomoda- ção dessa informante. Voltando um pouco a atenção para a informante 2, vejamos a seguinte pergunta e resposta: E – Você acha que as pessoas são julgadas pela maneira que falam? I2 – “Sim, principalmente pelo sotaque [...]. Eu acho que tem muito preconceito sim, ainda. Principalmente contra o nordestino, porque é um sotaque bem original, bem diferente de tudo que existe [...] justamente por ser tão diferente, as pessoas acham que é estranho, que é feio, que tá fora do padrão, enfim. Não existe um padrão”. Em sua resposta, ela dá ênfase ao aspecto do sotaque, reco- nhecendo que os sotaques pertencentes ao Nordeste não carregam muito prestígio. Dessa forma, esses sotaques sofrem preconceito em outras regiões, mas a informante 2, por sua vez, não se posi- ciona favorecendo nenhum sotaque sobre o outro, afirmando que “não existe um padrão”. Nesse sentido, ela não demonstra atitudes negativas para com os sotaques nordestinos, o que certamente é uma barreira a menos para o processo de acomodação linguística. 145 As atitudes que ela demonstrou a partir disso não inibem o processo, e por um conjunto de fatores como esse, percebemos a diferença nos índices de acomodação entre as duas informantes analisadas. As atitudes que ela demonstrou a partir disso não inibem o processo, e por um conjunto de fatores como esse, percebemos a diferença nos índices de acomodação entre as duas informantes analisadas. RESULTADOS Quando questionadas sobre suas opiniões acerca do modo de falar das pessoas em João Pessoa comparado às de sua cidade natal, Rio de Janeiro, elas tiveram isso a dizer: S U M Á R I O E – Quando você compara a fala (modo de falar) das pes- soas de sua terra, com a fala das pessoas da PB, você pode dizer que aqui as pessoas falam: a) depressa; b) muito depressa; c) devagar; d) arrastado. I1 – “Eu acho muito arrastado, algumas pessoas. Princi- palmente do interior daqui. Mas, é... depressa também. Tem uma coisas que falam assim que eu nem consigo entender direito... umas gírias, assim, daqui...” I2 – “Aqui as pessoas falam muito depressa [...]. Eu já me acostumei, mas no início foi difícil (risos)”. I2 – “Aqui as pessoas falam muito depressa [...]. Eu já me acostumei, mas no início foi difícil (risos)”. E – Você gostaria de falar igual aos paraibanos? Por quê? E – Você gostaria de falar igual aos paraibanos? Por quê? I1 – “Não... Eu acredito que traz muito da sua identidade, também, isso. [...]. Acho bonito, acho interessante o sota- que daqui, algumas coisas são bem diferentes, mas não...” I1 – “Não... Eu acredito que traz muito da sua identidade, também, isso. [...]. Acho bonito, acho interessante o sota- que daqui, algumas coisas são bem diferentes, mas não...” I2 – “Eu não veria problema nisso não”. I2 – “Eu não veria problema nisso não”. E – Você acredita que com o passar dos anos estará falando como paraibanos? E – Você acredita que com o passar dos anos estará falando como paraibanos? I1 – “Não”. I2 – “Sim. As expressões, gírias. Com certeza”. Notei uma contradição nas respostas da informante 1: ainda que ela não acredite que com o passar dos anos passará a falar como os paraibanos, ela acredita que sua fala já mudou e relata que seus pais percebem as mudanças. O que poderia explicar essa contradi- ção é o fato de ela não demonstrar desejo de mudar seu sotaque. 146 S U M Á R I O Há uma resistência e isso certamente é um fator inibidor da aco- modação, ligado à sua identidade e às suas atitudes linguísticas. RESULTADOS Por outro lado, a informante 2 não demonstra a mesma resistência a acomodar ao dialeto paraibano e afirma já ter se acostumado com o mesmo, apesar de, em um momento inicial, ter sentido dificuldade com a velocidade de fala desse dialeto. No caso dos informantes 9 e 11, que são irmãos do mesmo sexo e faixa etária, foi possível notar que um conjunto de atitudes e sentimentos desses informantes para com o dialeto pessoense e o carioca foi um dos maiores diferenciais entre os dois. Essas ati- tudes e sentimentos exerceram forte influência para o processo de acomodação linguística deles e a comparação de suas respostas revelaram o verdadeiro impacto desta variável. Vejamos as seme- lhanças de perfil desses informantes e a diferença entre os índices de acomodação dos mesmos: Tabela 2 – Informantes 9 e 11 INFORMANTE SEXO IDADE TEMPO DE EXPOSIÇÃO PERCENTUAL DE ACOMODAÇÃO 9 M 18 7 anos 14.1% 11 M 22 7 anos 32.8% Fonte: Possatti (2020) Tabela 2 – Informantes 9 e 11 Apenas ao acessar as atitudes desses informantes, se tor- nou possível levantar e corroborar hipóteses sobre as ocorrências da acomodação, assim como a velocidade do processo. No exemplo dado com os informantes 9 e 11, o tempo de exposição foi o mesmo e houve uma diferença significativa entre os índices de acomodação. No exemplo anterior com as informantes 1 e 2, o tempo de exposi- ção foi inversamente proporcional ao índice de acomodação, mesmo tendo sido selecionado como a variável mais favorecedora e estatis- ticamente relevante, como podemos ver na tabela a seguir: 147 Tabela 3 – Acomodação do /s/ (não-palatalização) com base na variável tempo de exposição TEMPO DE EXPOSIÇÃO APLICAÇÃO/TOTAL PERCENTUAL PESO RELATIVO Alto 193/508 38.0% 0.74 Baixo 39/387 9.2% 0.22 Fonte: Possatti (2020). Tabela 3 – Acomodação do /s/ (não-palatalização) com base na variável tempo de exposição S U M Á R I O De acordo com Laver et al. (1979) e Trudgill (1998), o tempo de exposição contribui de modo significativo para o processo de aco- modação linguística. Essa variável foi estratificada em baixo tempo de exposição (de 1 a 5 anos) e alto tempo de exposição (acima de 5 anos), e como podemos ver, o grupo pertencente ao alto tempo de exposição convergiu em uma frequência mais de quatro vezes maior. RESULTADOS Para entender a relevância dessa variável, basta olharmos para o peso relativo; há uma disparidade grande entre os dois grupos, sendo que valores acima de 0.5 são favorecedores da ocorrência do fenômeno (acomodação) e valores abaixo de 0.5 são inibidores do mesmo. Sendo assim, essa variável foi tida como a mais relevante e favore- cedora da acomodação linguística dos participantes entrevistados. Se voltarmos às comparações anteriormente apresentadas entre os entrevistados, um fato fica claro: há outras variáveis que se sobrepõem aos efeitos do tempo de exposição, e essas são as ati- tudes linguísticas e a identidade dos falantes. Giles et al. (1982), já afirmavam que as atitudes linguísticas dos falantes são de extrema importância para identificar a extensão da acomodação, a percep- ção e o grau de aceitação da mesma. 148 CONSIDERAÇÕES FINAIS Pude ter uma noção inicial do papel das variáveis controladas a partir da análise quantitativa dos dados, mas essa noção formava apenas uma fração da imagem completa. Alguns dos fatores que mais exerceram influência foram observados apenas a partir da aná- lise qualitativa, individual e comparativa. Essa análise mais próxima e mais contextual, com atenção àquilo que não é imediatamente óbvio, proporcionou uma compreensão mais ampla e refinada acerca do fenômeno e as variáveis influenciadoras. As atitudes linguísticas e a identidade dos falantes entrevistados demonstraram, por vezes, se sobrepor a variáveis como tempo de exposição, por exemplo. São essas atitudes, movidas pelas crenças e influenciadas por estereótipos e preconceitos, que fazem com que as pessoas jul- guem ou sejam julgadas pela sua maneira de falar e seu sotaque, o que por sua vez interfere no processo de acomodação linguística dos indivíduos; isso ficou evidente nos resultados da pesquisa. Alguns dos entrevistados que menos acomodaram expressa- ram não considerar o dialeto paraibano como sendo bonito. Alguns trechos, inclusive, revelaram crenças e visões de mundo bastante estigmatizadas em relação à Paraíba e seu povo. Alguns deles tam- bém expressaram gostar do seu dialeto de origem e expressaram o desejo de preservar o que consideram constituir parte de sua iden- tidade. Essas informações revelam um pouco de suas identidades e suas atitudes para com os dois dialetos. As discussões sobre atitudes linguísticas e acomodação dialetal têm um papel importante na construção de uma sociedade mais justa e inclusiva. Ao se conscientizar sobre as atitudes linguís- ticas que valorizam ou desvalorizam determinados dialetos ou sota- ques, é possível combater o preconceito linguístico e promover a diversidade cultural e linguística. Esse tipo de discussão pode ser 149 S U M Á R I O útil no campo pedagógico, ajudando professores a abordar questões de atitudes linguísticas em sala de aula e a promover a inclusão e o respeito à diversidade linguística. Para concluir, percebemos a influência das atitudes lin- guísticas, tema foco deste capítulo, para o processo de acomoda- ção linguística. A influência dessa variável se estendeu para todos os participantes da pesquisa, e, com um olhar próximo e atento, fui capaz de apontar essa variável, juntamente com algumas outras, como sendo peças-chave para entender quais são os fatores que regem a acomodação linguística, acelerando-a ou desacelerando-a. CONSIDERAÇÕES FINAIS Incentivo outros pesquisadores a também se atentarem a diferentes métodos de análise que encapsulam variáveis que são menos ime- diatamente evidentes, a fim de possibilitar uma melhor compreensão do fenômeno em estudo. REFERÊNCIAS AJZEN, Icek. Attitudes, Personality and Behavior. London: Open University Press, 1988. KERSWILL, Paul. A Sociolinguistic Study of Rural Immigrants in Bergen. Norway: Cambridge University, 1985. LABOV, William. The social stratification of English in New York City. Washington: Center of Applied Linguistics, 1966. AJZEN, Icek. Attitudes, Personality and Behavior. London: Open University Press, 1988 FREITAG, Raquel Meister Ko. (Re)Discutindo Sexo/Gênero na Sociolinguística. In: FREITAG, FREITAG, Raquel Meister Ko. (Re)Discutindo Sexo/Gênero na Sociolinguística. In: FREITAG, Raquel Meister Ko.; SEVERO, Cristine Gorski (Orgs.). Mulheres, Linguagem e Poder: Estudos de Gênero na Sociolinguística Brasileira. São Paulo: Blucher, 2015, p. 17 -74. Raquel Meister Ko.; SEVERO, Cristine Gorski (Orgs.). Mulheres, Linguagem e Poder: Estudos de Gênero na Sociolinguística Brasileira. São Paulo: Blucher, 2015, p. 17 -74. GILES, Howard. Accent mobility: a model and some data. Anthropological Linguistics, v. 15, p. 87-105, 1973. GILES, Howard. Accommodation theory: some new directions. In: SILVA, S. (Orgs.). Aspects of Linguistic Behavior. York, England: York University Press, 1980, p. 105-136. GILES, Howard; COUPLAND, Nikolas; COUPLAND, Justine. (Orgs.). Contexts of Accomodation: Developments in applied sociolinguistics. Cambridge: Cambridge University Press. 1991. 150 GILES, Howard; RYAN, Ellen Bouchard; SEBASTIAN, R. J. An integrative perspective for the study of attitudes toward language variation. In: GILES, Howard; RYAN, Ellen Bouchard. (ed.). Attitudes towards language variation: social and applied context. London: Edward Arnold, 1982, p. 1-19. KAUFMANN, Göz. Atitudes na sociolinguística. In: MELLO, Heliana; ALTENHOFEN, Cléo; RASO, Tommaso. (Org.). Os contatos linguísticos no Brasil. Belo Horizonte: UFMG, 2011. p. 121-137. KERSWILL, Paul. A Sociolinguistic Study of Rural Immigrants in Bergen. Norway: Cambridge University, 1985. LABOV, William. The social stratification of English in New York City. Washington: Center of Applied Linguistics, 1966. LABOV, William. [1972]. Padrões Sociolinguísticos. Trad.: Marcos Bagno, Marta Scherre e Caroline Cardoso. São Paulo: Parábola, 2008. LASAGABASTER, David. Attitude. In: AMMON, Ulrich. et al. (Orgs.) Sociolinguistics: An International Handbook of the Science of Language and Society. 2 ed. Berlin/New York: De Gruyter, 2004. p. 399. LAVER, John.; TRUDGILL, Peter. Phonetic and linguistic markers in speech. In: SCHERER, Klaus; GILES, Howard. (Orgs.). Social markers in speech. Cambridge: CUP, 1979. p. 1-32. POSSATTI, Lucas. Acomodação dialetal de cariocas residentes em João Pessoa: uma análise sociolinguística. 124 f. Dissertação (Mestrado em Linguística) – Universidade Federal da Paraíba, João Pessoa, 2020. SANKOFF, David; TAGLIAMONTE, Sali; SMITH, Eric. Goldvarb X: a variable rule application for Macintosh and Windows. Toronto: University of Toronto, 2005. SANKOFF, David; TAGLIAMONTE, Sali; SMITH, Eric. Goldvarb X: a variable rule application for Macintosh and Windows. Toronto: University of Toronto, 2005. TIMBERLAKE, Alan. Reanalysis and actualisation in syntactic change. In: LI, Charles. (Org.) Mechanisms of Syntactic Change. AustIn: University of Texas, 1977. TRUDGILL, Peter. Language contact and inherent variability: the absence of hypercorrection in East Anglian present-tense verb forms. In: TRUDGILL, Peter; CHESHIRE, Jenny (Orgs.). The sociolinguistics reader: multilingualism and variation. London: Published Arnold, 1998. p. 103-111. TIMBERLAKE, Alan. Reanalysis and actualisation in syntactic change. In: LI, Charles. (Org.) Mechanisms of Syntactic Change. AustIn: University of Texas, 1977. TRUDGILL, Peter. Language contact and inherent variability: the absence of hypercorrection in East Anglian present tense verb forms In: TRUDGILL Peter; TIMBERLAKE, Alan. Reanalysis and actualisation in syntactic change. In: LI, Charles. (Org.) Mechanisms of Syntactic Change. AustIn: University of Texas, 1977. Mechanisms of Syntactic Change. AustIn: University of Texas, 1977. TRUDGILL, Peter. Language contact and inherent variability: the absence of hypercorrection in East Anglian present-tense verb forms. In: TRUDGILL, Peter; CHESHIRE, Jenny (Orgs.). The sociolinguistics reader: multilingualism and variation. London: Published Arnold, 1998. p. 103-111. hypercorrection in East Anglian present-tense verb forms. In: TRUDGILL, Peter; CHESHIRE, Jenny (Orgs.). The sociolinguistics reader: multilingualism and variation. London: Published Arnold, 1998. p. 103-111. 151 Esse ato de fala, de “erguer a voz”, não é um mero gesto de palavras vazias: é uma expressão de nossa transição de objeto para sujeito - a voz liberta. bell hooks (2019) Paulo Freire, que nasceu no Recife em 19 de setembro de 1921 e faleceu em 1997, em 2012 foi reconhecido como patrono da Educa- ção Brasileira. Suas contribuições dizem respeito, principalmente, à alfabetização de jovens e adultos. LABOV, William. The social stratification of English in New York City. Washington: Center of Applied Linguistics, 1966. O professor teve experiências no nordeste brasileiro, em Angicos, onde em 1963 iniciou o projeto de alfabetização de pequenos agricultores, o projeto era chamado pelos fazendeiros da região de “praga comunista”. No período da ditadura militar, Freire ficou preso por 70 dias e depois passou cinco anos exilado no Chile. Escreveu várias obras mundialmente conhecidas, entre elas estão Educação como prática da liberdade (1967), Pedago- gia do oprimido (1968); Pedagogia da esperança (1992) e Pedagogia da autonomia (1997). Embora o educador tenha deixado um legado imenso para a educação no Brasil e no mundo, nos últimos anos tem sido alvo das Fake News. Sua obra tem sido associada à delírios fundamentalistas criados em torno da educação pública no Brasil, como a “ideologia de gênero”, “kit gay” e “doutrinação esquerdista”. O problema se agrava na medida em que o trabalho deste pensador é pouco discutido nos cursos de licenciatura e nos cursos de Letras, os mesmos cursos em que se estuda e discute Foucalt e Bakhtin, por exemplo - teóricos que em vários aspectos dialogam com o pensamento freireano -  o educador acaba sendo deixado de lado. Já há um tempo, os estudos decoloniais têm denunciado o modo como a colonialidade opera nas Universidades por meio da ausência de teóricos do Sul que pensam no e para Sul global nas discussões acadêmicas, Paulo Freire é um desses estudiosos. Sua obra é fundamentada em sua experiência dentro e fora do Brasil, mas sempre se volta para a prática educativa no Brasil,  na América Latina e em países de África. 153 Não podemos esquecer, e isso bell hooks (2017) nos ajuda a lembrar, que nossas leituras críticas precisam nos permitir apro- fundar e debater a produção de Freire, apontando seus equívocos e aprendendo com suas contribuições, e que possamos seguir ten- sionando estes equívocos para que criemos uma crítica construtiva, aproveitando o acúmulo das proposições libertadoras freireanas e avançando onde a própria construção masculinista patriarcal impe- diu Freire de avançar. Esse movimento é, também, um movimento de honrar sua produção intelectual e perceber o valor dela. As obras de Freire têm sempre como horizonte desequilibrar as localidades de oprimido e opressor por meio de uma educação libertadora. Para isto, Freire trata do papel do educador, que passa a ser educador/educando, e do educando, que passa a ser educando/ educador neste processo, que é sempre um processo dialético. LABOV, William. The social stratification of English in New York City. Washington: Center of Applied Linguistics, 1966. Nesta perspectiva a educação é sempre dialógica e troca, é necessário que a escola rompa com a postura descontextualizada e autoritária que provoca na/o aluna/o atitudes de anti-responsabilidade, anti-partici- pação e antidiálogo. Freire bem nos ensina que não devemos che- gar nos espaços sociais e nas lutas sendo objetos para, depois, nos tornarmos sujeitas/os, e a escola, como esse espaço político que é, deve ser lugar que estimula a atitude política, engajada e crítica. Pedagogia do Oprimido (1968) é considerada a principal obra do autor e um dos textos fundantes da pedagogia crítica. Nesta obra o professor trata da relação entre opressores e oprimidas/os, dos modos como essas relações acontecem e se perpetuam. Os opressores ao impossibilitarem o diálogo, ou seja, ao impossibilita- rem a humanização, tornam os oprimidos “quase coisa”. “Na medida em que, para dominar, se esforçam por deter a ânsia de busca, a inquietação, o poder de criar, que caracterizam a vida, os opressores matam a vida” (FREIRE, 2020, p. 65). Na perspectiva freireana, a construção do lugar de ser opri- mido coloca essas/es sujeitas/os em uma busca, movida pelo desejo 154 S U M Á R I O de manutenção do cistema, de se aproximarem dos sentidos pro- duzidos pelo ser opressor; numa tentativa de escape da violência, o desejo pela norma pode impulsionar uma busca sem fim pela fuga da violência. Freire (2020) nos diz que “quando a educação não é liber- tadora o sonho do oprimido é ser opressor”, e com isso aponta para esse desejo, que significa um determinado movimento: “a aposta nessas estruturas normativas como fonte de conforto e segurança [...] é um sinal evidente da falta de imaginação política interseccional [...], que estão limitados a lutar no interior do projeto de mundo do qual temos sido reiteradamente excluídas” (MOMBAÇA, 2021, p. 68) Uma educação libertadora, que supere a contradição opres- sores-oprimidas/os só é possível, em Freire, na medida em que as/os oprimidas/os sejam construídas/os não mais como objetos-abjetos. “Estes que oprimem, exploram e violentam, em razão de seu poder, não podem ter, neste poder, a força de libertação dos oprimidos nem de si mesmos.” (FREIRE, 2020, p. 41). Colocando Freire em diálogo com Jota Mombaça, talvez um caminho anterior necessário seja jus- tamente o de redistribuir a violência (MOMBAÇA, 2021). LABOV, William. The social stratification of English in New York City. Washington: Center of Applied Linguistics, 1966. Dizemos isso pois é a própria condição plena de conforto - seja ela social, eco- nômica, ontológica… - direito reconhecido apenas de um pequeno e seleto grupo de pessoas, reconhecidas como as mais humanas entre as humanas e as desumanizadas. Neste sentido, Freire (2020) afirma que a educação deve reconhecer a raiva direcionada às injustiças, ao desamor, à violência e práticas exploratórias. Para o estudioso, a raiva é importante para que ocorram as transformações. Paulo Freire diferencia a educação libertadora da educa- ção bancária, aquela que faz depósitos e transferências de conteú- dos para os educandos sem que os conhecimentos, necessidades e desejos destes educandas/os-educadoras/es sejam levados em conta no processo educativo. A educação bancária ignora a reali- dade e as condições das/os oprimidas/os uma vez que trabalha para a manutenção do status quo, trabalha para a manutenção do poder dos opressores. Já a pedagogia do oprimido “terá dois momentos 155 distintos. No primeiro momento, “os oprimidos vão desvelando o mundo da opressão e vão comprometendo-se, na práxis, com sua transformação;”. Já no segundo momento, a partir desta percep- ção da realidade e deste engajamento, “é transformada a realidade opressora, esta pedagogia deixa de ser do oprimido e passa a ser a pedagogia dos homens em processo de permanente libertação” (FREIRE, 2020, p. 57). A educação bancária, em cuja prática se dá a inconci- liação educador-educandos, rechaça este companhei- rismo. E é lógico que seja assim. No momento em que o educador “bancário” vivesse a superação da contra- dição já não seria “bancário”. Já não faria depósitos. Já não tentaria domesticar. Já não prescreveria. Saber com os educandos, enquanto estes soubessem com ele, seria sua tarefa. Já não estaria a serviço da desumaniza- ção. A serviço da opressão, mas a serviço da libertação. (FREIRE, 2020, p. 86-87) A manutenção da lógica monológica da educação bancária se dá também pelo medo que se tem da palavra consciente da/o oprimida/o. Para Grada Kilomba, quando o diálogo é possibilitado, “verdades que supostamente não deveriam ser ditas” vêm à tona. “Segredos como a escravidão, Segredos como o colonialismo, segre- dos como o racismo” são desvelados. (KILOMBA, 2019, p. 21) Por outro lado, a educação libertadora busca justamente a superação destes segredos através de um profundo e coletivo diálogo sobre  a realidade e as diferentes opressões que atravessam as existências das/os sujeitas/os, que são plurais e sócio-historicamente situadas/ os. LABOV, William. The social stratification of English in New York City. Washington: Center of Applied Linguistics, 1966. É necessário considerar, portanto, que educação é ação refle- xiva, é práxis política. Para Freire, educadoras/es-pesquisadoras/es devem cons- tantemente se perguntar quais são os motivos que os levam a estu- dar. Perguntas como “Para que estudo/ensino?”, “A favor de quem estudo/ensino?” e “Contra quem estudo/ensino?” devem ser nor- teadoras nas nossas propostas curriculares e nas nossas práticas 156 escolares. Estas considerações colocam à vista a própria compreen- são da educação enquanto esta coisa essencialmente política, que implica necessariamente em uma ou outra concepção de sujeita/o e do mundo. E nesse sentido, os movimentos sociais possuem apro- ximações gigantes com a própria produção do que é o pedagógico, se colocando de forma responsiva e propositiva em relação ao status quo. Dentro dos movimentos sociais as/os mais diversas/os sujeitas/ os se constroem e se produzem em comunidade e em aproximações, distanciamentos e reconhecimentos. Como resultado desse processo de autorreconheci- mento, projetos políticos são construídos, metodolo- gias são sistematizadas, novos modos de ações cole- tivas surgem: Pedagogias são gestadas. Pedagogias engajadas, que compartilham com a educação e com a sociedade novas possibilidades político-pedagógicas (PASSOS, 2022, p. 39). Assim, as perguntas anteriores retornam e nos colocam em um looping reflexivo: para além do que guia nossa prática, o que guia nosso comprometimento ético enquanto professoras/es de lín- gua quando temos como fundamento a compreensão de que nossa atuação é sempre política e ideologicamente marcada? Para ressaltar a importância das obras de Paulo Freire para o ensino de línguas e a necessidade da leitura de suas obras nos cursos de formação de professoras/es de línguas, trazemos alguns conceitos de Bakhtin, estudioso russo que fundamenta os documentos oficiais,  DCEs do Paraná, PCNs e BNCC, que tratam do ensino de línguas. Aproximações entre os pensamentos dos dois autores já foram feitas por alguns pesquisadores, como João Wanderley Geraldi, Fabio Scorsolini-Comin e Edite Marques Moura em sua tese de doutorado intitulada Paulo Freire e BakhtIn: Um diálogo possível. As histórias de vida dos dois autores apresentam situações em comum;a luta contra a ditadura, o exílio, as várias mudanças, 157 a espiritualidade e o interesse pela linguagem são temas que atra- vessam as experiências dos dois pensadores e que estão presen- tes em suas obras. Ambos tomam o dialogismo/dialogicidade como constitutivo do ser humano. Para Geraldi (2005, p. LABOV, William. The social stratification of English in New York City. Washington: Center of Applied Linguistics, 1966. 14): De modo extremamente resumido, podemos dizer que a linguagem, tanto para Paulo Freire quanto para Vygotsky e Bakhtin, tem uma função constitutiva dos sujeitos. Os três autores compartilham um ponto de partida: a dia- logia como espaço de construção do humano. Não há diálogo sem a construção de recursos expressivos, atra- vés dos quais pensamentos são organizados e expostos, compreendidos e modificados. S U M Á R I O Ao tratarem da dialogicidade das relações humanas, tanto Bakhtin quanto Freire atentam para as relações de alteridade. Para o primeiro pensador, a alteridade está relacionada à exterioridade, ao exercício da exotopia, que compreende o sair de si para se ver com os olhos do outro. Este exercício “cria a possibilidade de diá- logo, e o diálogo ajuda-nos a compreender uma cultura de maneira profunda. Pois qualquer cultura contém significados que ela própria desconhece, que ela própria não compreendeu; eles estão ali, mas como potencial.” (MORSON; EMERSON, 2008, p. 73). Neste mesmo raciocínio, o dialogismo é visão de mundo. A preocupação com a natureza dialógica do enunciado, portanto, é a essência das suas produções. Segundo Bakhtin (2015, p. 51): o discurso depara com a palavra do outro e não pode deixar de entrar numa interação viva e tensa com ele. Só o Adão mítico, que chegou com sua palavra primeira ao mundo virginal ainda não precondicionado, o Adão solitá- rio conseguiu evitar efetivamente até o fim essa orienta- ção dialógica mútua com a palavra do a contra no objeto. Quando Freire nos aponta a necessidade e a importância de estarmos atentas à subjetividade de nossas/os alunas/os, ele nos diz isso chamando nossa atenção para não compreendermos cada um/a dessas/es alunas/os como um “Adão mítico”; ele está nos atentando 158 S U M Á R I O para o cuidado de se voltar ao reconhecimento de que todas as pessoas que compõem o espaço escolar e a sala de aula carregam consigo experiências, cosmovisões e construções sociais e culturais diversas, que as compõem enquanto sujeitas/os no mundo. A escola possui uma prática comum de produzir respostas únicas em seus estudantes. Essa produção é, antes de tudo, fundamentada em uma base homogeneizadora e unificadora, que corta toda a capacidade imaginativa e plural do corpo discente quando o coloca em uma lógica de compreensão de uma única voz e reflexão adequada. LABOV, William. The social stratification of English in New York City. Washington: Center of Applied Linguistics, 1966. Paulo Freire, ao dizer que só é possível que a contradição opressor-oprimido/a seja superada através do diálogo, da dialogici- dade, da mesma forma, está, assim como Bakhtin, afirmando que é através do diálogo que se dá a alteridade e a constituição do sujeito. É importante ter em mente que quando, juntamente com os teóricos citados, estamos tratando de diálogo, não consideramos que o diá- logo seja algo pacífico. Pelo contrário, este diálogo é disputa e é dis- putado. Freire critica uma educação que interrompe e impossibilita as respostas das/os educandas/os justamente por considerar que, muitas vezes, assim como afirma Lugones (2006) quando trata do monólogo do colonizador, a escola também produz um monólogo. Para o Círculo de Bakhtin, o diálogo não se dá necessaria- mente de forma pacífica e amigável, pois as relações dialógicas tam- bém se constituem como o embate entre as muitas vozes sociais. Desta forma, assim como as forças centrípetas e centrífugas agem sobre a língua, nessas relações, agem forças que nos aproximam e nos afastam das palavras do outro. Conforme nossos discursos se aproximam de um outro, eles também se afastam de outros. Para Freire, dizer a palavra significa conhecer a realidade e dela participar criticamente. Neste sentido, Bakhtin e o círculo dizem que a palavra é o signo ideológico por excelência, a palavra carrega valores, visões de mundo. Para Volochinov, ao dizer uma palavra a 159 pessoa está dizendo “verdades ou mentiras, coisas boas ou más, importantes ou triviais” (VOLOCHINOV, 1988, p. 95). S U M Á R I O É possível fazer uma aproximação entre a palavra na pers- pectiva bakhtiniana e na concepção freireana, já que em ambas as perspectivas a palavra é práxis, conforme Freire, a palavra é ação- -reflexão. Paulo Freire, ao dizer que é necessário levar em conta a palavra das/os educandas/os (das massas) durante todo o pro- cesso de educação-diálogo, considera este conteúdo valorativo que a palavra carrega. Este diálogo, como exigência radical da revolução, res- ponde a outras exigências radicais – a dos homens como seres que não podem ser fora da comunicação pois que são comunicação. Obstaculizar a comunica- ção é transformá-los em quase “coisa” e isto é tarefa e objetivo dos opressores, não dos revolucionários” (FREIRE, 2020, p. 172). LABOV, William. The social stratification of English in New York City. Washington: Center of Applied Linguistics, 1966. Paulo Freire propõe uma educação libertadora que diminua a distância entre opressores e oprimidas/os, mas isto não significa uma aproximação por parte da/o oprimida/o à consciência do opres- sor, pelo contrário, é a partir de uma consciência de si e do outro que essa distância diminui. Uma vez que esta consciência de si per- mite a quebra da relação opressor-oprimida/o, permite a revolução. Assim as forças centrípetas (normatizadoras) e centrífugas (estrati- ficadoras) presentes na língua e na vida, devem ser foco de análise nas aulas de línguas para que na percepção dessas forças e dos modos pelos quais elas atuam, as/os oprimidas/os se libertem das palavras do opressor. É enorme o significado desse processo de luta com a palavra do outro e com sua influência na história da formação ideológica da consciência individual. Minha palavra e minha voz, nascidas da palavra do outro ou dialogicamente estimuladas por ela, mais cedo ou mais tarde começam a libertar-se do poder dessa palavra alheia. Esse processo se complexifica pelo fato de que as 160 diferentes vozes dos outros entram em luta pela consci- ência do indivíduo (assim como lutam na realidade social circundante). É tudo isso que cria o terreno propício para a objetivação experimentadora da palavra do outro (BAKHTIN, 2015, p. 143). S U M Á R I O CONSIDERAÇÕES FINAIS A intenção desta escrita é, antes de tudo, apresentar alguns apontamentos sobre as aproximações possíveis das produções frei- reanas e os campos da linguagem e do ensino de línguas no Brasil, fazendo um chamado para que nosso campo sinta a presença de Freire e das proposições que dialogam com seus pensamentos. Esse movimento é também um chamado para que possamos ampliar as leituras já existentes deste autor ao colocar suas reflexões em diálogo com debates que podem estar, até então, um pouco mais distantes. Os diálogos - e como já dissemos, nem sempre pacíficos - são um dos modos de produzir o novo, a alternativa, e colocar em campo novas possibilidades existentes. Afirmamos repetidamente nos campos da linguagem que a linguagem é poder. Falamos sobre as relações entre linguagem e poder e o entrelaçamento disto com a escola e a produção curri- cular. O que faz com que não dialoguemos com Freire? Ele dialoga conosco quando, por exemplo, aponta que: “Quem disse que este é o culto? E aí tu chegas à questão do poder: só quem tem poder define, só quem tem poder descreve, só quem tem poder perfila e é por isso que o opressor perfila o oprimido, por isso é que o opressor do nome à terra do oprimido” (FREIRE, 1985, p. 48). Nós sabemos que a escola é uma das instituições produtoras de políticas linguísticas, que produz, curricularmente inclusive, defi- nições e normativas de como ser e existir no mundo, e sabemos que 161 todas estas normatividades acontecem na e pela linguagem, assim como as desobediências a elas. Desta forma, entendendo o currículo como essa entidade reguladora, precisamos perceber como ele rege também a manutenção de práticas de silenciamento, impedindo esta pedagogia do diálogo: se o currículo está nas mãos de um seleto grupo que dita quais são as narrativas que representarão Outros, seria então justificável afirmar que máscaras de silenciamento continuam a existir e são perpetuadas por mecanismos pedagógicos que buscam “eleger – arbitrariamente – uma identidade específica como o parâmetro em relação ao qual as Outras identidades são avaliadas e hierarquiza- das” (SILVA, 2000, p. 83) (PASSOS, 2019, p. 199). REFERÊNCIAS BAKHTIN, Mikhail. Teoria do romance I: A estilística. Organização e tradução de Paulo Bezerra. São Paulo: Editora 34, 2015. FREIRE, Paulo. Pedagogia do oprimido. 74 ed. Rio de Janeiro/São Paulo: Paz e Terra, 1986. FREIRE, Paulo. Pedagogia da autonomia: saberes necessários à prática educativa. 65ª ed. Rio de Janeiro/ São Paulo: Paz e Terra 2020. FIGUEIREDO, Virginia. M. PIACENTI, Tânia. M. Conversa com Paulo Freire: Linguagem e Poder. Perspectiva. CED, Florianópolis, v. 1, n. 4, p. 47-51, jan./dez. 1985. GERALDI, João Wanderley. A linguagem em Paulo Freire. In: Educação, sociedade e culturas. n. 23, 7-20,2005. hooks, bell. Ensinando a transgredir: a educação como prática da liberdade. 2. ed. São Paulo: Editora WMF Martins Fontes, 2017. hooks, bell. Erguer a voz: pensar como feminista, pensar como negra. São Paulo: Elefante, 2019. KILOMBA, Grada. Memórias de Plantação: episódios de racismo cotidiano. Rio de Janeiro: Cobogó, 2019. CONSIDERAÇÕES FINAIS se o currículo está nas mãos de um seleto grupo que dita quais são as narrativas que representarão Outros, seria então justificável afirmar que máscaras de silenciamento continuam a existir e são perpetuadas por mecanismos pedagógicos que buscam “eleger – arbitrariamente – uma identidade específica como o parâmetro em relação ao qual as Outras identidades são avaliadas e hierarquiza- das” (SILVA, 2000, p. 83) (PASSOS, 2019, p. 199). S U M Á R I O O currículo, que é ideologicamente e politicamente com- posto, é objeto também de disputa. Nós, enquanto professoras/es de línguas, precisamos nos atentar a como disputar e romper com estes mecanismos pedagógicos do qual fala Maria Clara Araújo dos Passos na citação anterior. E ainda, se defendemos a palavra como ação reflexiva que é, como visão de mundo, como cultura e como ideologia, precisamos nos atentar também em quais são os sentidos que comunicamos a partir de nossas decisões políticas em atuação em sala de aula. Em uma entrevista concedida a Virgina M. Figuei- redo e Tânia M. Piacenteti, em 1985, Freire nos diz: É preciso deixar claro à criança, sem fazer nenhum tipo de discurso proselitista ou academicamente revolucionário, que não tem sentido nenhum, mas fazer, através do teste- munho e da explicação ligeira,com que a criança comece a perceber a relação entre linguagem e ideologia, lingua- gem e classe social, linguagem e poder. [...] Depois, é pre- ciso convencer as crianças de que elas precisam apren- der “nós chegamos” pra poder brigar melhor com a classe dominante (FREIRE, 1985, p. 50). Acessar a tecnologia que é a escrita, a leitura e o letramento é um dos modos de produzir estas disputas de que tanto falamos até 162 aqui; é produzir sentido sobre o mundo, e não apenas lê-lo de forma neutra e bancarista. O modo de atuação escolar que se encaminhe para uma formação crítica e engajada pode ser um dos modos de produzirmos alternativas a esse mundo que conhecemos. KILOMBA, Grada. Memórias de Plantação: episódios de racismo cotidiano. Rio de Janeiro: Cobogó, 2019. LUGONES, Maria: On complex communication. Hypatia. v. 21, n. 3, 2006. MOMBAÇA, Jota. Não vão nos matar agora. 1. ed. Rio de Janeiro: Cobogó, 2021. MORSON, Gary Saul; EMERSON Caryl. Mikhail BakhtIn: criação de uma prosaística/ tradução de Antônio de Pádua Danesi. São Paulo: EDUSP, 2008. PASSOS, Maria Clara Araújo dos. O currículo frente à insurgência decolonial: constituindo outros lugares de fala. Cad. Gên. Tecnol., Curitiba, v. 12, n. 39, p. 196- 209, jan./jun., 2019. 163 PASSOS, Maria Clara Araújo dos. Pedagogias da Travestilidades. Rio de Janeiro: Civilização Brasileira, 2022. SILVA, Edite Marques da. Paulo Freire e BakhtIn: um diálogo possível. 2011. 189 f. Tese (Doutorado em Letras) – Universidade Presbiteriana Mackenzie, São Paulo, 2011. VOLOCHINOV. Marxismo e filosofia da linguagem: problemas fundamentais do método sociológico na ciência da linguagem. Tradução do francês de Michel Lahud e Yara F. Vieira. 4. ed. São Paulo: Hucitec, 1988[1929]. VOLOCHINOV. Marxismo e filosofia da linguagem: problemas fundamentais do método sociológico na ciência da linguagem. Tradução do francês de Michel Lahud e Yara F. Vieira. 4. ed. São Paulo: Hucitec, 1988[1929]. VOLOCHINOV. Marxismo e filosofia da linguagem: problemas fundamentais do método sociológico na ciência da linguagem. Tradução do francês de Michel Lahud e Yara F. Vieira. 4. ed. São Paulo: Hucitec, 1988[1929]. S U M Á R I O 164 S U M Á R I O INTRODUÇÃO O tema proposto para investigação neste trabalho busca, por meio da análise da produção científica bibliográfica, compreender como as práticas de ensino realizadas pelo intérprete em sala de aula têm proporcionado a compreensão do aluno surdo em relação à aprendizagem da Língua Portuguesa. Nosso foco de pesquisa é compreender como o intérprete tem possibilitado ao aluno surdo a compreensão e aprendência da Língua Portuguesa, pois, compreendemos que a estrutura da Língua Portuguesa é complexa, e, trabalhar a polissemia dos termos com os alunos surdos têm sido um dos desafios enfrentados pelo intér- prete em sala de aula. Desta forma, justifica-se a pesquisa na compreensão de como o intérprete tem desenvolvido práticas de ensino que favoreça a compreensão do aluno surdo durante o processo de ensino apren- dizagem da Língua Portuguesa. Assim, compreendemos que o trabalho do intérprete vai além da tradução literal realizada em sala de aula com os alunos surdos, pois, é por meio do intérprete que a interação entre professor ouvinte e aluno surdo ocorre, portanto, a importância do intérprete em sala de aula tem destaque no que tange a educação inclusiva que per- mite a interação entre os sujeitos no contexto escolar. Destarte, que o intérprete ao trabalhar com a educação do surdo deve compreender como este constrói sua aprendizagem por meio de sua língua materna, a Libras, fato esse relevante para com- preender a relação entre significado e significante na construção de signos, para então estabelecer um entendimento preciso sobre a formação do signo linguístico para alunos surdos. Desta maneira, compreendemos que a prática de ensino realizada pelo intérprete 166 em sala de aula, deve ser bem embasada teoricamente, pois, irá cul- minar em um bom trabalho com os alunos surdos. REVISÃO TEÓRICA O objetivo principal deste trabalho é analisar e refletir sobre a importância das práticas de ensino desenvolvidas pelo intérprete em sala de aula na busca da compreensão do aluno surdo em rela- ção a aprendizagem da Língua Portuguesa. Para compreendermos a temática aqui proposta, faremos uma contextualização histórica visando entender como tem sido direcionado os projetos educa- cionais no atendimento e acesso da comunidade surda nas esco- las públicas do país. A luta dos surdos no país tem sido fundamental na busca de fortalecer e ampliar o debate em relação aos direitos das pessoas surdas, principalmente em relação ao acesso educacional. Destaca- mos, aqui, que essa luta se torna necessária uma vez que o sistema capitalista tem permeado a história com o aumento da desigualdade em relação às pessoas com deficiências. As políticas sociais desenvolvidas no Brasil nas últimas décadas, seguem orientadas pelos projetos neoliberais desenvolvi- dos pelo capital. A educação das pessoas surdas e o acesso dos surdos a educação tem marcado o embate entre Estado e a comu- nidade surda, de um lado o sistema capitalista, que visa ampliar e garantir mão de obra e lucro essencial para manter o sistema, do outro lado, a comunidade surda que busca seus direitos à educação, saúde, trabalho e moradia. É preciso compreender que as situações sociais vivi- das pelo segmento de pessoas com deficiência não se determinam apenas pelos fatos mais recentes, mas, sim, 167 são determinações históricas concretas, que imputam a respeito dessas compreensões agregadas de elementos pouco científicos, com resultados negativos para elas. Também são condições históricas e materiais que con- tinuam a determinar a atual realidade das pessoas com deficiência, podendo, inclusive, constituir marcos his- tóricos de emancipação humana de fato (CARVALHO; TURECK, 2014, p. 33). Corroborando com os autores Carvalho e Tureck (2014), compreendemos que a história e as condições sociais de reproduzir a vida são elementos indispensáveis para refletir a história do atendi- mento das pessoas com deficiência, e que a educação especial tem seus projetos educacionais norteados historicamente por políticas de segregação e exclusão. Os avanços das pesquisas em educação especial ocorrem a partir da aceitação da ciência como fonte de pes- quisa e orientação desta área. Pontuamos, que apesar do avanço das pesquisas em relação à educação especial, ainda não superamos o modelo de segregação e exclusão em relação às pessoas com defi- ciência na sociedade. REVISÃO TEÓRICA Portanto, é na contradição das relações sociais existentes dentro da sociedade capitalista, que os sujeitos buscam lutar por sua emancipação humana, nesse movimento podemos compreender a luta das pessoas com deficiência por sua emancipa- ção, por seus direitos. Conforme Torres (2020) é necessário que a comunidade surda entenda a importância de lutar por seus direitos, de buscar garantir perante a sociedade o direito à sua cultura, a sua identidade, a sua língua materna, o direito de interagir e pertencer a sociedade. A Língua de Sinais foi reconhecida por meio da lei nº 10.436, de 24 de abril de 2002. Apesar da lei ser promulgada, a luta persiste, pois na prática ainda vivenciamos a desigualdade de acesso e a falta de atendimento aos surdos em todos os espaços sociais (mercados, hospitais, bancos, escolas, aeroportos etc.). Apesar da lei existir, a maioria dos espaços sociais não disponibiliza intérpretes, de modo a 168 S U M Á R I O garantir a comunicação do surdo com os ouvintes, o que tem dificul- tado a vida e a convivência social dos surdos perante a sociedade. Em 2021, quase 20 anos após implantar a lei nº 10.436, a comunidade surda ainda luta pela implementação desta lei, pro- cesso esse que ocorre paulatinamente, motivo esse que nos faz refletir sobre a atual conjuntura política social e educacional em rela- ção ao atendimento das pessoas surdas no que tange ao aprendi- zado da Língua Portuguesa. No ano de 2021 tramitou no senado federal o projeto de lei – PL 4.909 de 2020, projeto de autoria do senador Flávio Arns. Este projeto visou realizar alterações na LDB nº 9.394/96, qualificando a educação bilíngue de surdos como modalidade de ensino indepen- dente, onde a educação bilíngue para surdos tenha a Língua Brasi- leira de Sinais – Libras como primeira língua e o português escrito como segunda língua. O projeto tem acirrado debates entre especia- listas em educação inclusiva e representantes da comunidade surda, projeto esse que originou a Lei 14.191 de 03/08/2021. A IMPORTÂNCIA DA PRÁTICA DO INTÉRPRETE NA APRENDIZAGEM DA LÍNGUA PORTUGUESA PARA ALUNOS SURDOS Em relação a educação, Souza et al. (2017) ressaltam que as dificuldades enfrentadas em sala de aula pelos alunos surdos são inúmeras e destaca a falta do profissional intérprete para o atendi- mento educacional necessário para alunos surdos. O fato aqui é que a educação, o ensino do aluno surdo tanto na sua língua materna como para o aprendizado de uma segunda língua, nesse caso 169 a Língua Portuguesa, necessita de atendimento de um profissional intérprete que saiba desenvolver seu trabalho no intuito de que o processo de ensino aprendizagem tenha êxito. Segundo Fernandes (2003) no Brasil é a partir da década de 90 que os estudos em relação ao ensino da Língua Portuguesa com base na Língua Brasileira de Sinais – Libras, ganha força, devido ao contexto social em que os movimentos políticos da comunidade surda se intensificaram e cobram por seus direitos. A discussão educacional pelo bilinguismo no Brasil é algo presente, o debate em prol de uma política educacional que forneça a comunidade surda um projeto educacional que reconheça sua cultura, sua língua materna e que possibilite o acesso dos surdos ao aprendizado tanto da Língua Brasileira de Sinais – Libras, como também, assegurem a Língua Portuguesa como segunda língua no currículo escolar. A Lei Federal 10.436/2002, regulamentada pelo Decreto Federal 5.626, em dezembro de 2005, em seu texto o decreto asse- gura aos alunos surdos o direito à aprendizagem da sua língua materna e a aprendizagem da língua portuguesa como segunda língua. “O decreto 5.625/2005 traz no texto a seguinte afirmação: o professor formado para atuar em Libras com alunos surdos deve ter também formação específica para o ensino da língua portuguesa como segunda língua” (LODI, 2013, p. 58). Segundo Lodi (2013), é por meio da língua materna, Libras, que o intérprete vai mediar os processos escolares e consequente- mente possibilitar ao aluno surdo compreender os conteúdos traba- lhados em sala de aula. Art. 14. As instituições federais de ensino devem garantir, obrigatoriamente, às pessoas surdas acesso à comunica- ção, à informação e à educação nos processos seletivos, nas atividades e nos conteúdos curriculares desenvolvidos em todos os níveis, etapas e modalidades de educação, 170 desde a educação infantil até à superior. A IMPORTÂNCIA DA PRÁTICA DO INTÉRPRETE NA APRENDIZAGEM DA LÍNGUA PORTUGUESA PARA ALUNOS SURDOS § 1º Para garantir o atendimento educacional especializado e o acesso pre- visto no caput, as instituições federais de ensino devem: I - promover cursos de formação de professores para: a) o ensino e uso da Libras; b) a tradução e interpretação de Libras - Língua Portuguesa; e c) o ensino da Língua Portuguesa, como segunda língua para pessoas surdas; II - ofertar, obrigatoriamente, desde a educação infantil, o ensino da Libras e também da Língua Portuguesa, como segunda língua para alunos surdos (BRASIL, 2005). Apesar da lei garantir o acesso à educação bilíngue para os surdos, na prática isso não se consolidou, a estrutura educacional brasileira não priorizou a política de formação de profissionais, pro- fessores e intérpretes, fato considerado primordial para possibilitar o atendimento educacional dos alunos surdos nas escolas do país. Conforme Souza (1998), o bilinguismo no Brasil é algo novo que precisa ser pensado e revisto, pois a autora nos traz vários moti- vos para repensar e reorganizar o projeto educacional em relação ao bilinguismo na educação. Assim, Souza (1998) aponta que há uma ausência de política linguística que consolide e amplie o acesso à educação bilíngue no país. Destacamos aqui, que em 2021 a reflexão apresentada por Souza no contexto social do ano de 1998 ainda é atual e pertinente, ou seja, de 1998 para 2021 houve pouco avanço em relação a educação bilíngue no país. Sabemos que o ensino da Língua Portuguesa como segunda língua é um desafio posto para o intérprete, principalmente, e, por- tanto, seu planejamento de trabalho, sua prática e suas metodologias em sala de aula, precisam ter como objetivo real a compreensão e a interação do aluno surdo com o conteúdo trabalhado pelo professor. Por isso, a importância do aluno surdo compreender e aprender a Língua Portuguesa como objeto de comunicação, pois esta fará a ponte entre a cultura surda e a cultura ouvinte. 171 Fernandes (2006) relata que são as práticas desenvolvidas em sala de aula pelos professores e intérpretes que possibilitam aos alunos surdos conhecer o mundo da escrita, e assim, apesar de não terem conhecimento dos sons, terão condições de com- preender e entender as situações desenvolvidas socialmente pelos sujeitos no dia a dia. A IMPORTÂNCIA DA PRÁTICA DO INTÉRPRETE NA APRENDIZAGEM DA LÍNGUA PORTUGUESA PARA ALUNOS SURDOS S U M Á R I O Outro fato relevante a ser considerado, é que a Língua Brasi- leira de Sinais - Libras tem estrutura diferente da Língua Portuguesa, portanto, o intérprete precisa conhecer e trabalhar conceitos das duas línguas concomitantemente com o aluno surdo. Considerando, que o surdo não possui em seu sistema referencial linguístico conhe- cimento sobre a língua portuguesa, portanto, cabe ao intérprete inte- ragir com o aluno surdo na busca da construção desse referencial linguístico. Aprender o português decorrerá do significado que essa língua assume nas práticas sociais (com destaque às práticas esco- lares) para as crianças e jovens surdos. E esse valor só poderá ser conhecido por meio da língua de sinais (FERNANDES, 2006, p.6). Desta maneira, o intérprete precisa ter como objetivo possi- bilitar a interação entre surdos e ouvintes, entre a Língua Brasileira de Sinais – Libras e a Língua Portuguesa, para que o aprendizado da Língua Portuguesa tenha êxito no processo de ensino aprendi- zagem. Compreendendo, que apesar das diferenças entre as duas línguas, o estudo sobre a linguagem no decorrer da história nos faz considerar e refletir que a língua como sistema de significação pode ser compreendida como expressão do pensamento; como instru- mento de comunicação e como forma de interação. À concepção de língua como lugar de interação corres- ponde a noção de sujeito como entidade psicossocial, sublinhando-se o caráter ativo dos sujeitos na produção mesma do social e da interação e defendendo a posição de que os sujeitos (re)produzem o social na medida em que participam ativamente da definição da situação na qual se acham engajados, e que são atores na atualização das imagens e das representações sem as quais a comu- nicação não poderia existir (KOCH, 2003, p. 15). 172 Assim, se a função social da escola, conforme Saviani (1986), é transmitir o conhecimento científico acumulado pela humanidade e formar sujeitos atuantes dessa história, a função do intérprete é possibilitar ao aluno surdo o acesso a esse conhecimento, é rea- lizar uma prática voltada à emancipação do sujeito, pois educar é um ato social. Diante desta perspectiva, o intérprete ao trabalhar a construção do conhecimento do aluno surdo em relação à Língua Portuguesa possibilita a esse aluno o acesso a escrita desta língua como meio de comunicação. A IMPORTÂNCIA DA PRÁTICA DO INTÉRPRETE NA APRENDIZAGEM DA LÍNGUA PORTUGUESA PARA ALUNOS SURDOS S U M Á R I O Aqui, refletimos a importância da escola como espaço de socialização do conhecimento e espaço de formação dos sujei- tos. A escola que idealizamos tem como base romper com a desi- gualdade social e priorizar o acesso educacional para todos den- tro de uma sociedade. As pesquisas desenvolvidas e compartilhadas por profes- sores e alunos surdos por meio da escrita têm sido a base para novas reflexões, para novas construções em relação ao debate da importância do aprendizado da Língua Portuguesa para a inte- ração do conhecimento. Dito isso, fica nítido que a prática desenvolvida pelo intér- prete em sala de aula tem contribuído para o processo de ensino aprendizagem do aluno surdo em relação a aprendizagem da Língua Portuguesa, pois o intérprete tem conhecimento da língua materna dos alunos surdos, fato necessário para auxiliar esses alunos a cons- truir novos conhecimentos. METODOLOGIA DA PESQUISA Este trabalho foi desenvolvido por meio de pesquisa biblio- gráfica e documental. Selecionamos diversos trabalhos que apresen- taram relevância em relação à temática de pesquisa nos aspectos econômicos, políticos, sociais e educacionais. 173 Nossa análise em relação à aprendizagem do aluno surdo tem amparo teórico na reflexão dos autores Capovilla e Rapahael (2004, p. 35): “Se a Libras é a língua natural e materna do Surdo, então, do ponto de vista lógico, espera-se que a eficácia da Libras para servir de ponte para a aquisição do Português seja maior do que a eficácia do Português para servir de ponte para a aquisição da Libras”. Como escopo da metodologia desse trabalho de pesquisa fizemos um levantamento e estudo analítico de trabalhos publicados na área da educação, utilizando-nos do método dialético na busca pela compreensão da problemática proposta (Compreender como as práticas de ensino realizadas pelo intérprete em sala de aula, tem proporcionado a compreensão do aluno surdo em relação a apren- dizagem da Língua Portuguesa). ANÁLISE E DISCUSSÃO DOS RESULTADOS Nossas reflexões em relação à temática analisada, contribuí- ram na compreensão de que o trabalho do intérprete tem sido essen- cial na interação dos alunos surdos para com o conhecimento da Língua Portuguesa, com a interação entre alunos surdos e professo- res ouvintes, permitindo assim, o acesso dos sujeitos envolvidos no processo educacional, para com a cultura linguística da comunidade surda e da comunidade ouvinte. Partindo deste pressuposto, nossa análise vai ao encontro do objetivo educacional de uma prática de ensino significativa, voltada a formação do sujeito histórico e social, prática esta realizada pelo intérprete ao proporcionar a interação social por meio do ensino da Língua Portuguesa para os alunos surdos de forma efetiva. Portanto, defendemos que o ensino da Língua Portuguesa em sala de aula para alunos surdos só terá resultados qualitativos e 174 S U M Á R I O significativos no processo de ensino aprendizagem, se o aluno surdo e o intérprete tiverem conhecimento da Libras, pois será por meio desta que o intérprete possibilitará aos alunos surdos a compreen- são dos conteúdos trabalhados em sala de aula. Nossa intenção ao trazer a temática para o debate é apenas como proposta inicial de pesquisa, pois compreendemos a comple- xidade do tema e portanto, acreditamos que a temática aqui pro- posta poderá ser proposta de pesquisas futuras. Apontamos como necessário que a Libras como língua materna da comunidade surda precisa fazer parte do conhecimento de todos os profissionais da educação, principalmente dos professo- res, pois a educação de alunos surdos em escolas públicas só será possível se os professores compreenderem a importância de conhe- cer a cultura do aluno surdo. Sabemos, que apesar da lei 10.436 ter sido implantada no país em 2002, garantido o direito do aluno surdo à educação bilíngue, essa lei ainda não teve sua implementação de maneira significativa. CAPOVILLA, Fernando C.; RAPHAEL, Walkiria D. Dicionário enciclopédico ilustrado trilíngue da língua de sinais brasileira, v. 1. 2 ed. São Paulo: Editora da Universidade de São Paulo, 2001. CARVALHO, José Roberto; TURECK, Lucia Terezinha Zanato. Algumas reflexões sobre a inclusão escolar de alunos com deficiência. In: TURECK, Lucia Terezinha Zanato et al. (Orgs.) A pessoa com deficiência na sociedade contemporânea: problematizando o debate. Programa Institucional de Ações Relativas às Pessoas com Necessidades Especiais – PEE. 2 ed. Cascavel: EDUNIOESTE, 2014, p. 33-52. REFERÊNCIAS BRASIL. Diário Oficial da República Federativa do Brasil. Decreto n. 5.626, de 22 de dezembro de 2005. Dispõe sobre a Língua Brasileira de Sinais - Libras, e o art. 18 da Lei no 10.098, de 19 de dezembro de 2000. Diário Oficial da República Federativa do Brasil: Brasília, 23 dez. 2005. BRASIL. Diário Oficial da República Federativa do Brasil. Decreto n. 5.626, de 22 de dezembro de 2005. Dispõe sobre a Língua Brasileira de Sinais - Libras, e o art. 18 da Lei no 10.098, de 19 de dezembro de 2000. Diário Oficial da República Federativa do Brasil: Brasília, 23 dez. 2005. CAPOVILLA, Fernando C.; RAPHAEL, Walkiria D. Dicionário enciclopédico ilustrado trilíngue da língua de sinais brasileira, v. 1. 2 ed. São Paulo: Editora da Universidade de São Paulo, 2001. CONSIDERAÇÕES FINAIS Nosso trabalho focou em analisar como as práticas de ensino realizadas pelo intérprete em sala de aula têm sido direcionadas na busca de proporcionar a compreensão do aluno surdo em relação à aprendizagem da Língua Portuguesa. Para alcançar nossos objeti- vos, realizamos por meio bibliográfico um diálogo com pesquisado- res da temática em questão que nos permitem compreender o traba- lho e as práticas de ensino desenvolvidas pelo intérprete ao mediar a aprendizagem da Língua Portuguesa para alunos surdos. Identificamos que as práticas de ensino realizadas pelo intérprete têm sido desenvolvidas sempre no intuito de superar 175 S U M Á R I O as dificuldades da compreensão do aluno surdo em relação ao con- teúdo trabalhado, a aprendizagem da Língua Portuguesa durante o processo de escolarização. Consideramos, que todo o trabalho desenvolvido pelo intér- prete durante o processo de ensino aprendizagem da Língua Por- tuguesa para alunos surdos tem como objetivo proporcionar um aprendizado qualitativo, que forneça subsídios necessários para que o aluno surdo consiga aprender a Língua Portuguesa como segunda língua e interagir com a comunidade ouvinte. Entendemos que a interação do aluno surdo no espaço esco- lar é importante e só será possível com a conscientização da comu- nidade escolar como um todo. A comunidade escolar deve buscar subsídios que favoreçam a interação com o aluno surdo, como com- preender e aprender a cultura da comunidade surda. Fato importante para que ocorra a interação entre surdo e ouvinte, comunidade esco- lar (pais, alunos, professores, todos os funcionários pertencentes a esse grupo escolar). Assim, nossas considerações destacam que a educação bilíngue no processo educacional dos alunos surdos tem como obje- tivo oportunizar ao aluno o conhecimento não apenas da Língua Por- tuguesa, mas que por meio da aprendizagem da Língua Portuguesa o aluno surdo consiga compreender e se desenvolver em todas as disciplinas escolares, como: matemática, física, química, biologia etc. Esta pesquisa demonstrou em seu resultado a necessidade de o intérprete compreender sua função social como educador, como mediador das relações sociais entre a comunidade surda e a comunidade ouvinte, portanto, ressaltamos a importância da conti- nuidade das pesquisas em relação ao Ensino da Língua Portuguesa para alunos surdos, pela relevância social da temática. 176 FERNANDES, Sueli. Práticas de letramento na educação bilíngue para surdos. Curitiba: SEED, 2006. KOCK, Ingedore Grunfeld Villaça. Desvendando os segredos do texto. 2 ed. São Paulo: Cortez, 2003. KOCK, Ingedore Grunfeld Villaça. Desvendando os segredos do texto. 2 ed. São Paulo: Cortez, 2003. LODI, Ana Claudia Balieiro. Educação bilíngue para surdos e inclusão segundo a Política Nacional de Educação Especial e o Decreto nº 5.626/05. Educ. Pesqui. São Paulo, v. 39, n. 1, p. 49-63, mar. 2013. A presente pesquisa foi realizada com apoio da Fundação Araucária e da UNICENTRO. Destaca-se também que esta pesquisa foi aprovada pelo Comitê de Ética em Pesquisa da Universidade Estadual do Centro-Oeste do Paraná (COMEP-UNICENTRO), sob o parecer 3.738.180, em 2 de dezembro de 2019. SAVIANI, Dermeval. Escola e democracia. São Paulo, Cortez, 1986. SAVIANI, Dermeval. Escola e democracia. São Paulo, Cortez, 1986. SOUZA, Maria Fernanda Neves Silveira de et al. Principais dificuldades e obstáculos enfrentados pela comunidade surda no acesso à saúde: uma revisão integrativa de literatura. Rev. CEFAC, v. 19, n. 3, p.395-405, 2017. SOUZA, Regina Maria de. Que palavra que te falta? Linguística, educação e surdez. São Paulo: Martins Fontes, 1998. TORRES, Denise Rosa. Considerações sobre a comunidade surda: aprendendo a despertar. Conteúdo Jurídico, Brasília. Disponível em: < http://bit.ly/3QT16N8>. Acesso em: 04 dez. 2020. 177 ZILIOTTO, Gisele Sotta; GISI, Maria Lourdes. As políticas educacionais e a educação de surdos. IV Seminário Internacional de Representações Sociais e Subjetividade e Educação – SIRSSE VI Seminário Internacional sobre Profissionalismo Docente – SIPD/ CÁTEDRA/ UNESCO. Anais. Paraná, 2015, p. 7662-7676. Disponível em: <https://bit. ly/3I8zeSx>. Acesso em: 21 de abril de 2021. ZILIOTTO, Gisele Sotta; GISI, Maria Lourdes. As políticas educacionais e a educação de surdos. IV Seminário Internacional de Representações Sociais e Subjetividade e Educação – SIRSSE VI Seminário Internacional sobre Profissionalismo Docente – SIPD/ CÁTEDRA/ UNESCO. Anais. Paraná, 2015, p. 7662-7676. Disponível em: <https://bit. ly/3I8zeSx>. Acesso em: 21 de abril de 2021. ZILIOTTO, Gisele Sotta; GISI, Maria Lourdes. As políticas educacionais e a educação de surdos. IV Seminário Internacional de Representações Sociais e Subjetividade e Educação – SIRSSE VI Seminário Internacional sobre Profissionalismo Docente – SIPD/ CÁTEDRA/ UNESCO. Anais. Paraná, 2015, p. 7662-7676. Disponível em: <https://bit. ly/3I8zeSx>. Acesso em: 21 de abril de 2021. S U M Á R I O 178 INTRODUÇÃO16 S U M Á R I O A Libras, segundo Streiechen (2017b, p. 27), “é um sistema lin- guístico legítimo e natural utilizado pela comunidade surda brasileira. [...] e, assim como qualquer outra língua, apresenta seus próprios componentes gramaticais”. A autora explica também que a Libras é uma língua autônoma, reconhecida pela Linguística, e que, devido às lutas e movimentos de pessoas surdas na busca pelos seus direitos, ela ganhou seu espaço na sociedade, sendo oficializada pela Lei nº 10.436 de 24 abril de 2002 (BRASIL, 2002). A referida Lei estabelece o reconhecimento da Libras como principal meio de comunicação dos surdos (STREIECHEN, 2017b). Embora o reconhecimento da língua de sinais tenha ocorrido desde 1960, Pizzio e Quadros (2011) ressaltam que ainda há signifi- cativa carência de pesquisas na área da Libras como L2. Nessa pers- pectiva, buscamos contribuir para a área com uma investigação sobre a aquisição/aprendizagem da Libras como L2 para o adulto, toman- do-se, especificamente, o contexto de cursos de licenciatura em História, Pedagogia e Letras (Português, Inglês e Espanhol), de uma universidade pública do Estado do Paraná. Elegemos, como objetivo principal: analisar as experiências de aprendizagem da Libras dos professores em formação e, como objetivos específicos: identificar as perspectivas dos acadêmicos acerca da aquisição/aprendizagem da Libras como L2 e discutir os elementos que influenciam no pro- cesso de aquisição/aprendizagem de L2 para adultos. O corpus de análise constitui-se de respostas a um questionário semiestruturado, aplicado a trinta e nove acadêmicos dos referidos cursos, o qual ver- sava sobre as experiências desses acadêmicos com a disciplina de Libras e sobre as suas percepções e os seus desafios para a aquisi- ção/aprendizagem da Libras como L2. 16 180 Partimos da hipótese de que quanto mais tarde se é exposto a uma língua, mais difícil torna-se a sua aquisição, pois, devido ao período crítico (12 anos de idade aproximadamente), o aprendizado de uma língua por adultos é mais desafiador (LENNENBERG, 1967; PIZZIO; QUADROS, 2011; STREIECHEN, 2017a). Nesse panorama, visamos estimular ouvintes a aprenderem a língua de sinais, de forma a diminuir as barreiras comunicativas com as quais os surdos se deparam diariamente. Desse modo, res- saltamos que os pressupostos aqui apresentados correspondem às teorias que serviram como base para o desenvolvimento desse tra- balho e para que pudéssemos obter nossas próprias conclusões. PRINCIPAIS TEORIAS SOBRE AQUISIÇÃO/ APRENDIZAGEM DE SEGUNDA LÍNGUA A literatura especializada na aquisição/aprendizagem de L2 aborda três grandes modelos: Inatista, Cognitivista e Sócio- -Construtivista. O modelo inatista baseia-se na teoria de Stephen Krashen (1982), que compõe cinco hipóteses, a saber: hipótese da aquisição- -aprendizagem, hipótese da ordem natural, hipótese do input com- preensível, hipótese do monitor e hipótese do filtro afetivo. A primeira hipótese traz uma distinção entre aquisição e aprendizagem. A aqui- sição requer uma comunicação de forma natural, como na aquisição da L1, e ocorre em ambiente informal, por um processo inconsciente, em que o aprendiz não se abstém aos erros gramaticais. Em contra- partida, a aprendizagem requer um conhecimento consciente das regras da língua; para isso, faz-se um estudo da língua alvo, de forma que o aprendiz se abstém aos erros gramaticais, em ambiente for- mal, como uma sala de aula. No entanto, como apontado por Gesser 181 S U M Á R I O (2010) e Mota (2008), essa hipótese não pode ser fortemente sus- tentada, pois o processo de forma consciente ou inconsciente pode variar de pessoa para pessoa. Na hipótese da ordem natural, as regras aplicadas na L1 e L2 acontecem de modo similar, contudo a ordem da aquisição não é a mesma, pois o processo não depende de como as regras são ensi- nadas (KRASHEN, 1982). Nessa perspectiva, Figueiredo (1995) des- taca que muitas pesquisas acerca da aquisição/aprendizagem da L2 estão correlacionadas a teorias acerca da aquisição da L1, de forma a enfatizar as semelhanças entre os dois processos. Alguns estudio- sos, como Cooper (1970) e Corder (1968), ressaltados por Figueiredo (1995), afirmam que a aquisição da L1 e L2 é semelhante entre adul- tos e crianças, porque quando a criança adquire sua língua materna ela apresenta algumas diferenças do adulto que fala a sua língua; o mesmo acontece com o adulto no aprendizado da L2, que apresenta diferenças com o nativo da língua alvo. A hipótese do input compreensível (ou insumo) enfatiza que para ocorrer aquisição é necessário que os aprendizes sejam expos- tos a um insumo compreensível, isto é, eles devem se expor a um nível de conhecimento na língua acima daquele que já possuem (i+1) (KRASHEN, 1982). A hipótese do monitor está relacionada ao aprendizado, a reforçar o conhecimento consciente e o fato de que o aprendiz tende a monitorar a sua produção linguística (KRASHEN, 1982). PRINCIPAIS TEORIAS SOBRE AQUISIÇÃO/ APRENDIZAGEM DE SEGUNDA LÍNGUA E por último a hipótese do filtro afetivo corresponde a um blo- queio mental (fatores emocionais) que impede os aprendizes de uti- lizar esse input, de tal forma que a aquisição só ocorrerá se o filtro afetivo estiver baixo (KRASHEN, 1982). Com base na última hipótese, Souza (2015) e Figueiredo (1995) dispõem que existem fatores internos e externos que contribuem para a aquisição/aprendizagem de línguas. Souza (2015) argumenta que, em comparação às crianças, os adultos enfrentam maiores difi- culdades no aprendizado de um novo idioma porque “sentem-se 182 S U M Á R I O pressionados a aprender essa língua em um período de tempo curto, sentindo-se muitas vezes desmotivados por não alcançarem seus objetivos” (SOUZA, 2015, p. 27). Para Bona (2013), fatores psicológi- cos (emocionais) podem contribuir nos desafios apresentados pelos adultos, pois esses geralmente têm mais medo de cometer erros. Em relação a fatores exógenos, Souza (2015), com base em Krashen (1982) e Vygotsky (1989), destaca que o ambiente contribui de forma positiva para o aprendizado, sobretudo quando as ativi- dades estimulam a criatividade do aprendiz e a interação com os demais. Para Krashen (1982), o processo de aquisição/aprendiza- gem só pode ocorrer em ambientes culturais que sejam propícios ao aprendizado, ressaltando, assim, a interação e a comunicação. No modelo Sócio-Construtivista mantém-se a ideia de que a linguagem tem uma estrutura biológica que distingue o comporta- mento humano, mas que ainda depende do ambiente externo para sua estruturação. Esse modelo está pautado na teoria de Vygotsky (1989), a qual defende que a interação e a linguagem são indissociá- veis. Diante disso, o autor ressalta que o homem necessita das rela- ções com o outro para aprender, isto é, os processos cognitivos são gerados por meio da interação entre o aprendiz e um participante de uma prática social. Em relação ao idioma, a troca de conheci- mento se torna um fator chave para que o aprendiz possa se familia- rizar com a língua alvo. O terceiro e último modelo, o cognitivo, aborda que as infor- mações linguísticas podem ser processadas de forma controlada ou automática, de maneira que as habilidades novas e automáticas (mais praticadas e exercitadas) são controladas, e aos poucos vão se adaptando de uma atenção focal para uma periferal. É um modelo que trata da questão mental, isto é, do funcionamento cerebral no processo de aprendizagem (GESSER, 2010). PRINCIPAIS TEORIAS SOBRE AQUISIÇÃO/ APRENDIZAGEM DE SEGUNDA LÍNGUA Algumas teorias, como a inatista de Chomsky (1976), reconhe- cem que depois de uma determinada idade, o processo de aquisição 183 torna-se mais difícil, e os desafios são maiores, considerando, assim, a existência de um período crítico (depois dos 12 anos). A hipótese do período crítico é um dos fatores negativos que são considerados na aquisição de línguas, principalmente quando se refere à L2. Bona (2013) e Pizzio e Quadros (2011) destacam que dados de aquisição de L2 indicam que quando as crianças são expostas a uma língua precocemente há sucesso no aprendizado em com- paração às pessoas que adquirem línguas após o período crítico, resultando em uma proficiência mais baixa. Diante do exposto, Pizzio e Quadros (2011) relatam que estudos realizados com surdos evi- denciaram que a aquisição tardia também compromete a aquisição da língua de sinais. De acordo com Santana (2007), Pizzio e Quadros (2011) e Streiechen (2017a), um dos primeiros a considerar essa suposição foi Lennenberg (1967). Baseando-se nos pressupostos de Chomsky (1976), de que a linguagem é inata, o autor propôs em seus estu- dos que, para que a aquisição de uma língua não seja prejudicada, é necessário que o aprendizado não ultrapasse o período crítico. A teoria levantada pelo autor ainda sugere que os dados linguísticos adquiridos nessa idade são armazenados em diferentes áreas do cérebro, e com o passar dos anos serão armazenados em apenas uma região cerebral, dificultando o aprendizado da língua, porém não o impossibilitando (SANTANA, 2007; STREIECHEN, 2017a). Ao se ter em vista que os adultos já possuem o conhecimento de sua língua natural, é difícil para o indivíduo reconhecer as estruturas de outra língua e tomá-las para si, principalmente uma língua que pos- sui características diferentes da sua língua, como é o caso da Libras. 184 ASPECTOS METODOLÓGICOS A presente pesquisa possui uma abordagem qualitativa, alicerçada em um estudo de campo, com caráter bibliográfico. Dados quantitativos são também utilizados para melhor funda- mentarmos as análises. Os dados da pesquisa foram coletados por meio de um questionário semiestruturado aplicado para trinta e nove acadêmi- cos, com faixa etária entre dezoito e quarenta e cinco anos de idade, sendo a maioria do gênero feminino, oriundos do 4º ano de História, 1º ano de Letras-Português, e 3º anos de Pedagogia, Letras-Inglês e Letras-Espanhol. O número de participantes variou entre os cursos. Obteve-se o total de participantes conforme a presença dos acadê- micos no dia da coleta de dados. Os participantes são identificados pela letra correspondente ao seu curso (H – História, P – Pedagogia, LP – Português, IE – Inglês e Espanhol), seguido por uma numeração que foi distribuída de forma aleatória. As respostas dos participantes foram separadas em dois subitens: um com foco na disciplina de Libras e outro na aquisição/ aprendizagem da Libras. No primeiro, apresentamos as perspectivas dos acadêmicos acerca da disciplina de Libras na formação de pro- fessores. Posteriormente, analisamos as perspectivas dos acadêmi- cos acerca da aquisição/aprendizagem da Libras como L2, de forma a discutir os elementos que influenciam no processo de aquisição/ aprendizagem de L2 para adultos. 185 PERSPECTIVAS DOS ACADÊMICOS SOBRE A DISCIPLINA DE LIBRAS No que se refere à obrigatoriedade da Libras, nos cursos de licenciatura, disposto pelo Decreto nº 5.626 (BRASIL, 2005), ressalta- mos que na maioria dos cursos é destinada uma carga horária mínima de 68 h/a e máximos de 102 h/a, com exceção do curso de Fonoau- diologia, que oferta a disciplina nos dois primeiros anos de curso. Os participantes foram questionados sobre a importância da disciplina para sua formação. As respostas tiveram um consenso absoluto, isto é, todos responderam que a Libras é importante, porém cada um com sua particularidade. As respostas se afunilaram para diferentes aspectos relacionados a essa língua. Alguns consideram que a Libras funciona como um suporte na formação do professor em sala de aula, como visto nas respostas abaixo: “Contribui com a formação, principalmente na ques- tão de atuação em sala de aula, potencializando os aprendiza- dos dos alunos” (H1). “Pois como futuro profissional da educação é interessante ter o maior conhecimento possível antes de entrar no ambiente escolar e servir como base para acontecimentos futuros” (H11). “É importante, extremamente importante para que no futuro sejamos capazes de trabalhar com o povo surdo nas salas de aula” (IE3). “A disciplina de libras é muito importante para a formação porque nos ajuda a entender mais sobre a cultura surda e praticar cada vez mais a inclusão” (IE6). 186 “Totalmente. Conhecer outras culturas por si só já é impor- tante, quando tratamos de uma ferramenta de comunicação e inclu- são essa importância se intensifica” (IE5). Conforme apontado por Streiechen e Fontana (2018), durante a disciplina de Libras, o acadêmico estará diante de temáticas impor- tantes no contexto histórico, social e cultural do povo surdo. Por- tanto, os assuntos tornam-se relevantes para sua formação, de modo que os futuros docentes compreendam seu papel diante do aluno surdo, em contextos escolares inclusivos. Diante das respostas dos participantes percebe-se que essa questão foi absorvida de forma satisfatória por eles, levando-os a compreender a importância do conhecimento da língua e da cultura para seu futuro como professor. Além disso, a maioria dos participantes ressaltou que a dis- ciplina é indispensável no preparo do professor para a recepção de alunos surdos. Considerando esse conceito, originaram-se as seguintes respostas: “Porque, sabemos que a área de ensino não é restrita ape- nas a alunos sem deficiência e iremos nos deparar nas escolas com diversas especialidades, assim, como alunos surdos” (H5). PERSPECTIVAS DOS ACADÊMICOS SOBRE A DISCIPLINA DE LIBRAS “Sim, visto que dentro das salas de aulas teremos diferentes tipos de aluno inclusive alunos com deficiências” (LP6). “Porque com a lei da inclusão de pessoas especiais nas escolas, pede um professor preparado para lidar e mediar conheci- mentos a alunos incluídos, assim, temos contato já em formação, nos prepara para o futuro” (IE8). “Considero importante pois nunca saberemos quando tere- mos um aluno especial na escola e devemos ter uma noção para podermos dar um início de conceito inicial” (P8). 187 “Sim, pois como futura pedagoga devemos estar prepara- dos para incluir os alunos com necessidades especiais no ambiente escolar, o ajudando no processo de ensino-aprendizagem” (P6). “Pois, enquanto futura professora, estarei me deparando com alunos que necessitam de atendimento especializado e preciso estar preparada para auxiliar esses educandos” (P3). Nesse mesmo âmbito, Streiechen (2017b) aponta que o conhecimento da Libras por acadêmicos de licenciatura, “visa a um novo dimensionamento curricular dessa língua e das práticas peda- gógicas em sala de aula”, de forma a incluir o aluno surdo, assim como possibilitar a interação entre professor e aluno. De acordo com a autora “a aquisição da Libras pelos acadêmicos dos cursos de licen- ciaturas, bacharelados e outros é um exemplo do processo de inclu- são” (STREIECHEN, 2017b, p.115), visto que os ouvintes têm acesso à cultura surda, com possibilidades de aprenderem à língua de sinais. Alguns participantes defendem que a aprendizagem da Libras é um aparato de conhecimentos, para além da universidade e da sala de aula, a vislumbrar novas oportunidades no mercado de trabalho, principalmente no que se refere à socialização com os sur- dos. Nesse quesito as respostas foram as seguintes: “Acredito que sendo a segunda língua oficial brasileira, se torna de suma importância na graduação e também fora dela” (H9). “Importantíssima! É uma área que cada vez tem mais demanda. Adoro poder ajudar quem precisa e às vezes meu conhe- cimento nessa área é muito pouco” (P1). “No meio da licenciatura é essencial o conhecimento em Libras, além da inserção cada vez maior da língua de sinais no nosso dia a dia”. (H4). “Isso é indubitável. A disciplina age enquanto um incentivo à aprimoramento da comunicação d (os, as) estudantes” (H8). 188 A oficialização da Libras, em forma de lei, e a obrigatoriedade da disciplina nos cursos de licenciatura e Fonoaudiologia, possibili- taram uma maior procura pelo aprendizado dessa língua. PERSPECTIVAS DOS ACADÊMICOS SOBRE A DISCIPLINA DE LIBRAS Assim, a Libras vem, paulatinamente, ganhando seu espaço com sua difusão nos diversos meios sociais, de forma a despertar o desejo das pes- soas ouvintes, tanto para objetivos profissionais como para estabe- lecer relações com os surdos, a fim de diminuir as barreiras comu- nicativas entre surdos e ouvintes. Também, o processo de inclusão, referente às pessoas com deficiências, ocasionou mudanças den- tro e fora das escolas, fazendo com que as pessoas busquem mais conhecimentos acerca desse assunto. A respeito da disciplina de Libras, os participantes foram questionados quanto à carga horária disponibilizada para os res- pectivos cursos. Diante disso, eles ponderaram que a carga horária disponível para a disciplina é muito baixa e ressaltaram que não é possível aprender a língua de sinais com uma carga horária tão redu- zida, de acordo com o que se observa nas respostas de H4 e H10: “não, 6 meses (1 semestre) é muito pouco para aprender, conhecer, ficar pelo menos num nível de conhecimento e prática” (H4); “não. Acredito que apenas um semestre não compreende a necessidade de conhecimento da disciplina” (H10). Dos trinta e nove participantes, trinta e quatro afirmaram que a carga horária não é suficiente, dois declararam que é suficiente e para três deles é parcialmente suficiente, conforme resposta de IE7: “para um aprendizado bem geral, creio que é suficiente, mas se fosse considerar um conhecimento mais concreto não é suficiente”. Nesse aspecto, Streiechen (2017b) destaca que essa é a prin- cipal queixa dos acadêmicos, e que a maioria das instituições ofe- rece a disciplina apenas em um dos anos do curso, com uma carga horária bastante reduzida. Nesse mesmo âmbito Barboza e Silveira (2015, p. 212) destacam que 189 [...] a formação do professor nas universidades ainda não tem qualidade plena, não oferece ampla bibliografia sobre a educação especial nas licenciaturas e sua formação prática é reduzida e/ou com carga horária insatisfatória. Essa falta de preparo leva os professores a uma aborda- gem extremamente oralista junto aos alunos surdos. S U M Á R I O Desse modo, podemos compreender que a carga horária da disciplina de Libras não satisfaz a demanda do contexto escolar, pois o futuro professor não terá condições de manter uma comunicação com seu aluno surdo, perpetuando, assim, as barreiras comunicati- vas, que por sua vez impossibilitam de se efetivar o vínculo profes- sor/aluno em sala de aula. PERSPECTIVAS DOS ACADÊMICOS SOBRE A DISCIPLINA DE LIBRAS Pode-se notar que, devido à baixa carga horária da dis- ciplina de Libras, o aprendizado da língua se torna muito básico, obtendo-se apenas um conhecimento sobre as estruturas da lín- gua e um pouco do seu vocabulário, como observado nas respostas destes participantes: “Não, existe a necessidade de uma ampliação do conteúdo, para maior fixação do conteúdo” (H1). “Não. O conteúdo é muito amplo e as poucas horas de estu- dos de libras na graduação são suficientes para uma noção básica, mas não profunda” (H5). “Não. Dá uma base para a compreensão da língua apenas” (LP7). “Não. Dá uma base para a compreensão da língua apenas” (LP7). Não. Dá uma base para a compreensão da língua apenas (LP7). “Não, considero uma carga horária muito insuficiente, creio que se tivéssemos uma carga horária maior, aprenderíamos mais conteúdos e sinais” (IE8). (LP7). “Não, considero uma carga horária muito insuficiente, creio que se tivéssemos uma carga horária maior, aprenderíamos mais conteúdos e sinais” (IE8). “Não, considero uma carga horária muito insuficiente, creio que se tivéssemos uma carga horária maior, aprenderíamos mais conteúdos e sinais” (IE8). “Não, pois não conseguimos aprofundar diversos assun- tos ou ver alguns, pois teve conteúdos que não deu tempo de 190 S U M Á R I O trabalhar em sala. Contudo o que foi estudado foi bem aprovei- tado, e entendido” (P8). De acordo com Carniel (2018), a implementação da Libras como componente obrigatório na formação docente nas institui- ções de ensino superior, regulamentado pelo Decreto nº 5.626/2005 (BRASIL, 2005), veio por meio de pouco mais de uma década de esforços e lutas da comunidade surda. No entanto, ainda há uma preocupação sobre a organização e os efeitos da disciplina na for- mação do futuro professor. A baixa disponibilidade de carga horá- ria para a disciplina representa um dos efeitos negativos sobre o aprendizado da língua, visto que, de acordo com o que foi levan- tado pelos participantes, o número de aulas não é suficiente, assim impossibilitando que o aprendizado da língua seja efetivo, mesmo no nível mais básico. Conforme destacado na seção anterior, a Libras é uma língua que possui seus próprios componentes gramaticais, de maneira que seu aprendizado requer um estudo completo. Nesse caso, com uma carga horária insuficiente, que não possa envolver toda a complexi- dade da língua, a efetividade de uma possível comunicação torna-se no mínimo falha. E isso se torna ainda mais complicado para o apren- diz adulto, ouvinte, levando em consideração a idade, assim como as questões ligadas ao emocional, como veremos no próximo subitem. Nota-se que os acadêmicos gostariam que a disciplina fosse ampliada e que tivesse um destaque em sua formação, como na res- posta de IE3: “Não, eu acredito que seja pouco. Gostaria que Libras ocupasse mais tempo da carga horária”. No entanto, ainda que seja pouca a carga horária, é de extrema importância que o acadêmico obtenha conhecimentos a respeito da Libras e aprenda sobre os aspectos da comunidade surda, visto que ele poderá ter contato com aluno surdo no dia a dia de sua carreira como docente. 191 AQUISIÇÃO E APRENDIZAGEM DA LIBRAS COMO L2 Os participantes foram questionados sobre o nível de dificul- dade para aprenderem a língua de sinais. No total de trinta e nove participantes, nove declararam que a Libras é uma língua difícil de aprender e um ressaltou que é muito difícil. No entanto, vinte e sete participantes acham que o nível de dificuldade é médio. Já, um parti- cipante declarou ser fácil e apenas um a considera muito fácil. Essa questão dispõe que o aprendizado da Libras apresenta um grau moderado de dificuldade, visto que a maioria dos partici- pantes a considera de nível médio. Ainda, podemos propor que o grau de dificuldade depende das diferenças individuais. De acordo com Gesser (2010), as diferenças individuais é um fator a ser consi- derado na aprendizagem de L2, visto que os aprendizes apresentam diferentes ritmos para aprender, assim como a forma de aprender e as estratégias usadas, entre outros aspectos. Gesser (2010) aponta que as salas de aulas apresentam heterogeneidade, portanto, tendo variedade de idade, gênero, profi- ciência e/ou conhecimento na língua, necessidades e objetivos dos alunos, logo, esses aspectos podem se tornar um determinante em relação à dificuldade no aprendizado da Libras. Outro fator apontado pela autora corresponde ao papel da instrução (professor e metodo- logia). Nesse sentido, alguns estudiosos como Brown (1994) e Yorio (1976), citados por Gesser (2010), determinam algumas variáveis sobre o aprendiz, de forma a colaborar com os métodos de ensino de outras línguas, estes pautados em questões cognitivas e afeti- vas. Gesser (2010) também destaca que algumas dessas variáveis estão presentes no contexto de aprendizagem de Libras, mas esse assunto nos leva a outro ponto de discussão que, a princípio, não trataremos nesta pesquisa. 192 De acordo com Pizzio e Quadros (2011), na aprendizagem de língua de sinais, como L2, o adulto depende da iconicidade para lem- brar os aspectos referentes à língua. O que acontece com sinalizantes iniciantes é que eles provavelmente se apropriam de elementos familiares que se aproximam em natureza às línguas de sinais, ou seja, gestos e pantomima. Isto pode ajudá-los a apren- der coisas básicas nas línguas de sinais, como posicionar os dedos, as mãos e os braços para a produção de um sinal e como meio de lembrar o significado de um sinal que eles estão vendo pela primeira vez. AQUISIÇÃO E APRENDIZAGEM DA LIBRAS COMO L2 [...] posterior- mente, os aprendizes de L2 abandonam essa estratégia, no momento em que aprendem a estrutura fonológica da língua de sinais (PIZZIO; QUADROS, 2011, p. 63-64). S U M Á R I O Nesse caso, podemos conceber que alguns participantes puderam se apropriar dos elementos familiares da sua língua e, desse modo, por meio dessa estratégia de aprendizado, puderam compreender alguns sinais de maneira que tornou o seu apren- dizado menos complexo. De acordo com Pizzio e Quadros (2011), as línguas de sinais apresentam as mesmas restrições que as demais línguas. Para as autoras, os aprendizes de Libras como L2 devem passar pelos mes- mos desafios que os aprendizes de línguas faladas. Por esse motivo, Streiechen (2017b) dispõe que o aprendizado de Libras requer um estudo sobre os parâmetros que norteiam a língua, portanto, é necessário um conhecimento acerca da construção de uma frase em Libras, entender que essa língua segue regras próprias, dife- rentes daquelas aplicadas às línguas faladas. Diferente da língua portuguesa e demais línguas orais, a língua de sinais utiliza o canal espaço-visual, isto é, as informações linguísticas em um processo de comunicação são realizadas no espaço, sendo pelas mãos, pelos movimentos corporais e pelas expressões faciais, e são percebidas visualmente por seus usuários (STREICHEN, 2017b). 193 Diante disso, os acadêmicos foram questionados sobre as dificuldades na aprendizagem da língua. Nesse caso, eles puderam selecionar as alternativas que correspondiam à sua dificuldade ao aprender a Libras: alfabeto manual; memorizar os vocabulários em língua de sinais; as expressões faciais e corporais; uso da estrutura sintática da língua de sinais; a coordenação motora na realização das configurações de mãos e outros de resposta aberta. Em relação ao alfabeto manual, apenas um participante declarou ser difícil de aprender. Streiechen (2017b, p. 55) ressalta que o alfabeto manual ou datilológico “é um recurso que os surdos uti- lizam para escrever nomes próprios ou de objetos que não tenham um sinal representativo na Libras”. O aprendizado do alfabeto em si pode não ser uma tarefa difícil para alguns aprendizes, porém, como aponta Gesser (2010), o aprendiz de Libras tem que fazer um movi- mento linguístico diferente do que está acostumado (da boca para as mãos), de forma que nesse processo um empenho cognitivo-motor enorme é empreendido. AQUISIÇÃO E APRENDIZAGEM DA LIBRAS COMO L2 Também há um empreendimento focal na decodificação do formato das mãos para a sua compreensão visual, e isso pode se tornar um desafio para alguns iniciantes. Quanto à memorização dos vocabulários em língua de sinais, dezessete participantes consideraram-na difícil. Gesser (2010) destaca que o aprendiz precisa entender como a língua de sinais funciona, para que assim ele possa incorporar as palavras em seu discurso. Em relação ao vocabulário é possível adicionar as estraté- gias como um meio de aprendizado. No entanto, alguns aprendizes não conseguem formular suas próprias estratégias de memorização, levando-os a terem mais dificuldades nesse aspecto. Mas essa ques- tão ainda está relacionada a aspectos individuais. No que diz respeito às expressões faciais ou marcadores não manuais e corporais, quatorze participantes acham que é mais difícil. Pizzio, Quadros e Rezende (2009) ressaltam que, para os usuários de línguas de sinais, as expressões faciais têm duas funções distintas: 194 S U M Á R I O expressar emoções e marcar estruturas gramaticais específicas, como, por exemplo, a interrogação. As pessoas ouvintes apresentam bastante dificuldade em compreender a importância que as expres- sões faciais têm para marcar características gramaticais. Gesser (2006, p. 158) dispõe que para o ouvinte “permanece sempre uma grande dificuldade em lidar com as expressões faciais; e devido à cultura vocal de uso de língua(gem) dos ouvintes, existe até certo receio em expressar-se linguisticamente com as mãos e com o corpo”. Em relação ao uso da estrutura sintática da língua de sinais, dezessete participantes acham-na difícil. Cotovicz, Streiechen e Antoszcyszen (2018) afirmam que toda aprendizagem de uma L2 implica em um conhecimento sobre o processo de formação das sentenças, isto é, um estudo sobre a sintaxe da língua. No caso da Libras é necessário que o aprendiz saiba organizar os sinais dentro da sentença, pois a estruturação das sentenças na língua de sinais ocorre por meio da manipulação dos sinais no espaço. Ainda, os autores destacam que outras características essenciais são obser- vadas na sintaxe da Libras, como os verbos, que ainda podem ser divididos em verbos simples ou sem concordância, verbos com con- cordância e verbos espaciais. Dessa forma, cada tipo de verbo tem um papel importante na construção sintática da Libras, isto é, em como se dá a organização dos sinais na formação de uma sentença. AQUISIÇÃO E APRENDIZAGEM DA LIBRAS COMO L2 Diante disso, destacam-se as concepções de Paiva (2014), nas quais a língua é vista como um conjunto limitado de estrutu- ras básicas, como as sintáticas, apresentando um grande número de variações. Dessa forma, “prevê-se que quanto mais diferentes forem as estruturas de duas línguas, mais difícil será a aquisição da segunda, o que faz com que uma comparação entre os dois idiomas possa prever as dificuldades em sua aquisição” (PAIVA, 2014, p. 14). Pizzio e Quadros (2011) ressaltam que, além do vocabulário em sinais, os aprendizes de uma língua de sinais devem também dominar a fonologia, a morfologia e a sintaxe, próprias dessa língua. 195 Figueiredo (1995) expõe que a diferença na aquisição de línguas entre a criança e o adulto está relacionada ao desempenho fonoló- gico, pois a L1 exerce uma influência na pronúncia da L2 do adulto. Gesser (2010) aponta que para os adultos os efeitos da L1 sobre a L2 são mais aparentes, especialmente com os iniciantes, visto que eles assumem que a língua alvo funciona da mesma forma que a sua L1. Pizzio e Quadros (2011) argumentam que “o problema é que muitas vezes os cursos de línguas de sinais ensinam vocabulário isolado, excluindo as relações espaciais, as construções com classificadores, entre outros” (PIZZIO; QUADROS, 2011, p. 64). É um fator preocu- pante, a fazer com que o aprendiz de L2 passe a usar a mesma ordem de palavras de sua L1, de forma a desestruturar a ordem correta da morfologia ou da sintaxe da língua alvo, assim, também dificultando a compreensão da mensagem pelo surdo. Quanto à coordenação motora, na realização das configu- rações de mãos, quatorze participantes declaram-na como difícil. De acordo com Streiechen (2017a), por ser uma língua que neces- sita de coordenação motora, as crianças apresentam mais facili- dade ao aprender a língua de sinais, comparado aos adultos que não possuem essa habilidade. Isso também nos leva a uma ques- tão já discutida anteriormente, sobre a mudança de modalidade de comunicação que o aprendiz está acostumado, da boca para as mãos, incluindo o corpo. Os participantes foram questionados se tinham algum conhecimento em Libras antes de ingressarem na universidade. Percebeu-se que, do total de trinta e nove participantes, dezenove declararam que nunca tiveram contato com a Libras antes da gradu- ação e vinte disseram que tiveram esse contato. AQUISIÇÃO E APRENDIZAGEM DA LIBRAS COMO L2 Dentre os que decla- raram já ter conhecimento na língua, a maioria alegou ter somente um conhecimento básico. De acordo com Gesser (2006, p. 70), “a maioria dos ouvin- tes que entra em contato com a língua de sinais geralmente passa 196 por uma situação de estranhamento. Isso acontece possivelmente devido ao desconhecimento que se tem do surdo, da surdez e da lín- gua de sinais”. Diante disso, é possível destacar que os acadêmicos não passaram por essa situação, visto que a maioria afirmou já ter algum conhecimento da língua, mesmo em nível mais baixo. Souza (2015) defende que a neurociência também vem con- tribuindo de maneira significativa ao que diz respeito aos processos de aprendizagem. Conforme apontado por Souza (2015), o indivíduo adulto possui algumas limitações biológicas, pois este já apresenta um nível X de desenvolvimento em seu Sistema Nervoso Central, diferente da criança, que ainda está em processo de desenvolvi- mento. Esse fato de certa forma interfere na percepção e assimilação dos sons, assim dificultando o aprendizado de idiomas para o adulto e facilitando para a criança. Ademais, Gesser (2010) destaca que “aprender qualquer lín- gua é tarefa árdua, que demanda dos alunos um empenho cogni- tivo muito grande, e o aprendizado de língua de sinais, para ouvin- tes iniciantes, é tarefa das mais árduas (JACOB, 1996 apud GESSER, 2010), levando em consideração os parâmetros referentes à língua, à modalidade, entre outros aspectos. No entanto, a interação com surdos ou mesmo com ouvintes, por meio da língua de sinais, é um fator relevante para o desenvolvimento e fluência nessa língua. De certa forma, as questões levantadas por estudiosos, acerca da interação social, é um aspecto que contribui de forma sig- nificativa na aquisição/aprendizagem de língua. Também aspectos relacionados à individualidade do aprendiz devem ser considerados, visto que alguma questão sobre o aprendizado de língua corresponde a aspectos psicológicos (motivacionais, emocionais e estratégicos). 197 S U M Á R I O CONSIDERAÇÕES FINAIS Por meio dessa pesquisa, constatamos que o aprendizado de Libras, como L2, pode variar de pessoa para pessoa, visto que os participantes apresentaram concepções diversas em relação ao seu aprendizado. Para alguns, a Libras é uma língua fácil de aprender e para outros é difícil. No entanto, o fator que mais colabora para os desafios no aprendizado da Libras são os parâmetros que nor- teiam a língua, isto é, as configurações de mãos, os movimentos, as expressões corporais, entre outros aspectos gramaticais que regem a língua de sinais. É importante enfatizar que as teorias, os modelos e as hipóte- ses sobre a aquisição/aprendizagem de L2 serviram para a compre- ensão do fenômeno em questão. Os resultados obtidos mostram que a questão da dificuldade pode não ser um fator referente somente à idade, e que muitas vezes o processo de aprendizagem da língua advém das particularidades de cada sujeito aprendiz. Por outro lado, percebemos que todos compreendem a importância da língua de sinais em nossa sociedade, e que esta é um dos instrumentos necessários para o processo de inclusão dos surdos. No entanto, a carga horária destinada aos cursos não é satis- fatória, de forma que os aprendizes conseguem obter um conheci- mento muito básico em relação ao uso da língua para a comunica- ção com um indivíduo surdo. Diante disso, é preciso uma reformula- ção na grade curricular dos cursos, principalmente nos cursos que apresentam uma carga horária inferior às demais, para que haja uma disponibilidade maior de conteúdo, incluindo a prática da língua. Ressaltamos que é importante que estejamos em contato com as mais diversas línguas e culturas, principalmente aquelas ao nosso redor, como a língua de sinais, observando a importância de seu conhecimento e uso, não somente por razões de inclusão, mas 198 principalmente por questões socioculturais. Em suma, esperamos incentivar os acadêmicos, e a comunidade em geral, a continuar a estudar a língua de sinais de modo que possamos contribuir com a qualificação de profissionais para atuarem na área da surdez. REFERÊNCIAS BARBOZA, Clévia Fernanda; SILVEIRA, Luciane Cruz. A importância da aprendizagem de Libras para a formação de professores bilíngues dentro de uma perspectiva inclusiva. Revista Espaço, Rio de Janeiro, s/v, n. 43, 192-218, jan./jun., 2015. BARBOZA, Clévia Fernanda; SILVEIRA, Luciane Cruz. A importância da aprendizagem de Libras para a formação de professores bilíngues dentro de uma perspectiva inclusiva. BONA, Camila de. 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Aquisição de segunda língua. Texto base do Curso de Letras Libras na modalidade de EaD. CCE, UFSC. Florianópolis, 2008. PAIVA, Vera Lucia Menezes de Oliveira e. A complexidade da aquisição de segunda língua: revisando e conciliando teorias. São Paulo: Parábola Editorial, 2014. PIZZIO, Aline Lemos.; QUADROS, Ronice Müller de. Aquisição da língua de sinais. Texto base do Curso de Letras Libras na modalidade de EaD. CCE, UFSC. Florianópolis, 2011. PIZZIO, Aline Lemos; QUADROS, Ronice Müller de; REZENDE, Patrícia Luiza Ferreira. Licenciatura e Bacharelado em Letras-Libras na modalidade EaD. CCE, UFSC. Florianópolis, 2009. SANTANA, Ana Paula. A idade crítica para a aquisição da linguagem. In: SANTANA, Ana Paula. Surdez e linguagem: aspectos e implicações neurolinguísticas. São Paulo: Plexus, p. 63-90, 2007. SOUZA, Arlei da Silva. O processo de aquisição de um segundo idioma em crianças e adultos. Estação científica, Juiz de Fora-MG, n. 14, jul./dez., 2015. STREIECHEN, Eliziane Manosso; FONTANA, Evelline Cristhine. Expectativa dos acadêmicos e os desafios dos professores de Libras no ensino superior. RE-UNIR, Porto Velho-RO, v. 5, n. 2, p. 100-120, 2018. 200 VYGOTSKY, Lev. Pensamento e Linguagem. Tradução: Jeferson Luiz Camargo. 2.ed. São Paulo: Martins Fontes, 1989. S U M Á R I O YORIO, Carlos Alfredo. Discussion of  “explaining sequence and variation in second language acquisition”. Language Learning, p. 59-63, 1976. YORIO, Carlos Alfredo. Discussion of  “explaining sequence and variation in second language acquisition”. Language Learning, p. 59-63, 1976. 201 Faz-se necessário pontuar que este artigo é fruto de recorte de um trabalho final de curso de graduação. A pesquisa pode ser apreciada, integralmente, em Mendonça (2021). INTRODUÇÃO O tema inclusão vem sendo discutido desde a década de 90, mas ainda há várias barreiras a serem desconstruídas, visto que, não só no campo social, mas também no educacional, essa pers- pectiva ainda é um desafio. A construção de um espaço educativo/ instrutivo com horizontes inclusivos requer modificações no que se refere ao ambiente, ou seja, a estrutura, como também às questões psíquicas, pois sabemos da existência dos pré-conceitos histori- camente construídos. Dessa forma, entendemos a necessidade de adotar novas práticas escolares, a fim de ressignificar as ações segregacionistas e atitudinais por parte dos profissionais que atuam nesse espaço. Logo, faz-se necessário enfatizar que uma formação adequada e continuada é indispensável para um fazer docente empático/huma- nista. Assim sendo, é importante ressaltar que cabe às instituições formadoras assumirem uma posição mais responsável sob esse viés, de forma que versem em suas salas de aula sobre essas temáticas visando contribuir para a construção de uma sociedade que respeite as singularidades e particularidades de cada sujeito. Por esse ângulo, realizamos a presente pesquisa17 no intuito de contribuir para uma construção de uma sociedade que respeite às diferenças. Na tentativa de corroborar com essa perspectiva, cria- mos um projeto que visou levar para sala de aula discussões acerca da inclusão do sujeito Surdo, de sua cultura e de sua língua. Esta, falada por uma minoria linguística, denomina-se Língua Brasileira de Sinais, a qual é tida como meio de expressão legal dos Surdos brasi- leiros após uma trajetória de muita luta. Faz-se necessário pontuar que este artigo é fruto de recorte de um trabalho final de curso de graduação. A pesquisa pode ser apreciada, integralmente, em Mendonça (2021). 17 203 Para tanto, elencamos como objetivo geral desta pesquisa discutir os resultados de uma intervenção pedagógica, que teve como intuito gerar a possibilidade de proporcionar o conhecimento de uma nova língua na educação básica, visando o respeito às particularida- des de cada indivíduo, enfatizando a importância da inclusão escolar. S U M Á R I O No que se refere ao desenvolvimento deste trabalho, ele se alicerça numa abordagem qualitativa, a qual analisa vivências indi- viduais em um meio social específico. É uma pesquisa-ação, a qual possibilita o entendimento e reflexão das práticas vivenciadas pelo pesquisador com ênfase no social, e também beneficia de forma direta e indireta os sujeitos envolvidos (BORTONI-RICARDO, 2008). INTRODUÇÃO Desse modo, justificamos que a motivação desse trabalho surgiu a partir de uma inquietação vivenciada no espaço escolar pela pesqui- sadora, onde ela passou a notar que um dos seus alunos sentia-se envergonhado por ter uma mãe Surda. Logo, tal acontecimento pas- sou a ser o fator crucial que a levou a realizar um projeto pedagógico conscientizador acerca da inclusão. O porquê social dessa temática, justifica-se por entendermos a sua relevância para a desconstrução dos preconceitos construídos acerca da pessoa com deficiência. Dessa forma, fez-se necessário propiciar reflexões acerca da temática na sala de aula para promo- ver o respeito às diferenças. Alega-se de modo científico a sua rele- vância para a construção de um fazer pedagógico ressignificado, a ponto de contribuir com esse ofício a partir de uma temática tão importante, mas pouco debatida. 204 S U M Á R I O O ENSINO DE LIBRAS: O QUE É PREVISTO NOS DOCUMENTOS OFICIAIS? A Lei Federal 10.436 de abril de 2002 reconhece a Língua Brasileira de Sinais (Libras) como oriunda das pessoas Surdas brasileiras, fruto de anos de resistências/lutas dessa comunidade. A partir desse reconhecimento, percebemos avanços no que se refere aos direitos linguísticos dos Surdos. Nessa direção, a Libras é vista como ferramenta importante para a comunicação dos Surdos, mas também, como uma conquista no que diz respeito à inclusão deles na sociedade. Após a aprovação da lei em 2002, tivemos a regulamenta- ção através de um decreto (este é um documento abrangente sobre a inserção da Libras como disciplina curricular) sobre a formação do professor, do profissional intérprete e tradutor, como também do direito de a pessoa Surda obter sua educação através de sua L1 e L2, preferencialmente na Libras (L1) e o direito à saúde. Assim, a Libras é ofertada no ensino superior a partir do decreto 5.626, aprovado em 22 de dezembro de 2005, que regulamenta a inserção dessa língua como componente curricular na grade dos cursos de licenciaturas. Vejamos o que diz o artigo 9º deste decreto: Art. 9º A partir da publicação deste Decreto, as institui- ções de ensino médio que oferecem cursos de formação para o magistério na modalidade normal e as instituições de educação superior que oferecem cursos de Fonoau- diologia ou de formação de professores devem incluir Libras como disciplina curricular (BRASIL, 2005, s/p). Este decreto (BRASIL, 2005, s/p) em seu parágrafo único, destaca que: “O processo de inclusão da Libras como disciplina curricular deve iniciar-se nos cursos de Educação Especial, Fono- audiologia, Pedagogia e Letras, ampliando-se progressivamente para as demais licenciaturas”. Desse modo, através dessa inserção, 205 S U M Á R I O proporcionou-se a difusão da língua na formação inicial docente, até então ainda desconhecida por alguns profissionais. Com isso, faz-se necessário prepará-los para atenderem as diferenças humanas e se atentarem para elas e, por isso, os docentes precisam ter domínio dos saberes para lidar com as diversas situações escolares, a fim de estabelecer uma educação efetiva e igualitária. Para tanto, o Plano Nacional de Educação - PNE, Lei nº 10.172/2001, destaca que “o grande avanço que a década da educa- ção deveria produzir seria a construção de uma escola inclusiva que garanta o atendimento à diversidade humana” (BRASIL, 2001, s/p). O ENSINO DE LIBRAS: O QUE É PREVISTO NOS DOCUMENTOS OFICIAIS? Nesse viés, entendemos que há a necessidade de investimento na formação de professores adequada para que eles possam conse- guir atender os anseios/realidades dessa perspectiva. A formação continuada também é indispensável nesse processo, e sobre isso Pimenta (2000, p. 17) ressalta a importância “de ressignificar os pro- cessos formativos a partir da reconsideração dos saberes necessá- rios à docência, colocando a prática pedagógica e docente escolar como objeto de análise”. Assim, para construirmos uma educação que seja oferecida para todos de forma igualitária, ou seja, na qual cada indivíduo seja respeitado independentemente de suas particularidades, necessita- mos salientar a importância no que se refere à formação de pro- fessores para atuarem nessa perspectiva. Segundo Fonseca (1995), existe uma necessidade de preparar docentes para esse ofício com urgência, porém, ele deixa claro que para os educadores consegui- rem transformar suas práticas, precisam de alguns aspectos como: ter uma formação continuada, ser equipados de recursos pedagó- gicos, como também obter uma orientação, visando adquirir novas competências e atingindo, assim, o êxito no seu ofício. A Libras, como componente curricular no ensino superior nos cursos de licenciaturas, permite ao docente em formação refletir sobre a prática pedagógica com os possíveis alunos Surdos que terá 206 S U M Á R I O em sua sala de aula, respeitando sua língua, sua cultura e as pecu- liaridades educativas. Essa disciplina é ofertada com a parte teórica, mas também com a parte prática. Esta, permite ao professor em for- mação um conhecimento básico na Libras, a exemplo da aprendi- zagem do seu nome e as saudações em Libras, entre outros. Vale salientar que este conhecimento básico não permitirá esse discente sair fluente na língua, ou plenamente capaz de lecionar nesta língua a um Surdo, mas já provoca uma curiosidade de ir à procura de um curso, de fazer pesquisas na área e afins. A expectativa desse componente curricular é também des- construir alguns (pré)conceitos e mitos acerca da surdez e da pes- soa Surda, além de proporcionar um primeiro contato com a Língua Brasileira de Sinais, bem como, possibilitar que eles levem para o seu futuro ambiente de trabalho reflexões acerca da deficiência e da perspectiva inclusiva. Sabemos que as aulas devem ter esse teor de discussão acerca da temática, como forma de respeito à diversidade. O ENSINO DE LIBRAS: O QUE É PREVISTO NOS DOCUMENTOS OFICIAIS? Ressaltamos que há uma necessidade de repensarmos com relação ao componente curricular em discussão, pelo fato de ser ofe- recida em um só semestre e ter uma carga horária reduzida, o que chamamos de lacuna, visto que, o aproveitamento poderia ser mais significativo se disponibilizassem uma duração maior de tempo. Quanto à implementação da Libras como disciplina no ensino regular da educação básica, infelizmente, ainda não temos na grade curricular. No entanto, em um momento futuro poderemos ter essa possibilidade, visto que a sua inserção poderá promover nesse espaço discussões pertinentes, evidenciando o conhecimento e reconhecimento de outra língua e cultura. Sabemos que nas grades curriculares do nosso país são existentes disciplinas como Francês, Espanhol ou Inglês, estas são ofertadas no intuito de oportunizar ao indivíduo conhecer uma segunda língua no ensino regular. 207 Dessa forma, entendemos que inserir a Libras nos currículos da educação básica também poderá ser viável nesse processo de ensino/aprendizagem e de inclusão. No que concerne, entretanto, à implementação de discussões sobre inclusão, principalmente do Surdo no contexto educacional e, especialmente, nas escolas regu- lares, não avançamos. Defende-se que “[...] a educação a que se tem direito deve promover os valores universais”, e entre estes estão, “[...] a igualdade entre as pessoas, o respeito à diversidade, à tolerância e a não dis- criminação” (UNESCO, 2004, p. 9), mas sabemos que ainda há uma carência muito significativa nesse sentido. Logo, como nos afirma Martins (2016, p. 27), “é necessário exercitar a vivência do respeito ao outro, entendendo que a diversidade não pode ser vista como uma barreira para a realização do ato educativo [...]”. A Base Nacional Comum Curricular (BNCC) do ensino fun- damental, complementa a fala de Martins (2016, p. 27) quando se refere à escola “como espaço de aprendizagem e democracia inclu- siva, deve se fortalecer na prática coercitiva de não discriminação, não preconceito e respeito às diferenças e diversidades” (BRASIL, 2018, p. 14). Sendo assim, entendemos que uma casa só é constru- ída de forma segura se ela tiver um alicerce, da mesma forma deve acontecer com o cenário inclusivo educacional, é da base que deve iniciar esse processo, para que futuramente possamos colher frutos dessas sementes plantadas no presente. Na seção seguinte, iremos apresentar como é possível realizar uma intervenção na educação básica, partindo de todas as questões que foram discutidas ao longo dos tópicos anteriores. 208 O PROJETO LIBRAS NA ESCOLA: CAMINHOS E POSSIBILIDADES Considerando o que discutimos ao longo do nosso estudo acerca da Inclusão Social, iremos apresentar neste espaço uma intervenção pedagógica que denominamos de Projeto Libras na Escola que foi planejada e posta em prática na tentativa de propor- cionar aos alunos do ensino fundamental I o conhecimento e reco- nhecimento de uma nova língua que possui peculiaridades diferen- tes da sua  língua materna. Além disso, nosso intuito durante todo o projeto foi refletir acerca das contribuições que a Libras traz para esse contexto educacional, como também mostrar que é possível agregarmos sua abordagem na sala de aula para alunos ouvintes, mesmo que esse trabalho ainda não seja previsto ou mesmo obriga- tório sob o viés legislativo. No que se refere à BNCC (BRASIL, 2018), temos temas con- temporâneos (Transversais/Integradores), os quais abordam temá- ticas que atravessam o campo educacional, ou seja, são discussões que atendem às demandas sociais atuais e, por isso, estão presentes no dia a dia dos sujeitos. Esclarecido isso, é importante frisar que: “cabe aos sistemas e redes de ensino, assim como às Escolas [...] incorporar aos currículos e às propostas pedagógicas a abordagem de temas contemporâneos que afetam a vida humana em escala local, regional e global, preferencialmente de forma transversal e integradora” (BRASIL, 2018, p. 19). Desta forma, esses pressupostos visam contribuir para um aluno-cidadão, participante de uma vida ativa e significativa na socie- dade. Nesse sentido, uma das competências específicas de Ciências Humanas para o ensino fundamental é “Compreender a si e ao outro como identidades diferentes, de forma a exercitar o respeito à dife- rença em uma sociedade plural e promover os direitos humanos” 209 (BRASIL, 2018, p. 357), isto é o que o documento nos orienta para uma construção de saber no que se refere à diversidade cultural. A Base reforça a necessidade do autoconhecimento, isto é, o aluno precisa entender o seu “eu” e o outro para que possa compre- ender seu papel no mundo e reconhecer que deve praticar o respeito com o outro sendo um ser consciente de suas ações. A partir dessas considerações, iremos apresentar a seguir a caracterização e con- textualização da pesquisa, o projeto em si e a descrição de suas eta- pas com algumas imagens para melhor semiotização dessa vivência. CARACTERIZAÇÃO E CONTEXTUALIZAÇÃO DA PESQUISA O lócus selecionado para nossa intervenção foi a sala de aula, por compreendermos que é neste espaço onde ocorrem diver- sas aprendizagens e interações. Para tanto, esta pesquisa se insere na abordagem qualitativa, pois esta, conforme Bortoni-Ricardo (2008, p.34) “[...] procura entender; interpretar fenômenos sociais inseridos em um contexto”, e se classifica como pesquisa-ação, que pode ser definida como [...] um tipo de pesquisa com base empírica que é con- cebida e realizada em estreita associação com uma ação ou ainda, com a resolução de um problema cole- tivo, onde todos os participantes estão envolvidos de modo cooperativo e participativo (THIOLLENT, 1985, p. 12  apud GIL, 2010, p. 42). [...] um tipo de pesquisa com base empírica que é con- cebida e realizada em estreita associação com uma ação ou ainda, com a resolução de um problema cole- tivo, onde todos os participantes estão envolvidos de modo cooperativo e participativo (THIOLLENT, 1985, p. 12  apud GIL, 2010, p. 42). Logo, de acordo com Gil (2010) este tipo de pesquisa, contri- bui de forma significativa e direta com a ação social. Sob essa ótica, será apresentada a seguir uma sequência de aulas e atividades que foram desenvolvidas em uma escola municipal no interior da Paraíba, 210 S U M Á R I O a qual atende aproximadamente 90 alunos em turmas de pré-escola (pré I e II) e fundamental (1° ano até o 5° ano), nos turnos manhã e tarde. Os participantes da presente pesquisa são alunos que residem em área urbana e periférica, e são pertencentes a uma classe social não privilegiada economicamente. Assim, a turma escolhida para a intervenção e a seleção do lócus, deram-se pelo fato de fazerem parte do contexto no qual atuei como cuidadora de uma criança com o espectro do Autismo, o que oportunizou um desenvolvimento melhor com os alunos, visto que, o contato com todos da turma era/foi frequente. É importante fri- sar que a realização desta intervenção foi autorizada pela instituição e que a referida, como já sinalizamos no início deste trabalho, fora idealizada a partir de uma vivência (um aluno/criança ouvinte que se sentia envergonhado por sua mãe ser surda) que foi notada no ambiente escolar e causou inquietação. Um outro ponto que precisa ser salientado, é que os discentes da instituição tiveram seu primeiro contato com a Libras a partir do projeto desenvolvido, o que despertou interesse por parte de todos. 18 O curso teve duração de dois anos e possibilitou otimizar o conhecimento básico da Libras prior- itariamente para os profissionais da educação e afins, numa tentativa de garantir a inclusão de forma efetiva no contexto social. CARACTERIZAÇÃO E CONTEXTUALIZAÇÃO DA PESQUISA Também, só foi possível realizar tal projeto, pelo fato de a pesquisa- dora ter obtido uma certificação de 240h concedida pela Fundação Centro Integrado de Apoio à Pessoa com Deficiência (FUNAD)18. O PROJETO E SUAS ETAPAS O projeto teve início no dia 19 (dezenove) de julho de 2019 (dois mil e dezenove) e finalizou no dia 13 (treze) de dezembro de 2019 (dois mil e dezenove), sendo dividido em quatro etapas 18 211 (totalizando 23 aulas ministradas). Estas foram formuladas no intuito de promover reflexões sobre a inclusão do sujeito Surdo no contexto social e escolar, além de uma conscientização sobre os preconceitos enraizados em nossa sociedade e o conhecimento de uma nova lín- gua que pode se tornar a L2. Vale salientar que o conteúdo das aulas foi organizado em comum acordo com a professora titular, que deu abertura para que escolhêssemos os temas. Antes de dar início às etapas do projeto, foram apresentadas aos alunos algumas informações sobre a Libras, sobre os Surdos, a surdez, o termo que deve remeter a essa minoria linguística, como também, foi tratado do assunto inclusão, o respeito e a empatia que devemos ter para com o próximo, independente- mente de sua cor, raça, língua, religião e afins. PRIMEIRA ETAPA Na etapa I, que foi composta por oito aulas, foi possível con- textualizar para os alunos alguns temas em Libras, foram eles: alfa- beto manual, nomes e números, cores, saudações, estações do ano, animais, revisão de todos esses temas e uma aula destinada a con- fecção de um cartaz. A seguir, iremos descrever como se deu cada aula, bem como o objetivo a ser alcançado em cada uma delas. A primeira aula focou, inicialmente, nos valores morais/éticos para efe- tivar a inclusão, dizer “não” ao preconceito e conscientizar sobre a importância da Libras no contexto social e escolar. Nosso intuito foi informar a cada aluno sobre a importân- cia de refletirmos sobre esses temas e de termos a partir deles um olhar mais humano e empático com o outro sujeito, independente- mente de suas singularidades e particularidades, esse foi o foco com ênfase ao respeito às diferenças. Na sequência, ministramos sobre 212 S U M Á R I O o alfabeto em Libras, iniciamos com a prática do alfabeto manual, em seguida, todos fizeram juntos e logo depois tivemos uma dinâ- mica com bexigas, em que cada uma possuía o nome de um aluno, e o participante que estourasse aquele balão faria o nome usando a datilologia19. Como forma lúdica, utilizamos a música infantil o “Abe- cedário da Xuxa” e, por fim, entregamos uma atividade para fixar no caderno com a intencionalidade de que eles tivessem o seu primeiro contato/conhecimento com a/da língua. Na segunda aula, iniciamos pedindo que cada aluno já apre- sentasse em datilologia seu próprio nome. Logo após, sugerimos um novo tema, este foram os números em Libras. Fizemos uma dinâ- mica, em que tínhamos um círculo com vários números dentro dos copos descartáveis e, quando o participante retirava um copo, nele continha um número, este teria que ser feito em Libras. Utilizamos a música infantil “Os índiozinhos” para trabalhar os números de 0 a 9 numa perspectiva lúdica, como também, expomos um vídeo20 de uma aula de Libras com essa temática realizada em um quadro do programa Carrossel animado transmitido pelo SBT. Por fim, fixamos no caderno uma atividade com os números em Libras para pintar. A terceira aula deu-se com a temática de cores em Libras. Iniciamos com um vídeo do Hugo21 (mostrando os sinais das cores), após esse primeiro momento, fizemos juntos em Libras para melhor visualizar e depois de forma individual. 19 É um sistema de representação das línguas orais e escritas que utiliza as mãos. 20 Rafaella Sessenta é uma professora de Libras, a qual participou do programa Carrossel animado (programa da rede de televisão SBT) apresentando os números em Libras no dia 23 de maio de 2012. Disponível em: <https://youtu.be/yfhrOdAHQfI>. Acesso em: 17 jan. 2023. 21 Hugo (intérprete 3D) é um personagem do aplicativo Hand Talk, o qual é um tradutor de Libras que visa proporcionar acessibilidade através das telas. O aplicativo permite converter textos, imagens e áudios para a Libras. PRIMEIRA ETAPA Cada aluno recebia em sua testa um objeto de uma cor estudada, o participante não podia ver a referida cor, mas precisava identificar através da realização do sinal que todos faziam em sua volta. 213 Para se tornar mais atrativo o tema, distribuímos uma base de isopor, pincéis e tintas guache, para que cada um pudesse fazer um desenho utilizando algumas cores estudadas, fixamos uma ativi- dade referente às cores – pintar e colocar abaixo a cor representada – no caderno e trabalhamos com a música “Gugudada” – a música das cores em Libras. – no caderno e trabalhamos com a música “Gugudada” – a música das cores em Libras. – no caderno e trabalhamos com a música “Gugudada” – a música das cores em Libras. A quarta aula teve como tema saudações em Libras. Deu-se a partir de uma breve discussão sobre os cumprimentos, logo após, foi apresentado o tema de forma prática. Em um outro momento, foi colocado em um recipiente vários papéis com palavras que se referiam ao tema, cada sujeito escolheu um, e ao abrir, esse fazia o sinal e todos os outros deveriam reconhecer, sem que precisasse da via oral auditiva, apenas do espaço-visual. Por fim, dividimos a sala em grupos, realizamos pequenos diálogos com o tema estudado e levamos uma atividade de pintura. Iniciou-se a quinta aula com os conceitos das estações do ano, depois com um vídeo do personagem Hugo do Hand Talk em Libras e a prática logo após. A professora titular nos ajudou a realizar uma oficina de origami, a qual cada um aprendeu a fazer uma flor, remetendo à estação da primavera. Por fim, foi entregue uma ativi- dade prática de origami para colorir e colar no caderno. Abordamos na sexta aula, o tema: os animais, adentramos, mostrando algumas imagens de animais e, logo após mostrando seus respectivos sinais. Depois, alguns participantes vieram até a frente para imitar algum animal, para que seus colegas fizessem o sinal em Libras. Depois, fizemos um ditado recortado e finalizamos a aula. Na sétima aula, fizemos uma revisão com todos os assuntos vistos anteriormente. Trabalhamos de forma breve, cada um deles, a fim de perceber os resultados obtidos deste primeiro passo do pro- jeto. PRIMEIRA ETAPA Para finalizar essa etapa, tivemos na oitava aula a confecção de um cartaz, foi separado grupos, e cada um ficou responsável para abordar uma das temáticas estudadas, com a finalidade de expor na sala para que eles pudessem sempre visualizar. 214 SEGUNDA ETAPA A etapa II, realizada em seis momentos: o primeiro, que con- tabilizou o horário de uma aula, foi destinado a todo corpo docente, discente e profissionais que atuam na escola, com o tema “Artefa- tos Culturais do povo Surdo”. Este foi o tema sorteado para o desfile cívico da cidade, e como estávamos à frente de um projeto de Libras, nos tornamos responsáveis por ele. A partir dos esclarecimentos feitos nessa palestra sobre a referida temática, abrimos um espaço para perguntas e iniciamos uma roda de conversa sobre o tema. Depois, exibimos em Libras “O Patinho Surdo” e finalizamos com uma apresentação interpretando o hino da cidade. Essa palestra foi pensada no intuito de todos terem consciência sobre o tema e que trouxessem reflexões para o campo educacional. O segundo momento (realizado em quatro aulas), foi desti- nado aos ensaios em Libras, do “Parabéns, Marí!”, porém, apenas duas integrantes puderam participar da apresentação. Por fim, no dia 18 (dezoito) de setembro de 2019 (dois mil e dezenove) foi realizado, em praça pública, o desfile, no qual trabalhamos os artefatos cultu- rais do povo Surdo, além da literatura surda e fizemos uma apresen- tação em Libras, fechando assim, esta etapa. Figura 1 – Desfile cívico (Representação da Literatura Surda) TERCEIRA ETAPA Na terceira etapa foram quatro aulas, durante as quais tive- mos o ditado da pipoca. Colocamos no quadro branco pipocas e ao lado um espaço, o qual deveria ser preenchido. Em papéis escreve- mos algumas palavras que se referiam aos temas da primeira etapa, dobramos e colocamos em uma sacola, depois agitamos e pedimos para que cada participante escolhesse um papel, ao abrir ele deveria fazer o sinal, se acertasse, colocaria o nome no espaço e tinha direito de pegar a pipoca que estava ao lado. Todos conseguiram realizar, alguns com ajuda dos colegas. Trabalhamos também com músicas em Libras, a exemplo de: “Borobletinha”, “O Sapo não Lava o Pé”, “Os Índiozinhos”, “Mariana”, “A Dona Aranha”. Todas estas foram sele- cionadas no intuito de revisar assuntos já vistos na primeira etapa. Por fim, juntos confeccionamos um painel que denominamos de “cantinho da Libras”. Figura 1 – Desfile cívico (Representação da Literatura Surda) Fonte: Mendonça (2021). Fonte: Mendonça (2021). 215 QUARTA ETAPA Na quarta e última etapa, tivemos cinco aulas: em quatro delas, realizou-se ensaios com toda a turma, com a música intitulada “Vem que está Chegando o  Natal” – Aline Barros em Libras, e, na quinta e última, finalizamos o projeto com apresentação do desenho animado “Min e as Mãozinhas”. A utilização dessa animação total- mente em Libras, foi pensada pelo fato de abordar temas sociais ao mesmo tempo que ensina os sinais, aproximando os sujeitos ouvin- tes da cultura surda, gerando assim, um interesse em conhecer melhor outra língua e cultura. O desenho apresenta alguns sinais, e não faz uso de legendas, tem repetições dos sinais, mas com função, elas são usadas dentro de uma conversação. 216 Nosso objetivo nessa aula foi proporcionar um entreteni- mento no reconhecimento dos sinais vistos no desenho, os quais eles já tinham sido capacitados durante o andamento do projeto. Tivemos como propósito tornar natural a visualização dos sinais no meio de uma conversa, ao finalizar cada episódio nós perguntáva- mos quais os sinais que apareceram no vídeo, e de forma muito autô- noma e rápida os educandos respondiam corretamente com a sina- lização e a palavra falada, o que mostrou que de forma significativa a concretização desse aprendizado. CULMINÂNCIA Após à conclusão de todas as etapas, foi proporcionado aos alunos um momento de confraternização para comemorarmos os resultados do nosso projeto houve a apresentação dos estudantes no pátio da escola, em que eles iniciaram com os sinais de “Oi” e “boa tarde” e, logo depois, realizaram a apresentação da música que ensaiaram na última etapa. Figura 2 – Apresentação da música “Vem que está chegando o Natal” Figura 2 – Apresentação da música “Vem que está chegando o Natal” Fonte: Mendonça (2021). Fonte: Mendonça (2021). 217 Para o encerramento do projeto, fizemos entrega de certifica- dos, como forma de gratidão e carinho pelo desempenho apresen- tado por eles. A culminância foi resultado de um processo permeado de muitas aprendizagens significativas, o que permitiu perceber a conclusão de um ciclo onde os resultados foram alcançados. Figura 3 – Festa de encerramento Fonte: Mendonça (2021). Figura 3 – Festa de encerramento S U M Á R I O Fonte: Mendonça (2021). CONSIDERAÇÕES FINAIS Um outro ponto positivo, foi perceber o respeito dentro da sala de aula com relação a inclusão de outros alunos com Autismo e paralisia cerebral, já não se referiam a eles de forma pejorativa, 219 S U M Á R I O e efetivamente os incluíam nas atividades diárias do contexto esco- lar. Outro ponto louvável foi a aceitação da escola e a relevância dada por todos os profissionais que compõem a instituição para aplica- bilidade do projeto. Vale ressaltar que a professora titular gostaria de disponibilizar um espaço maior para realização, mas haja vista, a escola ainda está vigente em um sistema curricular tradicional e conteudista, a professora não podia ceder o espaço desejado, mas ainda assim foi contra o sistema e deu abertura para que o projeto se realizasse, mesmo que não tenha acontecido com profundidade. Algo pontual que merece ser ressaltado, foi a percepção tida pela professora titular, a qual não permitiu que as aulas fossem margi- nalizadas no sistema escolar, pois o projeto fez parte do componente curricular desses alunos no ano vigente oficialmente, uma vez que, foi colocado na caderneta, garantindo assim todas as aulas registra- das do projeto. Sabemos que, enquanto professores pesquisadores e em formação, devemos trazer respostas para a sociedade por meio da pesquisa, e a partir da elucidação desse projeto, acreditamos que trouxemos contribuições não só para o espaço escolar, mas para toda sociedade. Sendo assim, enfatizamos a importância das pes- quisas objetivando trazer contribuições para o nosso meio social. CONSIDERAÇÕES FINAIS Levando em consideração o trajeto que promovemos ao longo desta pesquisa, a pesquisadora, enquanto futura docente, reconhece ainda mais que exerce um papel importante na socie- dade, o de ser uma agente social. Para tanto, compreende que ao professor é dada a responsabilidade de levar assuntos pertinentes, relevantes e indispensáveis para uma construção humanística na sua sala de aula. Neste ofício, é sabido que não basta mediar as gra- des curriculares, mas sim, formar alunos-cidadãos, conscientes de suas ações humanas e sociais. 218 Assim, compreendemos e reforçamos aqui, que não foi o nosso objetivo central fazer o estudante sair fluente na língua atra- vés do projeto, pois sabemos que a Libras contempla a modalidade espaço-visual, a qual se difere das línguas orais auditivas, além de termos o fator determinante que é a carga horária reduzida para aprendizagem de uma língua. Porém, o nosso intuito foi apresentar a eles uma outra modalidade de língua, ensinar variados conteúdos da Libras e trazer reflexões acerca da comunidade surda e no que se refere à inclusão. Identificamos através das práticas em sala de aula, que mais da maioria obteve um aproveitamento considerável, o que nos faz crer que a metodologia empregada corresponde à faixa etária. Nos sentimos gratificados por receber respostas tão positivas a partir de nossos estímulos, o que nos faz reforçar a importância e urgência de termos como disciplina obrigatória no ensino regular, o ensino da Libras como L2 na educação básica. Logo, pudemos observar que houve motivação por parte da pesquisadora e professora titular, que tentaram encontrar os melhores caminhos possíveis para essa aprendizagem, que de acordo com Vygotsky (1991), a motivação é um pré-requisito para aprendizagem. Foi perceptível uma ligação existente entre os alunos, quando alguém apresentava dificuldade em algum sinal, outros tentavam aju- dar, entendemos assim a existência do que Vygotsky (1991) chama de desenvolvimento proximal, ou seja, o aluno para desenvolver suas habilidades de forma plena, necessita de uma assistência. O projeto cumpre então um papel perante a sociedade, pois tivemos retornos de pais que acolheram a iniciativa junto a seus filhos, e observaram que eles haviam aprendido alguns sinais e utilizavam em casa, na tentativa de ensinar no contexto familiar. BRASIL. Ministério da Educação. Base Nacional Comum Curricular. MEC, 2018. Brasília, DF, 2018. FONSECA, Solange Gomes. Educação Especial. Porto Alegre: Artes Médicas, 1995. REFERÊNCIAS BORTONI-RICARDO, Stella Maris. O professor pesquisador: introdução à pesquisa qualitativa. São Paulo: Parábola Editorial, 2008. BRASIL. Decreto nº 5.626, de 22 de dezembro de 2005. Regulamenta a Lei nº 10.436, de 24 de abril de 2002, que dispõe sobre a Língua Brasileira de Sinais - Libras, e o art. 18 da Lei nº 10.098, de 19 de dezembro de 2000. Brasília: MEC, 2005. BRASIL. Política Nacional de Educação Especial na perspectiva da Educação Inclusiva. Brasília, 2008. Disponível em: <https://bit.ly/3JKX9sl>. Acesso em: 01 jan. 2023. 220 BRASIL. Lei nº 13.146, de 6 de julho de 2015. Institui a lei brasileira da pessoa com deficiência (Estatuto da Pessoa com Deficiência). Brasília, 2015. BRASIL. Lei nº 13.146, de 6 de julho de 2015. Institui a lei brasileira da pessoa com deficiência (Estatuto da Pessoa com Deficiência). Brasília, 2015. BRASIL. Ministério da Educação. Base Nacional Comum Curricular. MEC, 2018. Brasília, DF, 2018. FONSECA, Solange Gomes. Educação Especial. Porto Alegre: Artes Médicas, 1995. GIL, Antônio Carlos. Como elaborar projetos de pesquisa. São Paulo: Atlas, 2010. MARTINS, Iara F. de Melo. Linguagem, inclusão e ensino. In: LINS, Juarez Nogueira (Org.). Estudos na área de linguagem: ensino, pesquisa e formação docente. Recife: EDUFPE, 2016. p. 17-27. MENDONÇA, Joyce da Silva Cruz de Mendonça. Abrindo horizontes inclusivos: o ensino da Língua Brasileira de Sinais como L2 para crianças ouvintes de uma escola pública do interior da paraíba. 2021. 40 f. Trabalho de Conclusão de Curso (Graduação em Letras) – Universidade Estadual da Paraíba, Guarabira, 2021. PIMENTA, Selma Garrido. Formação de professores: saberes e a identidade da docência. In: PIMENTA, Selma Garrido (Org). Saberes pedagógicos e atividade docente. São Paulo: Cortez, 2000. p. 15-34. UNESCO. Políticas educativas e equidad. Reflexiones del Seminário Internacional. Santiago de Chile, 2004. Disponível em: <https://bit.ly/3HPmYoG>. Acesso em: 23 jan. 2023. VYGOTSKY, Lev Semionovitch. A formação social da mente: os desenvolvimentos dos processos psicológicos superiores. 4. ed. São Paulo: Martins Fontes, 1991. 221 S U M Á R I O INTRODUÇÃO A argumentação é inerente ao discurso humano, mas “é no texto que ela se expressa” (CAVALCANTE, 2016, p. 122). É partindo dessa premissa que situamos o estudo da argumentação sob o viés da Linguística Textual e objetivamos, neste capítulo, apresentar algumas reflexões acerca da produção do cartaz, no Ensino Funda- mental, com foco na argumentação para além da redação do Exame Nacional do Ensino Médio (ENEM). Para tanto, amparamos nossa discussão teórica nos postula- dos de Amossy (2020), Antunes (2003), Cavalcante e Custódio Filho (2010); Marcuschi (2008, 2010); Koch (1984), entre outros, de modo a manter pontos de contato entre a dinâmica argumentativa e o ensino de Língua Portuguesa. Ademais, caracterizamos esta pesquisa como qualitativa, uma vez que interpretaremos fatos a partir de um contexto específico (BORTONI-RICARDO, 2008), além disso, evidenciamos o caráter de pesquisa-ação deste estudo, tendo em vista que o corpus foi gerado a partir de uma prática interventiva do ensino de Língua Portuguesa em uma escola pública da rede estadual da Paraíba. Nesse contexto, especificamos, que nosso corpus é consti- tuído por dois cartazes, produzidos por estudantes de uma turma de 9º ano do Ensino Fundamental, da rede estadual da Paraíba. E, a temática, em evidência, isto é, a gravidez na adolescência, deve- -se ao fato de contemplarmos em nossas aulas de produção textual, pautas sociais que visam o engajamento cidadão dos nossos alunos. Vale ressaltar, ainda, que a análise será feita tendo como norte, sobretudo, as leituras sugeridas na disciplina Linguística Tex- tual do PPGL/UFPE, ministrada pela professora Suzana Cortez, no segundo semestre de 2022. E, quanto à estruturação deste capítulo, 223 inicialmente, apresentaremos a fundamentação teórica, na sequên- cia, a metodologia e a análise do corpus, em seguida, as considera- ções finais e, por fim, as referências que embasaram a pesquisa. ALGUNS APONTAMENTOS SOBRE AS NOÇÕES DE TEXTO, GÊNERO, ARGUMENTAÇÃO E O ENSINO DE LÍNGUA PORTUGUESA São grandes as contribuições dos estudos linguísticos frente às demandas do ensino de Língua Portuguesa, e, nesse cenário, des- tacamos uma das áreas importantíssimas, a saber, a Linguística do Texto (a qual denominaremos daqui em diante como LT), que surgiu na década de 60, e “pode ser definida como o estudo das opera- ções linguísticas, discursivas e cognitivas reguladoras e controlado- ras da produção, construção e processamento de textos escritos ou orais em contextos naturais de uso” (MARCUSCHI, 2008, p. 73). Na direção dessas ideias, entende-se que um olhar pedagógico voltado apenas ao aspecto formal da língua escrita não consegue dar conta de um trabalho eficiente no que tange à produção de textos. Em vista dessa perspectiva, consideramos fundamental refle- tirmos, brevemente, sobre alguns postulados da LT, que são extrema- mente caros para o processo de didatização da escrita, no domínio escolar, por exemplo, a noção de “texto” que, em alguns casos, ainda gira em torno de uma ideia estritamente linguística de modo que os estudantes identificam como “textual”, apenas, o código linguístico verbalizado. Todavia, conforme Marcuschi (2008, p.75) [...] “O texto não é simplesmente um artefato linguístico, mas um evento que ocorre na forma de linguagem inserida em contextos comunicativos”. 224 Nessa conjuntura, pensamos o “texto” sob um viés enuncia- tivo de modo que seu planejamento deve abarcar propósitos comu- nicativos, sujeitos sociais, entre outras demandas que perpassam o universo linguístico, o que permite ao estudioso da LT, estreitar os laços com outras áreas do conhecimento. Acerca da perspectiva enunciativa, vale ressaltar, segundo Bakhtin (1995, p. 113) que: S U M Á R I O Na realidade, toda palavra comporta duas faces. Ela é determinada tanto pelo fato de que procede de alguém como pelo fato de que se dirige para alguém. [...] A palavra é uma espécie de ponta lançada entre mim e os outros. Se ela se apoia sobre mim numa extremidade, na outra apoia-se sobre o meu interlocutor. A palavra é o território comum do locutor e do interlocutor. Nesse contexto, os sentidos de um texto são construídos e reconstruídos pelos sujeitos sociais, que tomam a linguagem como instrumento de ação no mundo. Desse modo, a tarefa de compreen- der e produzir textos, exige de nós, uma gama de saberes, que per- passam a dinâmica linguística. Segundo Cavalcante e Custódio Filho (2010, p. ALGUNS APONTAMENTOS SOBRE AS NOÇÕES DE TEXTO, GÊNERO, ARGUMENTAÇÃO E O ENSINO DE LÍNGUA PORTUGUESA 58) [...] “o texto emerge de um evento no qual os sujeitos são vistos como agentes sociais que levam em consideração o contexto sociocomunicativo, histórico e cultural para a construção de sentidos”. Nessa direção, o que fica em evidência nas pesquisas sob os direcionamentos da LT, não é uma ideia dicotômica tampouco hie- rárquica entre o tratamento dado ao texto pelo analista dessa área, tendo em vista as perspectivas textuais e discursivas, posto que: [...] é cada vez mais frequente, em LT, desconsiderar as fronteiras muito bem delimitadas entre texto e discurso e investir no entendimento de que essas duas instâncias se imbricam e, por vezes, se confundem, sem que isso sig- nifique a necessidade de que as disciplinas em torno das duas matérias estudem as mesmas coisas ou se juntem para formar uma única área de investigação. Os progra- mas investigativos de cada uma garantem naturalmente as especificidades, de modo que o diálogo entre elas não 225 implica perda de identidade, mas, sim, ganho explicativo e avanço-teórico metodológico (CAVALCANTE; CUSTÓ- DIO FILHO, 2010, p. 62). implica perda de identidade, mas, sim, ganho explicativo e avanço-teórico metodológico (CAVALCANTE; CUSTÓ- DIO FILHO, 2010, p. 62). Assim, compreender a linguagem humana, materializada em textos, encaminha-nos ao reconhecimento de que é sempre necessário dialogar com outras vozes, sem contudo, desconsiderar o escopo da área investigativa a qual estamos filiados, em nosso caso, a LT, “[...], uma disciplina constitutivamente aberta ao diá- logo, assentada na crença epistemológica de que é exatamente no diálogo que as coisas se aprimoram” (CAVALCANTE; CUSTÓDIO FILHO, 2010, p. 62). Muito se diz sobre a problemática que envolve o ensino de Língua Portuguesa com ênfase nas habilidades de leitura e escrita dos educandos, nesse contexto, é fundamental discutirmos sobre outra noção valiosa no que tange ao encaminhamento dos jovens para o estudo dos textos, a saber, os gêneros textuais. Uma vez que nossas atividades cotidianas são mediadas pela linguagem, entendemos os gêneros textuais como instrumen- tos que organizam nosso agir no mundo, daí a importância de inves- tigarmos esse objeto sob os escopos da forma e da função. Nessa dinâmica, vale ressaltar que tais gêneros são criações humanas para necessidades da vida em sociedade, desse modo, não apresentam caráter estático, isto é, caem em desuso e são ressignificados coti- dianamente. Nesse sentido, vejamos o dizer de Marcuschi (2010, p. 19): ALGUNS APONTAMENTOS SOBRE AS NOÇÕES DE TEXTO, GÊNERO, ARGUMENTAÇÃO E O ENSINO DE LÍNGUA PORTUGUESA Pensemos, por exemplo, na intensidade de gêneros que emergiram com o advento das tecnologias digitais, na perspectiva de um mundo cada vez mais globalizado. Nesse contexto, temos a conversa no WhatsApp, por exemplo, que, inicialmente, focava na escrita e, hoje, já assume outras configurações via áudio, tendo em vista a necessidade de uma comunicação mais apressada. Nesse sentido, vejamos o dizer de Marcuschi (2010, p. 19): 226 Já se tornou trivial a ideia de que os gêneros textuais são fenômenos históricos, profundamente vinculados à vida cultural e social. Fruto de trabalho coletivo, os gêne- ros contribuem para ordenar e estabilizar as atividades comunicativas do dia a dia. São entidades sociodiscursi- vas e formas de ação social incontornáveis em qualquer situação comunicativa. No entanto, mesmo apresentando alto poder preditivo e interpretativo das ações humanas em qualquer contexto discursivo, os gêneros não são instrumentos estanques e enrijecedores da ação criativa. Caracterizam-se como eventos textuais altamente male- áveis, dinâmicos e plásticos. Surgem emparelhados a necessidades e atividades socioculturais, bem como na relação com inovações tecnológicas, o que é facilmente perceptível ao se considerar a quantidade de gêneros tex- tuais hoje existentes em relação a sociedades anteriores à comunicação escrita. Nessa linha de raciocínio, Bakhtin (1992) e Marcuschi (2008) discorrem sobre a relação entre gênero, língua e atividade social. Vejamos em que consiste essa relação e os pontos de aproxima- ção entre os dois autores. Segundo Bakhtin (1992, p. 282), “Ignorar a natureza do enunciado e as particularidades do gênero que assina- lam a variedade do discurso em qualquer área do estudo linguístico leva ao formalismo e à abstração, desvirtua a historicidade do estudo, enfraquece o vínculo existente entre a língua e a vida.” Ao passo que para Marcuschi (2008, p. 149), “[...] a análise de gêneros engloba uma análise do texto e do discurso e uma descrição da língua e da visão da sociedade, e ainda tenta responder a questões de natureza socio- cultural no uso da língua de maneira geral”. Com base nos aportes teóricos de Bakhtin (1992) e Marcus- chi (2008), entendemos a relação entre gênero, língua e atividade social sob uma perspectiva contínua, primeiro (I) temos uma ativi- dade social (II) que emerge um gênero específico (III) e que para pro- duzirmos sob a modalidade oral ou escrita, necessitamos de saberes da língua enquanto sistema, mas também, enquanto artefato cultural. ALGUNS APONTAMENTOS SOBRE AS NOÇÕES DE TEXTO, GÊNERO, ARGUMENTAÇÃO E O ENSINO DE LÍNGUA PORTUGUESA 227 Por exemplo, se estou de saída, preciso avisar ao meu esposo acerca desse fato e não disponho de um aparelho telefônico naquele momento, escrevo um bilhete. Nesse contexto, tanto Bakhtin (1992) como Marcuschi (2008) evidenciam a necessidade de tomarmos como objeto de investi- gação analítica as situações sociais em que os gêneros emergem, como um caminho coerente para apropriação de um saber linguís- tico que envolva o gênero, o texto, o discurso, mas também, a vida social mediada por nossas atividades linguageiras. Por fim, vale discutir, um pouco, acerca da argumentação, dinâmica muito cara ao contexto escolar, sobretudo, quando se leva em conta o ideal de cidadania preconizado nos documentos oficiais, que parametrizam o ensino em nosso país. Nesse sentido, incial- mente, é necessário mencionar que todo texto apresenta, de certo modo, uma orientação argumentativa, isto é, nossos dizeres materia- lizados em textos escritos e/ou orais não são neutros. Nessa lógica, Koch (1984, p. 19-20) assevera que: A interação social por intermédio da língua caracteriza- -se fundamentalmente, pela argumentatividade. Como se dotado de razão e vontade, o homem, constantemente, avalia, julga, critica, isto é, forma juízos de valor. Por outro lado, por meio do discurso - ação verbal dotada de inten- cionalidade - tenta influir sobre o comportamento do outro ou fazer com que compartilhe determinadas de suas opiniões. É por esta razão que se pode afirmar que o ato de argumentar, isto é, de orientar o discurso no sen- tido de determinadas conclusões, constitui o ato linguís- tico fundamental, pois a todo e qualquer discurso subjaz uma ideologia na acepção mais ampla do termo. A neu- tralidade é apenas um mito: o discurso que se pretende “neutro”, ingênuo, contém também uma ideologia a da sua própria objetividade. Na direção dessas ideias, entende-se que se há uma orienta- ção argumentativa em todo texto, é possível trabalhar a argumentação 228 S U M Á R I O no Ensino Fundamental a partir de variados gêneros de texto. Em contrapartida, na contemporaneidade, muitas escolas, reduzem a lida com o fazer argumentativo às etapas finais da escolarização básica em virtude do ENEM, focalizando as práticas de letramento dos jovens na tessitura, exclusiva, do gênero redação do ENEM. Na perspectiva de perpassar os padrões formais da argumentação, há também “A noção de dimensão argumentativa [...], proposta para pensar em formas alternativas de argumentação que vão além das formas canônicas.” (AMOSSY, 2020, p. ALGUNS APONTAMENTOS SOBRE AS NOÇÕES DE TEXTO, GÊNERO, ARGUMENTAÇÃO E O ENSINO DE LÍNGUA PORTUGUESA 72), nesse contexto, é possível pensar a argumentação a partir de textos de caráter narrativo, como a fábula, por exemplo, entre outras possibilidades. Assim, entendemos a tarefa de argumentar como “a tenta- tiva de modificar, de reorientar, ou mais, simplesmente, de reforçar, pelos recursos da linguagem, a visão das coisas por parte do alocu- tário” (AMOSSY, 2020, p. 130). Nesse caso, é possível contribuir com o desenvolvimento da argumentação crítica do estudante do Ensino Fundamental, trabalhando diversos gêneros, por exemplo, história em quadrinhos, gêneros publicitários diversos, entre outros, uma vez que para Cavalcante (2019, p. 320) “Além da visão de argumentação como tentativa de influenciar o outro, aceitamos ainda o pressuposto de que só argumentamos por meio de gêneros”. Em suma, os gêne- ros de texto são instrumentos basilares para práticas de letramento escolar com foco na dinâmica argumentativa. Para finalizar essa breve reflexão teórica, é válido ressaltar que, enquanto   professores de Língua Portuguesa, embasados nas noções de texto, gênero e argumentação, aqui discutidas, precisa- mos considerar em nossas práticas de ensino da escrita, conforme os postulados de Antunes (2003, p. 45) que: Uma visão interacionista da escrita supõe, desse modo, encontro, parceria, envolvimento entre sujeitos, para que aconteça a comunhão das ideias, das informações e das intenções pretendidas. Assim, por essa visão se supõe que alguém selecionou alguma coisa a ser dita 229 a um outro alguém, com quem pretendeu interagir, em vista de algum objetivo. Enfim, é preciso rever algumas práticas mecânicas para o ensino da escrita que se distanciam dos usos reais que os estu- dantes fazem da língua enquanto agentes sociais. Desse modo, é fundamental que a escrita, também seja tomada, como uma forma de ação no mundo. A seguir, discorreremos sobre o percurso metodológico para coleta e análise dos textos que constituirão o corpus deste capítulo. METODOLOGIA E ANÁLISE DE TEXTOS Iniciamos esse capítulo a partir de uma abordagem qualita- tivo-interpretativa, à luz de Amossy (2020), Antunes (2003), Caval- cante e Custódio Filho (2010), Marcuschi (2008, 2010), entre outros, buscando respaldo teórico que aborde a temática aqui discutida. Vale ressaltar que, objetivando atender o caráter aplicado desse estudo, utilizaremos a abordagem qualitativa que, segundo Bortoni-Ricardo (2008, p. 34), “procura entender, interpretar fenômenos sociais inse- ridos em um contexto”. À medida que tomamos uma problemática vivenciada por nós, em sala de aula, como ponto de partida para produção cientí- fica, assumimos a postura de professor pesquisador, uma vez que, buscando solucionar tal problemática, além de construirmos conhe- cimento, melhoramos nossa prática pedagógica. Desse modo, Bor- toni-Ricardo (2008, p. 46) esclarece: O professor pesquisador não se vê apenas como um usu- ário do conhecimento produzido por outros pesquisado- res, mas se propõe também a produzir conhecimentos sobre seus problemas profissionais, de forma a melhorar 230 sua prática. O que distingue o professor pesquisador dos demais professores é seu compromisso de refletir sobre a própria prática, buscando reforçar e desenvolver aspectos positivos e superar as próprias deficiências. Para isso ele se mantém aberto a novas ideias e estratégias. S U M Á R I O É nesse contexto que situamos nosso estudo, isto é, uma pesquisa-ação, cujos sujeitos envolvidos desempenharam função ativa na construção do conhecimento. O lócus da pesquisa foi uma escola pública da rede estadual da Paraíba e os sujeitos participantes são estudantes com faixa etária entre 14 e 15 anos de uma turma do 9º ano do Ensino Fundamental. De modo a aplicar, em nossa análise, noções teóricas da disciplina LT, elegemos como especificidade um olhar para textos multissemi- óticos que refletem o posicionamento crítico de jovens estudantes acerca de uma temática de cunho social, isto é, a gravidez na ado- lescência. Nesse cenário, destacamos, então, que a argumentação crítica do estudante pode ser trabalhada para além da redação do ENEM, desde o Ensino Fundamental, a partir de práticas de escrita de gêneros textuais diversos. Em virtude da dimensão dessa produção acadêmica, sele- cionamos dois cartazes, produzidos pelos estudantes supracita- dos, de modo a constituir nosso corpus analítico. Vale ressaltar que tais dados são frutos de um projeto interventivo avaliado e apro- vado pelo Comitê de Ética em Pesquisa (CEP-UEPB) sob protocolo 53561516.0.0000.5187. METODOLOGIA E ANÁLISE DE TEXTOS E, a escolha pela temática das produções, isto é, a gravidez na adolescência, deu-se em virtude do nosso reconhe- cimento acerca da função social da escola: impulsionar os educan- dos a agir socialmente a partir dos saberes linguageiros adquiridos. Nessa lógica, os Parâmetros Curriculares Nacionais (PCN) de Lín- gua Portuguesa evidenciam que: Procurando desenvolver no aluno a capacidade de com- preender textos orais e escritos e de assumir a palavra, 231 produzindo textos em situações de participação social, o que se propõe ao ensinar os diferentes usos da lingua- gem é o desenvolvimento da capacidade construtiva e transformadora. O exercício do diálogo na explicitação, contraposição e argumentação de ideias é fundamental na aprendizagem da cooperação e no desenvolvimento de atitudes de confiança, de capacidade para interagir e de respeito ao outro. A aprendizagem desses aspectos precisa, necessariamente, estar inserida em situações reais de intervenção, começando no âmbito da própria escola (BRASIL, 1997, p. 40). Assim, o trabalho com uma escrita argumentativa cumpre um papel social colaborativo. Vejamos: Figura 1 – Cartaz 1 produzido por estudantes do 9º ano Figura 1 – Cartaz 1 produzido por estudantes do 9º ano Figura 1 – Cartaz 1 produzido por estudantes do 9º ano Fonte: Dados da pesquisa da autora (2016). Fonte: Dados da pesquisa da autora (2016). No cartaz 1, percebe-se que o posicionamento dos discen- tes gira em torno dos cuidados necessários para evitar uma gravi- dez não planejada, a saber, a utilização de preservativo ou da pílula anticoncepcional. Tais dizeres são embasados em orientações dos órgãos responsáveis pela saúde pública em nosso país. Nessa linha de raciocínio, constata-se que os educandos visam influenciar os interlocutores, ressaltando os encaminhamentos possíveis para 232 S U M Á R I O combater a problemática da gravidez precoce, ressaltando, por- tanto, métodos contraceptivos. Na direção dessas ideias, é perti- nente mencionar o escopo da argumentação, segundo Koch e Elias (2016, p. 34): “Argumentar é tentar influenciar nosso interlocutor por meio de argumentos cuja constituição demanda apresentação e organização de ideias, bem como estruturação e raciocínio que será orientado em defesa de uma tese ou ponto de vista, visando à ade- são do interlocutor.” Assim, verifica-se que há orientação argumentativa para além do gênero redação do ENEM e que é possível chegar a tal constata- ção quando se leva em conta os propósitos comunicativos de cada texto e os impactos que eles provocam nos interlocutores. Outro aspecto extremamente relevante, em textos dessa natureza, é a utilização da diversidade semiótica com vistas a chamar ainda mais a atenção do interlocutor além de intensificar a clareza semântica dos signos linguísticos verbalizados. Por exemplo, ava- liamos, como muito pertinente, a relação entre a imagem não verbal centralizada no cartaz, a qual apresenta várias bonequinhas de mãos dadas em prol de um mesmo objetivo e o escrito verbalizado, que fica abaixo da mesma, o qual diz o seguinte: “Todos lutando contra a gravidez na adolescência”. Ressaltando essa diversidade semiótica presente nos textos atuais, Rojo (2012, p. 1) afirma que: É o que tem sido chamado de multimodalidade ou multis- semiose dos textos contemporâneos, que exigem multi- letramentos. Ou seja, textos compostos de muitas lingua- gens (ou modos, ou semioses) e que exigem capacida- des e práticas de compreensão e produção de cada uma delas (multiletramentos) para fazer significar. Nessa direção, os estudantes utilizaram, ainda, uma espécie de panfleto, oriundo de busca na internet, que dizia: “Gravidez na adolescência... Figura 1 – Cartaz 1 produzido por estudantes do 9º ano É a melhor hora?” Após esse questionamento, havia duas alternativas a serem assinaladas: uma com uma mamadeira 233 S U M Á R I O e outra com um refrigerante Coca-Cola. A partir dessa leitura multis- semiótica, os discentes destacaram que a adolescência não é a fase ideal para assumir a responsabilidade de cuidar de um filho de modo a provocar a reflexão dos interlocutores. Por fim, os educandos fizeram um alerta, para tanto, empre- garam o modo verbal imperativo: “Não destrua seus sonhos, previ- na-se, pois assim, você irá conquistá-los.” Essa construção provoca uma reflexão sobre a importância da aprendizagem gramatical para produção de textos diversos. Nesse caso, o modo imperativo é usado para expressar ações que se exige do interlocutor, por meio de pedi- dos, sugestões e conselhos. S U M Á R I O Figura 2 – Cartaz 2 produzido por estudantes do 9º ano Fonte: Dados da pesquisa da autora (2016). Figura 2 – Cartaz 2 produzido por estudantes do 9º ano Fonte: Dados da pesquisa da autora (2016). No cartaz 2, os educandos mencionaram algumas consequ- ências da gravidez precoce, de natureza psicológica e fisiológica, que afetam negativamente a vida de adolescentes. Ademais, a sintonia entre os recursos não verbais e as palavras ao redor foi muito perti- nente, uma vez que ressaltou, a partir dos variados sinais de interro- gação, os diversos dilemas que a adolescente enfrenta ao longo do processo de gravidez. Nessa direção, entendemos que: “Argumentar 234 S U M Á R I O seria, assim, uma negociação persuasiva na tentativa de influenciar, de pôr em ação uma série de estratégias, dentre elas as textuais, para negociar, em uma determinada interação, certos pontos de vista, a partir dos quais se tenta influenciar o outro.” (CAVALCANTE, BRITO, GIERING & PINTO, 2019, p. 99). Nesse caso, é possível constatar que a intenção persua- siva pode ser materializada em textos diversos a partir de variados recursos semióticos e, que, nessa perspectiva, não basta informar o “outro” acerca de um ponto de vista, mas, convidar o “interlocutor” a defendê-lo também, uma vez que persuadir é “sensibilizar o outro a agir” (ABREU, 2009, p. 25). Os discentes afirmaram, ainda, que é direito de todo adoles- cente o acesso a informações referentes à orientação sexual, ressal- tando a importância do letramento nessa perspectiva. Figura 1 – Cartaz 1 produzido por estudantes do 9º ano Nesse con- texto, temos um encaminhamento interventivo frente à problemá- tica em pauta, o que remete a uma das competências cobradas no ENEM, a saber, a competência V – Elaborar proposta de intervenção para o problema abordado, respeitando os direitos humanos. Nessa perspectiva, evidenciamos que o trabalho com o desenvolvimento das habilidades critico-textuais dos jovens, pode ser focalizado a partir de práticas de letramento, como a produção do gênero cartaz de modo a evidenciar temáticas socioculturais. Desse modo, para Portela (2016, p. 32) “ensinar a argumentar pode ser entendido como ensinar a atuar socialmente, para que o indivíduo possa modificar a realidade que o rodeia através da razão e da emoção, provocando no outro uma reação.” Por fim, assim como no cartaz 1, os estudantes também fizeram um alerta, utilizando verbo no modo imperativo: “Não pule etapas... Tudo tem o seu tempo’’, apresentando assim, um con- selho para o interlocutor, ademais, ressaltamos a marcação do verbo “pule”, no presente, que segundo Koch e Elias (2014) serve para provocar reflexões. 235 Na direção dessas ideias, constatamos o quanto os recursos linguísticos e gramaticais, a exemplo, dos pontos de interrogação além do modo e tempo verbal, mencionados anteriormente, podem ser utilizados em favor dos propósitos argumentativos textuais do escrevente. Portanto, esses saberes devem ser trabalhados sob o viés enunciativo. Segundo Antunes (2003, p. 86): Regras de gramática, como o nome já diz, são normas, são orientações acerca de como usar as unidades da lín- gua, de como combiná-las, para que se produzam deter- minados efeitos, em enunciados funcionalmente inteligí- veis, contextualmente interpretáveis e adequados aos fins pretendidos na interação. Em suma, ao adotar uma perspectiva de língua interacionista, o professor de língua materna compreende que a aprendizagem das regras gramaticais deve ter como foco, o desenvolvimento das habi- lidades de leitura e produção textual do jovem estudante. CONSIDERAÇÕES FINAIS Chegamos ao fim desse texto, constatando que o nosso obje- tivo de apresentar algumas reflexões acerca da produção do cartaz, no Ensino Fundamental, com foco na argumentação para além da redação do ENEM foi alcançado, uma vez que a análise do corpus comprovou que é possível argumentar a partir de práticas de letra- mento do gênero supracitado. Nesse contexto, ressaltamos a perti- nência de explorar a multissemiose, como estratégia enunciativa, na produção de gêneros dessa natureza. Nessa direção, enfatizamos que, o desenvolvimento crítico- -argumentativo dos estudantes, pode e deve ser trabalhado, desde o Ensino Fundamental, a partir de práticas de letramento envoltas nos variados gêneros de texto. Desse modo, enquanto professores de 236 S U M Á R I O língua, precisamos reconhecer que há um percurso no processo de escolarização capaz de encaminhar o estudante para produzir com excelência gêneros argumentativos de impacto social, como a reda- ção do ENEM, por exemplo. E, nesse percurso há uma infinidade de gêneros que ao serem trabalhados, amparados em temáticas socio- culturais, podem render frutos a todos nós, apaixonados pelo saber linguístico e cientes que a leitura e a escrita devem ser tomadas como “objetos de ensino” sempre! Assim, esperamos que essas breves reflexões impulsionem o aprofundamento de pesquisas sobre a argumentação, uma temática tão cara não apenas para o domínio escolar, mas para nossa vivência em sociedade que é mediada por ações de linguagem. REFERÊNCIAS ABREU, Antônio Suárez. A arte de argumentar: Gerenciando Razão e Emoção. São Paulo: Ateliê Editorial, 2003. AMOSSY, Ruth. A dimensão argumentativa do discurso: questões teóricas e práticas. In: CAVALCANTE, Mônica Magalhães; BRITO, Mariza Angélica Paiva, (Orgs.). Texto, discurso e argumentação: Traduções. Campinas: Pontes, 2020, p. 71-96. ANTUNES, Irandé. Aula de português: encontro & interação. São Paulo: Parábola Editorial, 2003. BAKHTIN, Mikhail. Estética da criação verbal. São Paulo: Martins Fontes, 1992. BAKHTIN, Mikhail. Marxismo e filosofia da linguagem. São Paulo: Hucitec, 1995. BORTONI-RICARDO, Stella Maris. O professor pesquisador: introdução à pesquisa qualitativa. São Paulo: Parábola Editorial, 2008. BRASIL. Ministério da Educação e do Desporto. Secretaria de educação Fundamental. Parâmetros Curriculares Nacionais: Primeiro e segundo ciclos do ensino fundamental: língua portuguesa. Brasília: MEC/SEF, 1997. 237 CAVALCANTE, Mônica Magalhães; BRITO, Mariza Angélica Paiva; GIERING, Maria Eduarda Giering; PINTO, Rosalice Botelho Wakim Sousa. A negociação persuasiva para a análise da argumentação no discurso. Revista (Con)Textos, Vitória (UFES), v. 13, n. 25, p. 99-116, 2019. CAVALCANTE, Mônica Magalhães; BRITO, Mariza Angélica Paiva; GIERING, Maria Eduarda Giering; PINTO, Rosalice Botelho Wakim Sousa. A negociação persuasiva para a análise da argumentação no discurso. Revista (Con)Textos, Vitória (UFES), v. 13, n. 25, p. 99-116, 2019. CAVALCANTE, Mônica Magalhães; CUSTÓDIO FILHO, Valdinar. Revisitando o Estatuto do Texto. Revista do GELNE, Piauí, v. 12, n. 2, p. 56-71, 2010. CAVALCANTE, Mônica Magalhães. Abordagens da argumentação na Linguística Textual. ReVEL, edição especial vol. 14, n. 12, p. 106-124, 2016. CAVALCANTE, Mônica Magalhães. Por uma análise argumentativa em linguística textual. In: VITALE, María Alejandra; PIRIS, Eduardo Lopes; CARRIZO, Alicia Eugenia; AZEVEDO, Isabel Cristina Michelan de (Orgs.). Estudios sobre discurso y argumentación. Coimbra: Grácio Editor, p.319-338, 2019. COPPI, Danielle dos Santos Mendes. Projeto de letramento: uma concepção social da escrita aplicada ao ensino de Língua Portuguesa. 106 f. Dissertação (Programa de Mestrado Profissional em Letras – PROFLETRAS) – Universidade Estadual da Paraíba, Guarabira, 2016. KOCH, Ingedore Villaça; ELIAS, Vanda Maria. Ler e escrever: estratégias de produção textual. São Paulo: Contexto, 2014. KOCH, Ingedore Villaça; ELIAS, Vanda Maria. Escrever e argumentar. São Paulo: Contexto, 2016. KOCH, Ingedore Villaça. Argumentação e linguagem. 5 ed. São Paulo: Cortez, 1984. KOCH, Ingedore Villaça. Argumentação e linguagem. 5 ed. São Paulo: Cortez, 1984. MARCUSCHI, Luiz Antônio. Produção textual: análise de gêneros e compreensão. São Paulo: Parábola Editorial, 2008. MARCUSCHI, Luiz Antônio. Gêneros textuais: definição e funcionalidade. In: DIONISIO, Angela Paiva; MACHADO, Anna Rachel; BEZERRA, Maria Auxiliadora (Orgs.). Gêneros textuais e ensino. São Paulo: Parábola Editorial, 2010. p. 19-38. PORTELA, Daniela Cristina Sales. A construção discursiva da argumentação em sala de aula: uma proposta de jogo digital como ferramenta de ensino-aprendizagem. 191 f. (Dissertação de mestrado) PROFLETRAS – UFMG. Belo Horizonte, 2016. ROJO, Roxane. Pedagogia dos multiletramentos: diversidade cultural e de linguagens na escola. In: ROJO, Roxane; MOURA, Eduardo (Orgs.). Multiletramentos na escola. São Paulo: Parábola Editorial, 2012, p. 11-31. 238 2 Este capítulo tem origem em um texto primeiro publicado como relatório final do Programa In- stitucional de Bolsa de Iniciação Científica (2019), desenvolvido pela graduada em Letras Portu- guês-Inglês Juliana Ferreira Benke e orientado pela professora Caroline Kretzmann. 3 O programa atende acadêmicos que precisam aperfeiçoar conhecimentos do ensino básico em leitura, interpretação e produção de textos, bem como no emprego da norma padrão da língua. INTRODUÇÃO22 A argumentação está presente em diversos contextos de atuação humana e, por esse motivo, há a importância de ser tra- balhada com os estudantes de todos os níveis de ensino, estimu- lando-os a ler, compreender e produzir textos criticamente sobre a realidade que os cerca e sobre os usos da língua em determinadas situações (KOCH; ELIAS, 2016). De acordo com Koch e Elias (2006), a argumentação é uma atividade humana, pois se encontra nas mais diversas práticas sociais. No Exame Nacional do Ensino Médio (Enem), no eixo de “Lin- guagens e códigos”, um texto dissertativo-argumentativo é solicitado aos estudantes. Chama a atenção o resultado do ano de 2017, de acordo com o Instituto Nacional de Estudos e Pesquisas Educacionais Anísio Teixeira (2017), entre 4,7 milhões de participantes, 302.974 mil candidatos receberam nota zero em suas redações, principalmente, por fugirem ao tema, que, na prova desse ano, foi “Desafios para a formação educacional de surdos no Brasil”. Esse fator demonstra um déficit dos estudantes ao produzirem textos de base argumentativa, o qual se repete ano após ano nos resultados do exame. Com o objetivo de auxiliar os discentes da graduação com dificuldades referentes aos conhecimentos do ensino básico, a Pon- tifícia Universidade Católica do Paraná (PUCPR) oferece aulas de nivelamento nas áreas de Língua Portuguesa23, Matemática, Física e Química. Nessa oferta, os professores auxiliam os graduandos em grupos de estudos, além de serem promovidas atividades específicas 22 23 240 S U M Á R I O após um diagnóstico feito inicialmente para identificar quais serão as habilidades a serem desenvolvidas nos encontros. As professoras atuantes no programa de nivelamento, em suas aulas, perceberam que a dificuldade dos estudantes, ao produ- zirem um texto do gênero dissertativo-argumentativo, era elevada. Portanto, a pesquisa é uma forma de mapear essas dificuldades e propor adequações na sequência didática para o trabalho com lei- tura e interpretação desse gênero, de forma que as habilidades dos discentes sejam desenvolvidas. Este estudo apresenta conceitos sobre argumentação e categorias argumentativas com base na Nova Retórica, de modo a estabelecer uma categorização da estrutura do gênero disserta- tivo-argumentativo; descreve a proposta didática para o ensino de gêneros textuais de Dolz, Noverraz e Schneuwly (2004); expõe como o gênero dissertativo-argumentativo é trabalhado nas aulas do pro- grama de nivelamento e, a partir disso, desenvolve uma nova pro- posta de sequência didática para o gênero. APORTE TEÓRICO Na fundamentação teórica da pesquisa, a fim de desenvol- ver uma sequência didática para o gênero dissertativo-argumenta- tivo, são levantadas as características composicionais e estilísticas desse texto, conforme exposição de Köche, Boff e Marinello (2013). São tomados como base os conceitos de argumentação e cate- gorias argumentativas de Fiorin (2015) e Wachowicz (2012). Esses conceitos ajudam na compreensão da utilização da argumentação e no emprego dos tipos de argumentos. Em seguida, é delineada a noção de sequência didática, a partir da proposta de Dolz, Noverraz e Schneuwly (2004). 241 GÊNERO E TIPO TEXTUAL Marcuschi (2007) afirma que os gêneros textuais contribuem para a ordenação e estabilização das atividades comunicativas do cotidiano. Por serem atividades sócio-comunicativas e formas de ação social, não são instrumentos que enrijecem a ação criativa, pelo contrário, são eventos textuais maleáveis, dinâmicos e plásticos. Comparando-se com a atividade comunicativa anterior ao século VII, a escrita alfabética multiplicou os gêneros textuais, per- mitindo a aparição de novos gêneros e formas de comunicação. Com isso, Marcuschi (2007) afirma que os gêneros são caracterizados por suas funções comunicativas, cognitivas e institucionais. Ao fazer a distinção entre tipo textual e gênero textual, o autor afirma que não é possível haver comunicação verbal sem ser por um gênero, consequentemente vendo a língua como uma atividade social, histórica e cognitiva. Marcuschi (2007) apresenta o tipo tex- tual como uma expressão que designa um tipo de sequência teori- camente definida pela natureza linguística de sua composição. Já a expressão gênero textual se refere a textos materializados que apre- sentam características sócio-comunicativas definidas por conteú- dos, propriedades funcionais, estilo e composição característica. O autor também explica o domínio discursivo que designa uma esfera ou momento de produção discursiva ou atividade humana a qual proporciona o surgimento de discursos específicos. Ainda, o autor afirma que todo texto é heterogêneo, tipo- logicamente variado, ou seja, pode haver mais de uma sequência linguística nele. Essas sequências caracterizam os tipos textuais a partir de traços linguísticos dominantes: narrativa, descritiva, argu- mentativa, injuntiva e expositiva. 242 S U M Á R I O TEXTO DISSERTATIVO-ARGUMENTATIVO É um gênero textual constituído a partir de um posiciona- mento e utilizado por estudantes, geralmente, da educação básica. Para definir dissertação, Köche, Boff e Marinello (2013) citam Del- force (1992), o qual afirma que o trabalho dissertativo se constitui da ação de construir uma problemática e dar uma opinião sobre ela. As autoras também citam Delcambre e Darras (1992), elas afirmam que o ato de dissertar é mostrar seu ponto de vista e defendê-lo por meio de argumentos. Esse gênero textual é considerado heterogêneo, uma vez que utiliza, como base, a tipologia textual dissertativa, mas, ao mesmo tempo, pode incluir outros tipos, como a narrativa e a descritiva, que dão suporte à argumentação. Köche, Boff e Marinello (2013) afir- mam que o processo de desenvolvimento da opinião é tão impor- tante quanto o ponto de vista, já que o autor do texto precisa uti- lizar argumentos para persuadir o leitor a seu favor. Nesse mesmo sentido, Koch e Elias (2016) afirmam que a diversidade de argumen- tos utilizados para sustentar uma posição, como fatos, exemplos e dados estatísticos, podem influenciar nas chances de convenci- mento do interlocutor. Para as autoras, a dissertação escolar precisa de uma ques- tão que será discutida. O título é um dos elementos que devem apresentar ao leitor, de maneira simples, o que será lido, ou seja, um título que chame a atenção e esteja conectado com o tema. Sua estrutura precisa apresentar e contextualizar o tema, a situação-pro- blema, para que o leitor entenda o que virá a seguir. Na discussão do problema, o autor do texto deve construir sua opinião sobre o assunto e utilizar argumentos que fundamentam e defendem sua escolha. Logo em seguida, como solução-avaliação, o produtor res- ponde ao problema apresentado, reafirmando seu ponto de vista ou formulando uma solução. 243 S U M Á R I O A ARGUMENTAÇÃO Os fundadores da nova retórica, Perelman e Olbrechts-Tyteca (1996), definem a argumentação como o campo do verossímil, ao contrário de Descartes, que considerava quase como falso tudo que não era comprovado pela lógica matemática. Ao contrário, a análise dos autores abrange as provas chamadas dialéticas, termo caracte- rizado por Aristóteles como a arte de raciocinar a partir de opiniões geralmente aceitas. Segundo os autores, o raciocínio dialético diz respeito ao verossímil ao invés de tratar de proposições necessárias. Em relação à argumentação, Wachowicz (2012) aborda as sequências argumentativas de Adam (2001) e cita a defesa de Perel- man e Olbrechts-Tyteca (1996) ao afirmarem que a argumentação é a arte de persuadir um auditório específico ou universal. De acordo com Wachowicz (2012), o ambiente de produção de uma argumenta- ção possui quatro elementos básicos: o auditório, os fatos, os valores ou juízos que são atribuídos a eles e, no meio do caminho, os argu- mentos. Os últimos podem seguir, a princípio, dois critérios: a união dos fatos entre si, gerando argumentos pelo processo de ligação; e o distanciamento desses fatos, criando os argumentos pelo processo de dissociação. Neste trabalho, com base em Fiorin (2015), serão desenvolvidos os argumentos por processo de ligação, divididos em quase-lógicos, baseados na estrutura do real e os que fundamentam a estrutura do real. Ao abordar os tipos de argumento por ligação, Fiorin (2015) começa pelos quase-lógicos, introduzindo a definição. No campo argumentativo, a definição é o estabelecimento de uma relação de equivalência que tem como objetivo dar sentido a um certo termo, além de caracterizar um elemento por suas características ou fun- ção, podendo mesclar os dois modos. As definições, por exemplo, podem servir como estratégias argumentativas quando apresentam uma característica de algo ou alguém com a intenção de ressaltar 244 S U M Á R I O uma qualidade ou um aspecto negativo, servindo a propósitos per- suasivos. Diferentemente da definição, a comparação precisa de dois elementos para trazer à tona suas diferenciações ou aproximações entre eles, dando concretude a uma abstração. Ainda, como estratégia argumentativa, é possível analisar a relação entre as partes e o todo, além da natureza de suas caracterís- ticas para que seja possível que um argumento se torne válido. Fiorin (2015, p. 129) afirma que “o todo pode ser estruturado ou não-estru- turado. A ARGUMENTAÇÃO Este é uma coleção de elementos, que, em conjunto, constitui o todo, que é a soma das partes [...]”. O site Politize publicou uma matéria que continha argumentos prós e contras à maioridade penal. O exemplo a seguir demonstra o tipo de argumento de inclusão e divisão a favor da redução, os quais se valem da relação parte e todo: “Porque a maior parte da população é a favor. O Datafolha divulgou recentemente pesquisa em que 87% dos entrevistados afirmaram ser a favor da redução da maioridade penal” (BLUME; CHAGAS, 2015). Dentro dos argumentos quase-lógicos, ainda há o argu- mento probabilístico, o qual foi fundado na lógica quantitativa e que leva em consideração a maioria. Como exemplo desse tipo argumen- tativo, o jornal online El País publicou uma notícia em que a fome é um crime cometido mundialmente: “Os dados mais recentes da FAO indicam que, após quase uma década de declínio, o número de pessoas afetadas pela fome no mundo aumentou novamente, com 815 milhões de habitantes sofrendo de desnutrição crônica em 2016” (SILVA; ESQUIVEL, 2018). Depois de dar exemplos de argumentos quase-lógicos, Fio- rin (2015) inicia os argumentos fundamentados na estrutura da rea- lidade com a implicação e a concessão. Argumentos baseados na implicação falam das realidades, do que pode acontecer já que é possível. Por outro lado, os que são baseados na concessão, que- bram expectativas criadas, por exemplo, de algo que aconteceu embora não fosse possível. 245 Ainda nos quase-lógicos, o argumento de causalidade opera a partir da apresentação dos fenômenos ocorridos. A causa imediata é a razão mais próxima de determinado evento ter sido produzido e a causa longínqua é aquela que ocasionou determinado evento, porém precedida de vários outros fatos. Como afirma Fiorin (2015, p. 152): “um mesmo fenômeno apresenta uma multiplicidade de causas e o enunciador escolhe aquela ou aquelas que interessam para os propósitos comunicativos”. Outro tipo de argumento utilizado são os fatos que, em deter- minados momentos, podem ser contestáveis – os que contêm maior complexidade –, e os incontestáveis – os quais são menos significa- tivos. Por exemplo, alguém ser assaltado é um fato de menor com- plexidade e que não pode ser questionado; já as motivações para o assalto podem ser decorrentes das diferentes interpretações para o fato, portanto, questionáveis. Assim, os fatos podem ser discutidos por sua objetividade, neutralidade e se realmente possuem valor causal. A ARGUMENTAÇÃO Inseridos na estrutura do real, existem os argumentos de coexistência, sendo um deles o argumento de autoridade (argumen- tum ad verecundiam). Ele é caracterizado pela “relação” de um atri- buto com a essência ou de um ato com o indivíduo, baseando-se no apelo à modéstia, ao respeito e à reverência. O enunciador de uma fala abandona a tese principal para colocar a si como modelo, como prova, levando a plateia a aceitar seu ponto de vista devido a sua autoridade, credibilidade, conhecimento ou integridade. Existem dois tipos desse argumento: o da ordem do saber e o do domínio de poder. Fiorin (2015) destaca que esse argumento é utilizado ape- lando à reverência ou ao respeito. Ao tratar dos argumentos que fundamentam a estrutura do real, o autor aborda o argumento pelo exemplo e por ilustração. Aquele é utilizado a partir de casos particulares ou de possíveis repetições de casos idênticos; este reforça uma tese que já foi aceita. O argumento pelo exemplo utiliza da generalização a partir de casos 246 S U M Á R I O particulares; já a ilustração apresenta, primeiro, o princípio geral para que os casos particulares sejam mostrados para dar veracidade à tese. Os argumentos descritos são tomados como base para ela- boração da sequência didática de produção do gênero artigo de opinião. Por ser um gênero de tipologia essencialmente argumenta- tiva, é necessário instrumentalizar os estudantes com o aprendizado de diferentes estratégias argumentativas para que estejam aptos a produzir o gênero. SEQUÊNCIA DIDÁTICA Para a elaboração da sequência didática do gênero disser- tativo-argumentativo, é apresentada a proposta dos autores Dolz, Noverraz e Schneuwly (2004). Nesse modelo, os autores propor- cionam aos professores um método de ensino a fim de melhorar a capacidade dos estudantes de dominar e produzir um gênero textual - seja escrito ou oral - conforme a situação ou o propósito comuni- cativo mais adequado. De acordo com Dolz, Noverraz e Schneuwly (2004, p. 97), “sequência didática é um conjunto de atividades escolares organi- zadas, de maneira sistemática, em torno de um gênero textual oral ou escrito”. Focados em gêneros textuais, os escritores apresentam a sequência didática para que os professores possam trabalhar gêne- ros com que os alunos ainda não tenham tido contato ou que tenham dificuldade ao serem trabalhados. Esse procedimento de ensino é dividido em quatro partes: apresentação da situação, que tem por objetivo externar detalhada- mente as atividades que serão produzidas pelos alunos em torno do gênero textual a ser trabalhado; a produção inicial, baseada no desenvolvimento de um primeiro texto; os módulos, cuja função é a 247 S U M Á R I O de trabalhar as dificuldades encontradas pelos alunos com o objetivo de aquisição de conhecimento aprofundado do gênero, suas carac- terísticas e linguagem técnica; e a produção final, a qual oferta a possibilidade de o estudante colocar em prática os conhecimentos adquiridos durante as atividades executadas. Com base nessa proposta didática, Gomes e Souza (2015) realizaram uma pesquisa com o objetivo de veicular a análise linguís- tica no ambiente escolar, sendo esse também um tema de estudo dos autores de Genebra. Gomes e Souza (2015) afirmam que é fun- ção da escola ajudar com o desenvolvimento das habilidades textu- ais orais e escritas dos estudantes, bem como deve haver a intera- ção entre esse processo de desenvolvimento e as práticas de análise linguística (AL). Ao afirmarem que as práticas de AL estão escassas nas escolas, Gomes e Souza (2015) propõem a sequência didática como fio condutor para o desenvolvimento da AL. O estudo de gêneros textuais e a AL estão ligados, já que esta proporciona, por meio do método interacionista, a reflexão metalinguística, um ato consciente e sistematizado. SEQUÊNCIA DIDÁTICA Ou seja, se forem colocados em prática os conhecimentos prévios do aluno, se houver o estudo das dificuldades encontradas e reflexão sobre a linguagem, o discente será capaz de escrever, reescrever, falar, reelaborar o texto de uma melhor forma por meio da mediação e ajuda do professor. ANÁLISE Levando em conta o conceito de pesquisa-ação, que, além de compreensão, visa a interceder numa situação a fim de modifi- cá-la (SEVERINO, 2017), a pesquisa foi realizada a partir da análise das aulas das professoras do programa de nivelamento em língua portuguesa e de um levantamento das dificuldades dos estudantes. 248 Com isso feito, foi elaborada uma sequência didática para as aulas de produção do texto dissertativo-argumentativo com base no refe- rencial teórico da pesquisa. Com isso feito, foi elaborada uma sequência didática para as aulas de produção do texto dissertativo-argumentativo com base no refe- rencial teórico da pesquisa. As aulas do programa de nivelamento são elaboradas a partir de uma sequência de atividades voltadas para a produção de deter- minado gênero textual. O Quadro 1, a seguir, apresenta a sequência de atividades desenvolvidas e aplicadas pelas professoras do pro- grama para produção do texto dissertativo-argumentativo: Quadro 1 – Sequência de atividades desenvolvidas e aplicadas pelas professoras do programa de nivelamento em língua portuguesa Conteúdo Descrição Avaliação diagnóstica Produção de um texto dissertativo-argumentativo para que fosse avaliado o conhecimento prévio de cada aluno sobre a produção do gênero. Videoaula sobre estratégias argumentativas A videoaula apresenta as condições de produção de um texto dissertativo- argumentativo, como quem são os autores, quem são os possíveis leitores, como e por que se escreve etc. Além disso, os tipos de argumentos para esse texto também são apresentados e exemplificados. Leitura e interpretação de artigo de opinião Apresentação do artigo de opinião “Nossos jovens heróis mimados”, os alunos deveriam interpretá-lo e, em seguida, responder algumas atividades relacionadas ao texto. Descobrir qual o tema, o ponto de vista do autor, seus argumentos e a conclusão eram alguns dos propósitos do exercício. Análise linguística das avaliações diagnósticas Após a realização da avaliação diagnóstica, foi entregue aos alunos um texto dissertativo-argumentativo para que analisassem a linguagem e, em seguida, produzissem uma reescrita. Uma folha de exercícios foi entregue aos discentes para que encontrassem elementos fundamentais do texto, como o ponto de vista do autor, os tipos de argumentos utilizados e, por fim, a conclusão. Exercícios de coesão Nesta aula, a coesão foi o objetivo principal. Foram apresentados aos alunos recursos coesivos para conceder fluidez ao texto e estabelecer sentidos. Exercícios de fixação, como interpretação das relações de sentido, foram dados aos estudantes. ANÁLISE Quadro 1 – Sequência de atividades desenvolvidas e aplicadas pelas professoras do programa de nivelamento em língua portuguesa 249 Formas de iniciar a argumentação e estratégias argumentativas Foram expostos, em slides, exercícios que desenvolviam a criação da argumentação, esses slides apresentavam textos e, em seguida, perguntas sobre o propósito comunicativo e as estratégias argumentativas de cada um deles. Produção de novo texto dissertativo- argumentativo Proposta de produção de texto dissertativo-argumentativo sobre “A garantia dos direitos da mulher na sociedade contemporânea: avanços e desafios”. Fonte: as autoras (2019). Fonte: as autoras (2019). Após a correção das avaliações diagnósticas por parte das professoras do programa, foram selecionados 32 textos para uma avaliação quantitativa do desempenho dos estudan- tes, conforme Tabela 1. Tabela 1 – Análise quantitativa do desempenho dos estudantes na avaliação diagnóstica Critérios Nota dos estudantes (32 alunos): texto expositivo- argumentativo Macroestrutura textual Conteúdo Insuficiente Regular Bom Ótimo Ø 15 14 3 Coerência 4 16 11 1 Microestrutura textual Coesão 1 14 17 Ø Linguagem Ø 11 18 3 Fonte: as autoras (2019). Tabela 1 – Análise quantitativa do desempenho dos estudantes na avaliação diagnóstica O primeiro item, conteúdo, diz respeito à estrutura macro de um texto, ou seja, se há respeito à temática proposta, apresenta ou não ponto de vista, argumento, conclusão e título coerentes. A maio- ria da turma não apresentou o tema de forma adequada, além de expor argumentos inconsistentes. O segundo item, coerência, pre- sente também na estrutura macro, tem como objetivo avaliar se o 250 S U M Á R I O aluno é capaz de apresentar progressão de suas ideias na escrita, sem a repetição de informações, além de analisar a capacidade do desenvolvimento de argumentos consistentes e condizentes com o ponto de vista. Os alunos que obtiveram marca “regular” não apre- sentaram as características do item, ou seja, utilizaram argumentos inconsistentes, repetitivos e pouco desenvolvidos. Na estrutura micro, o primeiro item é a coesão, o qual avalia a habilidade de conectar as partes do texto por meio de variados recursos coesivos, do repertório adequado e da utilização apro- priada de pontuações. Mesmo tendo utilizado bons recursos coesi- vos, os estudantes repetiram esses recursos para conectar as ideias do texto, ou seja, a escrita não é desenvolvida adequadamente. O último item, a linguagem, analisa o emprego da norma culta da lín- gua, o nível de linguagem, a adequação vocabular e sem repetições. ANÁLISE Grande parte da turma obteve marca “boa”, porém misturaram lin- guagem formal com informal, tiveram alguns erros ortográficos e continuaram a repetir palavras. Dos 32 alunos inscritos no programa de nivelamento em língua portuguesa, apenas um não obteve essas marcas, pois seu texto fugiu ao tema proposto. Na nova proposta de produção do texto dissertativo-argu- mentativo, aplicada ao final da sequência de atividades, os estudan- tes deveriam discorrer sobre o tema “A garantia dos direitos da mulher na sociedade contemporânea: avanços e desafios”. Na produção, a professora solicitou aos estudantes que respeitassem a proposta do texto dissertativo-argumentativo, que utilizassem dois argumentos para a sustentação do ponto de vista, elaborassem uma conclusão e título. Para a correção, foram utilizados os seguintes critérios: con- teúdo (desenvolvimento do texto dentro da temática, apresentação de ponto de vista, argumentação, conclusão e título adequado); coe- rência (progressão e unidade textual sem repetições, argumentação consistente e condizente com o ponto de vista); coesão (articulação das partes do texto utilizando recursos coesivos adequados e varia- dos, além de empregar a pontuação adequadamente); e linguagem 251 (utilização da norma culta da língua, linguagem adequada ao con- texto de produção e vocábulos apropriados e sem repetições). Para a proposição de melhorias na sequência didática de ensino do texto dissertativo-argumentativo no programa, foram ana- lisados 18 textos dos 32 produzidos pelos estudantes os quais foram divididos entre os grupos “bom”, “regular” e “insuficiente” (seis exem- plares de cada grupo). Os textos considerados bons possuíram notas entre 8,0 e 9,5; os médios entre 7,0 e 7,5; e os insuficientes obtiveram nota entre 5,0 e 6,5. ANÁLISE A análise dos textos considerados bons, grupo 1, é apresentada no Quadro 2: Quadro 2 – Análise do atendimento aos critérios de correção do texto dissertativo- argumentativo – grupo 1 Texto Indicadores Insuficiente Regular Bom Ótimo Observações 1 Conteúdo x Atendeu a proposta e a estrutura do gênero Coerência x Não desenvolve argumentação consistente Coesão x Repetições de vocábulos; problema de clareza Linguagem x Emprego de linguagem inadequada ao gênero Texto Indicadores Insuficiente Regular Bom Ótimo Observações 2 Conteúdo x Título inadequado Coerência x Argumento incompleto, não explica uma afirmação Coesão x Emprego da pontuação Linguagem x Crase; acentuação Quadro 2 – Análise do atendimento aos critérios de correção do texto dissertativo- argumentativo – grupo 1 252 Texto Indicadores Insuficiente Regular Bom Ótimo Observações 3 Conteúdo x Título inadequado Coerência x Argumento incompleto e sem consistência Coesão x Emprego inadequado de termo coesivo Linguagem x Emprego de linguagem inadequada ao gênero Texto Indicadores Insuficiente Regular Bom Ótimo Observações 4 Conteúdo x Falta de organização textual nos parágrafos Coerência x Falta de termos coesivos Coesão x Emprego inadequado da pontuação Linguagem x Ausência de concordância verbal Texto Indicadores Insuficiente Regular Bom Ótimo Observações 5 Conteúdo x Atendeu a proposta e a estrutura de forma incompleta Coerência x Conclusão sem clareza Coesão x Repetição de vocábulos; emprego de pontuação; falta de termos coesivos Linguagem x Concordância verbal inadequada Texto Indicadores Insuficiente Regular Bom Ótimo Observações 6 Conteúdo x Atendeu a proposta e a estrutura do gênero Coerência x Uso adequado e consistente de informações Coesão x Poucas inadequações no uso de pontuação Linguagem x Erro no uso de citação; concordância verbal inadequada Fonte: as autoras (2019) Fonte: as autoras (2019). 253 s bons, os erros recorrentes ção, dificuldade em empreg s e desenvolver os argument 3, é apresentada a análise do critérios de correção do texto diss ativo – grupo 2 Nos textos considerados bons, os erros recorrentes foram: inadequação no uso de pontuação, dificuldade em empregar dife- rentes estratégias argumentativas e desenvolver os argumentos para defesa do ponto de vista. Em seguida, no Quadro 3, é apresentada a análise do grupo considerado regular, grupo 2. ANÁLISE Quadro 3 – Análise do atendimento aos critérios de correção do texto dissertativo- argumentativo – grupo 2 Texto Indicadores Insuficiente Regular Bom Ótimo Observações 1 Conteúdo x Conhecimento de senso comum (introdução clichê); inadequação de paragrafação Coerência x Falta de estratégias argumentativas Coesão x Inadequações pontuais no emprego de termos coesivos. Linguagem x Uso da 1ª p.s. (na linguagem acadêmica não é aceito o uso da 1ª p.s. Texto Indicadores Insuficiente Regular Bom Ótimo Observações 2 Conteúdo x Uso inadequado de termos introdutórios ao tema Coerência x Não emprega argumentação consistente Coesão x Unidade textual prejudicada por falta de articulação Linguagem x Acentuação; letra parcialmente ilegível; conjugação verbal inadequada Quadro 3 – Análise do atendimento aos critérios de correção do texto dissertativo- argumentativo – grupo 2 254 Texto Indicadores Insuficiente Regular Bom Ótimo Observações 3 Conteúdo x Conhecimento de senso comum (introdução clichê) Coerência x Conclusão sem clareza Coesão x Uso inadequado de pontuação Linguagem x Ausência de concordância verbal Texto Indicadores Insuficiente Regular Bom Ótimo Observações 4 Conteúdo x Conclusão sem clareza Coerência x Argumentação incompleta e incoerente Coesão x Uso inadequado de pontuação e elementos coesivos Linguagem x Linguagem redundante; erro de acentuação Texto Indicadores Insuficiente Regular Bom Ótimo Observações 5 Conteúdo x Atendeu a proposta e a estrutura de forma incompleta Coerência x Não desenvolve argumentação consistente Coesão x Inadequações pontuais no uso de termos coesivos Linguagem x Uso de termos inadequados ao gênero Texto Indicadores Insuficiente Regular Bom Ótimo Observações 6 Conteúdo x Conclusão sem clareza Coerência x Argumentação inadequada; falta de clareza Coesão x Uso inadequado de pontuação Linguagem x Utiliza linguagem informal Fonte: as autoras (2019) Fonte: as autoras (2019). Fonte: as autoras (2019). 255 Novamente os usos inadequados de pontuação foram observados nos textos, agora, do grupo regular. Além desse pro- blema, foi visto também que os estudantes regulares possuem muita dificuldade em empregar estratégias argumentativas para fundamentação da argumentação e utilizam linguagem inadequada para o gênero proposto. ANÁLISE A análise dos textos considerados insuficientes, grupo 3, é apresentada no Quadro 4: Quadro 4 – Análise do atendimento aos critérios de correção do texto dissertativo- argumentativo – grupo 3 Texto Indicadores Insuficiente Regular Bom Ótimo Observações 1 Conteúdo x Conclusão desconectada da tese defendida; não desenvolve sua opinião Coerência x Frases desconexas e soltas; não conecta as informações durante o desenvolvimento da argumentação Coesão x Uso inadequado de pontuação e elementos coesivos Linguagem x Problema de concordância verbal Texto Indicadores Insuficiente Regular Bom Ótimo Observações 2 Conteúdo x Fuga parcial, pois não discorre sobre o tema de maneira específica Coerência x Não possui progressão e unidade textual Coesão x Uso inadequado de pontuação Linguagem x Ausência de concordância verbal e citação incompleta Quadro 4 – Análise do atendimento aos critérios de correção do texto dissertativo- argumentativo – grupo 3 256 Texto Indicadores Insuficiente Regular Bom Ótimo Observações 3 Conteúdo x Título inadequado Coerência x Repetições; argumentação incompleta Coesão x Uso inadequado de pontuação e elementos coesivos Linguagem x Uso inadequado de pontuação e de conjugação do verbo Texto Indicadores Insuficiente Regular Bom Ótimo Observações 4 Conteúdo x Título inadequado; falta clareza e objetividade no texto Coerência x Argumentação pouco desenvolvida, mas apresenta estratégia argumentativa; problemas de repetição Coesão x Uso inadequado de pontuação e elementos coesivos Linguagem x Linguagem informal empregada; ortografia inadequada; repetição de vocábulos e de argumentação Texto Indicadores Insuficiente Regular Bom Ótimo Observações 5 Conteúdo x Fuga parcial do tema Coerência x Falta de clareza e progressão de ideias Coesão x Repetição de informações e de palavras; problemas de pontuação Linguagem x Uso de termos da oralidade 257 Texto Indicadores Insuficiente Regular Bom Ótimo Observações 6 Conteúdo x Problemas na paragrafação do texto; não apresenta estratégias argumentativas suficientes na argumentação Coerência x Falta de clareza na argumentação Coesão x Problemas de pontuação; ponto de vista pouco elaborado e truncado Linguagem x Uso da 1ª p.s. (linguagem acadêmica não utiliza 1ª p.s.; problemas de concordância Fonte: as autoras (2019). Texto Indicadores Insuficiente Regular Bom Ótimo Observações 6 Conteúdo x Problemas na paragrafação do texto; não apresenta estratégias argumentativas suficientes na argumentação Coerência x Falta de clareza na argumentação Coesão x Problemas de pontuação; ponto de vista pouco elaborado e truncado Linguagem x Uso da 1ª p.s. (linguagem acadêmica não utiliza 1ª p.s.; problemas de concordância Fonte: as autoras (2019). ANÁLISE Texto Indicadores Insuficiente Regular Bom Ótimo Assim como os outros dois grupos, os textos produzidos pelos estudantes com menor desempenho também apresentam ina- dequações no uso da pontuação, ocasionando problemas na coesão textual, além de terem dificuldade no desenvolvimento da argumen- tação e da progressão textual. Outro problema encontrado nesse grupo foi o uso de linguagem inadequada ao gênero textual proposto (o emprego da 1ª pessoa do singular e termos da oralidade). Portanto, a partir da análise dos textos e da similaridade das inadequações encontradas, são apresentadas sugestões de aperfei- çoamento da sequência didática analisada, principalmente, no que se refere às estratégias argumentativas, a como articular as ideias selecionadas para defesa do ponto de vista, à pontuação com intuito de melhorar a coesão textual, ao planejamento do texto e à lingua- gem que é própria do texto dissertativo-argumentativo. Nessa proposta de aperfeiçoamento, a sequência será divi- dida em quatro partes, com base nas orientações de Dolz, Noverraz e Schneuwly (2004). A primeira, a apresentação da situação, obje- tiva expor aos estudantes, detalhadamente, a proposta de aula e as 258 S U M Á R I O atividades que serão efetuadas por eles. Nessa primeira atividade, os alunos deverão ser levados a relembrar ao gênero em questão com o objetivo de estudar a tipologia textual nele predominante; a estrutura composicional; o propósito comunicativo e o público-alvo. O profes- sor pode escrever no quadro “dissertação argumentativa” e, a partir do que os estudantes responderem que caracteriza esse gênero, ele registrará quais são essas características. Finalizada essa interação, o discente pode explicar o porquê de algumas características serem ou não pertencentes do texto dissertativo-argumentativo. Após essa introdução, devem analisar dois textos bem avaliados e produzidos no Enem e buscar as principais características: título, tema, problema, ponto de vista, argumentos e conclusão. Além disso, precisam verifi- car qual é o nível de linguagem empregado pelos autores e a pessoa discursiva utilizada. Finalizada a apresentação da situação, deve ser proposta aos estudantes a produção inicial com o tema de alguma edição recente do Enem. Terminada a primeira aula, cuja função era de apresentar a situação e fazer com que os estudantes produzissem seu primeiro texto, a segunda e a terceira aulas devem ter o objetivo de traba- lhar as dificuldades24 encontradas pelos estudantes. 24 As atividades propostas na AL precisam sempre ser adaptadas conforme as dificuldades dos gru- pos em que a sequência didática for aplicada. ANÁLISE No primeiro momento da segunda aula, os discentes podem fazer uma nova aná- lise da estrutura composicional de uma redação do Enem, seguida de uma atividade sobre o emprego de estratégias argumentativas adequadas à defesa de um ponto de vista. Na terceira aula, deve ser realizada uma atividade de análise linguística elaborada com base nas principais dificuldades de conteúdo, coerência, coesão e lingua- gem demonstradas nas produções dos estudantes. No quarto encontro, os estudantes deverão fazer a reescrita do texto produzido na primeira aula, colocando em prática os conhe- cimentos adquiridos durante as atividades executadas e a partir das correções do professor. 24 259 CONSIDERAÇÕES FINAIS O objetivo de sugerir o aprimoramento da sequência didá- tica para o trabalho com leitura e interpretação do texto dissertati- vo-argumentativo para os estudantes do programa de nivelamento de língua portuguesa foi cumprido neste trabalho. Isso somente foi possível porque já havia uma produção inicial elaborada pelos discentes, que foi analisada e teve categorizados os principais pro- blemas encontrados. Trata-se de um reforço do que orientam Dolz, Noverraz e Schneuwly (2004) com relação à necessidade de o pro- fessor solicitar uma primeira produção a partir da qual a sequência didática será desenvolvida. No entanto, há necessidade de aplicação da sequência suge- rida para verificação da sua validade e execução dos ajustes neces- sários. Dessa forma, nesta pesquisa, destaca-se o levantamento das dificuldades comuns dos estudantes ao produzirem um texto disser- tativo-argumentativo, que podem servir como base para a elabora- ção de sequências didáticas para ensino do gênero. Isso pode ser feito sem deixar de se considerar a realidade de cada turma. KOCH, Ingedore Villaça; ELIAS, Vanda Maria. Ler e escrever: estratégias de produção textual. São Paulo: Contexto, 2006. KÖCHE, Vanilda Salton; BOFF, Maria Benetti; MARINELLO, Adiane Fogali. Leitura e produção textual: gêneros textuais do argumentar e expor. 6. ed. Editora Vozes, 2013. MARCUSCHI, Luiz Antônio. Gêneros textuais: definição e funcionalidade. In: DIONÍSIO, Angela Paiva; MACHADO, Anna Rachel; BEZERRA, Maria Auxiliadora (Orgs.). Gêneros textuais e ensino. 5. ed. Rio de Janeiro: Lucerna, 2007. PERELMAN, Chaïm; OLBRECHTS-TYTECA, Lucie. Tratado da argumentação – A Nova Retórica. Tradução de Maria E.G.G. Pereira. São Paulo: Martins Fontes, 1996. REFERÊNCIAS BENKE, Juliana Ferreira; KRETZMANN, Caroline. Sequência didática para leitura e produção de texto dissertativo-argumentativo. Curitiba, Pontifícia Universidade Católica do Paraná, 2019. Relatório de Iniciação Científica (não publicado). BLUME, Bruno André; CHAGAS, Inara. Redução da maioridade penal: argumentos contra e a favor. Politize, 2015. Disponível em: <http://bit.ly/3JMgCZb>. Acesso em: 02 dez. 2018. BLUME, Bruno André; CHAGAS, Inara. Redução da maioridade penal: argumentos contra e a favor. Politize, 2015. Disponível em: <http://bit.ly/3JMgCZb>. Acesso em: 02 dez. 2018. DOLZ, Joaquim; NOVERRAZ, Michèle; SCHNEUWLY, Bernard. Sequências didáticas para o oral e a escrita: apresentação de um procedimento. In: SCHNEUWLY, Bernard; DOLZ, Joaquim. (Orgs.). Gêneros orais e escritos na escola. Campinas: Mercado de Letras, 2004, p. 95-128. DOLZ, Joaquim; NOVERRAZ, Michèle; SCHNEUWLY, Bernard. Sequências didáticas para o oral e a escrita: apresentação de um procedimento. In: SCHNEUWLY, Bernard; DOLZ, Joaquim. (Orgs.). DOLZ, Joaquim; NOVERRAZ, Michèle; SCHNEUWLY, Bernard. Sequências didáticas para o oral e a escrita: apresentação de um procedimento. In: SCHNEUWLY, Bernard; DOLZ, Joaquim. (Orgs.). Gêneros orais e escritos na escola. Campinas: Mercado de Letras, 2004, p. 95-128. a escrita: apresentação de um procedimento. In: SCHNEUWLY, Bernard; DOLZ, Joaquim. (Orgs.). Gêneros orais e escritos na escola. Campinas: Mercado de Letras, 2004, p. 95-128. p ç p q g Gêneros orais e escritos na escola. Campinas: Mercado de Letras, 2004, p. 95-128. 260 INSTITUTO NACIONAL DE ESTUDOS E PESQUISAS EDUCACIONAIS ANÍSIO TEIXEIRA. INSTITUTO NACIONAL DE ESTUDOS E PESQUISAS EDUCACIONAIS ANÍSIO TEIXEIRA. Sinopse Estatísticas do Exame Nacional de Ensino Médio 2017. Brasília: Inep, 2018. Disponível em: <http://bit.ly/3n6PMlS>. Acesso em: 9 fev. 2023. KOCH, Ingedore Villaça; ELIAS, Vanda Maria. Escrever e argumentar. São Paulo: Contexto, 2016. KOCH, Ingedore Villaça; ELIAS, Vanda Maria. Ler e escrever: estratégias de produção textual. São Paulo: Contexto, 2006. FIORIN, José Luiz. Argumentação. São Paulo: Contexto, 2015. GOMES, Andréia de Fátima Rutiquewiski; SOUZA, Sweder. Os módulos da sequência didática e a prática de análise linguística: relações facilitadoras. Revista (Con)Textos Linguísticos, v. 9, n. 14, p. 8-22, 2015. SEVERINO, Antônio Joaquim. Metodologia do trabalho científico. 23. ed. São Paulo: Cortez, 2007. SEVERINO, Antônio Joaquim. Metodologia do trabalho científico. 23. ed. São Paulo: Cortez, 2007. SILVA, José Graziano da; ESQUIVEL, Adolfo Pérez. A fome é um crime. El País, 2018. Disponível em: <http://bit.ly/3Lz74SM>. Acesso em: 03 dez. 2018. WACHOWICZ, Teresa Cristina. Análise linguística nos gêneros textuais. Curitiba: Intersaberes, 2012. 261 S U M Á R I O INTRODUÇÃO Nos últimos três anos, a sociedade se deparou com as difi- culdades referentes ao enfrentamento da pandemia da Covid-19. Com isso, o sistema educacional brasileiro sofreu um impacto no que tange ao cotidiano das escolas, ocasionando o fechamento tem- porário destas, visto que houve a necessidade de isolamento social, conforme recomendação e medidas protocoladas pelo Ministério da Saúde (MS) e pela Organização Mundial da Saúde (OMS). Considerando o contexto pandêmico em que a educação se inseriu, inúmeros desafios surgiram em meio a toda comunidade escolar, fazendo com que houvesse uma ressignificação do ensino, pautada em novas abordagens pedagógicas ofertadas pela tecno- logia. Sendo assim, professores e alunos, distantes fisicamente das salas de aula, foram chamados a vivenciar um “novo normal”, tri- lhando por caminhos de adversidades, capazes de permitir novas maneiras de ensinar e aprender em meio à crise sanitária. Ao observar a problemática exposta, isto é, esse aconteci- mento mundialmente histórico, fomos chamados, enquanto educa- dores, a assumir uma postura de reflexão, a fim de mostrar a possi- bilidade de o ensino ser contornado em meio a tantas novidades, as quais a escola tem buscado se adaptar. Diante disso, surgiu, então, a motivação de promover com os educandos de uma turma do 6º ano do ensino fundamental de uma escola pública da cidade de Belém-PB, um projeto de letramento, de forma remota, que buscasse acompanhar os desafios que fizeram parte desta realidade que tem sido enveredada por inúmeros profissionais da educação e alunos. Nessa direção, este capítulo tem por objetivo apontar de que forma o uso dos gêneros textuais/discursivos nas aulas de língua portuguesa pode mediar uma conscientização do valor social da escrita. É importante informar ao/à leitor/a que este artigo é fruto 263 S U M Á R I O de recorte de uma pesquisa de trabalho final de graduação. Neste estudo, especificamente, tratamos de analisar uma sequência de produções de cartazes conscientizadores que agrupam uma das eta- pas desenvolvidas no projeto de letramento realizado por via remota, que teve como propósito a conscientização sobre os impactos da pandemia no cotidiano das pessoas. O trabalho pode ser apreciado, integralmente, em Oliveira (2021). Desse modo, a pesquisa justificou-se pelo fato de que a abor- dagem trazida para discussão foi ao encontro das principais provo- cações e inquietações que consideramos serem pertinentes para o desenvolvimento do projeto. INTRODUÇÃO Ou seja, ao contextualizarmos uma temática que foi vivenciada em tempo real, tivemos a oportunidade de observar de que forma o “novo normal”, trazido pela pandemia, impactou a realidade de todo um coletivo, isto é, escola, professo- res, alunos e sociedade. Para atingir os objetivos pré-estabelecidos, tomamos como metodologia a abordagem qualitativa de caráter descritivo-inter- pretativista que, de acordo com Bortoni-Ricardo (2008), busca-se a interpretação dos fatos sociais diante de algum contexto específico. Nesse sentido, este projeto de letramento se configurou como uma pesquisa-ação, por se tratar de uma proposta direcionada e aplicada a uma realidade específica, na qual houve uma intervenção signifi- cativa, na tentativa de que as observações e inquietações apresen- tadas provoquem interesses reflexivos diante do que a comunidade escolar tem vivenciado. Em termos de organização, o texto está dividido em três seções. Na primeira, estabelecemos os procedimentos metodológi- cos que situam o desenvolvimento da pesquisa. A segunda seção traz a análise da quarta etapa do projeto de letramento, que é a pro- dução escrita dos cartazes conscientizadores. Por fim, enfatizamos algumas considerações reflexivas, bem como algumas referências utilizadas nesse estudo. 264 UM OLHAR METODOLÓGICO: NATUREZA DA PESQUISA, LÓCUS E PARTICIPANTES Considerando a natureza qualitativa da pesquisa, que acon- tece através da interpretação de fenômenos presentes em um con- texto social, abordaremos o caráter descritivo-interpretativista, visto que à medida que descrevemos o processo de aplicação das ativi- dades realizadas durante o trajeto construtor das etapas, poderemos tecer atribuições de sentidos válidos que nos conduzam a compre- ender as significações conferidas pelos participantes do projeto e pelas pessoas no tocante às práticas sociais. Nesse contexto, impulsionado pelo olhar sensibilizado para compreender uma problemática encontrada na realidade social, o professor assume uma postura de professor pesquisador, que busca entender e encontrar possíveis caminhos para uma resolução de determinado problema posto em sua prática pedagógica. É nesse sentido que compactuamos com os ensinamentos de Bortoni-Ricardo (2008), quando a autora apresenta o perfil do professor pesquisador como sendo aquele que não se considera apenas como um sujeito que se utiliza dos conhecimentos que são produzidos por outros pesquisadores, mas que se destina à produ- ção de conhecimentos sobre os obstáculos enfrentados em sua prá- tica docente, na tentativa de melhorá-la. Desse modo, a diferença existente entre um professor pesquisador e outros profissionais da educação, pode ser identificada mediante o compromisso firmado de refletir sobre a sua práxis, com o intento de traçar uma dimen- são positiva dessa reflexão, a fim de buscar uma superação de suas falhas. Logo, ele permite a abertura de ideias e estratégias que pos- sam contribuir nesse processo. Partindo dessa premissa, situamos a abordagem do nosso trabalho que se direciona para uma pesquisa aplicada, ou seja, houve 265 S U M Á R I O uma ação direta desenvolvida pelos participantes do projeto, em que assumiram a postura de protagonistas na construção do conheci- mento através do intermédio do professor. O lócus da pesquisa direcionou-se para uma escola da rede municipal de ensino púbico na cidade de Belém-PB. A instituição atende um público de aproximadamente 450 alunos, distribuídos em 15 turmas dos anos finais do ensino fundamental (6º ao 9º ano) e 02 turmas da EJA (ciclos III e IV), que funcionam com uma média de 20 a 30 alunos por turma. A escola funciona com o atendimento ao alunado nos turnos manhã, tarde e noite. UM OLHAR METODOLÓGICO: NATUREZA DA PESQUISA, LÓCUS E PARTICIPANTES Diante disso, os participantes da pesquisa são alunos perten- centes a uma turma de 6º ano que totaliza 30 alunos com faixa etária entre 10 e 11 anos, que residem na zona urbana e rural da cidade e pertencem, economicamente, a uma classe de baixo valor aquisitivo. Diante do cenário conflituoso e desigualitário do ensino remoto que as escolas no país enfrentam, com relação ao acesso e à aquisição de aparatos tecnológicos, só foi possível desenvolver o projeto com 12 (doze) alunos. Vale ressaltar que a maioria dos participantes pos- suía seus próprios equipamentos (celulares), e alguns utilizavam os aparelhos dos pais. Nesse sentido, por estarmos percorrendo esse caminho de adversidades no âmbito da educação, em que a migração para o ensino remoto tem sido o meio de se chegar aos nossos alunos, foi necessário utilizar os recursos oportunizados pela tecnologia (pla- taforma Google Meet), para que houvesse uma reunião de apresen- tação prévia com os participantes diante da proposta do projeto. É importante destacar que houve uma autorização prévia via What- sApp (áudios explicativos) dos pais dos alunos para participarem do projeto, como também a criação de um grupo no mesmo aplicativo, com o intuito de facilitar a comunicação entre todos. Além disso, um termo de autorização institucional foi integralizado em nossa pes- quisa, a fim de formalizar a execução do projeto. 266 Desse modo, a turma foi selecionada pelo fato de os alu- nos poderem, possivelmente, compreender o momento difícil que a sociedade tem enfrentado e, principalmente, por estarem na condi- ção de aprendizes, buscando aprender em meio às problemáticas trazidas pela pandemia que, por vezes, têm desestimulado e barrado o percurso de muitas aprendizagens no ensino remoto. Diante desse cenário e impactados pelo “novo normal” imposto pela crise pandê- mica, intitulamos o projeto intervencionista como: Ser paciente salva vidas: a importância da empatia em tempos de Pandemia, na tentativa de promover reflexões acerca dessa realidade vivenciada por todos nós, a fim de mobilizar os estudantes a uma ação protagonizante. LETRAMENTOS EM CENA: A APROPRIAÇÃO DE UMA ESCRITA SIGNIFICATIVA Considerando todo o percurso de conhecimento sobre a abordagem temática, chegamos à parte prática do projeto, isto é, o momento das produções escritas. Assim sendo, na manhã do dia 15 de abril de 2021, foi feita uma recuperação do propósito do projeto: conscientizar-nos e, possivelmente, conscientizar outras pessoas. Nessa etapa, tratamos de apresentar e discutir sobre o gênero tex- tual cartaz, assim como produzir um cartaz de conscientização, apli- cando na escrita os conhecimentos que foram construídos ao longo das etapas – ter consciência, empatia, cuidados necessários etc. Inicialmente, instigamos os alunos a tomarem gosto pelo material que ia ser produzido. Alguns deles relataram não saberem desenhar, mas na oportunidade, perguntamos se já tinham visto algum cartaz. A resposta foi positiva. Inclusive, uma das participan- tes recordou que já tinha produzido um cartaz em outro ano esco- lar. Com a proposta da confecção do gênero, os participantes foram 267 convocados a agir diante da realidade que estamos enfrentando, a fim de chamar a atenção das pessoas para a conscientização através de suas produções, uma vez que [...] a língua usada nos textos – dentro de determinado grupo – constitui uma forma de comportamento social. Ou seja, as pessoas cumprem determinadas atuações sociais por meios verbais, e tais atuações – a exemplo de todo o social – são tipificadas, estabilizadas; por outras palavras, são sujeitas a modelos, em que a recorrência de certos elementos lhes dá exatamente esse caráter de estabele- cido, de típico, de regular [...] (ANTUNES, 2009, p. 54). S U M Á R I O Após esse momento inicial, foi apresentado de forma geral o que seria o cartaz, onde o encontramos, qual sua função social, quais seriam seus objetivos e características, entre outros aspectos. Em seguida, foi compartilhado com eles exemplos de cartazes prontos, para que pudessem visualizar como são estruturados com os aspec- tos linguísticos verbais e não verbais, compreendendo que seriam eles os protagonistas dessa linda produção. É necessário salientar que os alunos sugeriram que as produções fossem realizadas em duplas, para facilitar o caminho a ser trilhado por eles, mas como a proposta do projeto visava à conscientização deles e, posterior- mente, de outras pessoas, ficou compreendido que as produções seriam individuais. Deste modo, concluímos a quarta etapa, na cer- teza que o objetivo proposto tinha sido alcançado. LETRAMENTOS EM CENA: A APROPRIAÇÃO DE UMA ESCRITA SIGNIFICATIVA Após o encontro no Google Meet, a intermediação foi feita pelo professor a partir de um acompanhamento individualizado do andamento das produções dos participantes. Foi enviado ao What- sApp de cada participante, uma imagem sugestiva referindo-se ao contexto pandêmico, a fim de que tivessem uma base para a produ- ção do cartaz de conscientização. Além disso, à medida que ia inte- ragindo com eles, sugestões foram pontuadas, assim como ideias e aperfeiçoamentos, conforme o que fora apresentado no encontro 268 sobre o gênero. Sendo assim, um prazo foi estabelecido até a con- clusão dos cartazes. Foi possível perceber o empenho e a dedicação dos partici- pantes nos dias de acompanhamento. Eles se envolveram com cada detalhe dos cartazes, que sugeriam as suas próprias ideias para que pudessem, possivelmente, ser acrescentadas ou aperfeiçoadas ao interior do cartaz. Essa autonomia demonstra o resultado de uma mediação significativa do professor, haja vista que a tarefa do profes- sor, segundo Street (2014, p. 152), ”[...] é permitir o conhecimento das formas tradicionais de leitura e escrita – as formas letradas dominan- tes, os gêneros da prosa expositiva e do texto argumentativo-disser- tativo, as convenções da escrita de cartas a organizações comerciais – a fim de empoderar seus alunos”. Depois do processo da escrita, encaminhamo-nos para a fina- lização do projeto, na semana seguinte, com a culminância. Diante do desenvolvimento da intervenção, a partir das reflexões sobre a conscientização, os participantes buscaram se apropriar da escrita significativa, ao abordarem a sua função social aplicada ao gênero textual cartaz, uma vez que “[...] em seus textos, as pessoas realizam seus fins comunicativos e, não, na possibilidade de se estabelecer um sistema uniforme para a classificação da imensa variedade de gêneros” (ANTUNES, 2009, p. 56-57). Desse modo, compreendemos que os participantes puderam assumir uma postura de agentes de letramento diante do contexto de ensino remoto. Assim, as produções finais deste trabalho podem ser visuali- zadas na sequência a seguir, que apresentam um percurso significa- tivo através dos desenhos criados mediante o contexto de pandemia, e trazem recomendações reflexivas de conscientização também cria- das pelos participantes, por meio da utilização de aspectos linguísti- cos próprios do gênero trabalhado, como mensagens informativas e a utilização de verbos no modo imperativo. Além disso, é necessário considerar que, pelo nível de escolarização, por vezes, o estudante 269 sente dificuldade de materializar seus anseios por escrito. LETRAMENTOS EM CENA: A APROPRIAÇÃO DE UMA ESCRITA SIGNIFICATIVA No entanto, o recurso não verbal facilita e estimula o desenvolvimento das habi- lidades de leitura do educando. O primeiro cartaz dessa sequência conscientizadora traz um alerta para a sociedade. Vejamos: Figura 1 - Cartaz - O mundo está doente Fonte: Dados da pesquisa (OLIVEIRA, 2021). Figura 1 - Cartaz - O mundo está doente S U M Á R I O Fonte: Dados da pesquisa (OLIVEIRA, 2021). Neste cartaz 1, podemos visualizar uma construção multisse- miótica, que nos leva a compreender de que forma o mundo encon- tra-se desde o momento que foi acometido com a infecção do novo coronavírus. Os curativos remetem à ideia de que a sociedade está doente, e que isso se dá pelo fato de as pessoas estarem com a ‘humanidade baixa’, ou seja, a falta da empatia e da não conscientiza- ção evidencia que o planeta se encontra em uma Unidade de Terapia Intensiva (UTI), machucado e que chora pelas perdas irreparáveis que estão acontecendo no mundo afora, e recebe a dose da vacina, a fim de se recuperar. Assim, o cartaz chama a atenção das pessoas para a colaboração com o lugar em que moramos. Desse modo, o cartaz a seguir vem tratar sobre a conscientização da sociedade. 270 Figura 2 - Cartaz – Conscientização Fonte: Dados da pesquisa (OLIVEIRA, 2021). S U M Á R I O Fonte: Dados da pesquisa (OLIVEIRA, 2021). Fonte: Dados da pesquisa (OLIVEIRA, 2021). O cartaz 2, nomeado como “conscientização”, nos ajuda a semiotizar de que forma podemos tomar conhecimento do contexto pandêmico que tem assolado o mundo todo. O megafone, instru- mento utilizado para reforçar a entonação do nosso dizer, reflete o desejo que as pessoas ouçam e compartilhem desse ideal. Isso se dá a partir da ênfase dada à palavra ‘ATENÇÃO’, em que a mensagem reflexiva e conscientizadora exposta no cartaz permite que sejamos conduzidos às informações pertinentes sobre esse momento difícil que estamos enfrentando, além de algumas orientações necessárias de prevenção. O terceiro cartaz aborda a prática da empatia. 271 Figura 3 - Cartaz – Empatia Figura 3 - Cartaz – Empatia Fonte: Dados da pesquisa (OLIVEIRA, 2021). S U M Á R I O Fonte: Dados da pesquisa (OLIVEIRA, 2021). LETRAMENTOS EM CENA: A APROPRIAÇÃO DE UMA ESCRITA SIGNIFICATIVA No cartaz 3, consideramos a importância da prática de se colocar no lugar do outro, ou seja, nesse tempo de sofrimento, em que muitas pessoas têm perdido suas vidas de forma rápida atra- vés do contágio com o vírus letal, a empatia precisa ter visibilidade no que tange à compreensão responsável e protetora das pessoas que amamos, mas também daquelas desconhecidas. Essa prática pode acontecer a partir das atitudes que tomamos, impulsionadas pelo agir do coração e mediadas pela mente. As várias cores nas letras que formam a palavra ‘EMPATIA’ dentro de um balão repre- sentativo, simbolizam as singularidades das pessoas e, ao mesmo tempo, suas integrações. O isolamento social é o conteúdo abor- dado no próximo cartaz. 272 Figura 4 - Cartaz – Fique em casa Figura 4 Cartaz Fique em casa Fonte: Dados da pesquisa (OLIVEIRA, 2021). Fonte: Dados da pesquisa (OLIVEIRA, 2021). Ficar em casa é a principal recomendação que o cartaz 4 faz para todos neste momento de crise sanitária. O enfoque dado ao cuidado que devemos ter conosco e com as demais pessoas que convivemos ou não, é primordial para que possamos, possivelmente, cumprir com nossa parte de nos mantermos em casa, obedecendo as orientações dos órgãos competentes, a fim de que essa situação difícil passe. A distância entre as pessoas é discutida no quinto cartaz. Figura 5 - Cartaz – Distanciamento social Fonte: Dados da pesquisa (OLIVEIRA, 2021). Figura 5 - Cartaz – Distanciamento social Fonte: Dados da pesquisa (OLIVEIRA, 2021). 273 A interrupção temporária de várias ações que costumeira- mente eram praticadas socialmente como, por exemplo, festejar, abraçar, viajar etc., chama a atenção para o distanciamento social. Não tem sido fácil manter-se distante das pessoas que fazem parte do nosso dia a dia. Desse modo, o cartaz 5 traz como foco a evita- ção de aglomerações em tempos de pandemia, para que as pes- soas, praticando a empatia de forma consciente, possam preservar a saúde de todos. É importante ter esse cuidado! Assim, o cartaz a seguir traz uma reflexão sobre a prática de higienização das mãos. Figura 6 - Cartaz – Lavagem das mãos Fonte: Dados da pesquisa (OLIVEIRA, 2021). Figura 6 - Cartaz – Lavagem das mãos Fonte: Dados da pesquisa (OLIVEIRA, 2021). Higienizar as mãos é uma prática pessoal usada desde quando tomamos conhecimento da importância de manter limpo o nosso corpo. LETRAMENTOS EM CENA: A APROPRIAÇÃO DE UMA ESCRITA SIGNIFICATIVA Assim, a utilidade da água e do sabão tem sido exce- lente no que se refere à eliminação do vírus nesse tempo de isola- mento social. É nesse sentido que o cartaz 6 reforça esta missão, de lavar bem e de forma eficaz as nossas mãos, para destruir esse inimigo invisível, que tem prejudicado e tirado a vida de muitas pes- soas, sem escolha de faixa etária. A importância do uso de máscaras é o foco do sétimo cartaz. 274 Figura 7 - Cartaz – Uso de máscara Fonte: Dados da pesquisa (OLIVEIRA, 2021). Fonte: Dados da pesquisa (OLIVEIRA, 2021). O acessório bastante recomendado neste tempo de isola- mento social é o uso de máscara de proteção facial. Sendo assim, tal acessório, usado de forma correta, exerce uma função que contribui para que a proliferação do vírus não tome proporções exacerbadas. Como pode ser visualizado no cartaz 7, é um ato de amor e zelo para com a nossa vida e daqueles que amamos. A prática do uso de más- cara precisa e deve ser adotada pelas pessoas, a fim de colaborar para o fim do vírus letal na sociedade. A abordagem do oitavo cartaz versa sobre a utilização do álcool em gel. Figura 8 - Cartaz – Uso do álcool em gel Fonte: Dados da pesquisa (OLIVEIRA, 2021). Figura 8 - Cartaz – Uso do álcool em gel Fonte: Dados da pesquisa (OLIVEIRA, 2021). 275 A utilização do álcool em gel, assim como a lavagem das mãos, reforça a importância da eliminação do vírus em nosso corpo. O cartaz 8 traz a reflexão de que, higienizando as mãos através desse recurso prático, podemos de forma recíproca, nos proteger e uni- dos colaborarmos com a sociedade nessa luta em favor da vida. As medidas de prevenção são o meio de vencer, através da unidade consciente e empática, este tempo difícil. Desse modo, as gotas de esperança (vacina) são sinalizadas no cartaz a seguir: Figura 9 - Cartaz – Esperança Fonte: Dados da pesquisa (OLIVEIRA, 2021). Figura 9 - Cartaz – Esperança Fonte: Dados da pesquisa (OLIVEIRA, 2021). Considerando a pertinência colaborativa da ciência na socie- dade, o cartaz 9 permite uma reflexão esperançosa diante do cenário caótico que vive o planeta, a fim de que a pandemia acabe. Dessa forma, o desenvolvimento da(s) vacina(s) nos faz acreditar, asserti- vamente, que dias melhores virão. LETRAMENTOS EM CENA: A APROPRIAÇÃO DE UMA ESCRITA SIGNIFICATIVA Nesse sentido, a função social da escrita evidencia que não basta apenas refletir, mas é preciso pro- vocar o outro a assumir uma postura de colaboração e de combate, haja vista que todos unidos pela eficácia da vacina, poderemos ter uma sociedade imunizada e livre desse vírus letal. Logo, teremos uma batalha vencida, como vem mostrar o décimo cartaz. 276 Figura 10 - Cartaz – Batalha vencida Figura 10 - Cartaz – Batalha vencida Figura 10 - Cartaz – Batalha vencida Fonte: Dados da pesquisa (OLIVEIRA, 2021). S U M Á R I O Fonte: Dados da pesquisa (OLIVEIRA, 2021). Fonte: Dados da pesquisa (OLIVEIRA, 2021). A partir da colaboração consciente das pessoas e da eficácia da vacina, o mundo esmaga o vírus. Ou seja, a força da coletividade pode vencer a batalha contra a Covid-19. Nesse sentido, o cartaz 10 reforça para a sociedade a orientação de continuarmos cumprindo com as medidas necessárias de prevenção, para que o inimigo invi- sível não torne a assolar o planeta e, dessa forma, possamos bre- vemente retornar à vida normal. Homenagear é uma atitude que expressa gratidão, como podemos ver no décimo primeiro cartaz. 277 Figura 11 - Cartaz – Homenagem aos profissionais de saúde Figura 11 - Cartaz – Homenagem aos profissionais de saúde Figura 11 - Cartaz – Homenagem aos profissionais de saúde Fonte: Dados da pesquisa (OLIVEIRA, 2021). S U M Á R I O Fonte: Dados da pesquisa (OLIVEIRA, 2021). Fonte: Dados da pesquisa (OLIVEIRA, 2021). O cartaz 11 atribui uma singela homenagem àqueles que têm estado na linha de frente em favor da vida desde o início da Pande- mia da Covid-19. A mensagem de reflexão contida no interior do car- taz mobiliza a sociedade a agradecer aos profissionais da saúde, que estão nesta missão super poderosa, diariamente, medindo esforços pela saúde de tantas pessoas que se encontram na luta pela con- servação da vida. A todos vocês, nossa gratidão! Assim, o décimo segundo cartaz traz a possibilidade de que esperançar-se é preciso, para que os bons dias retornem ao nosso cotidiano. 278 Figura 12 - Cartaz – Dias melhores Fonte: Dados da pesquisa (OLIVEIRA, 2021). Figura 12 - Cartaz – Dias melhores Figura 12 - Cartaz – Dias melhores S U M Á R I O Fonte: Dados da pesquisa (OLIVEIRA, 2021). Fonte: Dados da pesquisa (OLIVEIRA, 2021). A expectativa da volta às aulas presenciais é o foco principal do cartaz 12. Sabemos que o cotidiano das escolas foi afetado pela chegada inesperada da pandemia, mas que com persistência, resis- tência e novas descobertas, a educação não parou. Dessa maneira, assim como a criança que olha para o alto e para frente, tenhamos a esperança que os dias melhores chegarão e, enquanto eles não chegam, vamos prosseguindo na busca e na construção do conhe- cimento. A educação é quem pode transformar uma sociedade, a fim de que seja um lugar saudável, próspero e emancipatório! CONSIDERAÇÕES FINAIS O percurso trilhado nesta pesquisa nos conduziu à com- preensão das possibilidades alcançadas por meio da intervenção direta que fizemos em uma realidade escolar. Trazemos à tona o alcance do objetivo proposto, enxergando que é possível abor- dar a ressignificação da escrita no ensino remoto, embora esse 279 contexto apresenta desafios, conflitos e dificuldades para a comuni- dade escolar como um todo. Foi permitido a todos os envolvidos no projeto adentrar numa abordagem que considera o significativo e emancipatório no que tange às habilidades leitoras e escritoras. Nesse sentido, o uso dos gêneros textuais/discursivos propostos na intervenção mediou, de fato, uma conscientização e apropriação da escrita para uma aplica- bilidade social, visto que é no mundo e pelo mundo que os sujeitos sociais agem por meio da linguagem. Com isso, a experiência no projeto de letramento impactou os participantes, levando-os a reconhecer que podem fazer muito com aquilo que possuem consigo, ao passo que se permitam a cola- borar com a sociedade de que fazem parte. Assim, esperamos que a experiência vivenciada pelo professor iniciante e todos os envolvidos neste projeto de letramento impacte outras realidades, a fim de que outros projetos de letramento possam ter vida e gerar grandes frutos emancipatórios nas escolas brasileiras. Viva os letramentos sociais! REFERÊNCIAS ANTUNES, Irandé. Língua, texto e ensino: outra escola possível. São Paulo: Parábola Editorial, 2009. BORTONI-RICARDO, Stella Maris. O professor pesquisador: introdução à pesquisa qualitativa. São Paulo: Parábola Editorial, 2008. BORTONI-RICARDO, Stella Maris. O professor pesquisador: introdução à pesquisa qualitativa. São Paulo: Parábola Editorial, 2008. OLIVEIRA, Gabriel Fernandes de. Novo normal, até quando? Um projeto de letramento para os desafios do ensino remoto do 6º ano do ensino fundamental em tempos de Pandemia. 2021. 65 f. Trabalho de Conclusão de Curso (Graduação em Letras) – Universidade Estadual da Paraíba, Guarabira, 2021. STREET, Brian. Letramentos sociais: abordagens críticas do letramento no desenvolvimento, na etnografia e na educação. Tradução de Marcos Bagno. São Paulo: Parábola Editorial, 2014. 280 S U M Á R I O INTRODUÇÃO Dialogando com diferentes propostas que versam sobre a importância de incentivar o processo de um letramento racial crí- tico desde os primeiros anos de vida das crianças, acredita-se que a educação de base é uma etapa essencial para a formação de um cidadão consciente das diferenças raciais que os cercam e os constituem. Para além do campo da linguagem e da Educação das Relações Étnico-Raciais, o respectivo artigo considera que há uma carência de materiais didáticos direcionados para bebês e crianças bem pequenas e que tratem do tema mencionado. Diante desta problemática, a pesquisa buscou responder a seguinte questão: Como os Pré-Livros, ou Livros de Imagens, podem se transformar em um veículo de enfrentamento e desconstrução do Racismo desde as primeiras fases da educação infantil? Nessa ótica, tem-se, como objetivo geral, construir reflexões acerca das potencia- lidades dos livros de imagens como agentes de mudança no campo das diversidades. Quanto aos objetivos específicos, o artigo busca apresentar o conceito de “Pré-livros”, fazer um levantamento biblio- gráfico sobre os livros-objetos e refletir sobre a possibilidade de esta- belecer um letramento racial crítico através dos livros de imagens. Ainda que a priori pareça complexo estabelecer relações entre a pré-escola e práticas antirracista, faz-se necessário demarcar que o racismo é uma construção social e quanto mais cedo crianças e adolescentes reconhecerem as diferenças raciais e étnicas, mais satisfatório será o processo de enfrentamento desta ideologia agres- siva e violenta no futuro. Sobre o conceito de racismo, mesmo sabendo que não é um conceito estanque e fechado, mas em constantes transformações, será entendido aqui “[...] como um fenômeno enraizado em ideolo- gias, doutrinas ou conjuntos de ideias que atribuem uma inferioridade 282 natural a determinados grupos com origens ou marcas adstritas específicas.” (CAMPOS, 2017, p. 1), e que, infelizmente, é estruturante da sociedade brasileira. Refletir sobre o racismo na educação infantil justifica-se devido a urgências e agências que cercam essa etapa educacional e que muitas vezes desencadeiam situações onde os educadores não estão preparados para resolver ou mediar. Em vista disso, um bom ponto de partida e uma estratégia viável seria introduzir ele- mentos pertencentes a culturas negras no cotidiano da educação infantil e pré-escolas, seja por meio da linguagem, apresentações textuais, valorização de narrativas orais negro-centradas, ou da alfa- betização e letramentos. INTRODUÇÃO Esse caminho pode ser trilhado pelas linhas da Pretagogia, “[...] uma pedagogia do fazer, da práxis, onde valo- riza-se a ancestralidade, tradição oral, corporeidade, religiosidade, território e circularidade como fontes e produtoras de saberes legí- timos.” (ALVES, 2019, p. 42). Fazer docente que valoriza, sobretudo, os discursos e as linguagens produzidas por indivíduos e grupos minorizados, a fim de não colaborar para que narrativas cotidianas e excludentes se perpetuem. Um exemplo bastante comum, e que resulta em incontáveis armadilhas discursivas e identitárias, é apresentar aos educandos, enquanto crianças, o lápis de cor salmão como se fosse o padrão da cor de pele, de qualquer pele e de todas as peles. Obviamente os discentes não conseguem ainda na primeira infância reconhecer as especificidades da linguagem, mas certamente, através dela, irão internalizar esse discurso e reproduzi-lo, compreendendo a partir dessa situação, que todas as outras cores de pele, para além dessa referência, estão fora da normalidade. Ao usar o termo ‘discurso’, proponho considerar o uso de linguagem como forma de prática social e não como ati- vidade puramente individual ou reflexo de variáveis situ- acionais. Isso tem várias implicações. Primeiro, implica ser o discurso um modo de ação, uma forma em que 283 as pessoas podem agir sobre o mundo e especialmente sobre os outros, como também um modo de representa- ção. (FAIRCLOUGH, 2008, p. 91). as pessoas podem agir sobre o mundo e especialmente sobre os outros, como também um modo de representa- ção. (FAIRCLOUGH, 2008, p. 91). S U M Á R I O Para o autor, linguagem e discurso estão associados às práti- cas sociais, sendo o discurso o elemento atrelado às representações e transformações das múltiplas relações e demonstrações de poder. Assim, com base em uma perspectiva social, elege-se, aqui, a linguagem como um campo promissor para a luta antirracista, considerando que a multiplicidade de linguagens desencadeia uma pluralidade de meios e formas de construir conhecimentos, de modo que “ao fazermos o uso da linguagem, recorremos a maneiras par- ticulares de representar, de agir e identificarmos o mundo e a nós mesmos. E, para tanto, lançamos mão de discursos, gêneros e estilos específicos” (SALES, 2012, p. 31). A linguagem é também um campo de disputas hegemônicas que colaboram para manutenção ou des- construção de desigualdades nos mais variados âmbitos. INTRODUÇÃO Apontando para a relação entre racismo e discurso, mate- rializado diante de diferentes linguagens, Van Dijk (2001) traz refle- xões acerca dessa interação, que segundo ele o discurso, quando é disseminado, seja por meio de textos escritos ou de natureza oral, possui um papel fundamental na propagação do racismo na atua- lidade, uma vez que “na América Latina o racismo está enraizado no colonialismo e nas subsequentes formas de dominação social, econômica e cultural pelas elites (mais) brancas” (VAN DIJK, 2008, p. 14). Sob essa indagação, o linguista explica que: Muitas práticas de racismo cotidiano, tais como as formas de discriminação podem até certo ponto ser explicadas, legitimadas ou sustentadas discursivamente de outro modo. Em outras palavras, a maioria dos membros dos grupos dominantes aprende a ser racista devido às for- mas de texto e de fala numa ampla variedade de eventos comunicativos (VAN DIJK, 2008, p. 15). 284 Em diálogo com Kleiman (1995) Van Dijk situa que o racismo, sob a ótica do processo de ensino-aprendizagem, pode promover tanto assimilação quanto o aprendizado desse fenômeno em si, que para ele é algo amplamente discursivo. Neste sentido, tendo como base o livro de Gabriel Nasci- mento (2019) intitulado “Racismo linguístico: os subterrâneos da linguagem e do racismo”, onde o autor observa que “um dos primei- ros signos impostos pelo colonialismo foi a assimilação ao homem branco através da linguagem como forma de o sujeito negro ser supostamente aceito pela branquitude” (NASCIMENTO, 2019, p. 70), compreende-se a vasta funcionalidade da linguagem no seio das disputas sociais e raciais, bem como seu potencial multiplicador do racismo na sociedade. Para Nascimento a linguagem não só é utilizada para gerar exclusão, como também é usada para a promoção de políticas públi- cas que se ocupam em excluir indivíduos negros. Isso ocorre, porque no Brasil “o colonialismo pode se manifestar (enquanto coloniali- dade) na linguagem e no sentido de se transformar em uma agenda política de opressão, produtora e reprodutora de desigualdades” (NASCIMENTO, 2019, p. 76). A linguagem é usada como um nicho de particularidades e possibilidades em plenitude, logo, o ler, escrever, ouvir, dizer, nar- rar, falar, escutar e se expressar, são reconfigurados de acordo com o contexto em que são apresentados e (re)significados nas intera- ções sociais habituais. INTRODUÇÃO Diante da versatilidade e adaptabilidade da linguagem, tais escritos foram estruturados a fim de sinalizar para os impactos do racismo linguístico na educação infantil e como a linguagem visual, pautada em sentenças imagéticas ou conjuntos iconográficos de fotografias e/ou pinturas, pode ser utilizada como um mecanismo de enfrentamento nessa acepção. 285 As imagens possuem um potencial formativo e informativo inestimável, possibilitando desde a primeira infância que os edu- candos tenham diferentes experiências ao serem “transportados” para uma gravura, cenário ou cena diferente do seu cotidiano. O que explica o fato de livros infantis, direcionados às crianças bem peque- nas, ainda que sejam lidos pelos pais ou responsáveis, tenham como foco a apresentação visual, destacando cores, traços e texturas. S U M Á R I O Observando o perfil dos materiais infantis literários e didá- ticos, de modo geral, nota-se que há outros elementos neles imbu- ídos, mas o destaque são linguagens imagéticas, que não envolve apenas o ver e enxergar, mas ao sentir, perceber e construir sentidos a partir do que é retratado e, sobretudo, do que não é retratado, mas está em entrelinhas. Haja vista que Quando alguém observa num livro uma figura qualquer e a nomeia, mesmo que não seja capaz de ler o texto que a acompanha (quando ele existe), está começando a compreender o sistema de símbolos presente nos livros, no qual se baseia a cultura escrita, quando lê as figuras, mesmo sem ler a palavra, de alguma forma já lê. (TEBET et.al, 2018, p.105). Por esse ângulo, nas próximas laudas, a linguagem será tra- tada como um espaço fluido de Poder, seja ela escrita, oral ou visual, porque se há o reconhecimento de uma língua instituída, há códigos e símbolos que deram a ela esse estatuto de legitimidade. Como já descrito, a linguagem aqui enfocada é a linguagem visual, refletida a partir de construções imagéticas e estampadas em livros de imagens, ou o que Bruno Munari definiu enquanto Pré-Li- vros, os quais possuem a capacidade de: Explorar as possibilidades visuais e tácteis do livro como objeto, através do uso de materiais presentes na produção de um livro, de diversas naturezas, e ques- tionando se é possível criar um livro apenas utilizando as matérias primas que podem constituir um livro, 286 pondo de lado o texto, uma das primeiras coisas em que se pensa quando se pensa num livro. (MUNARI, 1988, apud FADIGAS, 2018, p. 19). INTRODUÇÃO Os livros objetos, livros de imagens ou Pré-livros são pro- duzidos em formato pequenos e, no modelo que está sendo tra- tado nessa pesquisa, foram inventados pelo Italiano Bruno Munari em 1950, reeditados em 1980, e além da visão, o tato, audição e até olfato eram explorados. S U M Á R I O Figura 1 – Livro ilegível Fonte: MUNARI (1981). Figura 1 – Livro ilegível Fonte: MUNARI (1981). Fonte: MUNARI (1981). Sobre as discussões teóricas que sustentam a presente pes- quisa, são marcadas principalmente por explanações de Nilma Lino Gomes (2002), Petronilha Beatriz Gonçalves Silva (2003), Ferreira (2014) e Tebet et al. (2018), todas Educadoras, Professoras e pesqui- sadoras que desenvolvem trabalhos e orientam pesquisas no âmbito da Educação das Relações Étnico-Raciais, Letramento Racial Crítico, Literatura e Identidades Étnico-Raciais. 287 METODOLOGIA A abordagem metodológica utilizada nessa investigação deu-se por intermédio de três etapas iniciais, sendo a primeira o levantamento bibliográfico nas bases de dados da Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES). Tal ponto de partida explica-se, pois “Qualquer trabalho científico inicia-se com uma pesquisa bibliográfica, que permite ao pesquisador conhe- cer o que já se estudou sobre o assunto.” (FONSECA, 2002, p. 32). Na sequência, realizou-se a leitura e fichamento das obras encon- tradas e que tratam do tema. Na terceira etapa ocorreu a análise de parte dos trabalhos encontrados, a fim de problematizar suas poten- cialidades na prática. Trilhou-se tal caminho, levando em consideração que “A prin- cipal vantagem da pesquisa bibliográfica reside no fato de permitir ao investigador a cobertura de uma gama de fenômenos muito mais ampla do que aquela que poderia pesquisar diretamente” (GIL, 2002, p. 45), aspecto relevante e que dialoga com a proposta desse estudo, pois o problema de pesquisa exposto nessa investigação requer dados muito esparsos. Essa etapa foi necessária para construir o Estado da Arte do respectivo trabalho, que é a parte central da investigação e escrita, porque visa “Expor resumidamente as principais ideias já discutidas por outros autores que trataram do problema, levantando críticas e dúvidas, quando for o caso” (RAMOS; SANTOS 2009, p. 66). Após levantamento bibliográfico, construção do Estado da Arte, que incorre em apresentar as discussões teóricas sobre o assunto, seguiu-se para as problematizações e reflexões acerca das produções encontradas, trabalho que se concretizou por intermédio da pesquisa qualitativa, que tem relação com interpretações e leitu- ras bastante subjetivas, sendo suas principais características: 288 [...] objetivação do fenômeno; hierarquização das ações de descrever, compreender, explicar, precisão das rela- ções entre o global e o local em determinado fenômeno; observância das diferenças entre o mundo social e o mundo natural; respeito ao caráter interativo entre os objetivos buscados pelos investigadores, suas orienta- ções teóricas e seus dados empíricos; busca de resulta- dos os mais fidedignos possíveis; oposição ao pressu- posto que defende um modelo único de pesquisa para todas as ciências. (SILVEIRA; CÓRDOVA, 2009, p. 32). Marcada por uma visão mais aberta de um fazer analítico plural e que considera relações humanas em perspectiva social, a pesquisa qualitativa “[...] não se preocupa com representatividade numérica, mas, sim, com o aprofundamento da compreensão de um grupo social, de uma organização, etc.” (SILVEIRA; CÓRDOVA, 2009, p. 31). METODOLOGIA Sob essa mesma ótica, compreendendo o âmbito das relações étnico-raciais, linguagens e educação infantil. REVISÃO BIBLIOGRÁFICA/ ESTADO DA ARTE Os trabalhos e pesquisas que tratam dos Pré-livros/Livros- -objetos, nortearam as problematizações apresentadas na sequên- cia. E a fim de articular as ideias e contribuições de diferentes auto- res com a proposta desse estudo, faz-se necessário frisar que uma das características desses livros de imagens é a preocupação não só com a forma, mas com o conteúdo e estética. Essa estratégia sus- tenta-se pela máxima de Lupton e Phillips onde o “Usar cores com valores contrastantes tende a precisar mais as formas, assim como a combinação de cores de valores próximos suaviza a distinção entre os elementos” (LUPTON; PHILLIPS, 2008, p.74). 289 Em diálogo com tal linha pedagógica e artística, a respeito de produzir material didático interessante aos olhos das crianças, Eli- zabeth Romani, em sua Dissertação de Mestrado intitulada “Design do Livro-Objeto Infantil” (2011) analisou a ludicidade desses mate- riais. Para ela, “O livro-objeto é compreendido como um produto de expressão artística passível de reprodução cuja narrativa é explorada por meio da manipulação.” (ROMANI, 2011, p.5). Manipulação que leva a apreender e produzir sentidos e significados a partir do des- pertar dos cinco sentidos. Pensando nesse processo do primeiro contato e manuseio da obra, a autora explica que: O formato caracteriza o aspecto físico do livro-objeto. Este tem o poder de incentivar a fantasia, porque é a primeira impressão que o leitor terá do objeto. O formato associado ao tamanho tem a capacidade de expressar sensibilidade, por exemplo: livros menores expressam sutileza e delicadeza. (ROMANI, 2011, p.10). Para a autora, cada Livro-objeto possui uma tipografia única e peculiar, dependendo do caso, mas ela expõe tal panorama diante dos livros infantis, visto que sua pesquisa explora a relação do projeto gráfico com a estrutura lúdica desses materiais, o que se relaciona com a proposta dessa pesquisa, que busca sinalizar para a estrutura lúdica e didática desses livros, em uma expectativa antirracista Ana Maria Ramos também apresenta a dimensão educativa e formativa dos livros-objetos e de acordo com ela esse material define-se pelo encontro entre livro-brinquedo, livro-álbum e livro-ar- tista (RAMOS, 2021). A dimensão lúdica e educativa desses materiais é abordada por Diana Maria Ferreira Martins, que em sua Tese de Doutorado denominada “O Lugar do Livro-Brinquedo na infância e na Litera- tura: Arquitetura, (Inter)Texturas e Outros Desafios” construiu, no âmbito dos Estudos Literários, uma reflexão histórico-conceptual 290 sobre os livros-brinquedo. 25 Nesse primeiro momento, as produções escolhidas para dar forma ao presente artigo foram de Fadigas (2018), Ramos (2021), Romani (2011), Xavier (2015), Santos (2018), Abreu (2021) e Martins (2019), algumas foram utilizadas apenas para construir o Estado da Arte e outras para auxiliar a responder a problemática deste artigo. REVISÃO BIBLIOGRÁFICA/ ESTADO DA ARTE Após a problematizar o conceito de livro- -brinquedo, a autora ainda buscou colaborar para a historiografia da literatura portuguesa infantil, mas antes de desenvolver essa discus- são, traz o histórico dos primeiros livros-objetos criados. De acordo com Martins “[...] a História do livro-objeto terá tido início na Idade Média (avançando paralelamente com a dos livros convencionais e do nascimento da literatura para a infância no século XVIII)” (2019, p.89). A autora explica que esses primeiros livros possuíam abas e diferentes dispositivos de manuseio, geralmente eram destinados aos campos da Teologia, Medicina e Astronomia. Sobre os diferentes formatos e estilos desses materiais didá- ticos e/ou literários, no contexto atual, tem grande influência das produções elaboradas nos séculos passados e “Ainda que inscritos numa tendência recente da literatura para a infância, a verdade é que a sua origem remonta a publicações de cariz didático dirigidas a um público adulto, comuns no séc. XVI, que tiram partido de sobreposi- ções[...].” (MARTINS, 2019, p.90). Mas a inserção de imagens, colagens e figuras moveis foi um estilo que se mantém até os dias atuais, pois o visual, a estética e ilusões de ótica, eram priorizados nessas obras. Nesse sentido, “[...] é possível constatar que a exploração de efeitos óticos não é recente, remontando a um período exórdio ao cinema e a uma série de contribuições que o fizeram nascer.” (MAR- TINS, 2019, p.97). A autora faz um paralelo entre brinquedo ótico e o livro-brinquedo, na medida em que identifica semelhança entre ambos, pois “Os brinquedos óticos nascem, não raras vezes, dessa demanda pela ilusão de movimento, que o ser humano deseja desde a pré-história.” (MARTINS, 2019, p.98). As reflexões apresentadas até o momento estabelecem que os Pré-livros, criados por Bruno Munari, são, na realidade, um dos diversos tipos de livros-objetos, podendo ainda ser entendido tam- bém como um livro-brinquedo. Tanto um, quanto outro possuem grande potencial instrutivo, formativo e sua natureza visual é um traço 291 presente em todos os materiais que são classificados como livros-ob- jetos. A percepção acerca do livro-brinquedo é basilar nessa pesquisa, pois foi desenvolvida e pensada prioritariamente para as crianças. RESULTADOS E DISCUSSÕES A pesquisa teve início com a busca por Dissertações de Mestrado disponíveis no Banco de Teses e Dissertações da CAPES, porém, o maior desafio encontrado foi a vasta quantidade de tra- balhos associados aos marcadores apresentados, que se digitados em conjunto, separado apenas por ponto e vírgula, apresentou mais de vinte mil obras. Logo, a busca precisou ser redefinida, a fim de desenvolver uma estratégia que fosse viável pensar a leitura de pelo menos parte dos trabalhos encontrados e seus resumos. Sendo assim, optou-se por uma investigação mais restrita que se constituiu a partir dos descritores: Pré-Livros; Letramento Racial Crítico; Infância. E a partir desses signos as buscar resultaram em um levantamento de duas mil setecentas e setenta e uma Dis- sertações de Mestrado e diante desse montante realizou-se novas buscas por meio da leitura do título e depois dos resumos, caso o resumo apresentasse conexão com o tema deste artigo, seguiria para leitura do trabalho, fichamentos, problematizações e diálogos com a pesquisa aqui elaborada. Ao todo, sete trabalhos foram selecionados no Banco de Teses e Dissertações da CAPES25. O primeiro foi de João Paulo Xavier, que através da produção bibliográfica denominada “Letramento Visual Crítico: Leitura, interpretação e apropriação das imagens dos livros 25 292 S U M Á R I O didáticos”, buscou identificar como as representações da diversidade étnica, cultural e social brasileira foram feitas na coleção de livros didáticos High Up, utilizada pela maioria das escolas estaduais na cidade de Belo Horizonte e aprovada pelo Programa Nacional do Livro e do Material Didático (PNLD) em 2015. Embora Xavier (2015) não tenha analisado Pré-Livros, e suas problematizações tenham se dado em torno de uma coleção especí- fica, ele aborda os conceitos de letramento crítico (LC) e letramento visual (LV), discussões substanciais para o trabalho aqui construído. Sobre a definição de LC, o autor não se restringe a uma ou outra corrente teórica, mas explana que “os conceitos de letramento crítico (LC) e a valorização dos multiletramentos tornam-se importantes por evidenciar de forma mais objetiva as múltiplas possibilidades para as práticas docentes” (XAVIER, 2015, p. 13). Bebendo em outra fonte, tem-se ainda a seguinte percepção acerca do respectivo conceito. RESULTADOS E DISCUSSÕES [...] os letramentos não se restringem apenas a saber ler e escrever, mas abarcam a capacidade de aplicar conheci- mentos socioculturais específicos em contextos significa- tivos de uso com o objetivo de atuar e modificar possíveis iniquidades sociais que, em grande medida, são perpetu- adas através de discursos (ABREU, 2021, p. 50). Inscritos sob o campo de leitura, interpretação, reflexão e ação, o letramento crítico tem a capacidade de abarcar outras expe- riências dos indivíduos, respondendo a aspectos sociais e culturais que desencadeiam os mais variados processos discursivos. Voltando à Dissertação de Mestrado de João Paulo Xavier, nota-se que o autor organiza uma articulação entre o Letramento Visual (LV) e a semiótica, ciência de suma importância para auxiliar na compreensão, interpretação e entendimento de dos mais varia- dos sistemas de signos. Assim, Xavier, com base em Ferraz (2014), descreve o LV como “[...] um subcampo dos multiletramentos, pois aborda a imagem em suas múltiplas formas e considera, assim, a mul- timodalidade e o uso de diferentes mídias e tecnologias no processo 293 de significação” (XAVIER, 2015, p. 52). Integrando o LC e o LV, o pes- quisador problematiza, então, a definição de um Letramento Visual Crítico, que seria a coadunação desses dois campos de aprendiza- gens e saberes formativos. Em uma caracterização mais precisa, Letramento Visual Crítico (LVC) é a perspectiva teórica que explora o texto imagético em movimento ou estático e busca compreender como seus elementos fotográfi- cos, detalhes cromáticos, texturas, enquadramento, foco, ângulo, entre outros aspectos, podem retratar realidades e discursos. (XAVIER, 2015, p. 59). S U M Á R I O Essa tipologia de letramento renderia bons resultados se fosse pensada a partir de Pré-Livros que evidenciassem cultu- ras negras do cotidiano, pois além de despertar no leitor, adulto ou criança, uma sensibilidade promovida por meio da explosão de cores, traços, estilos e formas, os possibilitaria fazer análises mais profundas do material a partir de suas próprias realidades, o que os levaria a perceber as diversidades que os cercam através das ima- gens, elementos chave no processo de construção de conhecimento de bebês e crianças bem pequenas em seus primeiros contatos com a literatura. RESULTADOS E DISCUSSÕES O primeiro contato com Pré-livros racializados (negros) pode ser também o primeiro passo para que futuramente os peque- nos leitores desenvolvam uma criticidade em relação ao que veem, manuseiam e leem, uma vez que O LVC se preocupa em levar o leitor a três níveis de aná- lise profunda: no primeiro, realizar uma leitura desses detalhes visuais; no segundo, interpretar criticamente esses aspectos, as escolhas, as possíveis intenções e expectativas da autoria desse texto imagético ao produzi- -lo dessa forma; no terceiro, refletir e discutir quais efeitos de sentido podem ser construídos a partir da imagem, quais podem ser os impactos dela sobre o leitor, quais vozes foram retratadas ou silenciadas por essa imagem e como a leitura, a interpretação e a apropriação dessa imagem podem empoderar o leitor a problematizar as situações retratadas. (XAVIER, 2015, p. 59). 294 E a fim de buscar outros trabalhos que forneçam subsídios teórico-metodológico para responder o questionamento levantado nesse artigo, chegou-se ao trabalho de Bruno Nascimento dos San- tos, denominado de “Raça, Racismo e Questão Racial no Ensino de História: Uma Análise a partir dos Livros Didáticos”, tal Dissertação fora defendida pelo Mestrado Profissional em Ensino De História (PROFHISTÓRIA) em 2018. Semelhante ao trabalho de Xavier, no sentido de investigar livros didáticos disponibilizados pelo governo, a pesquisa de Santos (2018) trabalha com três coleções distribuídas pelo Programa Nacio- nal do Livro e do Material Didático (PNLD) para o ensino médio do triênio 2015-2017. Interessado em analisar a abordagem da questão racial na sociedade brasileira, o trabalho mencionado dialoga com a intencio- nalidade da pesquisa aqui desenhada, porque o autor acredita “que a abordagem da questão racial, na Educação Básica, contribuirá para a desconstrução de estereótipos naturalizados sobre (e pela) a popu- lação negra que pavimentam as discriminações e a desigualdades raciais.” (SANTOS, 2018, p. 7). Os livros didáticos problematizados por Bruno Nascimento dos Santos foram separados por unidades e as reflexões foram sis- tematizadas de acordo com assuntos que atravessasse questões raciais no país, seja por meio de acontecimentos históricos passados ou contemporâneos. De acordo com a Professora e pesquisadora Janice Gonçalves, refletindo sobre lugares de memória, a palavra vetor “indica aquilo que porta algo, assim como transmite, aponta ou, ainda, orienta” (GONÇALVES, 2015, p. 17). RESULTADOS E DISCUSSÕES O autor não constrói uma abordagem que esmi- úce imagens, mas no terceiro capítulo de seus escritos, ao tratar da coleção “História Sociedade & Cidadania”, Santos (2018) faz a última análise da Dissertação e cita que há na coleção de livros didático que ele investiga, na unidade IV, mais precisamente, cinco imagens de comunidades quilombolas brasileiras e esse conjunto imagético trata da invisibilidade da população quilombola no Brasil. Ressaltando o potencial iconográfico dessa linguagem visual, o autor situa que 295 A proposta da unidade, de acordo com os questionamen- tos que acompanham ambas as fontes, é de, além de estabelecer uma relação imediata entre imagem e texto, induzir à atenção, a partir desse ponto de partida da uni- dade, para as lutas de diversos grupos por terra e liber- dade na América. (SANTOS, 2018, p. 90). As imagens possuem uma força de impacto significativa no processo de ensino-aprendizagem, logo, podem ser entendidas como vetores26 didáticos promissores no âmbito do Letramento Racial Crítico, conceito que “tem uma compreensão poderosa e complexa da forma como raça influencia as experiências sociais, econômicas, políticas e educacionais dos indivíduos e dos grupos” (SKERRETT, 2011, p. 314). As discussões sobre Letramento Racial Crítico não foram contempladas nas Dissertações apresentadas até o momento, o que trouxe à tona a necessidade de realizar novas buscas ao Banco de Teses e Dissertações da CAPES, dessa vez procurando apenas pes- quisas que versem sobre o respectivo conceito, atividade ou tema. Ao fazer tal investigação, o próprio site ofereceu um recorte temporal mais restrito (2010-2016) e filtrou mil duzentos e seis (1206) trabalhos que foram atravessados pelo então conceito. Um aspecto bastante interessante nessa etapa de levanta- mento bibliográfico refere-se à grande quantidade de obras catalo- gadas pela Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) sobre as múltiplas formas de letramento e que não estão disponíveis para consulta na própria plataforma, pois são produções anteriores à criação da Plataforma Sucupira que fora ide- alizada em 2012. Tal informação leva a reflexões sobre a vasta quan- tidade de Dissertações produzidas nas décadas anteriores sobre o assunto, que, a princípio, de acordo com a leitura dos títulos des- 26 296 S U M Á R I O ses trabalhos registrados, foram aos poucos sendo substituídos por temas que propunham problematizações acerca de Letramentos Digitais, novas tecnologias e o advento da Internet na modernidade. RESULTADOS E DISCUSSÕES Esse fato levou à uma fragmentação nas buscas e tal impasse ampliou o campo de investigação, fazendo com que as atenções fos- sem direcionadas ao Google Acadêmico, segundo sítio eletrônico usado para novos levantamentos. A pesquisa bibliográfica na plataforma do Google Acadêmico rendeu resultados mais objetivos e considerando que as definições acerca dos conceitos de Letramento, Letramento Crítico, Letramento Visual, Letramento Visual Crítico e Letramento Racial Crítico já foram mobilizados neste estudo, buscou-se pelo descritor Pré-Livro na pla- taforma mencionada e a partir deste marcador obteve-se duzentos e noventa e seis (296) resultados, embora alguns tenham se repe- tido, pois o sistema de busca não reconhece que muitas vezes há o mesmo trabalho, mas armazenado sob diferentes formatos. As buscas nesse sítio eletrônico não tipificam as obras, tam- pouco as separam de acordo com suas características. Assim, inse- ridos no montante total de bibliografias disponíveis, estavam pro- duções de diferentes naturezas, dentre elas haviam livros, artigos, Monografia, Teses, Dissertações e muito mais. Antes de seguir, é preciso pontuar que o conceito de Pré- -Livro é bastante explorado na área de Designer e por isso, é mais comum que autores que constroem reflexões em torno desses materiais sejam pesquisadores, professores e estudantes vinculados a essa grande área, como é o caso de Márcio Duarte, que em seu artigo O Livro e Suas Silenciosas Narrativas, pontua que os Pré-Livros são “[...] assim chamados pela sua forma, busca inserir o repertório e a experimentação em um nível extremo, criando situações abstratas e únicas, sugerindo direções e narrativas inusitadas a cada vez que se folheia, ou melhor, que se manuseia o livro” (DUARTE, 2016, p. 4). 297 Na sequência, Duarte explana que tais materiais são diversos e repleto de significação e sentidos, que pensam, sobretudo, na intera- ção entre leitor e a obra em um sentido mais específico que vai além de folhear páginas e do manuseio, mas perpassa diferentes percep- ções, aguçando a curiosidade e desencadeando diferentes reflexões sobre o que se vê, toca e nota. Tais livros, Na sequência, Duarte explana que tais materiais são diversos e repleto de significação e sentidos, que pensam, sobretudo, na intera- ção entre leitor e a obra em um sentido mais específico que vai além de folhear páginas e do manuseio, mas perpassa diferentes percep- ções, aguçando a curiosidade e desencadeando diferentes reflexões sobre o que se vê, toca e nota. RESULTADOS E DISCUSSÕES Tais livros, [...] são caracterizados por serem constituídos em sua totalidade de formas e texturas, objetos que isolados não possuem um significado literário, mas ao ser trabalhado juntamente ao seu contexto formal, adquire um conjunto de informações possível de verbalizações infinitas, sendo limitada apenas pelo seu interlocutor (DUARTE, 2016, p. 5). As discussões elencadas por Márcio Duarte dialogam com as perspectivas teóricas de Cassia Letícia Carrara Domiciano, que inclusive é referenciada por ele em seu artigo apresentado recente- mente. Doutora do Instituto de Estudos da Criança e estudiosa dos livros sem textos, Domiciano esclarece que nesse tipo de material “apenas a imagem e a materialidade do livro comunicam” (DOMI- CIANO, 2006, p. 1). Destacando o trabalho dos Designer e dos ilus- tradores como autores possíveis dessas obras, a autora comenta que A imagem tem papel fundamental nos livros infantis. Os livros sem texto primam pela exploração deste elemento e tentam potencializar, não somente o poder das imagens, mas também, em muitos deles, da materialidade do livro em si. A narração de uma estória pode ser feita somente através do texto visual (DOMICIANO, 2006, p. 5). Sob esse prisma, encontra-se o fio condutor que estabelece relação entre a temática desse estudo, a Educação das Relações Étnico-Raciais e os Pré-Livros ou Livro de Imagens. E ainda que não se tenha encontrado um trabalho que os problematize em um seg- mento antirracista, nota-se a evidência de pesquisas que, há pelo menos duas décadas, dão destaque às diversidades por meio das linguagens, seja ela oral, visual ou escrita. 298 PRÉ-LIVROS COMO AGENTE DE DESCONSTRUÇÃO DO RACISMO LINGUÍSTICO A estratégia em relacionar diferentes tipos de letramentos justifica-se na medida em que se percebe as imagens como um meio de conectar diferentes olhares sobre culturas distintas, assim, o Pré-Livro pode, sim, ser compreendido como um agente de des- construção, sobretudo, no que se refere aos ensinamentos norteados pelas africanidades, conceito que “[...] refere-se às raízes da cultura brasileira que têm origem africana” (SILVA, 2003, p.26), em uma linha de pensamento afrodiaspórica e que representa uma integração e interação entre culturas, cosmovisão, linguagens, costumes e tradi- ções que atravessaram o atlântico e chegaram até o Brasil, man- tendo suas agências e essências. Falamos então de uma herança trazida pelos africanos para o Brasil que chamamos africanidades, elementos das diversas culturas vindas da África que se encontram em nosso país assim como as afrodescendências consti- tuídas das manifestações culturais erigidas pelos afrodes- cendentes brasileiros ressignificando e reprocessando o saber ancestral dando origem a uma gama de manifesta- ções afro-brasileiras (OLIVEIRA, 2013, p. 17). As africanidades são práticas negro-centradas que se valori- zadas em sala de aula possibilitam aos educandos negros um pro- cesso de valorização de suas identidades e identificações negras, nesse segmento Nilma Lino Gomes (2002), explana que A escola pode ser considerada, então, como um dos espa- ços que interferem na construção da identidade negra. O olhar lançado sobre o negro e sua cultura, no interior da escola, tanto pode valorizar identidades e diferenças quanto pode estigmatizá-las, discriminá-las, segregá-las e até mesmo negá-las. (GOMES, 2002, p. 39). 299 A escola pode promover a valorização de culturas minoritá- rias de inúmeras maneiras e uma delas é escolhendo bons materiais didático e prezando pelo desenvolvimento de um Letramento Racial Crítico em todas as instâncias escolares, pois “[...] para termos uma sociedade mais justa e igualitária, temos que mobilizar todas as iden- tidades de raça branca e negra para refletir sobre raça e racismo e fazer um trabalho crítico no contexto escolar em todas as disciplinas do currículo escolar” (FERREIRA, 2014, p. 250). S U M Á R I O Esse movimento deve ser constante, levando a mudanças significativa, principalmente no que se refere ao Racismo Linguís- tico, que de acordo com Gabriel Nascimento está diretamente rela- cionado à Racialização, compreendida como “[...] a enunciação que permite formar raça enquanto enunciado nas hierarquias de poder do sistema-mundo” (NASCIMENTO, 2019, p. 67). PRÉ-LIVROS COMO AGENTE DE DESCONSTRUÇÃO DO RACISMO LINGUÍSTICO Para Nascimento, as identidades são racializadas e através delas, a linguagem pode ser uma ferramenta usada para sustentar o Racismo no país, porque a linguagem e o preconceito racial manifestam-se por meio do ato da fala e do que é falado. Assim pensar a necessidade de produzir e democratizar livros de imagens/livros sem textos/Pré-Livros sobre culturas negras e africanas, ressalta uma importante inflexão nessa direção, porque os alunos teriam por intermédio desses materiais, a capacidade de produzir seus próprios textos e elaborar suas próprias falas a partir do que veem e os Professores seriam mediadores nesse processo de construção de um Letramento Racial Crítico Visual. CONSIDERAÇÕES FINAIS Este artigo foi elaborado com a intenção de estabelecer conexões entre teoria e prática e sinalizar para as principais possi- bilidades e discussões acerca dos livros-objetos, livros de imagens, 300 livros com imagens e principalmente dos Pré-Livros, refletindo sobre o letramento racial crítico para crianças bem pequenas. A menção à literatura para bebês e crianças bem pequenas foi considerada após a reflexão da Professora Gabriela Tebet et al. (2018) que em seu artigo “Espaços que Gritam: Criação Coletivas de Outras Formas de Livros e de Leituras para Bebês e Crianças que não Lêem Letras”, questiona quais narrativas são constituídas em espaço escolar e quais as culturas representadas nesse contexto. A problematização principal de Tebet et al. e que se conecta à proposta desse estudo refere-se à seguinte pergunta: “É possível pensar em formas de leitura para e por bebês e crianças que ainda não lêem letras?” (TEBET et al., 2018, p.102). Tem-se, a partir desta indagação a possibilidade de destacar as Literaturas para bebês, diversidades e culturas minorizadas já na fase pré-letras. Para além do levantamento bibliográfico, desenhou-se neste trabalho, diferentes caminhos para promover a construção, uso e a implantação de Pré-Livros que prezem pela diversidade desde a primeira infância. Assim, as ideias predominantes aqui expostas apontaram para o potencial formativo e cognitivo do trabalho e aná- lise de/com imagens. Em certa medida, a pesquisa respondeu ao questionamento levantado e concluiu que os Pré-Livros, ou Livros de Imagens, podem se transformar em um veículo de transformação e desconstrução do Racismo linguístico, ao passo que as imagens são repletas de signi- ficações e significados e impactam bebês, crianças bem pequenas, adolescentes e adultos. Ao longo desses escritos, reconheceu-se diferentes pro- postas metodológicas acerca do uso dos “Pré-livros” como material didático, livro-brinquedo, vetor de desconstrução do racismo linguís- tico e agente de transformação. 301 Diante das leituras, levantamentos, reflexões e problemati- zações construídas, identificou-se uma carência nos estudos sobre Pré-Livros voltados para a Educação das Relações Étnico-Raciais, logo, o artigo sinaliza também para a urgência em políticas públi- cas vinculadas ao Plano Nacional do Livro e do Material Didático (PNLD), que evidenciem as diversidades como um campo de estu- dos e metodologias específicas. Por fim, espera-se que a atual pes- quisa abra caminhos para novas investigações no âmbito do letra- mento racial crítico construído por intermédio do livro de imagens. REFERÊNCIAS CAMPOS, Luiz Augusto. Racismo em três dimensões: uma abordagem realista-crítica. Revista Brasileira de Ciências Sociais, Rio de Janeiro, v. 32, n. 95, p. 2-19, 2017. 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Revista da ABPN, Goiânia, v. 6, n. 14, p. 236-263, jul./dez. 2014. FONSECA, João José Saraiva da. Metodologia da pesquisa científica. Fortaleza: UEC, 2002. Apostila. GIL, Antônio Carlos. Como elaborar projetos de pesquisa. São Paulo: Atlas, 2002. 302 GOMES, Nilma Lino. Educação e identidade negra. Revista Aletria, Belo Horizonte, v. 9, p. 38-47, 2002. GONÇALVES, Janice. Lugares de memória, memórias concorrentes e leis memoriais. Revista Memória em Rede, Pelotas, v. 7, n. 13, p. 15-28, jul./dez. 2015. KLEIMAN, Angela B. Modelos de Letramento e as Práticas de Alfabetização na Escola. In: KLEIMAN, Angela B. (Org.). Os significados do letramento: uma nova perspectiva sobre a prática social da escrita. Campinas: Mercado de Letras, 1995, p. 15-61. LUPTON, Ellen; PHILLIPS, Jennifer C. Novos fundamentos do design. Tradução de Cristian Borges. São Paulo: Cosac Naify, 2008. MARTINS, Diana Maria Ferreira. O lugar do livro-brinquedo na infância e na literatura: arquitetura, (inter)texturas e outros desafios. 2019. 536 f. Tese (Doutorado em Estudos da Criança) – Instituto de Educação da Universidade do Minho, Braga, 2019. MUNARI, Bruno. Das Coisas Nascem Coisas. Lisboa: Edições 70, 1988. NASCIMENTO, Gabriel. Racismo linguístico: os subterrâneos da linguagem e do racismo. Belo Horizonte: Letramento, 2019. OLIVEIRA, Alexsandra Flávia Bezerra de. Feira Livre de Bodocó: Memória, Africanidades e Educação. 151 f. Dissertação (Mestrado) − Universidade Federal do Ceará/Faculdade de Educação, Fortaleza, 2013. RAMOS, Ana Maria. Livro-objeto: o relevo da materialidade no processo de leitura. 2021. REFERÊNCIAS Disponível em: <https://bit.ly/3DQidK3>. Acesso em: 13 jan. 2023. RAMOS, Ieda Cristina Alves. SANTOS, Daniel Labernarde dos. Estrutura do Projeto de Pesquisa. In: GERHARDT, Tatiana Engel; SILVEIRA, Denise Tolfo. (Orgs.). Métodos de Pesquisa. São Paulo: Editora da UFRGS, 2009, p. 65-87. 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Dissertação (Mestrado em Linguística Aplicada) – Faculdade de Letras da Universidade Federal de Minas Gerais, Belo Horizonte, 2015. 304 15 Maísa Cardoso AS AFINAL, O QUE É LER? ALGUMAS CONCEPÇÕES E PROVOCAÇÕES SOBRE A PRÁTICA DA LEITURA E A FORMAÇÃO DO LEITOR DOI:10.31560/pimentacultural/2023.97778.15 S U M Á R I O INTRODUÇÃO Em tempos de corrida por aumentos do Índice de Desenvol- vimento da Educação Básica (IDEB), gestores públicos tentam de tudo para aumentar índices e, sem dúvida, melhorar o nível de lei- tura dos estudantes é uma pauta fundamental. Sem me ater à ética dessa corrida de índices, não é preciso lembrar que não é de hoje o consenso sobre a importância e a necessidade da leitura. É comum ouvirmos afirmações como “preciso ler mais”, “devo começar a ler”, “meu filho odeia ler”, “nossos estudantes não gostam de ler”. Sem dis- cutir a veracidade dessa questão, vamos também concordar com a premissa: ler é uma das práticas importantes na formação intelectual do estudante e uma das responsabilidades impostas à escola pela sociedade há tempos. Assim, é de comum acordo que a leitura é o caminho para o desenvolvimento intelectual e para a obtenção de uma melhor posição no mercado de trabalho, entre outras generali- dades comuns sobre os benefícios dessa atividade. Entretanto, indo além da questão do benefício, como edu- cadores, compreendemos a real amplitude das conceções da prá- tica leitora? A maioria relaciona o ato de ler ao sentar-se num lugar tranquilo e, respeitosamente, digerir um livro, de preferência, bem extenso. Essa imagem está fundamentada na concepção de leitura como ritual, mencionada por Goulemot (1996, p. 109), ao recordar a exigência cerimonial de suas professoras frente à leitura; uma pos- tura lânguida era entendida como falta de apreço à atividade. Essa visão não mudou muito. Nos dias de hoje, ainda se prega o ler idí- lico. Consequentemente, não é espantoso que a maioria dos “meros mortais” rejeite a leitura, por considerar que se trata de uma prática altamente refinada, ou um sacrifício para poucos. Essa imagem des- considera a complexidade e a profundidade das concepções de lei- tura, bem como suas implicações no ensino. 306 Assim, este capítulo visa discorrer sobre o fenômeno da lin- guagem no que tange à prática da leitura, com base na abordagem da Linguística Aplicada, focando principalmente na importância de se refletir sobre as concepções da prática da leitura a fim de atuarmos como educadores(as) de modo significativo na formação leitora dos estudantes, da qual somos, em boa parte, responsáveis. Acredita- mos que estarmos a par dessa discussão pode nos favorecer no trato com a prática em sala de aula, além de desmistificarmos algumas questões que envolvem o ato de ler. INTRODUÇÃO Ainda, em tempos de aplicativos de leitura, alguns adotados por instituições escolares, com rankings de leitura e trilhas literárias, ousamos dizer que, somente buscando compreender estudos sobre o ato de ler, suas concepções é que poderemos, de fato, atuar como formadores de leitores competentes. Localizamo-nos na LA, dada a constante autorreflexão pró- pria da área, sendo os linguistas aplicados os “que tratam de diferen- tes problemas da vida social” (KLEIMAN, 1998, p. 54), daí nossa pre- ocupação com a discussão sobre o ato de ler na escola, a instituição praticamente responsável pela formação do leitor, principalmente, nas classes desfavorecidas. Quanto à organização, este capítulo teórico pretende explo- rar algumas concepções de leitura, tratar da relação entre leitura e o leitor e, por fim, abordar a importância da escola e da família na formação do leitor. CONCEPÇÕES DE LEITURA Em sentido lato, podemos afirmar que a leitura está além da palavra, pois compreende também sinais não-linguísticos. “Pode-se ler a tristeza nos olhos de alguém, a sorte na mão de uma pessoa ou o passado de um povo nas ruínas de uma cidade”, 307 S U M Á R I O afirma Leffa (1996, p.10), que concebe a leitura como um processo de representação, efetuado por meio de uma triangulação mediada por elementos indiretos à realidade. Quando olhamos para as ruínas de uma cidade, vemos as ruínas, porém podemos ler seu abandono, o desprezo de seus conterrâneos ao bem público etc. “Ler é, na essên- cia, olhar uma coisa e ver outra.” (LEFFA, 1996, p.10). Essa é a leitura de mundo, citada por Paulo Freire (1982). Contudo, como lembra Aguiar (2004), a leitura de mundo, indispensável para a leitura da palavra, não pode ser a única a que o sujeito tenha acesso. Por isso, vamos evocar outras definições de leitura discutidas em Solé (1998), Dell’Isola (1996), Kleiman (1993), relacionadas principalmente à compreensão de textos escritos. Entendemos, com Solé (1998), que ler é “compreender e interpretar textos escritos de diversos tipos e com diferentes inten- ções e objetivos” (SOLÉ, 1998, p. 18). Assim, a leitura consiste em um trabalho subjetivo, uma vez que é sempre produção de sentido, e não reconstituição de sentido ou “descoberta” do sentido desejado pelo autor, abordagem ainda comum em exercícios escolares. Ler é constituir sentido, conforme Goulemot (1996). Dell’Isola completa, afirmando que, a partir do significado oferecido pelo texto, “o leitor escolhe uma dimensão dentro da multiplicidade de possibilidades que a linguagem permite, dando ao texto um sentido que é fruto de uma leitura idiossincrática” (DELL’ISOLA, 1996, p. 72). Esse conceito difere radicalmente do de leitura como deco- dificação, mencionado por Kleiman (1993, p. 20), que consiste na compreensão da leitura como um processo baseado somente na superfície textual. Para ler, em tal acepção, bastaria ser alfabetizado. A decodificação constitui, evidentemente, um dos níveis de leitura; entretanto, ele não é o único. Se assim fosse, haveria um descom- prometimento com a interpretação, com os sentidos e vozes pre- sentes no texto, como se este não fosse polissêmico. A leitura assim compreendida pressupõe um leitor submisso, controlado pela voz do 308 texto, que é decodificada e reproduzida, em um movimento mecâ- nico e acrítico. Adotamos neste trabalho as concepções de linguagem apontadas por Geraldi (1984): 1) Lingua- gem como forma da expressão do pensamento; 2) A linguagem como instrumento de comuni- cação e 3) Linguagem como forma de interação (GERALDI, 1984: 41-47). CONCEPÇÕES DE LEITURA Trata-se de uma concepção autoritária de leitura, que, desconsiderando o leitor, seu contexto e suas experiências, entende ser o texto portador de uma única e verdadeira interpretação; o leitor, por sua vez, é aquele que consegue chegar a essa interpretação. A autora considera que, ao contrário: são os elementos relevantes ou representativos os que contam, em função do significado do texto, a experiência do leitor é indispensável para construir o sentido, não há leituras autorizadas num sentido absoluto, mas apenas reconstruções de significados, algumas mais e outras menos adequadas, segundo os objetivos e intenções do leitor (KLEIMAN, 1993, p. 23). são os elementos relevantes ou representativos os que contam, em função do significado do texto, a experiência do leitor é indispensável para construir o sentido, não há leituras autorizadas num sentido absoluto, mas apenas reconstruções de significados, algumas mais e outras menos adequadas, segundo os objetivos e intenções do leitor (KLEIMAN, 1993, p. 23). Esses diferentes modos de pensar a leitura estão relaciona- dos ao desenvolvimento histórico da pesquisa sobre esse assunto, no qual os focos se alternam, enfatizando ora o autor, ora o texto, ora o leitor (BATISTA, 1991). A ênfase a um ou outro elemento é norteada pela concepção de linguagem27 subjacente à concepção de leitura. Da concepção de linguagem como expressão do pensa- mento decorre o entendimento de que ler é reconhecer o que o autor quis dizer no texto. Trata-se da leitura que se baseia na observação de dados biográficos do autor, seu contexto e intenções, relacio- nando tudo isso ao texto. De acordo com a concepção de linguagem como instru- mento de comunicação, o sentido encontra-se integralmente no texto; elementos exteriores, como experiências de vida do leitor e o contexto, entre outros, não auxiliariam na constituição do sentido do texto. Essa concepção salta aos olhos na afirmação de que ler é “extrair o significado do texto” (LEFFA, 1996, p. 12), o que pressupõe 27 309 S U M Á R I O um leitor persistente e sem história a quem cabe extrair o significado único, preciso e claro, contido no texto. A consideração da subjetividade na linguagem levada a suas máximas consequências deslocou a ênfase para a figura do leitor que, de acordo com sua história, suas experiências, seus objetivos e seu contexto sociocultural atribui significado ao que lê. CONCEPÇÕES DE LEITURA Esse modo de pensar a leitura que caminha do leitor ao texto é denominado leitura por atribuição (LEFFA, 1996). Nessa concepção, os conheci- mentos de todo leitor são importantes, pois cada um lerá o texto de acordo com sua bagagem de experiências. A leitura do texto muda à medida que muda seu leitor. Essa prática de leitura é feita a partir de hipóteses que são levantadas pelo leitor, as quais ele procura com- provar, justificar. Portanto, a ênfase recai apenas no que o leitor tem a dizer sobre o texto. Leffa (1996) destaca que, ao centrar-se em apenas um dos elementos do processo – leitor, texto ou autor – a concepção de leitura torna-se limitada. Isso porque o sentido do texto não está somente no autor, em suas intenções, como se o leitor pudesse adi- vinhar-lhe os pensamentos; nem é totalmente dependente do texto, pois não há uma extração do sentido, mas uma reprodução feita pelo leitor do assunto tratado no texto. Conforme o autor, o conte- údo não passa do texto para o leitor, mas se reproduz no leitor, sem deixar de permanecer no texto. O leitor, por sua vez, não pode ser a fonte de todo sentido, já que limitaria suas interpretações às suas vivências, permitindo ainda que o mesmo texto pudesse ser lido de todas as diferentes formas que seus leitores desejassem, sem neces- sidade de comprovação. Na mesma direção, outros autores concluem que a conceitu- ação de leitura não pode ser parcial, enfatizando apenas o leitor ou apenas o texto: “A leitura acontece ao desencadear-se o processo criativo em que o sujeito e linguagem interagem permanentemente, uma vez que o texto nunca está acabado, não é produto, antes, 310 dispositivo de produção” (DELL’ISOLA, 1996, p. 73). O texto é enun- ciação projetada pelo autor, continuada e perpetuada pelo leitor num processo em que um exerce influência sobre o outro. Trata-se da leitura coprodutora do texto, aquela intersticial entre o autor, mentor do texto; o texto em si, enquanto manifestação de linguagem, e o sujeito- -leitor, cuja objetividade cognitiva lê, literalmente, a pro- dução contida no texto-forma e cuja subjetividade é inte- racional com a linguagem do emissor, para produzir um novo texto-conhecimento, advindo tanto dos caracteres denotativo e conotativo quanto dos espaços lacunares do texto original (DELL’ISOLA, 1996, p. 74). 28 Adotaremos as expressões “leitor competente”, “bom leitor”, “leitor maduro” e “leitor modelo” para nos referimos ao leitor cuja prática de leitura corresponde ao conceito de leitura como interação, já abordado neste capítulo. 29 De acordo com Smith (1999, p. 39), dois tipos de memória são importantes na leitura: Memória de Curto Prazo é a memória instantânea, uma memória temporária que nos possibilita encontrar sentido para aquilo que estamos fazendo no momento. Diferencia-se da Memória de Longo Prazo, que mantém “guardados” os eventos ocorridos há mais tempo. 30 O modelo teórico do processamento “descendente” propõe que, para compreender textos, o leitor parte principalmente dos seus próprios conhecimentos e expectativas – do geral para o particular (MEURER, 1988, p. 264). 31 Já o modelo teórico do processamento “ascendente” propõe que, para compreender textos, o leitor parte do símbolo escrito e, sequencialmente, atribui significado a palavras, frases, orações, parágrafos e textos completos, num movimento do particular para o geral sem basear-se em seus conhecimentos (MEURER, 1988, p. 258). CONCEPÇÕES DE LEITURA S U M Á R I O Pensar a leitura desse modo é dar relevância para o papel do leitor, do autor, do texto e para o processo de interação entre leitor e texto. Trata-se da leitura entendida como interação, um processo complexo que não corresponde somente à decifração de palavras, mas compreende também a associação lógica, o encadeamento e o relacionamento das ideias ali presentes, a assimilação das ideias do autor e sua relação com os conhecimentos prévios do leitor sobre o assunto, e a tomada de posições (AGUIAR, 2004). As palavras de Batista (1991, p. 23) resumem a concepção de leitura que assumimos neste trabalho: A leitura é, portanto, um aspecto, dentre outros, de uma relação de interlocução. Compreendê-la, delimitá-la, con- tê-la dentro de seus limites é, portanto, paradoxalmente, não considerá-la em si mesma, mas em suas relações com os demais aspectos dessa relação interlocutiva: o texto, o autor e as práticas histórico-sociais nas quais essa relação de interlocução se constituiu. A concepção de leitura como interação corresponde a um tipo de leitor, com características próprias, isto é, um leitor compe- tente, do qual tratamos a seguir. 311 A LEITURA E O LEITOR A atividade complexa em que a leitura se constitui, enquanto ato de interação, exige competências variadas do leitor. Este não se resume, como vimos, nem a um mero decodificador, nem a um sujeito inteiramente autônomo, que leva em conta apenas sua pró- pria história de vida, na interpretação de um texto. Neste tópico, procuraremos listar algumas das caracte- rísticas essenciais a um leitor competente28, cujo desempenho encontra-se intrinsecamente relacionado à prática de leitura enten- dida como interação. Tratando da leitura assim concebida, Solé (1998) destaca que ela exige uma postura ativa do leitor. Assumimos, dessa forma, a concepção de leitor desvinculada da ideia de passividade, enten- dendo-o, ao contrário, como coprodutor, agente do texto lido. O leitor interage com o texto quando, por exemplo, ativa seus conhecimentos prévios, buscando, em sua memória de longo prazo29, as informações de que precisa para confirmar as hipóteses feitas ao longo da leitura; quando duvida do texto, questiona-o, des- denha dele, sem exceder-se em suas adivinhações (num processa- mento descendente30). Também interage quando não se deixa limitar pelo texto (num processo ascendente31). 29 Já o modelo teórico do processamento “ascendente” propõe que, para compreender textos, o leitor parte do símbolo escrito e, sequencialmente, atribui significado a palavras, frases, orações, parágrafos e textos completos, num movimento do particular para o geral sem basear-se em seus conhecimentos (MEURER, 1988, p. 258). 312 Meurer (1988, p. 264) afirma que as pesquisas têm apresen- tado o leitor maduro como aquele que usa os processos ascendente e descendente, sem depender exclusivamente de um ou de outro. Isso porque a compreensão do que se lê está além da soma dos símbolos escritos, e se o leitor se fixar apenas no símbolo escrito, nos detalhes, poderá “ficar preso” a eles, não sendo capaz de apre- ender a ideia, em seu aspecto global. Da mesma forma, se confiar em demasia em suas próprias convicções, em sua capacidade de inferir sobre o conteúdo, pode deixar de lado informações (co)tex- tuais pertinentes para a ativação de esquemas necessários para a compreensão adequada do texto. Portanto, o bom leitor não deve confiar demais em uma ou outra fonte de informação, mas utilizar ambas, na prática da leitura. Apesar de o leitor não depender exclusivamente de suas experiências para a compreensão do texto, a capacidade de trans- portar experiências e conhecimentos prévios para o que lê é de fun- damental importância na leitura. Kleiman (1989, p. A LEITURA E O LEITOR 13) enfatiza esse aspecto, afirmando que a utilização do conhecimento prévio se dá na interação dos diversos níveis de conhecimentos disponíveis para o leitor, ao longo de sua vida. E é esse conhecimento adquirido que determina, durante a leitura, as inferências que o leitor fará, com base em marcas formais do texto. Segundo a autora, “são vários os níveis de conhecimento que entram em jogo durante a leitura”. O primeiro deles é o conhecimento linguístico, que tem papel fundamental no processo. A esse respeito, declara: “o conhecimento linguístico [...] é um componente do cha- mado conhecimento prévio sem o qual a compreensão não é pos- sível” (KLEIMAN, 1989, p. 14). Ele abrange a pronúncia da língua e o seu uso, e tem um papel central no processamento do texto, agindo como um “controlador”, que permite agrupar as palavras em unida- des maiores, constituintes da frase, e escolher quais outros agrupa- mentos serão formados a partir das unidades já formadas. 313 Também importante aspecto do conhecimento prévio é o conhecimento textual, uma vez que os textos sociais estão incorpo- rados a gêneros, cuja estrutura determina o tipo de leitura que pode ser feita. O leitor competente não se comporta do mesmo modo, ante diferentes gêneros. Por exemplo, a partir do reconhecimento de que se está diante de uma peça publicitária, tem uma postura: não fica à mercê da “oferta quentíssima”, pois sabe que há um jogo de apela- ções e manipulações originárias do consumismo. Um outro componente do conhecimento prévio é o chamado conhecimento de mundo, do qual um leitor maduro também faz uso ao ler. Ele está relacionado a um conhecimento que pode ser adqui- rido formal (conhecimento de mundo enciclopédico) ou informal- mente (conhecimento parcial ou estruturado) e se refere a tudo o que temos na memória sobre determinados assuntos, situações típi- cas de nossa cultura, que ativamos pelo simples contato com algum mínimo elemento que o lembre. Essas “lembranças”, denominadas esquemas, direcionam nossas expectativas (KLEIMAN, 1989). Para que haja compreensão, o leitor ativa esse conhecimento, a partir de marcas formais presentes no texto. Ele busca, em sua memória de longo prazo, informações relevantes ao que está lendo, a fim de fazer relações e reconstruções, tecendo significados. Além da necessidade de ativar os conhecimentos prévios, o leitor competente é capaz de fazer leituras de acordo com seus objetivos (SOLÉ, 1998). A LEITURA E O LEITOR Ele não é o mesmo leitor sempre, pois não lê os diversos textos da mesma forma, mas utiliza, para cada gênero, estratégias que o façam atingir objetivos pré-determinados. Dessa forma, ele pode fazer uma leitura seletiva, quando precisa encontrar um endereço telefônico em uma lista; será mais detalhista e obedecerá a uma sequência, caso esteja diante de uma receita de bolo. Não manterá a mesma postura, ao folhear uma revista semanal ou um jornal, pois lerá apenas o que lhe convém, passando “olhos” sobre aquilo que não lhe interessar. 314 Porém, tomará nota, sublinhará o texto, buscará fazer relações com o que sabe, quando objetivar apreender um conhecimento. Como diversos são os textos, diversos são os objetivos com que o leitor maduro vai a eles (SOLÉ, 1998). Tais objetivos determinam as estratégias de leitura, bem como a postura física e intelectual que será adotada perante os textos. Contradizendo a leitura idílica, que prevê uma postura ereta e respeitosa, Goulemot (1996, p. 110) assinala que, frente ao texto, podemos adotar diferentes atitudes corporais: “sentado, deitado, alongado, em público, solitário, em pé...”. Objetivos de leitura diversos acompanham posturas diversas, pois uma leitura por prazer acarreta, evidentemente, uma postura física mais relaxada. Essas características de leitor competente denotam que ele é, acima de tudo, um crítico usuário da língua e, sem dúvida, cons- ciente de seu papel na sociedade. Nesse sentido, a escola, uma das principais instituições promotoras da igualdade social e agência de letramento, deve objetivar a formação desses leitores. É o que discutiremos a seguir. A ESCOLA, A FAMÍLIA E A FORMAÇÃO DO LEITOR Os Parâmetros Curriculares Nacionais do Ensino Fundamen- tal destacam a importância da escola na formação de leitores com- petentes, questionando, desde as séries iniciais, quais textos seriam adequados a essa atividade leitora (BRASIL, 1997). Criticam as fra- ses curtas dos livros voltados ao público iniciante, afirmando que “as pessoas aprendem a gostar de ler quando, de alguma forma, a qualidade de suas vidas melhora com a leitura.” (BRASIL, 1997. p. 29). O documento oficial toca apenas de leve na questão do gosto pela 315 leitura, ressaltando mais a necessidade da formação de leitores com- petentes, o que, sob sua ótica, acarretará na formação de escritores: O trabalho com leitura tem como finalidade a formação de leitores competentes e, consequentemente, a forma- ção de escritores, pois a possibilidade de produzir tex- tos eficazes tem sua origem na prática de leitura, espaço de construção da intertextualidade e fonte de referên- cias modalizadoras. A leitura, por um lado, nos fornece a matéria-prima para a escrita: o que escrever. Por outro, contribui para a constituição de modelos: como escrever. (BRASIL, 1997, p. 41). S U M Á R I O Formar bons leitores é desenvolver a capacidade de ler, o gosto e o compromisso com a leitura, e a escola só dará conta de tal responsabilidade se motivar os alunos, se mobilizá-los interna- mente, mostrando-lhes a autonomia que a leitura oferece e quão interessante pode ser essa atividade. Esse resultado demanda, além de esforço, recursos materiais, e, acima de tudo, um exercício de conscientização a respeito do poder transformador da leitura sobre o mundo do educando (BRASIL, 1997). A Base Nacional Curricular Comum (BNCC) (BRASIL, 2018) amplia os objetos de leitura para além dos gêneros impressos já consagrados pela escola e inclui gêneros multissemióticos e mul- timidiáticos. Assim, contempla também as produções de lingua- gem produzidas e veiculadas pelas atuais tecnologias digitais de informação e comunicação. Uma escola que se fundamenta nessa formação tem caracte- rísticas bastante peculiares, de acordo com Silva (1983). Em primeiro lugar, possui uma biblioteca cujo acervo atende às necessidades de professores e de alunos. Os serviços bibliotecários são dinâmicos e fazem parte dos projetos da escola e das decisões curriculares. Em tal escola, a biblioteca não é vista como um lugar “sagrado”, mas como um espaço em que o educando se sente “à vontade”, onde tem liberdade para escolher e até sugerir livros. Antologias por temas e por autores, da editora Ática, que buscam selecionar textos de autores renomados da literatura brasileira e universal. A ESCOLA, A FAMÍLIA E A FORMAÇÃO DO LEITOR Nesse ambiente, 316 os funcionários conhecem seu papel de incentivadores da leitura. O currículo, por sua vez, atenta para a necessidade de contemplar ativi- dades de leitura, como espaço para pesquisas, debates e discussões. Os educadores de uma instituição como essa também são, evidentemente, leitores. Não se restringem a sugerir livros, mas os leem e falam deles para seus alunos. Além disso, incentivam o uso da biblioteca, ensinam a fazer pesquisas e apontam outras fontes para os educandos, além daquelas utilizadas na sala de aula. Sem dúvida, essas atitudes favorecem o aluno, que acaba por tornar-se mais autônomo em seu aprendizado, além de desenvolver sua capa- cidade de escolher gêneros, temas, autores, que vão definindo seu caminho enquanto leitor. Destacamos as palavras de Solé (1998, p. 90) dirigidas àque- les que pretendem um ensino-aprendizagem de leitura: ler é muito mais do que possuir um rico cabedal de estra- tégias e técnicas. Ler é sobretudo uma atividade voluntá- ria e prazerosa, e quando ensinamos a ler devemos levar isso em conta. As crianças e os professores devem estar motivados para aprender e ensinar a ler. Por isso, a formação do leitor competente também não deve menosprezar o caráter de fruição que a leitura pode ter. Essa função é constantemente destacada em coleções escolares clássicas vol- tadas ao público adolescente, como “Para gostar de ler”32 (ou “De conto em conto”), nas quais identificamos um discurso que procura incentivar o leitor para os caminhos da leitura, para a fruição, des- tacando que “são histórias pequenas no tamanho, mas enormes na emoção” e ainda que “em cada uma delas o prazer e a reflexão espe- ram pelo leitor” (DE CONTO, 2002, p. 3). Antologias por temas e por autores, da editora Ática, que buscam selecionar textos de autores renomados da literatura brasileira e universal. 32 317 Um exemplo de iniciativa no sentido de estabelecer novos diálogos com este público atual foi a coleção “Descobrindo os clássi- cos”, que teve como objetivo principal inserir em uma história atual o tema de romances clássicos, a fim de incentivar o conhecimento da literatura clássica, de um modo interessante para o adolescente. Por exemplo, em “Dona Casmurra e seu Tigrão”33 (JEF, 2005), a lingua- gem, o contexto, o enredo e outros aspectos envolvidos na criação da história têm estreita relação com o “habitat” do adolescente. O livro conta a história de Barrão, lutador de jiu-jítsu, que sofre com a incerteza da traição. Na história, precisa ler Dom Casmurro para conseguir passar de ano, quando conhece uma biblio- tecária esquisita que o ajuda a entender tanto o livro quanto sua própria história (JAF, I. Dona Casmurra e seu Tigrão. 2. ed. São Paulo: Ática, 2008). A ESCOLA, A FAMÍLIA E A FORMAÇÃO DO LEITOR Uma das funções da leitura literária é, sem dúvida, provocar o prazer, o lúdico, instalar-nos, através do texto lido, num ambiente ficcional altamente envolvente e maravilhoso, capaz de nos trans- portar de um espaço para outro inimaginável. E as razões do prazer provocado pela leitura são numerosas e pessoais, relacionadas à experiência emocional desencadeada em cada um. Embora, efetivamente, uma das funções mais importantes do texto literário seja saciar “uma espécie de necessidade universal de ficção e de fantasia, que de certo é coextensiva ao homem” (CAN- DIDO, 1972, p. 804), essa leitura por prazer pode ser desencadeada por gêneros que vão de uma revista em quadrinhos até uma biogra- fia, desde que a leitura seja escolhida por critérios pessoais. A propó- sito, Solé (1998, p. 97) afirma que é “fundamental que o leitor possa ir elaborando critérios próprios para selecionar os textos que lê, assim como para avaliá-los e criticá-los”. Mas, o despertar para o prazer da atividade leitora, seja de textos literários ou não, nem sempre tem sido tratado de modo ade- quado pelas instituições que se apresentam como responsáveis pela formação de leitores: a família e a escola. A esse respeito, Rousseau já aconselhava àqueles que desejassem formar o leitor: “[...] faça que a leitura sirva a seus prazeres, e logo ele se entregará a ela sem que você tenha de intervir” (ROUSSEAU apud CERIZARA, 1990, p. 132). 33 318 A constituição do gosto pela leitura decorre da formação de “bons leitores”, condição indispensável ao desenvolvimento social e à realização individual. Dessa maneira, é preciso que o educando esteja convencido das vantagens que “o ato de ler propícia para a vida individual e a construção social” (AGUIAR, 2004). O aluno que rejeita qualquer contato com os textos em sala de aula necessita convencer-se de que praticando a leitura terá mais dinamismo para suas leituras pessoais, ao assistir a um filme ou ler instruções de um jogo. Ainda precisará lembrar-se que, exercitando sua capacidade leitora, terá melhores resultados em um teste ou em um estágio, e nas outras disciplinas, que demandam leituras de comandos, inter- pretações de problemas e entendimentos de conceitos diversos. Por meio da leitura de ficção, poderá “viajar” e viver experiências em lugares aos quais jamais chegaria na vida real. Considerando a necessidade de tornar o ambiente escolar favorável à leitura, Magalhães & Alçada (1990, p. A ESCOLA, A FAMÍLIA E A FORMAÇÃO DO LEITOR 18) enfatizam que “é na escola que se aprende a ler”. A escola é o local privilegiado de contato com os livros”. Essa afirmação é verdadeira, sobretudo para as classes sociais que têm menor acesso aos meios de leitura, fora do contexto escolar. Nesses casos, a responsabilidade da escola na formação desses leitores se intensifica. Contudo, será mais fácil para a escola cumprir esse obje- tivo, se contar com o auxílio do contexto social do aluno: família, instituições que frequenta, grupo de amigos que incentivam a lei- tura e/ou são leitores contribuem decisivamente no processo de formação do leitor. Abordando o assunto, Charmeux (1995, p. 117) salienta o papel dos pais na formação de leitores que gostem de ler, ou seja, o “cará- ter positivo da relação afetiva que une os pais e a coisa escrita é um elemento importante para um aprendizado desejado pela criança”. Essa afirmação é corroborada por pesquisas de psicologia social, 319 S U M Á R I O que afirmam ser grande parte da aprendizagem humana decorrente da observação do comportamento de outras pessoas (SILVA, 1983). Algumas maneiras de tornar positiva a presença do livro na família é indicada por Charmeux (1995). A primeira é a presença fun- cional do objeto livro nos atos da vida familiar cotidiana. A criança observa que a leitura exerce funções variadas no seu dia a dia, ao verificar que membros da família fazem uso do escrito, quando recebem uma correspondência, quando lêem instruções para o uso de um aparelho doméstico ou produzem legendas de fotos, convi- tes, cartas e e-mails. Partilhar as situações de prazer com o escrito é outro impor- tante aspecto apontado, no qual tem especial relevância a tradicional leitura realizada pelos pais, antes de dormir. Essa atividade memorá- vel, devido ao afeto que a caracteriza, torna-se sobremaneira impor- tante para o histórico do leitor. Conduzida, desde cedo, ao mundo da ficção, das histórias, das hipóteses, das imagens que acompanham o escrito nessa fase, a criança, muito provavelmente, verá o texto como uma fonte de prazer, e sentir-se-á motivada a se tornar uma leitora independente. As visitas a bibliotecas e livrarias são referidas como outras maneiras de partilhar a leitura, em família. A exploração conjunta des- ses locais acaba por ampliar a visão da criança, gerando curiosidade e interesse por mundos diferentes do seu, atitudes que facilitarão o desenvolvimento do senso crítico. A ESCOLA, A FAMÍLIA E A FORMAÇÃO DO LEITOR Em consequência dessas visitas, pode-se oferecer livros, como presentes, o que valoriza o objeto da leitura, tornando a obra marcante e individual, para ser lida diver- sas vezes e em lugares variados. Essa abertura cultural pode levar a outras leituras, como as da TV, do noticiário, da leitura das revistas e gibis, em uma espécie de estímulo à leitura do desconhecido, do diferente (CHARMEUX, 1995). 320 Quanto ao acompanhamento da leitura na escola, especifica- mente, Charmeux (1995) ressalta que devem os pais se inteirarem do trabalho da criança, de forma interessada e amiga, e nunca “autoritá- ria”. Para tanto, sugere a autora que os pais explorem as informações sobre as leituras realizadas pela criança na escola, sem exagero, sem invadir o espaço do filho. Acompanhar os progressos da criança implica observar as competências adquiridas e o trabalho que o professor vem reali- zando, atitudes que podem auxiliar na construção do conhecimento, na compreensão, análise e solução das dificuldades, na abertura de caminhos para a construção dos sentidos dos textos. Dessa maneira, a formação de leitores competentes, prioritariamente a cargo da escola, será muito menos laboriosa num contexto social que lhe seja favorável. CONSIDERAÇÕES FINAIS Este capítulo visou retomar algumas concepções essenciais de leitura, com base nos estudos linguísticos principalmente nos estudos decorrentes da década de oitenta e noventa. Acredito que as provocações aqui postas inserem-se no bojo do que a LA propõe, assinalada na “intenção de transgredir, política e teoricamente, os limites do pensamento e da ação tradicionais, não somente entrando em território proibido, mas tentando pensar o que não deveria ser pensado, fazer o que não deveria ser feito” (PENNYCOOK, ano). Nesse sentido, ao invés de aceitarmos prontamente as “formações advindas de secretarias, automáticas e devidamente desejosas de aumentos de IDEB, ou mesmo, inserir na sala de aula o aplicativo de leitura adquirido pela secretaria, é preciso dar uma passo atrás e pensarmos, questionarmos e colocarmos em discussão quais con- cepções de leitura estão sendo mobilizadas nessas ações, por nós e 321 S U M Á R I O por  estes gestores educacionais que executam essas ações antes de nós e - mais problemático ainda - sem contar com nossa opi- nião de educadores. Esse questionamento passa por sermos leitores competentes também. Por fim, reforçamos com nosso estudo que formar leitores é uma  construção que visa promover/despertar/construir a criticidade no uso da linguagem, no seu aspecto socio-discursivo, está para além da decodificação de textos. Ler é assumir posicionamentos, se arriscar, buscar comprovações, relacionar repertórios e construir novos. Além disso, para formar leitores é necessário mais agentes que os presentes na educação, é preciso que a família e outras ins- tâncias sociais acolham esse objetivo. Como provocação, é preciso, porém, ressaltar que, ao tratar da formação de leitor, é necessário retomar a formação do formador de leitor, portanto, a de se considerar os inúmeros cursos de licen- ciatura de Pedagogia e Letras que “pipocam” por aí sem uma aná- lise de qualidade razoável. Muitos professores de Língua Portuguesa e pedagogos saem das universidades e faculdades sem o devido preparo para formar leitores. Daí, ainda é possível encontrar educa- dores(as) que concebem a leitura como atividade sagrada – silen- ciosa e apenas relacionada aos gêneros literários pertencentes ao cânone definido por alguns – ou apenas como atividade lúdica que não necessita de auxílio ou orientações “senta e lê”. CONSIDERAÇÕES FINAIS Outra obstáculo na busca pela formação de leitores é o desa- fio de contar com mais agentes sociais além da escola, haja vista que a família, as instituições religiosas, as secretarias de cultura também serem responsáveis por essa formação – aliás, um desafio grandioso é a construção de boas parcerias entre escola e família, como tam- bém escola e agentes culturais, pois se o agente cultural produz o livro mas não dialoga com seus leitores potenciais, os estudantes, as consequências estão por aí: livros sem leitores. 322 Ainda, como provocação às instituições escolares, na maior parte, estão longe de serem modelos de locais de leitura – uma vez que a biblioteca, via de regra, ou está fechada ou não tem aten- dimento, principalmente quando se trata de escolas públicas de Ensino Fundamental ou colégios para Ensino Médio. Bem certo que os aplicativos estão chegando (no Paraná, em 2022, chegou o Leia Paraná/Odilo), mas a formação leitora e crítica passa por qualidade e não apenas quantidade de livros lidos, ou seja,  é preciso que o pro- fessor(a) esteja apto para orientar, compartilhar da prática da leitura com seus estudantes, ajudando-os a construírem repertórios social- mente críticos e linguisticamente ricos. A leitura no mundo digital é bem-vinda e pode atender a este leitor contemporâneo que está acostumado com o hipertexto que “é uma forma híbrida, dinâmica e flexível de linguagem que dia- loga com outras interfaces semióticas, adiciona e acondiciona à sua superfície formas outras de textualidade” (MARCUSCHI & XAVIER, 2010, p. 208). Daí resgatar as concepções de leitura e polemizar o que está sendo posto pelos gestores educacionais, muitas vezes, sem a devida atenção pedagógica e educacional faz-se necessário. Aliás, no aplicativo paranaense lançado, até o momento, em março de 2023,  não possui obras literárias de autores brasileiros, ou seja, nas escolas paranaenses, onde caberá a literatura brasileira se não consta no próprio aplicativo de leitura proposto pelo estado e sendo exigido seu uso? Assim, os estudos das obras que são cobradas em vestibulares de universidades estaduais e federais, para as quais nosso aluno de escola pública tem cota social, ainda deverá ser uma “luta” do professor indo à caça de obras: nas precárias bibliotecas das escolas ou na esperança de encontrar um “pdf” perdido na web para ser compartilhado com a turma. No mínimo, questionável essa situação. CONSIDERAÇÕES FINAIS Sem dúvida, os desafios são muitos, tantas perguntas, mas se pudermos começar resgatando um pouco os estudos das con- cepções do ler e suas implicações, acreditamos que alguns passos já estão sendo dados nessa longa trajetória. 323 Por fim, finalizamos essa discussão na perspectiva da LA, enquadrando nosso trabalho como “construção de uma verdade contingente, cuja natureza movente e fluída” possibilita sempre revi- sitá-lo e considerar perspectiva diferente e mudanças na pesquisa (LOPES, 2009, p. 37). REFERÊNCIAS AGUIAR, Vera Teixeira de. Conceito de Leitura. In. CECCANTINI, João Luis Cardoso Tápias (Org.). Pedagogia Cidadã: cadernos de formação. São Paulo: UNESP, 2004, p. 61-75. BATISTA, Antônio A. G. Sobre leitura: notas para a construção de uma concepção de leitura de interesse pedagógico. Em Aberto. Brasília, n. 52, p. 21-38, 1991. BRASIL. Secretaria de Educação Fundamental. Parâmetros curriculares nacionais: terceiro e quarto ciclos do ensino fundamental: língua portuguesa/ Secretaria de Educação Fundamental. – Brasília : MEC/SEF, 1997. CANDIDO, Antonio. Literatura e a formação do homem. Ciência e cultura. São Paulo, v.24, n. 9, set. 1972, p. 804. CERIZARA, Beatriz. Rousseau: a educação na infância. São Paulo: Scipione, 1990, p. 132-142. CHARMEUX, Eveline. Aprender a ler: vencendo o fracasso. 2. ed. São Paulo: Cortez, 1995. COLEÇÃO DE CONTO EM CONTO. São Paulo: Ática, 2002. COLEÇÃO DE CONTO EM CONTO. São Paulo: Ática, 2002. DELL’ISOLA, Regina Lúcia Péret. A interação sujeito-linguagem em leitura. In: Magalhães, Izabel (Org.). As múltiplas faces da linguagem. Brasília: UNB, 1996, p. 69-75. FREIRE, Paulo. Pedagogia do Oprimido. 17 ed. Rio de Janeiro, Paz e Terra, 1987. GOULEMOT, Jean Marie. Da leitura como produção de sentidos. In: CHARTIER, Roger (Org.). Práticas de Leitura. Tradução: Cristiane Nascimento. 2 ed. São Paulo: Estação Liberdade, 2001, p. 109-125. JAF, Ivan. Dona Casmurra e Seu Tigrão. São Paulo: Ática, 2005. JAF, Ivan. Dona Casmurra e Seu Tigrão. São Paulo: Ática, 2005. 324 LEFFA, Vilson J. Aspectos da leitura: uma perspectiva psicolinguística. Porto Alegre: Sagra/DC Luzzatto, 1996, p. 10-18. KLEIMAN, Angela. Leitura: ensino e pesquisa. Campinas. Pontes/Ed. Unicamp, 1993, p.12-54. MAGALHÃES, Ana. Maria. ALÇADA. Isabel. Ler ou não ler: eis a questão. Porto Alegre: Kuarup, 1990. MARCUSCHI, Luiz. A. XAVIER, Antônio. C. Gêneros textuais emergentes no contexto da tecnologia digital. In: MARCUSCHI, Luiz. A. XAVIER, Antônio. C. (Orgs.). Hipertexto MARCUSCHI, Luiz. A. XAVIER, Antônio. C. Gêneros textuais emergentes no contexto da tecnologia digital. In: MARCUSCHI, Luiz. A. XAVIER, Antônio. C. (Orgs.). Hipertexto e gêneros digitais: novas formas de construção de sentido. 2. ed; - Rio de Janeiro: da tecnologia digital. In: MARCUSCHI, Luiz. A. XAVIER, Antônio. C. (Orgs.). Hipertexto e gêneros digitais: novas formas de construção de sentido. 2. ed; - Rio de Janeiro: Lucerna, 2005. p. 13-14 e gêneros digitais: novas formas de construção de sentido. 2. ed; - Rio de Janeiro: Lucerna, 2005. p. 13-14 MEURER. José. Luiz. Compreensão de linguagem escrita: aspectos do papel do leitor. In: BOHH. REFERÊNCIAS I; VANDRESEN, P. (Orgs.). Tópicos de Linguística Aplicada. Florianópolis: UFSC, 1988. p. 258- 269. LOPES, Luiz. Paulo da M. (2009). Linguística Aplicada como lugar de construir verdades contingentes: sexualidades, ética e política. Gragoatá, v. 14, n. 27, 30 dez. 2009. PENNYCOOK, Alastair. Uma Linguística Aplicada Transgressiva. In MOITA-LOPES, Luiz. Paulo (Org.). Por uma linguística aplicada indisciplinar. São Paulo: Parábola, 2006. p. 67-83. SOLÉ, Isabel. Estratégias de leitura. Trad. Claudia Schilling. 6. ed. Porto Alegre: Artes Médicas, 1998. 325 16 José Cristovão Maia Lucena Marreiro Leonardo Carvalho de Oliveira HISTÓRIAS EM QUADRINHOS OMO INCENTIVO À LEITURA: UMA REVISÃO BIBLIOGRÁFICA DOI:10.31560/pimentacultural/2023.97778.16 S U M Á R I O INTRODUÇÃO O presente artigo trata das histórias em quadrinhos (HQs) como uma ferramenta no ensino e aprendizado escolar. Como escreve Santos (2001, p. 47), “a criança que não lê nem História em Quadrinhos tampouco se sentirá disposta a enfrentar textos didá- ticos, literários e informativos”. Essas produções seriam o incentivo inicial para o contato das crianças com outros textos. Além disso, as HQs são exemplos de textos multimodais que combinam palavras e imagens para representar uma narrativa. Muitas vezes, as HQs têm sido associadas ao entretenimento, leitura fácil e até ociosidade. Esta mentalidade pode afetar seu uso na educação escolar. Há uma longa história dessas produções sendo empregadas para educação. Um volume crescente de pesquisas, projetos e atividades tendo como objeto as HQs demonstram o inte- resse no tema (VERGUEIRO, 2010; RAMOS, 2017). O objetivo desta pesquisa é fazer uma revisão bibliográfica sobre o uso de gêneros multimodais - HQs - como incentivo à leitura para crianças do Ensino Fundamental I. Entre os objetivos específi- cos, destacamos: 1) observar como a Base Nacional Comum Curri- cular (BNCC) trata as possibilidades educacionais das histórias em quadrinhos; 2) demonstrar alguns elementos que compõem as HQs e 3) caracterizar as histórias em quadrinhos como recurso de incen- tivo à leitura em sala de aula. Este trabalho realiza uma revisão bibliográfica sobre a temá- tica. De acordo com Prodanov e Freitas (2013), a revisão bibliográ- fica consiste na seleção de trabalhos relacionados ao objeto de pesquisa, como documentos, artigos acadêmicos, pesquisas de pós-graduação, entre tantas outras produções. Para compreen- der o papel das HQs na educação, portanto, passamos a buscar 327 “contribuições dos autores dos estudos analíticos constantes dos textos” (SEVERINO, 2007, p. 122). Alguns conceitos são importantes para uma melhor compre- ensão da pesquisa, como: leitura (SANTOS; MORAES; LIMA, 2018), multimodalidade (KRESS; VAN LEEUWEN, 2001; ROJO, 2011; ROJO; BARBOSA, 2015) e HQs (EISNER, 2001; 2005; IANNONE; IANNONE, 2004; VERGUEIRO, 2010). O capítulo está dividido em quatro tópicos: o primeiro trata da importância da leitura e dos usos de HQs em sala de aula de acordo com a BNCC; no segundo tópico são apresentadas algumas observações sobre os gêneros multimodais; os elementos que com- põem as histórias em quadrinhos são abordados no terceiro tópico; por fim, no último, reunimos alguns apontamentos sobre como as HQs podem incentivar o hábito da leitura. LEITURA E HQS A leitura é uma das habilidades mais importantes que uma pessoa pode aprender. Ela cria possibilidades de ganhar conheci- mento e compreensão os quais de outra forma não estariam dispo- níveis. Estimula o desenvolvimento de pensamento crítico e a forma- ção de opiniões. Além disso, a leitura ajuda no aperfeiçoamento de habilidades de escrita e na comunicação de forma eficaz (SANTOS, MORAES e LIMA, 2018). A leitura está inter-relacionada com o processo educacional. Ler é a identificação dos símbolos e a associação de significados apropriados a eles, requer identificação e compreensão. As habi- lidades de compreensão ajudam a criança a entender o sentido das palavras isoladamente e em um contexto. Nesse processo, as HQs se mostram uma poderosa ferramenta, pois articulam imagens 328 e palavras escritas para produzir significado. Esses elementos são organizados em uma determinada maneira e ordem para desenvol- ver uma narrativa (RAMOS, 2010). A legislação educacional explicita a importância da leitura. A BNCC é um documento que contém as diretrizes de aprendizagem para todos os alunos e as alunas no Brasil, desde a Educação Infantil até o Ensino Médio. A BNCC se baseia no princípio de que todos os educandos devem ter acesso a uma educação de qualidade que os prepare para o sucesso na vida, independentemente da sua origem social ou econômica (BRASIL, 2017). O documento estabelece as diretrizes mínimas sobre o que os alunos e as alunas devem ser capazes de fazer em cada série, e a leitura é uma parte importante para atender a esses padrões. Para ajudar os educandos a atingirem as metas de leitura estabelecidas, os docentes precisam oferecer oportunidades para que os estudan- tes pratiquem a leitura e criem um hábito. Leitura no contexto da BNCC é tomada em um sentido mais amplo, dizendo respeito não somente ao texto escrito, mas também a imagens estáticas (foto, pintura, desenho, esquema, gráfico, diagrama) ou em movimento (filmes, vídeos etc.) e ao som (música), que acompanha e cos- significa em muitos gêneros digitais (BRASIL, 2017, p. 72). Em vários pontos, ao longo da BNCC, é possível encontrar referências a diferentes possibilidades de ensino e leitura. De acordo com o documento, a aproximação e familiaridade da criança com as manifestações da língua colaboram com o amadurecimento de um senso crítico e estimulam a prática de leitura. LEITURA E HQS Os conhecimentos sobre os gêneros, sobre os textos, sobre a língua, sobre a norma padrão, sobre as diferen- tes linguagens (semioses) devem ser mobilizados em favor do desenvolvimento das capacidades de leitura, produção e tratamento das linguagens, que, por sua vez, devem estar a serviço da ampliação das possibilidades 329 de participação em práticas de diferentes esferas/ cam- pos de atividades humanas (BRASIL, 2017, p. 65). As HQs também são citadas na BNCC para desenvolver as “capacidades de leitura” dos estudantes. Existe uma associação das HQs ao que foi denominado de “Campo Artístico-Literário”. Esse campo é compreendido da seguinte maneira no documento: Campo de atuação relativo à participação em situações de leitura, fruição e produção de textos literários e artísticos, representativos da diversidade cultural e linguística, que favoreçam experiências estéticas. Alguns gêneros deste campo: lendas, mitos, fábulas, contos, crônicas, canção, poemas, poemas visuais, cordéis, quadrinhos, tirinhas, charge/cartum, dentre outros (BRASIL, 2017, p. 96). Entre as habilidades esperadas que sejam desenvolvidas na disciplina de Língua Portuguesa para o Ensino Fundamental I, refe- rente à “leitura de narrativas visuais”, a BNCC apresenta: “Construir o sentido de histórias em quadrinhos e tirinhas, relacionando ima- gens e palavras e interpretando recursos gráficos (tipos de balões, de letras, onomatopeias).” (BRASIL, 2017, p. 97). As HQs são encaradas como uma das ferramentas a serem empregadas por professoras e professores para criar outros meios para a aprendizagem em sala de aula. As HQs estão presentes em várias disciplinas escolares e, se bem empregadas, podem facilitar o ensino e fomentar a leitura. GÊNEROS MULTIMODAIS Gêneros multimodais utilizam várias formas de linguagem para transmitir uma mensagem. Esta possibilidade de construção de textos é cada vez mais utilizada, pois se mostra eficaz na difusão de determinadas ideias. Além disso, permite que as pessoas sejam 330 S U M Á R I O criativas e explorem várias formas de expressão. Esses textos desa- fiam os leitores e leitoras à medida que trabalham em vários sistemas de signos para construir significado (ROJO, 2011). A leitura e a escrita impressas são e sempre foram multi- modais. Requerem a interpretação de elementos visuais presentes nos textos, como os espaços, cores, fontes ou estilos e, cada vez mais, imagens, e outros modos de representação e comunicação. Uma abordagem multimodal permite que esses recursos semióticos sejam considerados e compreendidos como algo além de estético. Em um contexto escolar, chamar a atenção das crianças para os vários componentes de textos multimodais é um aspecto impor- tante do ensino de leitura. Textos multimodais exigem que os leito- res e leitoras trabalhem em vários sistemas de interpretação e usem diferentes estratégias para compreendê-los. Eles podem incluir uma combinação de elementos escritos e visuais, e podem ser produ- zidos em mídia impressa ou digital. Rojo e Barbosa (2015, p. 108) definem o texto multimodal como “aquele que recorre a mais de uma modalidade de linguagem ou a mais de um sistema de signos, ou símbolos (semiose) em sua composição”. Todos os textos são multi- modais, envolvendo a interação e integração de vários códigos semi- óticos (KRESS; VAN LEEUWEN, 2001). Há muitos benefícios em empregar textos multimodais. Eles podem tornar informações complexas mais compreensíveis e cap- turar a atenção de leitores e leitoras de uma maneira que um texto construído de outra forma não poderia. A HQ é um exemplo de um meio multimodal, à medida que o texto e a imagem trabalham juntos na construção de significados. Os textos utilizados dentro e fora da escola são geralmente acompanhados por imagens visuais e elementos de design, além do texto impresso. Os leitores e leitoras interagem com textos impres- sos que contêm elementos multimodais, como livros ilustrados, tex- tos informativos, revistas e jornais, bem como textos digitais que 331 trazem hiperlinks, imagens de vídeo, música, efeitos sonoros e designs gráficos (FERNANDES, 2020). Existem diferentes possibilidades de textos multimodais e, de fato, as pessoas os utilizam todos os dias. GÊNEROS MULTIMODAIS As HQs representam parte desse campo e, na medida em que podem atuar como análogos a outros tipos de textos multimodais, os quadrinhos abrem uma série de possibilidades para pensar a alfabetização multimodal. A COMPOSIÇÃO DAS HQS Como os objetos são organizados e posicionados em uma imagem visual é chamado de composição. A disposição e o posicio- namento de vários objetos determinam sua importância relativa e como eles interagem com outros elementos em uma imagem. As HQs são uma produção que cria uma narrativa por meio de imagens e palavras. Apesar de serem elementos distintos, ima- gens e textos são efetivos para a difusão de mensagens. Outro ponto importante referente às HQs, a comunicação também quando há apenas ilustrações na história. Podemos dizer que as HQs, como demais formas de arte, compõem o imaginário coletivo de uma comunidade, transmitindo significados que um grupo em uma dada época outorga a determinados acontecimentos, pessoas, hábitos, valores etc. (IANNONE; IANNONE, 2004). A combinação de palavras e imagens – multimodalidade – funciona para criar significado de maneiras muito particulares; em um texto multimodal, o significado é criado por meio do textual e do imagético. A articulação dos recursos textuais com os elementos imagéticos expande o entendimento acerca de situações e perspec- tivas propostas de uma maneira que, se separados, qualquer um des- ses códigos teria mais trabalho para alcançar (VERGUEIRO, 2010). 332 Tendo em vista que essa combinação texto/imagem aparece nas HQs com um desenvolvimento peculiar, o que se caracteriza muito mais do que uma mera complementação de informações de uma linguagem a outra, mas na emergência de uma outra modalidade de interpretação. Sobre a leitura, Eisner afirma que Tendo em vista que essa combinação texto/imagem aparece nas HQs com um desenvolvimento peculiar, o que se caracteriza muito mais do que uma mera complementação de informações de uma linguagem a outra, mas na emergência de uma outra modalidade de interpretação. Sobre a leitura, Eisner afirma que O processo de leitura dos quadrinhos é uma extensão do texto. No caso do texto, o ato de ler envolve uma con- versão de palavras em imagens. Os quadrinhos ace- leram esse processo fornecendo as imagens. Quando executados de maneira apropriada, eles vão além da conversão e da velocidade e tornam-se uma só coisa (EISNER, 2005, p. 9). S U M Á R I O Em sua forma mais simples, uma página de HQ é constitu- ída por dois ou três quadros (retângulos pretos, cujas bordas são chamadas de moldura), inseridos sobre um fundo branco, a página. Cada quadro é (eventualmente) separado por um espaço vazio pro- jetado para dividir duas imagens. A COMPOSIÇÃO DAS HQS A leitura dos quadros acontece na mesma ordem de um livro convencional: da esquerda para direita e de cima para baixo. Os quadros comumente trazem fragmentos de conversações ou atividades cuja duração excede a de um instante capturado em um registro fotográfico (EISNER, 2001). A duração dos quadros não é determinada somente pelos componentes verbais, mas também por elementos visuais, como, por exemplo, linhas de movimento como pode ser observado abaixo: Figura 1 – Ação em um quadro Fonte: Sousa (2007). Figura 1 – Ação em um quadro Figura 1 – Ação em um quadro Fonte: Sousa (2007). Fonte: Sousa (2007). 333 Podemos também destacar nos quadrinhos acima a ausên- cia de palavras. Eisner (2001, p. 16) aponta que é “possível contar histórias somente com as imagens, sem ajuda de palavras, mas é preciso ter uma lógica na sequência das imagens para alcançar sua finalidade”. Nessas situações, os educadores e educadoras devem se atentar para a narrativa visual e buscar estimular a criatividade da turma para que expressem o que acontece na história. Outro elemento importante para as HQs são os balões, entendidos como o espaço no qual está contida a maior parte do texto verbal. Balões são usados para expressar a oralidade ou pen- samento (EISNER, 2001). Figura 2 – Tipo de balões Fonte: Divertudo. Disponível em: <http://bit.ly/3lDPmTD>. Acesso em: 27 nov. 2022. Fonte: Divertudo. Disponível em: <http://bit.ly/3lDPmTD>. Acesso em: 27 nov. 2022. Observamos diferentes modalidades de balões: 1) balão de fala, o que traz os diálogos dos personagens; 2) balão de pensa- mento, explicita o que se passa pela mente do personagem naquele momento (formato de nuvem) e 3) balão trêmulo, que transmite algum tipo de sentimento intenso, no caso a raiva. A legenda é o outro elemento dos quadrinhos que con- tém texto verbal. Esse texto representa a voz do narrador, que, 334 fundamentalmente, tem a função de acrescentar informação à men- sagem transmitida no quadro. Abaixo exemplos de legendas: Figura 3 – Legendas em um quadro Fonte: Sousa (2002). Figura 3 – Legendas em um quadro Fonte: Sousa (2002). Fonte: Sousa (2002). Nos quadrinhos, os efeitos sonoros podem ser uma forma escrita da modalidade verbal. Os vocábulos onomatopeicos são fre- quentemente integrados à composição da imagem, e sua representa- ção pode ter qualidades semânticas e imagéticas, como letras de um enunciado raivoso aparecendo em chamas para enfatizar uma metá- fora da raiva. A COMPOSIÇÃO DAS HQS Abaixo, exemplo de onomatopeias dentro da narrativa: Figura 4 – Onomatopeias Fonte: Gestão Educacional. Disponível em: <http://bit.ly/40aPU2d>. Acesso em: 27 nov. 2022. Fonte: Gestão Educacional. Disponível em: <http://bit.ly/40aPU2d>. Acesso em: 27 nov. 2022. 335 As HQs são compostas por diferentes elementos multimo- dais que podem ser observados nas maneiras como os textos estão dispostos nos balões; nos diferentes formatos das fontes que for- mam a fala dos personagens; nas composições que tornam as cenas inteligíveis; em expressões faciais, em síntese, nos modos de repre- sentação que constituem a HQ. HQS EM SALA DE AULA Por meio dos quadrinhos, os professores e professoras podem motivar os alunos e as aulas a criarem o hábito da leitura de forma mais divertida e interessante. Ao examinar as HQs como textos multimodais e lê-las como um exercício de multiletramentos ou letramentos multimodais, podemos compreender não apenas as práticas letradas que cercam os quadrinhos em particular, mas sobre as práticas letradas que cercam todos os textos multimodais. Em sua composição, portanto, os quadrinhos são multimodais, possibili- tando hábitos que vão além da leitura baseada em palavras (CARVA- LHO, DANTAS e AGUIRRE, 2019; SILVA; VIEIRA, 2018). No primeiro ciclo do ensino fundamental (1º a 5º anos), as crianças deixam “de ver a si mesma[s] como centro do mundo e passa[m] a incorporar os demais a seu meio ambiente, ou seja, evo- luindo em termos de socialização” (VERGUEIRO, 2010, p. 24). Come- çam a reconhecer especificidades de diferentes pessoas e grupos. Os alunos e alunas podem trabalhar com as HQs, assim como serem incentivados a elaborar atividades mais bem desenvolvidas. Tarefas baseadas em HQs podem funcionar como um recurso para aperfeiçoar o conhecimento de gramática e vocabulário em sala de aula. O uso de HQs pode trazer benefícios para a aprendiza- gem da língua portuguesa. Entre alguns benefícios, podemos citar: 336 1) ajudam a melhorar o vocabulário (RAMOS, 2010); 2) tornam o pro- cesso de aprendizagem divertido e significativo (SILVA et al., 2022) e 3) criam hábitos de leitura (CANGUÇU; KORBES, 2011). O uso de HQs estimula e facilita o ensino de vocabulário. Elas ajudam os alunos e as alunas a aprenderem indiretamente por meio das HQs. Elas ajudam os/as estudantes a melhorar seu vocabulá- rio por meio da relação entre as imagens e as legendas. Os alunos e alunas também podem aprender sinônimos, antônimos, figuras de linguagem, acrescentando novas palavras ao seu repertório e familiarizando-se com seu uso. Em sala de aula, a leitura de HQs também pode ajudar a aproximar os/as estudantes de outros textos (AMARAL; GOMES, 2013). O uso de HQs pode tornar o processo de aprendizagem diver- tido e atrativo para as crianças. Os/as discentes poderiam descrever as imagens nas HQs usando adjetivos, por exemplo. Simultanea- mente, se lembrarão do adjetivo porque ele é combinado com a visu- alização do quadro, estimulando o desenvolvimento do vocabulário. Dessa forma, os estudantes, pela leitura de quadrinhos, são constantemente instados a exercitar o seu pensa- mento, complementando em sua mente os momentos que não foram expressos graficamente, dessa forma desenvolvendo o pensar lógico. Além disso, as histórias em quadrinhos são especialmente úteis para exercícios HQS EM SALA DE AULA Na era da internet, os leitores e as leitoras tendem a encontrar informações resumidas online em vez de ler um texto para encontrar essas mesmas informações. Diante dessa situação, as formas de lei- tura têm se transformado. Para aproximar os alunos e alunas de tex- tos em sala de aula e criar um hábito de leitura, o docente pode usar a HQ para ajudar nessa tarefa. As HQs podem ajudar no desenvolvimento de habilidades de leitura, melhorando a compreensão do que é lido, por meio do uso de imagens e palavras. Dessa forma, os estudantes, pela leitura de quadrinhos, são constantemente instados a exercitar o seu pensa- mento, complementando em sua mente os momentos que não foram expressos graficamente, dessa forma desenvolvendo o pensar lógico. Além disso, as histórias em quadrinhos são especialmente úteis para exercícios 337 de compreensão de leitura e como fontes para estimular os métodos de análise e síntese das mensagens (VER- GUEIRO, 2010, p. 24). S U M Á R I O Assim, as imagens funcionam como auxílio para tornar o texto mais compreensível. Elas também poderiam motivar os alunos e as alunas estimulando estratégias de leitura e facilitando a leitura do contexto do texto com o apoio das imagens. O uso de imagens dá suporte às palavras ou gramática utilizadas nos diálogos, tornando o texto mais compreensível. A compreensão e a competência leitora, portanto, podem ser aprimoradas. As pessoas que leem HQs também consomem outros pro- dutos culturais, como periódicos, livros e jornais, sem ignorar as informações obtidas na internet, por meio de redes sociais e sites de notícias. Como afirma Santos (2001, p. 47), “a utilização de qua- drinhos pode ser de grande valia para iniciar o jovem no caminho que leva à consolidação do hábito e do prazer de ler”. O hábito de leitura, portanto, pode ser ampliado e incentivado por meio das HQs e seu emprego em sala de aula, assegurando que muitos estudantes reconheçam as vantagens da leitura e deparando-se com menores inconvenientes para dedicar-se às leituras com intenção de estudo. Além dos benefícios, as HQs também têm seus desafios no processo de ensino. É fundamental, portanto, conhecer os desafios do uso de HQs para que estratégias sejam pensadas para maximizar seu uso durante as aulas. O/a docente precisa desconstruir a per- cepção de que HQs são apenas entretenimento. HQS EM SALA DE AULA Professores e professoras precisam organizar os objetivos de se trabalhar com uma HQ durante a aula (sem ignorar o tempo disponível para a realização da atividade), em seguida, pensar nas estratégias didáticas que serão utilizadas para envolver os educan- dos naquilo que está sendo proposto no plano pedagógico. 338 As imagens visuais não são mais incluídas como ilustração do texto impresso, mas adicionam à história e ao significado geral novas e variadas dimensões. Na HQ, as imagens não são subservientes ao texto impresso, mas um elemento essencial para compreensão da narrativa. Desta forma, os alunos e as alunas podem começar a perceber as ligações entre diferentes formas de usar a linguagem. CONSIDERAÇÕES FINAIS As HQs oferecem os meios para o desenvolvimento da prá- tica de leitura, interpretação e outras vantagens educacionais, como sugerido na BNCC (BRASIL, 2017). O uso de recursos didáticos alternativos, como, por exemplo, as HQs, para auxiliar os alunos e alunas na construção de conhecimento e viabilizar uma compreen- são mais ampla quanto aos conceitos aplicados para o desenvolvi- mento do aprendizado. Os professores e professoras poderiam orientar seus edu- candos e educandas a observarem como as HQs funcionam e os elementos que são importantes para a narrativa, desconstruindo estereótipos. Educadores e educadoras poderiam introduzir a cul- tura popular em sua sala de aula com facilidade e eficácia por meio das HQs. Ao incorporar a cultura popular no currículo, os professores e as professoras superariam a separação que muitos alunos sentem entre suas vidas dentro e fora da escola. Nesta revisão percebemos que, à medida que as culturas e tecnologias de comunicação estão se tornando mais visuais, a educação está sendo cada vez mais entendida como multimodal. As HQs são um formato de publicação multimodal apropriado para essa cultura de educação em mudança e elas têm o potencial de serem utilizadas no processo de ensino, incentivando as crianças a ter contato com textos. 339 Sugerimos que mais pesquisas sobre as HQs na educação sejam realizadas, o que pode ajudar educadores e educadoras a per- ceberem outras possibilidades educacionais em sala de aula. REFERÊNCIAS AMARAL, Elisângela Leal da Silva; GOMES, Nataniel dos Santos. Uso dos quadrinhos para o ensino de gramática: uma análise preliminar. Cadernos do CNLF, v. 27, n. 4, p. 438-455, 2013. AMARAL, Elisângela Leal da Silva; GOMES, Nataniel dos Santos. Uso dos quadrinhos para o ensino de gramática: uma análise preliminar. Cadernos do CNLF, v. 27, n. 4, p. 438-455, 2013. BRASIL. Base Nacional Comum Curricular. Brasília, 2017. BRASIL. Base Nacional Comum Curricular. Brasília, 2017. CANGUÇU, Claudineia Pereira; KORBES, Lenita Maria. O incentivo da leitura por meio de histórias em quadrinhos. Revista Eventos Pedagógicos, v. 2, n. 1, p. 50-60, 2011. CARVALHO, Gisely Karla de Medeiros; DANTAS, Charlise Katiene Ferreira de Mendonça; AGUIRRE, Moisés Alberto Calle. Letramento: entre contos e histórias em quadrinhos. Holos, v. 7, p. 1-13, 2019. EISNER, Will. Quadrinhos e arte sequencial. Tradução de Luis Carlos Borges. 3 ed. São Paulo: Martins Fontes, 2001. EISNER, Will. Quadrinhos e arte sequencial. Tradução de Luis Carlos Borges. 3 ed. São Paulo: Martins Fontes, 2001. EISNER, Will. Narrativas gráficas. Tradução de Leandro Luigi Del Manto. São Paulo: Devir, 2005. FERNANDES, Cláudia Regina Ponciano. Em prol de textos multimodais no contexto escolar: quais, como e por quê? Fórum Linguístico, v. 17, n. 2, p. 4919-4927, 2020. IANNONE, Leila Rentroia; IANNONE, Roberto. O mundo das histórias em quadrinhos. São Paulo: Moderna, 2004. KRESS, Gunther; VAN LEEUWEN, Theo. Multimodal discourse. The modes and media of contemporary communication. London: Arnold, 2001. PRODANOV, Cleber Cristiano; FREITAS, Ernani. Metodologia do trabalho científico: métodos e técnicas da pesquisa e do trabalho acadêmico. Novo Hamburgo: Feevale, 2013. RAMOS, Paulo. Os quadrinhos em aulas de língua portuguesa. In: RAMA, Ângela; VERGUEIRO, Waldomiro. (Org.). Como usar as histórias em quadrinhos na sala de aula. 4 ed. São Paulo: Contexto, 2010, p. 65-85. IANNONE, Leila Rentroia; IANNONE, Roberto. O mundo das histórias em quadrinhos. São Paulo: Moderna, 2004. KRESS, Gunther; VAN LEEUWEN, Theo. Multimodal discourse. The modes and media of contemporary communication. London: Arnold, 2001. PRODANOV, Cleber Cristiano; FREITAS, Ernani. Metodologia do trabalho científico: métodos e técnicas da pesquisa e do trabalho acadêmico. Novo Hamburgo: Feevale, 2013. RAMOS, Paulo. Os quadrinhos em aulas de língua portuguesa. In: RAMA, Ângela; VERGUEIRO, Waldomiro. (Org.). Como usar as histórias em quadrinhos na sala de aula. 4 ed. São Paulo: Contexto, 2010, p. 65-85. 340 RAMOS, Paulo. Tiras no ensino. São Paulo: Parábola Editorial, 2017. RAMOS, Paulo. Tiras no ensino. São Paulo: Parábola Editorial, 2017. S U M Á R I O ROJO, Roxane. Pedagogia dos multiletramentos: diversidade cultural e de linguagens na escola. In: ROJO, Roxane; MOURA, Eduardo. (Orgs.). Multiletramentos na escola. São Paulo: Parábola, 2011, p. 11 - 32. ROJO, Roxane; BARBOSA, Jacqueline Peixoto. Hipermodernidade, multiletramentos e gêneros discursivos. São Paulo: Parábola Editorial, 2015. SANTOS, Roberto. Aplicações da história em quadrinhos. Comunicação & Educação, n. 22, p. 46-51, 2001. SANTOS, Acácia Aparecida; MORAES, Mayara; LIMA, Thatiana Helena. Compreensão de leitura e motivação para aprendizagem de alunos do ensino fundamental. Psicologia E l Ed i l 22 93 101 2018 SANTOS, Acácia Aparecida; MORAES, Mayara; LIMA, Thatiana Helena. Compreensão de leitura e motivação para aprendizagem de alunos do ensino fundamental. Psicologia Escolar e Educacional, v. 22, p. 93-101, 2018. SEVERINO, Antônio Joaquim. Metodologia do trabalho científico. São Paulo: Cortez, 2007. SEVERINO, Antônio Joaquim. Metodologia do trabalho científico. São Paulo: Cortez, 2007. SILVA, Jeniffer Aparecida; VIEIRA, Mauricéia Silva de Paula. Tiras cômicas e charges: potencialidades para promover o letramento multimodal. Revista Práticas de Linguagem, v. 8, n. 2, p. 195-211, 2018. SILVA, Andréa Cristina Teixeira et al. História em quadrinhos na aprendizagem das séries iniciais. Revista Ibero-Americana de Humanidades, Ciências e Educação, v. 8, n. 5, p. 1907-1920, 2022. SOUSA, Maurício de. Turma da Mônica. Água boa para beber. São Paulo: Maurício de Sousa Editora, 2002. SOUSA, Maurício de. Almanaque da Mônica. n. 27. São Paulo: Panini, 2007. SOUSA, Maurício de. Almanaque da Mônica. n. 27. São Paulo: Panini, 2007. VERGUEIRO, Waldomiro. A linguagem dos quadrinhos: uma “alfabetização” necessária. In: RAMA, Ângela; VERGUEIRO, Waldomiro. (Orgs.). Como usar as histórias em quadrinhos na sala de aula. 4. ed. São Paulo: Contexto, 2014, p. 31-64. 341 RESISTÊNCIA TRANS EM UMA PERSPECTIVA DISCURSIVA: A CONTRARRELIGIÃO DE URIAS EM DIABA DOI:10.31560/pimentacultural/2023.97778.17 S U M Á R I O INTRODUÇÃO Neste capítulo, analisamos trechos da canção Diaba, da can- tora e modelo trans Urias, que marcam um combate direto a dis- cursos religiosos que pretendem o apagamento da identidade trans, bem como aqueles que se colocam como uma espécie de contrar- religião, a partir da contradição e da ressignificação de elementos depreciativos, segundo o discurso religioso contestado. Para tanto, tomamos como base teórica a Análise de Discurso (AD) de linha francesa, especificamente aquela inaugurada por Michel Pêcheux (1997), perpetuada, no Brasil, pelos estudos de Eni Orlandi (1999). A importância concedida ao sujeito e suas diversas posições em relação ao discurso na AD pecheutiana fornece um excelente embasamento teórico para o estudo da canção Diaba, uma vez que o eu-lírico da obra perpassa todas as posições-sujeito estabelecidas por Pêcheux no contexto do discurso religioso. Levando em conta as condições de produção da canção, percebe-se que o combate ao discurso religioso (que se infiltra no discurso político) cujo objetivo é segregar integrantes da população LGBTQIA+ (a qual nós, auto- res, integramos) do convívio social ainda é necessário, sobretudo para a população trans, que sofre violências sistematizadas sim- plesmente por existir. Este trabalho, então, propõe, por meio da AD, a ideia de acolhimento de qualquer pessoa pertencente ao espectro da diver- sidade sexual, ao promover e celebrar o discurso de existência desse grupo, em detrimento ao que algumas ideologias religiosas e políticas defendem. A fim, então, de entender como o discurso permite a forma- -sujeito Diaba a partir de elementos do discurso religioso, estrutu- ramos o presente trabalho da seguinte forma: após esta introdu- ção, apresentamos os pressupostos teóricos da AD que sustentam 343 a presente pesquisa; na sequência, comentamos os elementos da fé cristã presentes na canção Diaba e, por fim, explicamos a posição- -sujeito criada por Urias em relação a esse discurso religioso. S U M Á R I O PRESSUPOSTOS TEÓRICOS Como já mencionado, utilizaremos as lições de Michel Pêcheux e Eni Orlandi como embasamento para esta análise. Começo, pois, trazendo à tona a ideia de ideologia. Para Pêcheux (1997), ideologias surgem e se retroalimentam da/na luta de classes. São conjuntos de atitudes práticas que estão em constante embate, sendo que, ao menos uma delas, está em posição de dominân- cia (PÊCHEUX, 1997). A seu tempo, Orlandi (1999, p. 46) esclarece que o “indivíduo é interpelado em sujeito pela ideologia para que se produza o dizer”. O indivíduo torna-se sujeito devido ao processo de interpe- lação. Por exemplo, no discurso político, o sujeito inscrito no apa- relho ideológico político, mais especificamente quando ele é filiado a um determinado partido, compactua com as ideias que o partido defende. Nesse caso, para Althusser (1985), ele está participando de certas práticas regulamentadas das ideias que o partido “oferece”. Essa escolha livre do sujeito em querer filiar-se ao partido “x” e não ao partido “y” ocorre no momento em que ele se depara com as ideologias de ambos. [...] a interpelação do indivíduo em sujeito de seu discurso se realiza pela identificação do sujeito com a formação discursiva que o domina, identificação na qual o sentido é produzido como evidência pelo sujeito e, simultanea- mente, o sujeito é “produzido como causa de si”. [...] Que- remos dizer com isso que é no non-sens (sic) das repre- sentações, que “não se mostram para ninguém, que se configura o lugar do sujeito que toma posição em relação 344 a elas, aceitando-as ou rejeitando-as, colocando-as em dúvida etc. Em suma, “o sujeito” se produz nesse “não- -sujeito” constituído por um amontoado de representa- ções “desprovidas de sentido”, e essa produção é acom- panhada precisamente por uma imposição de sentido às representações (PÊCHEUX, 1997, p. 238, grifo do autor). Isso significa, em outras palavras, que não existe discurso produzido fora de um contexto ideológico. Como apenas os seres humanos, ou melhor, os sujeitos, são capazes de produzir discurso, o sujeito está, obrigatoriamente, inserido em uma ideologia discur- siva (PÊCHEUX, 1997; ORLANDI, 1999). É por essa razão que Orlandi (1999, p. 50), explica que o sujeito é “ao mesmo tempo livre e sub- misso. Ele é capaz de uma liberdade sem limites e uma submissão sem falhas: pode tudo dizer, contanto que se submeta à língua para sabê-la. Essa é a base do que chamamos de assujeitamento”. PRESSUPOSTOS TEÓRICOS Essa contradição entre liberdade e submissão gera os movi- mentos do sujeito em relação ao discurso ideológico (ORLANDI, 1999). Ao se encontrar inserido em determinado discurso, o sujeito pode, segundo Pêcheux (1997), caracterizar-se como “bom sujeito” ou “mau sujeito”. Antes de comentar a forma-sujeito de Pêcheux (1997) é necessário explicar que na perspectiva discursiva que estamos ins- critos, a noção de sujeito advém da sustentação da reconfiguração de três regiões do conhecimento: o Marxismo, pela não transparên- cia da história, a Psicanálise, pela não transparência do sujeito e a Linguística, pela não transparência da língua. Nessa linha, Pêcheux (1997, p. 149) destaca que “o funcionamento da Ideologia em geral como interpelação dos indivíduos em sujeitos (e, especialmente, em sujeitos de seu discurso) se realiza através do complexo de forma- ções ideológicas [...] e fornece ‘a cada sujeito’ sua ‘realidade’”. Dessa forma, o sujeito repete dizeres inscritos em formações discursivas distintas que, para Pêcheux (1997), é o interdiscurso, 345 pois este é atravessado pela memória do dizer e esse processo faz com que o interdiscurso seja apagado, provocando um efeito de transparência da realidade. “Todo indivíduo humano, isto é, social, só pode ser agente de uma prática se se revestir da forma de sujeito. A ‘forma-sujeito’, de fato, é a forma de existência história de qualquer indivíduo, agente das práticas sociais” (PÊCHEUX, 1997, p. 150, grifo do autor). Ou seja, a identificação do sujeito ao Sujeito (universal da Ideologia) é vista materialmente na língua. Os processos discursivos se mostram materializados na lín- gua como pensamentos inéditos, o sujeito interpelado se dá de dis- tintos modos que Pêcheux (1997) o distingue da seguinte maneira: será “bom sujeito” aquele que adequar seu discurso, sem questiona- mentos, à ideologia em que se encontra inserido. Ou seja [...] consiste numa superposição (um recobrimento) entre o sujeito da enunciação e o sujeito universal, de modo que a “tomada de posição” do sujeito realiza seu assu- jeitamento sob a forma do “livremente consentido”: essa superposição caracteriza o discurso do “bom sujeito” que reflete espontaneamente o Sujeito [...] (PÊCHEUX, 1997, p. 199, grifo do autor). Essa primeira modalidade de identificação entendemos como uma identificação plena do sujeito da enunciação com a for- ma-sujeito. Poderíamos citar, como exemplo de bom sujeito, pessoas LGBTQIA+ que aceitam discursos religiosos e políticos contrários às suas próprias existências e apoiam medidas como a “cura gay”. PRESSUPOSTOS TEÓRICOS Já o “mau sujeito” é aquele que, ainda que inserido em deter- minado discurso ideológico, começa a questioná-lo, implicando uma colisão entre o próprio discurso e aquele em que se inscreve. Nas palavras do filósofo: [...] caracteriza-se o discurso do “mau sujeito”, discurso no qual o sujeito da enunciação “se volta” contra o sujeito uni- versal por meio de uma “tomada de posição” que consiste, desta vez, em uma separação (distanciamento, dúvida, [...] caracteriza-se o discurso do “mau sujeito”, discurso no qual o sujeito da enunciação “se volta” contra o sujeito uni- versal por meio de uma “tomada de posição” que consiste, desta vez, em uma separação (distanciamento, dúvida, 346 questionamento, contestação, revolta...) com respeito ao que o “sujeito universal” lhe “dá a pensar”: luta contra a evidência afetada pela negação, revertida a seu próprio terreno (PÊCHEUX, 1997, p. 199-200, grifo do autor). S U M Á R I O O “mau sujeito” instaura um questionamento, mas não rompe com a formação discursiva da forma-sujeito. O sujeito não é consti- tuído mais em sua unidade, mas em sua divisão. Também podemos chamar essa modalidade de contra-identificação do sujeito. Em rela- ção à população LGBTQIA+, pode-se dar como exemplo pessoas que aceitam a própria existência, mas não questionam medidas polí- ticas e/ou religiosas que as segregar. Ocorre que, por vezes, essa colisão de discursos é tão forte que o sujeito rompe com a ideologia em que se encontra. A isso Pêcheux chama de “desidentificação” (PÊCHEUX, 1997). Todavia, como já mencionamos, o sujeito está sempre assujeitado a uma ideologia, pois não existe discurso fora dela (ORLANDI, 1999). É por isso que a desidentificação implica, obrigatoriamente, a inscrição em uma ideologia contrária. Nessa modalidade, o sujeito da enunciação rompe com a forma-sujeito da formação discursiva em que se ins- creve e se identifica com outras. A partir, então, dessa configuração sujeito-discurso-ideolo- gia que analisaremos trechos da canção Diaba, de Urias, sobretudo no que tange ao discurso religioso cristão. CONDIÇÕES DE PRODUÇÃO Segundo Orlandi (1999, p. 30), as condições de produção englobam tanto o “contexto imediato” como o “contexto sócio-his- tórico, ideológico” em que determinado discurso é proferido. Passa- mos, então, a apresentar as condições de produção de Diaba. 347 Urias é uma mulher trans que descobriu a identidade femi- nina a partir da arte drag. Ex-produtora da cantora drag Pabllo Vittar, atingiu certa fama com versões de canções nacionais e internacio- nais. A partir desse sucesso, surge Diaba, primeiro single autoral de Urias, que celebra a sua existência trans e, mediante contradições, despreza o discurso religioso que pretende marginalizá-la. Lançado em 2019, Diaba aparece em um contexto em que a política nacional discutia uma questão importante para a comunidade das pessoas lésbicas, gays, bissexuais, transexuais, travestis, queers, intersexo, assexuais e das demais identidades do espectro da diversi- dade (LGBTQIA+): a criminalização de atos de homofobia. Em junho daquele ano, o Supremo Tribunal Federal, no julgamento da ação direta de inconstitucionalidade por omissão (ADO) nº 26 (BRASIL, 2019), equiparou ao crime de racismo, previsto na Lei nº 7.716/1989 (BRASIL, 1989), a violência às pessoas do espectro da diversidade. A atuação da Suprema Corte foi motivada pela omissão do Poder Legislativo nacional em regulamentar a questão. O julga- mento, no entanto, não foi unânime: dos 11 ministros, três posiciona- ram-se contrariamente a essa equiparação, sob o argumento de que não cabe ao Judiciário legislar. Além desse argumento jurídico, outro aspecto foi levantado no julgamento, provocado pela Associação Nacional de Juristas Evan- gélicos do Brasil (ANAJURE, 2019): a proteção à liberdade religiosa. A partir da atuação da associação mencionada, o STF entendeu que manifestações religiosas, ainda que contrárias à comunidade LGB- TQIA+, não seriam consideradas crime, pois estariam enquadradas na liberdade de culto, prevista no art. 5º, VI, da Constituição da Repú- blica (BRASIL, 1987). Esse embate entre a homofobia e a liberdade religiosa dá a tônica para a discussão que travamos no presente trabalho: o dis- curso contrarreligioso de Urias enquanto Diaba. 348 O “BOM SUJEITO” PROFANO A primeira menção ao mundo religioso ocorre logo após a apresentação de Urias, na seguinte passagem: “U-ri-as! / Muito prazer / Eu sou o oitavo pecado capital” (URIAS et al., 2019). Ao se apresentar como o “oitavo pecado capital”, a cantora se inscreve no discurso cristão, ainda que do lado profano dessa crença. A ideia de pecado capital mobiliza a memória cristã do século XVIII no Brasil, época em que as determinadas atitudes eram consi- deradas tão avessas ao discurso religioso que deveriam ser punidas com a morte, “por serem gestadas pelas desordens das paixões ou pelos instintos desordenados” (FLECK, DILLMAN, 2013, p. 12). São eles: a avareza, a gula, a inveja, a luxúria, a soberba, a preguiça e a ira. Urias, ao se apresentar como o “oitavo pecado capital”, se inscreve no discurso cristão, se equiparando aos demais, como um “novo pecado capital” que “surgiu” na sociedade. Quer dizer, ela não é uma can- tora, um sujeito que vende sua força de trabalho produzindo músicas, mas sim um pecado, algo imoral para/segundo o discurso cristão. Esse fato mobiliza a memória, que, enquanto noção para a AD, não é individual, mas coletiva, segundo Pêcheux (2010). Ela representa a estabilização do discurso que possibilita repetições. Memória deve ser entendida aqui não no sentido direta- mente psicologista da “memória individual”, mas nos sentidos entre- cruzados da memória mítica, da memória social inscrita em práticas, e da memória constituída do historiador. O risco evocado de uma vizinhança flexível de mundos paralelos se deve de fato à diversidade das condições supostas com essa inscrição: é a dificuldade – com a qual é preciso um dia se confrontar – de um campo de pesquisa que vai da referência explícita e produtiva à linguística, até tudo o que toca as disciplinas de interpretação (PÊCHEUX, 2010, p. 50). 349 Com a autodenominação “oitavo pecado capital”, Urias esta- belece que sua existência, enquanto mulher trans, mobiliza a memó- ria de contradição máxima com a fé cristã, pois, assim como os peca- dos capitais existentes, é punível com a morte. Tal memória opera o interdiscurso com a realidade brasileira referente a pessoas trans, visto que o país está no topo da lista no número de assassinatos de pessoas dessa comunidade há 13 anos, apesar da “proteção legal” conferida pela já citado entendimento do STF acerca da criminaliza- ção da homotransfobia (PINHEIRO, 2022). O “BOM SUJEITO” PROFANO S U M Á R I O Nos versos que seguem, Urias mantém a resignação quanto a seu lugar de desprestígio em relação à fé cristã: “Eu sempre fui vista por muitos como o mal / Possuo você, possuir você / Diaba” (URIAS et al., 2019). O “mal”, para a ideologia cristã, mais especifica- mente a católica, é o desvirtuamento total dos dogmas dessa reli- gião: a abertura da vida aos vícios e pecados (SARTIN, 2016). O grau mais profundo desse mal são os demônios, “anjos que pagaram com a queda o uso perverso de sua liberdade” (SARTIN, 2016, p. 455). Os demônios, como a Diaba, são os responsáveis pelo ato de possessão, que ocorre, entre outros casos, quando a “vítima” leva “uma vida dissoluta no pecado, e impenitente”. Assim, ao se inscre- ver no discurso cristão, Urias, adotando a forma bom-sujeito, escla- rece que ela é a forma mais grave de pecado para essa fé (pecado capital), configurando-se na Diaba que pode praticar a possessão dos cristãos que também se desviam da vida virtuosa pregada por essas religiões. 350 PRIMEIRA CONTRADIÇÃO: DIABA ENQUANTO “MAU SUJEITO” No decorrer da canção, entretanto, essa posição “bom sujeito” começa ser abalada. Até aqui, o uso de verbos na primeira pessoa do singular demonstrou uma aceitação pacífica desse lugar desvalorizado, segundo a fé cristã. A partir dos versos “Sua lei me tornou ilegal / Me chamaram de suja, louca e sem moral” (URIAS et al., 2019), interpretamos que o uso do pronome possessivo de ter- ceira pessoa “sua” e a indeterminação do sujeito causada pela con- jugação do verbo “chamar” na terceira pessoa do plural dão indícios de questionamento dessa fé, pois Urias, ao deixar de usar a primeira pessoa verbal, afasta-se de tal ideologia, contra-identifica-se. Surge, então, o primeiro movimento na canção: Urias deixa a forma “bom sujeito” e assume a de “mau sujeito”. É interessante notar que a artista aproxima o discurso reli- gioso do legal nos versos citados. Urias, nesse caso opera a memó- ria de prescrição e punição, que estão presentes tanto no discurso legal (lei, crime e pena) quanto no religioso (dogma, pecado e puni- ção/penitência). Urias demonstra, assim, que a atuação do Estado e da Igreja, ainda que sejam esferas distintas na sociedade brasileira do século XXI, pode se aproximar de forma bastante perigosa para aqueles que são marginalizados por esses sistemas. Ora, no Brasil, o Estado (seja o português, seja o brasileiro), desde o período colonial até a ditadura militar, reprimiu veemente- mente, com base na legislação de cada época, qualquer comporta- mento que desviasse do padrão cis-heteronormativo (MEMÓRIAS DA DITADURA, 201?). Apenas em 1988, com a atual Constituição da República (BRASIL, 1988), o sistema jurídico brasileiro deixou de oprimir diretamente a comunidade LGBTQIA+, sendo que a prote- ção veio apenas, como visto, em 2019. 351 E mesmo nesse contexto de proteção, como já mencionado, representantes da fé cristã, de vertente evangélica, posicionaram-se contra essa proteção estatal. Não obtendo êxito em impedir a cri- minalização da homofobia, essas lideranças religiosas conseguiram resguardar o direito de posicionar-se contra a comunidade sob o véu da liberdade de crença. Entendemos, portanto, que a adoção de uma linguagem impessoal a partir desses versos demonstra o questionamento do sistema jurídico e religioso estabelecido. Ainda, o uso dos tempos pretérito perfeito em “tornou” e do pretérito imperfeito em “chama- ram” permite a interpretação de que essas atitudes não serão mais toleradas pela alegoria Diaba, reforçando a lógica de contraidentifi- cação nesses discursos. PRIMEIRA CONTRADIÇÃO: DIABA ENQUANTO “MAU SUJEITO” Sendo assim, “o efeito daquilo que defini- mos como o interdiscurso continua a determinar a identificação ou a contraidentificação do sujeito com uma formação discursiva, na qual a evidência do sentido que lhe é fornecida, para que ele se ligue a ela ou a rejeite” (PÊCHEUX, 1997, p. 166). Urias só está “permitida” ou “autorizada” a enunciar esses versos, pois está inscrita na formação discursiva cristã, porém, com afastamentos, que produzem questio- namentos, dúvidas, mas não está desvinculada. SEGUNDA CONTRADIÇÃO: A FORMA-SUJEITO RELIGIOSA E A ACEITAÇÃO Seguindo com a nossa análise, há o seguinte trecho: “Vão ter que me engolir por bem ou por mal”. Inscrita, ainda, na ideologia cristã, Urias demonstra que o Outro precisa aceitá-la nesse discurso, seja por bem (“bom sujeito”), seja por mal (“mau sujeito”). Pêcheux (1997) situa a discussão sobre o sujeito a partir de sua forma histórica. Desse modo, a forma-sujeito, como o modo de 352 S U M Á R I O ser sujeito na história, possibilita a inscrição do sujeito em uma for- mação discursiva específica. Esta, por sua vez, determina as possi- bilidades de dizer e de não dizer desse determinado sujeito, em sua posição. É desse modo, portanto, que falamos em posição-sujeito. Compreendemos, pois, na esteira das teorizações de Pêcheux (1997), que, discursivamente, existem, no interior das formações discursivas, posições que os sujeitos ocupam em sua relação com a forma histó- rica de existência dessa formulação. O “bom sujeito”, nesse caso, é aquele que acolhe o “peca- dor”. Entende-se, aqui, como “bom sujeito” porque segue a máxima derivada da exegese bíblica: “Deus ama os pecadores, mas odeia o pecado” (ABIBLIA.ORG, 2022). Já o “mau sujeito” seria aquele que, ao rejeitar a existência trans de Urias, vai contra os ensinamentos cristãos de acolhimento, questionando-os. É interessante verificar que a segunda contradição mobili- zada por Urias, diferentemente da primeira, não se aplica a si, mas opera sobre as seguidoras e seguidores da fé cristã. Ou seja, ao con- tradizer o “bom sujeito” que aceita o lugar de repressão na ideolo- gia religiosa em apreço, tornando-se, portanto, o mau sujeito que contesta esse lugar. Urias cria para cristãos e cristãs duas escolhas que terão o mesmo resultado: a aceitação de sua existência, seja ela pela via do “bom sujeito” (acolhimento do pecador), seja o de “mau sujeito” (descumprimento desse dogma). TERCEIRA CONTRADIÇÃO: A DESIDENTIFICAÇÃO O último movimento contraditório ocorre nos seguintes ver- sos: “Com toda educação / Foda-se sua crença” (URIAS el al., 2019). Para além da óbvia contradição semântica (educação x foda-se), 353 S U M Á R I O esses dois versos que desconstroem todo o encaixe na ideologia cristã que Urias trouxe ao longo da canção. Ora, ao entoar “foda- -se sua crença”, Urias rompe com a ideologia na qual, até então, tentava se inserir. Aí está o terceiro movimento ideológico de Urias: ela não deixa de ser Diaba, pois repete o verso várias vezes, sendo esse, inclusive, o título da canção. No entanto, ocorre a desidentificação, que é “uma tomada de posição não-subjetiva”, proposta por Pêcheux (1997, p. 201, grifo do autor), com o discurso religioso que até então permeou sua existência. Ou seja, o sujeito da enunciação rompe com a forma-sujeito da formação discursiva em que se inscreve e se iden- tifica com outras formações discursivas. O rechaço à ideologia cristã implica a mais importante con- tradição de Urias em Diaba, uma vez que ao mesmo tempo em que se insere nesse discurso tanto enquanto bom sujeito como mau sujeito, a desidentificação rompe com essa ideologia ao referir-se com desprezo a ela. Agora, segundo Pêcheux (1997), é impossível falar em “desassujeitamento”, uma vez que não há sujeito não inter- pelado por um discurso. CONSIDERAÇÕES FINAIS O posicionamento contrarreligioso apresentado por Urias em Diaba soma-se à crescente ideologia de resistência da comunidade trans no Brasil (cf. CARVALHO, 2015). A desidentificação com o dis- curso religioso junta as pessoas integrantes dessa comunidade em um contradiscurso que, ao mesmo tempo que as acolhe e as protege, marca sua (r)existência de forma clara e violenta, se assim for preciso. A violência aqui mencionada é discursiva: o processo de inserção no discurso religioso apenas para com ele se desidentificar 354 S U M Á R I O ocorre sempre por meio de termos profanos, que incomodam aque- les que se identificam efetivamente na ideologia cristã. Além de Diaba, podemos citar a tese de doutoramento da Professora Doutora Megg Rayara Gomes de Oliveira (primeira travesti a atingir essa titu- lação acadêmica na Universidade Federal do Paraná), intitulada O diabo em forma de gente: (r)esistências e gays, afeminados, viados e bichas pretas na educação (OLIVEIRA, 2017), sobretudo a subseção “Bye bye igreja: nada de medo, nada de culpa, nada de pecado!” (OLIVEIRA, 2017, p. 189). Percebe-se que  Urias e a professora Megg Rayara adotaram argumento discursivo similar (Diaba - diabo) para romperem com a ideologia religiosa sobre seus corpos e suas vidas. Mencionamos, também, a cantora, atriz e apresentadora Linn da Quebrada, que se identifica como travesti. Em seu primeiro álbum, Pajubá, o discurso contrarreligioso é encontrado em Submissa do 7º dia (QUEBRADA, 2017). Ao mesmo tempo em que rejeita a ideolo- gia cristã que tenta negar a sua existência, inscrevendo-a em um discurso de submissão e de morte, Linn desvela, de forma obscena, a contradição dos homens que se inserem no discurso cristão cis- -heteronormativo perante a sociedade, mas que buscam relações sexuais com travestis para satisfazerem seus desejos mais profanos. Por fim, com base em nossa análise, é possível dizer que independentemente de proteção ou perseguição estatal, aceitação ou repressão religiosa, as pessoas trans e a comunidade LGBT- QIA+ sempre vão ter um discurso próprio: o da (r)esistência. Como efeito de fim para este trabalho, utilizamos a fala da professora Megg Rayara que sintetiza bem esse discurso: “A bicha não pode ser elimi- nada, ainda que se faça de morta, faça de conta que está em vias de ser eliminada. Quando menos se espera, ela desfaz os laços de fita que tentam aprisioná-la e se lança maravilhosa mundo afora” (OLI- VEIRA, 2017, p. 198). 355 REFERÊNCIAS ALTHUSSER, Louis. Aparelhos Ideológicos de Estado: nota sobre os aparelhos ideológicos de Estado. 2 ed. Rio de Janeiro: Grall, 1985. ALTHUSSER, Louis. Aparelhos Ideológicos de Estado: nota sobre os aparelhos ideológicos de Estado. 2 ed. Rio de Janeiro: Grall, 1985. BRASIL. [Constituição (1988)]. Constituição da República Federativa do Brasil. 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A Igreja Católica, a possessão demoníaca e o exorcismo: velhos e novos desafios. Temporalidades, v. 8, n. 2. Belo Horizonte, 2016. URIAS; HODARI; GORKI; MAFFALDA; ZEBU. Diaba. Rio de Janeiro: Warner Chapell, 2019. Disponível em: < https://youtu.be/_r83_ualtpM>. Acesso em: 19 nov. 2022. 357 Esta forma pode ser vista em Pires (2014). INTRODUÇÃO O presente capítulo foi inspirado por um dos sucessos de Chico Buarque – a música Apesar de Você – escrita em 1970, lan- çada inicialmente como compacto simples; posteriormente, sendo regravada em 1978, quando finalmente entrou para o álbum Chico Buarque (1978). O grande agente responsável por isso foi o período da Ditadura Militar no Brasil, regime que durou de 1964 a 1985, per- manecendo durante 21 anos instaurando censuras em obras de arte de diversos artistas34. Como a obra de Chico, muitas outras de outros músicos, como Caetano Veloso e Gilberto Gil, foram barradas pela censura da época, exigindo alterações, exclusões e silênci(ament)o. Ainda, dentro do disco que leva seu nome, de 1978, Chico agrupou diversos de seus sucessos, entre os quais estão músicas de protesto contra a Ditadura Militar, como Cálice e Apesar de Você, nas quais estão implícitos discursos que indicam a falta de liberdade na época. Assim, partindo disso, abordaremos noções que existem dentro da Análise de Discurso (AD), como a formação discursiva, a memória discursiva e o silenciamento. Estas noções serão de grande importância para nosso gesto de análise em alguns trechos da música de Chico, nos quais pode- mos identificar cenários de censura, esperança e religião. A escolha da música se deu por interesse particular das autoras, a partir de tro- cas anteriores acerca dos conteúdos presentes em Apesar de Você e outras canções do gênero MPB. Alguns estudos anteriores foram analisados, logo é possível notar que a temática já foi apresentada socialmente mesmo que com diferentes enfoques. Em nosso caso, procuramos enfatizar a presença de tais cenários no estudo somente da canção já mencionada, ao invés de utilizar diversos materiais de 34 359 pesquisa. Consideramos que a importância de nosso material esteja em sua contemporaneidade e o modo como muitas das análises podem ser aplicadas nos dias atuais. Assim, dentro da perspectiva discursiva, os fundamentos utilizados para a efetivação deste estudo são, principalmente, as teorias de Eni Orlandi (1993, 2013) e Michel Pêcheux (2010, 2014a, 2014b). UMA BREVE TEORIZAÇÃO ACERCA DAS NOÇÕES DISCURSIVAS A AD tem como principal objeto de estudo a exploração dos mecanismos da determinação histórica dos processos de significa- ção. Seu fundador foi Michel Pêcheux (1938-1983), um linguista e filósofo que ilustrou os efeitos de sentido entre a linguagem e a ide- ologia, que concebe discurso como “efeitos de sentidos (e não trans- missão de informação) entre os interlocutores” (PÊCHEUX, 2014a, p. 82). Isso significa que o sentido (sempre) pode ser outro, conforme Orlandi (2013), a partir da tomada de posição do sujeito e a ideologia se manifesta na ordem da língua. Segundo Pêcheux (2014b), o sujeito está sempre interpelado pela ordem da ideologia e da língua, já que as relações sociais tidas por estes influenciam em seus discursos, como já apontado anterior- mente. Ademais, se temos o sujeito constantemente interpelado a sua ideologia, logo em seu discurso, aquele está constantemente para- lelo ao sentido. No caso, é a partir da colocação de Althusser (2008, p. 219): “reconhecimento mútuo entre os sujeitos e o Sujeito, e entre os próprios sujeitos, e o reconhecimento do sujeito por si mesmo” que Pêcheux discorre sua teoria da formação do sujeito-discurso. Ele afirma que “o funcionamento da Ideologia em geral como inter- pelação dos indivíduos em sujeitos (e, especialmente, em sujeitos 360 de seu discurso) se realiza através do complexo das formações ide- ológicas [...] e fornece ‘a cada sujeito’ sua ‘realidade’” (PÊCHEUX, 2014b, p. 149). Isso significa que, para Pêcheux (2014b, p. 150), trata-se de recuperar as “formas travestidas e ‘fantasmagóricas’ inerentes à subjetividade” discutidas por Lacan e, posteriormente, por Althusser. Já a noção de formação discursiva, Pêcheux (2014b, p. 146- 147, grifos do autor) da seguinte maneira Chamaremos, então, formação discursiva, aquilo que, numa formação ideológica dada, isto é, a partir de uma posição dada numa conjuntura dada, determinada pelo estado da luta de classes, determina o que pode e deve ser dito (articulado sob a forma de uma arenga, de um sermão, de um panfleto, de uma exposição, de um programa etc.). Dessa maneira, o sujeito, ao enunciar seu discurso, está filiado a uma formação discursiva, tendo em vista que as condições de produção produzem determinação sobre/do(s) sentido(s) da formação discursiva. Ao compreender a essencialidade da formação discursiva, temos, também, as condições de produção que podem ser com- preendidas como o local em que o discurso está sendo produzido. UMA BREVE TEORIZAÇÃO ACERCA DAS NOÇÕES DISCURSIVAS Mais especificamente, seriam os discursos inscritos em determina- das condições, no caso em determinados contextos sócio-histórico ideológico (ORLANDI, 2013). Junto dos aspectos formativos de discurso e ideologia, pro- curamos entender a funcionalidade do indivíduo interpelado pelo sujeito e sua ideologia, a qual é uma noção trabalhada por Althusser (2008, p. 96) e, basicamente, trata da maneira como a ideologia pode apontar os indivíduos a sujeitos. Nas próprias palavras do autor: “toda ideologia interpela os indivíduos concretos enquanto sujei- tos concretos, através do funcionamento da categoria do sujeito”. Portanto, ainda de acordo com Althusser (2008), os indivíduos 361 S U M Á R I O já são sujeitos desde o princípio, pois a ideologia já os interpelou anteriormente, havendo esta continuidade. Outra noção que dará suporte em nossa análise é a de esquecimento. De modo geral, o discurso proposto nunca é original, de acordo com Pêcheux (2014b), uma vez que o sujeito “esquece” que sua fala está ancorada em conceitos e ideologias prévias carac- terizando a noção apresentada. Dentro da AD temos dois tipos de esquecimentos, o 1 e o 2: Concordamos em chamar de esquecimento n° 2 o ‘esque- cimento’ pelo qual todo sujeito falante ‘seleciona’ no interior da for- mação discursiva que o domina, isto é, no sistema de enunciados, formas e sequências que nela se encontram em relação de pará- frase [...]. Por outro lado, apelamos para a noção de ‘sistema incons- ciente’ para caracterizar um outro ‘esquecimento’ o esquecimento n° 1, que dá conta do fato de que o sujeito-falante não pode, por defini- ção, se encontrar no exterior da formação discursiva que o domina (PÊCHEUX, 2014b, p. 161). O sentido de uma palavra apaga seu caráter material, fazendo com que ela seja percebida como algo que remete a diferentes dis- cursos. O discurso pertence à ordem da ideologia, ou seja, ao expres- sar algo, as palavras, os discursos estão carregados de ideologia(s). O silenciamento, é outra noção que nos dará suporte, isso porque se torna fundamental para a construção da análise da canção buarqueana aqui analisada. Para Orlandi (1993), o silenciamento se dá de dois modos: estar em silêncio em si, muitas vezes, usando de tal como respostas para determinadas colocações e o silenciamento do “não-dito”, do implícito. Isso porque, de acordo com a autora, “todo dizer é uma relação fundamental com o não-dizer [...]. O sentido não para, ele muda de caminho” (ORLANDI, 1993, p. UMA BREVE TEORIZAÇÃO ACERCA DAS NOÇÕES DISCURSIVAS 12-13). Ele atravessa (n)as palavras, que podem indicar outro sentido ou até mesmo que aquilo que é mais importante nunca se diz. 362 Por fim, a última noção aqui que será utilizada é a de memó- ria discursiva. Esta trabalha, como seu próprio nome indica, com a memória do sujeito em situações sociais e comuns em sociedade, usando do interdiscurso para se dar no indivíduo, este enquanto sujeito, evidentemente. Pêcheux (2010, p. 30), aborda esta noção delimitando-a da seguinte maneira: “memória deve ser entendida aqui não no sen- tido diretamente psicologista ‘memória individual’, mas nos sentidos entrecruzados da memória mítica, da memória social inscrita em prá- ticas e da memória construída do historiador”. Logo, a memória dis- cursiva é uma fala já trazida anteriormente, mas adaptada, se assim podemos colocar, ao contexto social mais próximo do falante. É a partir desse formulado teórico, portanto, que nos emba- samos para discutir os processos de sentido em “Apesar de Você”. A seguir, fazemos algumas reflexões acerca deste objeto e, também, trazemos o percurso metodológico, nosso gesto de análise. A CANÇÃO DE PROTESTO, A CONTEXTUALIZAÇÃO DO COMPOSITOR E DA LETRA DE “APESAR DE VOCÊ” Dentro da Música Popular Brasileira (MPB), no período da Ditadura Militar, especialmente entre os anos de 1976 e 1978, perí- odo mais rígido do Regime Militar, houve um considerável número de cantores e compositores que faziam de maneira direta ou metafó- rica críticas ao governo do país. Artistas como Geraldo Vandré, João Bosco, Aldir Blanc e Taiguara, escreviam canções de um gênero cha- mado de canção de protesto. 363 Junto com eles, se inclui Francisco Buarque de Hollanda – ou Chico Buarque, como é comumente conhecido – é um dos maiores nomes da MPB. Nascido no ano de 1944, no Rio de Janeiro, tem uma discografia composta por aproximadamente oitenta discos, mui- tos deles com grande importância e relevância na história da MPB, assim como Noite dos mascarados, Cálice e outros de seus singles. Um deles foi Apesar de Você, música lançada em 1970, auge da Dita- dura Militar, em um primeiro momento e relançada em seu álbum intitulado com o nome do compositor: Chico Buarque, em 1978. Com características marcadas do samba carioca, o qual crescia demasia- damente no Brasil nos anos 60 e 70, se tornando um hino da MPB e do que, nos anos seguintes, seria colocado como gênero protesto. A Ditadura Militar se deu durante 21 anos, iniciando em 1964 por um golpe civil-militar – uma vez que houve, de certa forma, apoio popular da unidade conservadora do país – e terminando em 1985. Antes do golpe, o presidente brasileiro era o conhecido Jango – João Goulart –, o qual propunha ideais inovadores ao núcleo nacional, como a reforma agrária e outras chamadas de base. Ao declarar tais vontades à sociedade, os grandes latifundiários e parlamenta- res tradicionalistas combateram tais propostas, levando ao que seria conhecido na história como o Golpe Militar de 64. Este se deu, de maneira sucinta, por meio do envio de tanques do exército ao Rio de Janeiro, local em que o então presidente estava, e tomada de poder por Castello Branco – o primeiro dos cinco militares que viriam a ser os presidentes do Brasil. A partir deste “marco” passaram a serem defasadas as par- ticularidades já conhecidas da Ditadura Militar como os atos ins- titucionais (AI) que traziam a censura, tortura e preconceito como idealizadores do que seria o maior genocídio brasileiro. A CANÇÃO DE PROTESTO, A CONTEXTUALIZAÇÃO DO COMPOSITOR E DA LETRA DE “APESAR DE VOCÊ” Logo, censuraram-na, destruindo todas as cópias ainda disponíveis na gravadora, deixando, somente, a gravação original que seria relançada em 1978. A CANÇÃO DE PROTESTO, A CONTEXTUALIZAÇÃO DO COMPOSITOR E DA LETRA DE “APESAR DE VOCÊ” No final dos anos 60, Costa e Silva criou a pior e mais amedrontadora atmosfera na sociedade, criando o famoso AI-5, o qual tinha como princípio a restrição dos direitos políticos e da liberdade de expressão, usando da violência como precursora dos movimentos feitos pelo governo, 364 S U M Á R I O se assim podemos colocar. Estes foram os “anos de chumbo”, parte do ambiente em que Chico Buarque compôs a música aqui analisada. O compositor estava de exílio com sua família na Itália e foi convencido por alguns colegas e amigos de retornar ao Brasil, pois, segundo eles, muitos pormenores melhoraram. Ao voltar, Buarque observou que o que lhe foi dito não se dava de modo total: Eu vim realmente entender o que estava acontecendo quando cheguei de volta em 1970. Era uma barra muito pesada, vés- peras de Copa do Mundo. Foi um susto chegar aqui e encontrar uma realidade que eu não imaginava. Em um ano e meio de distância dava para notar. Aqueles carros entulhados com o ‘Brasil ame-o ou deixe-o’ ou ainda ‘Ame-o ou morra’ nos vidros de trás. Mas não tinha outra. Eu sabia que era o novo quadro, independentemente de cho- ques ou não. ‘Muito bem é aqui que eu vou viver’. Que realmente eu já estava aqui de volta. Então fiz o Apesar de Você (BUARQUE, 1979). Foi durante o governo Médici, de 1970, que a canção foi com- posta levando em consideração perspectivas pessoais e sociais tam- bém, uma vez que usou da experiência de muitos para moldar sua canção e fazê-la uma grande afronta silenciosa à ditadura. Inicialmente, a música passou pelas análises dos censores, uma vez que o “você” da canção pode ser confundido com um par amoroso autoritário. Assim como o próprio compositor colocou ao ser indagado e interrogado posteriormente pelos militares acerca do interlocutor de Apesar de Você: “É uma mulher muito mandona, muito autoritária”, respondeu Buarque. Para nós, é fácil observar a dicotomia presente no sujeito a quem a música é direcionada, mas, inicialmente, não foi assim, circulando bastante nas mídias, em especial com os jovens. No entanto, mais adiante, os censores con- sideraram do que realmente poderia se tratar o cântico depois de uma nota colocada em algum jornal, afirmando que este seria uma 365 homenagem ao presidente Médici. PERCURSO METODOLÓGICO E GESTO DE ANÁLISE A escolha do corpus da pesquisa, a letra de canção de MPB Apesar de Você (1970) de Chico Buarque, foi feita a partir do inte- resse em discutir sobre as condições de produção e os discursos utilizados no discurso buarqueano. Apesar de Você foi determinada, já que apresenta diversos aspectos possíveis de análise discursiva, considerando também as temáticas sociais importantes. Durante a seleção dos constituintes de análise, notamos alguns níveis da aná- lise presente na canção, auxiliando na delimitação deliberada sobre o seguimento dado ao estudo. A análise da canção é feita partindo de três princípios gerais: a censura, a esperança e a religião, presentes grandemente na can- ção de Buarque. Ao estudarmos a letra, notamos outros detalhes passíveis de exploração, no entanto, pensando em esferas maiores de Apesar de Você optamos pela divisão a princípio denotada. Os outros aspectos mencionados são o discurso autoritário, a resistên- cia a qual é presente em grande parte da canção e a invenção, no sentido da Ditadura Militar controlar questões que não necessitam de controle de fato. 366 S U M Á R I O ANÁLISE DE APESAR DE VOCÊ Logo no começo da música de Chico, a letra nos dá sinais de censura, podendo ser percebida já nos primeiros versos: “Hoje você é quem manda / não tem discussão não / falou tá falado / a minha gente hoje anda falando de lado / e olhando pro chão, viu / você que inventou esse estado”. Aqui, quando o eu-lírico da can- ção menciona “você”, tanto no primeiro como no último verso acima mencionado, o efeito de sentido produzido, segundo o contexto em que foi escrita, é que se refere à Ditadura Militar; contudo, conseguiu ser gravada sem ser censurada, pois o significado desse sujeito, na época, implicava à interpretação de que o “você” fosse, na verdade, uma mulher. Somente depois de fazer muito sucesso é que foi vetada pelo Governo Federal, pois foi quando finalmente se deram conta do significado imerso na música. Ainda, além da questão da posição-sujeito, existe nos trechos da música um silenciamento por parte do sujeito que se expressa. Segundo as diretrizes de Orlandi (1993), se trata do silêncio da opres- são. Neste caso, ainda usando os fundamentos de Orlandi, o silêncio não fala, mas significa, que é o caso dos versos de Chico Buarque mencionados acima. Sendo assim, está presente nos discursos que são falados para “não dizer”, ou seja, para não sustentar outro discurso. Assim, no final da música, quando o eu-lírico diz “Você vai se dar mal (lá-lá-iá, lá-lá-iá) / Etc e tal / Lá-lá-iá, lá-lá-iá (lá-lá-iá, lá-lá-iá)”, o “você” ainda se refere ao regime militar, enquanto os ver- sos repetidos “Lá-lá-iá” podem estar significando que quem fala foi silenciado e substitui as palavras que realmente queria utilizar, mas que poderiam ofender o interlocutor, neste caso, o sujeito do regime militar, e por ele ser punido ou censurado ao proferir tais palavras. Desse modo, assim como Milton Nascimento no álbum “Milagre dos Peixes” (1973) – no qual várias músicas foram gravadas apenas com vocalizes, pois as letras haviam sido censuradas – Chico Buarque 367 S U M Á R I O usa do mesmo subterfúgio para demonstrar que nesta letra, e espe- cialmente neste trecho, é evidenciado o processo de silenciamento e apagamento daquilo que gostaria de ser dito e que pela força da censura do poder instituído não se deixou. ANÁLISE DE APESAR DE VOCÊ Preeminentemente, a esperança no canto de Chico Buar- que se dá após a primeira parte da música, logo após as expressões principais de censura da Ditadura Militar na condição social antes delimitada foram situadas na canção. A partir dos versos “Como vai proibir / Quando o galo insistir / Em cantar” podemos definir o iní- cio da formação discursiva esperançosa: o primeiro verso denuncia como tal período político-social era apresentado à comunidade que não cumpria as regras determinadas pelos militares, logo expõe as condições de produção para a composição da canção no geral. Esta denúncia se dá de modo bastante incisivo no momento inicial da canção, em que o compositor traz a realidade da censura e repreensão, contudo não nos estenderemos a isso pois já foi tratado anteriormente. A terminologia “proibir” declara a falta de inocên- cia nas decisões tidas pelo Governo, uma vez que a formação dis- cursiva inscrita por Buarque procura desdobrar e enfatizar aquelas condições precedentes. O segundo e terceiro versos dialogam também com a ideia da censura e também da impossibilidade de comunicar ou expressar, no geral, a tortura e criminalidade – ambas impostas por aqueles que exibe um país econômico e socialmente estável ao exterior. Isso se dá por conta da interpelação de ideologia recorrente no período da Ditadura Militar, no qual os conservadores estabeleciam relações com suas notáveis concepções, fazendo que o estabelecimento do sujeito enquanto opressor social se desse por completo. Ou seja, o sujeito seria aquele que censura, o qual tortura e mata. Já pensando em Buarque, o mesmo ocorre. A ideologia pela qual o compositor é interpelado é contrária à defendida pelos 368 S U M Á R I O ditadores, esses seriam os liberais sustentados por defender a liber- dade de expressão. Em ambos os casos, salientamos que a memória discursiva pode ser notada, pois o discurso utilizado pelos dois polos políticos – ditatorial e democrático – já ocorreram de distintas formas em momentos anteriores, lembrando ainda que a memória discur- siva traz o aspecto do interdiscurso no discurso (ORLANDI, 2013). Ao considerarmos tal noção junto da noção de paráfrase, em uma perspectiva discursiva é “o ‘sentido’ de um texto, de uma frase, e, no limite, de uma palavra, só existem em referência a outros textos, frases ou palavras que constituem seu ‘contexto’” (PÊCHEUX & LÉON, 2014, p. ANÁLISE DE APESAR DE VOCÊ 165), lembramos também do esquecimento 1, o qual, em nosso gesto de interpretação, seria o uso do discurso pelos militares e de Buarque, visto que mesmo antagônicos pertencem a alguém antes de utilizados pelos sujeitos. A estrofe termina com os seguintes versos: “Água nova bro- tando / E a gente se amando / Sem parar”, em que podemos anali- sar com grande evidência, a esperança e a resistência no discurso do cantor. O discurso inicial traz a ideia de que novas ideologias, menos fascistas e opressoras, emergirão na sociedade cedendo espaço àqueles considerados fora do padrão sempre cultuado. É interessante notar que a formação discursiva utilizada pelo cantor está interligada à fala dos jovens, ou aqueles que não concordavam com o “movimento” ditatorial, obviamente reafirmando a formação discursiva inscrita e desenvolvida durante toda a música de Chico. O segundo verso apresenta essa mesma função de espe- rança na canção, e no discurso como um todo, já que no período militar comumente as demonstrações de afeto por parte dos casais homoafetivos eram desconsideradas. “Sem parar” faz um comple- mento à ideia anterior, sendo relacionada às condições de produção do discurso da luta contra a homofobia. Contudo, podemos ir além. O censo e tortura privaram muitas culturas, representações e ide- ais a circularem no meio social, por não se adequarem à formação 369 S U M Á R I O ideológica trazida de uma memória discursiva de ódio e violência. Chico Buarque, ao explorar a pequena frase, coloca que a Ditadura não enfreará o que é distinto do pregado por eles mesmos. O refrão icônico da canção, o qual esteve bastante presente nas mídias sociais no ano de 2018 e rememorada em 2022, também traz muito da esperança já mencionada: “Apesar de você / Amanhã há de ser / Outro dia”. Primeiramente, gostaríamos de salientar que o sujeito “você” deu a possibilidade da música passar pelo censo ditatorial, pois a interpretação fica aberta o suficiente para escolher o interlocutor da frase, uma mulher ou a própria Ditadura. Em nossa análise podemos inferir para quem é de fato dire- cionada a música mas em tempos como aqueles essa pluralidade de significados, possibilitada pelo léxico e condições de produção das canções, auxiliaram grandes nomes a conversar com seu público sem serem assassinados pelos ditadores. ANÁLISE DE APESAR DE VOCÊ Assim, o compositor exter- naliza que independente dos opressores na comunidade, ainda há possibilidade de esperança, pois há possibilidade de acontecer efeti- vamente. Isso se dá por conta da memória discursiva, mais uma vez. A Ditadura Militar foi um movimento recorrente nos países no início do século XX, ainda mais pensando na Segunda Guerra Mundial – a qual carregou nomes como Adolf Hitler e Benito Mus- solini, ditadores conhecidos –, no entanto mesmo sendo um perí- odo de violência foi finalizado. Pois de acordo com a própria memó- ria discursiva sabemos que “tudo tem um começo, um meio e um fim”, logo, os ouvintes de Apesar de Você puderam, de fato, crer na colocação do cantor. Os versos seguintes complementam nossa análise, exatamente por trazer outros aspectos que dialogam com a memória mencionada. Acompanhado da primeira repetição do refrão na música, temos: “Inda pago pra ver / O jardim florescer / Qual você não que- ria”, em que a confiança em um futuro mais solene e menos bruto se 370 S U M Á R I O mostra. É interessante notar os detalhes que o artista utilizou para estar mais perto de seu público e mais distante possível dos militares: o uso da fala coloquial (“inda”), pois não comum à elite – exatamente aqueles que desejavam a perpetuação das ideias conservadoras que os favoreciam. Podemos notar a funcionamento do esquecimento 2 de Pêcheux (2014b) nessa “apropriação” da fala ao escolher as termi- nologias utilizadas, desenvolvendo esta relação silenciosa e vigiada com seus ouvintes. Já o segundo discurso também usa da memória discursiva, pois entendemos, por conta das tantas vezes que foram proferidas tais ideias, que “após uma tempestade, as flores desabro- cham”. Ou ainda que este jardim é impedido de desabrochar com as armas e violência, o que se deu demasiadamente durante a Ditadura, e portanto, entendemos que o jardim renascer seria o fim da violên- cia, o fim da tempestade. Em “Qual você não queria” retomamos a ideia do sujeito do discurso, com suas múltiplas possibilidades de interpretação. No entanto, o que não trouxemos seria a noção do silenciamento na palavra “você”. Os escritores que decidiam se posicionar contra o governo não o podiam fazer de modo explícito, isso já é compre- endido por nós. Ao utilizarem termos que, desacompanhados, não induzissem a nenhum tipo de formação ideológica contrária ao Regime, os manifestantes utilizavam do silenciamento, pensando no não-dito. ANÁLISE DE APESAR DE VOCÊ Não explorar algo na canção foi uma decisão, já que a escolha dos termos teve de ser cautelosa para não defasar o que de fato queria se dizer, contudo foi o suficiente para que fosse interpre- tada de diversas maneiras pelo público. Finalmente, o discurso de esperança é trazido novamente no artigo em “Como vai abafar / Nosso coro a cantar / Na sua frente”, localizado em uma posição ao final da canção. Notamos que a menção ao ato de cantar, no segundo verso, está interligada com o contexto do próprio Chico Buarque e com sua própria condição de produção, uma vez que o compositor também dá a voz à Apesar de Você e à inúmeras músicas que fazem parte da MPB e de seu original 371 S U M Á R I O propósito. Dessa forma, ao mencionar o “coro”, Buarque pro- cura mostrar que há um grande grupo de pessoas que não fler- tam com o conservadorismo exacerbado da Ditadura Militar, pelo contrário. E ao trazer o verbo “cantar”, traz sua própria inscrição enquanto sujeito-cantor. O último princípio analisado por nós seria o do discurso reli- gioso presente fortemente na canção buarqueana, sendo possível notá-lo em: “Você que inventou este estado / E inventou de inventar / Toda a escuridão / Você que inventou o pecado / Esqueceu-se de inventar / O perdão”. O primeiro verso traz o mesmo sujeito analisado no princípio anterior, logo a mesma explicação é aplicada aqui uma vez que essa autoridade é imposta pelo próprio sujeito. Uma auto- ridade similar à tida por Deus, com “-D” maiúsculo já que é o “Todo Poderoso” de acordo com a cristandade, portanto, entendemos que o governo se colocava à população como Deus em sociedade, ou melhor como Deus na terra. A partir da elaboração althusseriana, “o discípulo se reco- nhece no mestre”, podemos fazer uma inferência ao discurso reli- gioso em que ao questionar Deus, o sujeito é penalizado com a morte, assim como no período da Ditadura Militar brasileira, em que ao questionar o regime, o sujeito também é penalizado com a morte. A partir desse entendimento, é possível notar como se repete pelos versos e estrofes seguintes, marcando a soberania e superio- ridade emplacada. Além disso, se é plausível que um ser tenha a possibilidade de inventar, como se dá no segundo verso, tem de possuir grande poderio, realmente sendo similar a um Deus. ANÁLISE DE APESAR DE VOCÊ Aqui, Chico Buarque mostra toda sua barganha ao enfatizar como os opressores realmente percebiam a si mesmos dentro do contexto ditatorial e opressor. Para mais, observamos a dicotomia da escuridão e da cla- reza presente na canção, enquanto este sendo sinônimo de puro 372 S U M Á R I O e coerente, aquele é de impuro e referente ao maligno. Esta dicoto- mia se aplica em diversas estruturas preconceituosas sociais, pois é uma memória discursiva que reproduz uma formação discursiva do bem e do mal bastante estruturada pela Igreja durante a Idade Média e por filósofos do mesmo período, como Maquiavel. Em nossa análise, Buarque usa desta dicotomia para mostrar que a tal soberania criou o mal àqueles considerados fora de um padrão estético-social aceito, tendo a luz, portanto, os semelhantes da soberania somente. A marcação mais evidente do discurso reli- gioso apresentado em Apesar de você seria a menção ao pecado, na qual o compositor enfatiza que essa similaridade com o papel da Igreja foi imposta pelos próprios ditadores para que, assim, tivessem a possibilidade de justificar suas opressões. Por fim, Chico Buarque termina a estrofe mostrando a realidade da Ditadura àqueles que apresentavam ideologias distintas: a falta do perdão. Não pensamos que o perdão devesse ser inventado de fato pelos autoritários, exata- mente por acreditarmos que não devessem apresentar nenhum tipo de poder. O perdão de que Chico fala seria o desrespeito e a falta de prudência com o poderio colocado às suas responsabilidades e utilizadas sem sensatez. Essa canção foi retomada de modo potente no cenário de 2022, por conta da polarização política nas eleições para Presidente da República neste mesmo ano. Dois representantes fortes foram colocados à frente: Luiz Inácio Lula da Silva e Jair Messias Bolsonaro, ambos oponentes de distintas colocações político-sociais. O último conquistou seu público a partir da já conhecida ideia dos valores da família tradicional brasileira – “Deus, pátria e família”, o qual ressoa de uma memória discursiva bastante similar ao que a aristocracia francesa propagava durante a Revolução Francesa –, defendida pela elite e por grande parte da população, pois entendemos que o Brasil é um país conservador o qual pouco luta pela melhoria das condi- ções existenciais das minorias. ANÁLISE DE APESAR DE VOCÊ 373 Lula por outro lado, o qual foi Presidente da República em dois momentos, dialoga com uma ideologia menos centralizada em valores de fato, trazendo seu foco em estratégias para a melhoria da população em si mesmo que em determinados aspectos flerte com um posicionamento mais neoliberal – em específico quando se trata de aspectos econômicos do país. Um dos aspectos mais mar- cantes da eleição foi a apropriação da bandeira brasileira pelos cha- mados “bolsonaristas”, usando do verde e amarelo em seus carros e casas para manifestar sua posição política. Enquanto os chamados “petistas” usam do vermelho para se manifestarem, isso por conta da cor da bandeira do Partido dos Trabalhadores (PT) ou ainda, se pensarmos em memória discursiva, por conta da bandeira comu- nista. Esta eleição não foi marcada somente pelas destoantes cores no cenário nacional, mas pela polarização política inflexível tida por muitos no Brasil. S U M Á R I O CONSIDERAÇÕES FINAIS Doravante às análises discursivas dadas em nosso capítulo, utilizando de fundamentações e noções da própria AD, pudemos analisar que Apesar de Você traz significados múltiplos em sua inter- pretação pela ciência já apontada. Independente do momento histó- rico de que instauramos como referência inicial, neste caso o período ditatorial no Brasil (1964-1985), a canção ressoa na sociedade por conta das memórias discursivas da formação ideológica e também das formações discursivas apresentadaas na canção de Buarque. Ao notarmos que este discurso é repetido em momentos atuais, nota- mos que presenciamos a memória se aplicar nas condições de pro- dução – pensando no contexto social (re)produzido por este discurso. Ao trazermos Michel Pêcheux a partir da tradução única de Eni Orlandi, a qual iniciou a vertente dos estudos semânticos da AD 374 S U M Á R I O ao Brasil, e outros autores que nos auxiliaram a desenvolver e estru- turar nosso capítulo como Althusser, pudemos incrementar nossa análise sob a música buarqueana. Chico Buarque utilizou de sua voz enquanto símbolo social e sua popularidade para dar esperança a um povo que se mostrava desacreditado de qualquer milagre – este nunca criado pelo Deus ditador, o qual não experienciou de fato seus reais efeitos, por ser blindado diante de suas acometidas estratégias. Outrossim, as can- ções buarqueanas, assim sendo a própria Apesar de Você, exploram o efetivo objetivo da MPB na sociedade brasileira: promover a cons- cientização acerca das decisões que interferem no coletivo. Não só pensando em Ditadura Militar, mas na pluralidade de decisões socio- políticas que interferem diretamente na (sobre)vivência de minorias. E com belas técnicas e jogos de palavras, Buarque dissimulou o censo a fim de fazer com que a MPB concretizasse seus propósitos. REFERÊNCIAS ALTHUSSER, Louis. Sobre a reprodução. 2 ed. Tradução de Guilherme João de Freitas Teixeira. Petrópolis, RJ: Vozes, 2008. BUARQUE, Chico. Chico Buarque e a história em Apesar de Você. Disponível em: <http://bit.ly/3J8Ci1E>. Acesso em: 06 nov. 2022. ORLANDI, Eni Puccinelli. As formas do silêncio: no movimento de sentidos. 3 ed. Campinas, SP: Editora da Unicamp, 1993. ORLANDI, Eni Puccinelli. Análise de Discurso: princípios & procedimentos. 11 ed. Campinas, SP: Pontes, 2013. PÊCHEUX, Michel; LÉON, Jacqueline. Análise sintática e paráfrase discursiva. In: ORLANDI, Eni Puccinelli (Org.). Análise de Discurso: Michel Pêcheux. 4 ed. Campinas, SP: Pontes, 2014, p. 159-172. 375 PÊCHEUX, Michel. Análise automática do discurso (AAD-69). In: GADET, Françoise; HAK, Tony (Orgs.). Por uma análise automática do discurso: uma introdução à obra de Michel Pêcheux. Tradução de Bethania S. Mariani et al. 5 ed. Campinas, SP: Editora, da Unicamp, 2014a, p. 59-158. , ( ) , ç ; , Tony (Orgs.). Por uma análise automática do discurso: uma introdução à obra de Michel Pêcheux. Tradução de Bethania S. Mariani et al. 5 ed. Campinas, SP: Editora, da Unicamp, 2014a, p. 59-158. PÊCHEUX, Michel. Papel da Memória. In: ACHARD, Pierre et al. Papel da Memória. 3 ed. Tradução de José Horta Nunes. Campinas, SP: Pontes, 2010. S U M Á R I O PÊCHEUX, Michel. Semântica e Discurso: uma crítica à afirmação do óbvio. Tradução de Eni Puccinelli Orlandi et al. Campinas, SP: Editora da Unicamp. 2014b. PIRES, Thiago Vieira. Ditadura militar brasileira e produção ideológica: um estudo de caso com militares que atuaram no período ditatorial. Cantareira, n. 28, p. 16-35, jan./ jun. 2014. POLITIZE. Ditadura Militar no Brasil. Disponível em: <http://bit.ly/3Hb18M2>. Acesso em 06 nov. 2022. 376 19 Samuel Barbosa Silva SO SOBRE A EDUCAÇÃO MORAL DO MOVIMENTO ESCOLA SEM PARTIDO SIÇÃO ÀS DISCUSSÕES ÊNERO/SEXUALIDADES DOI:10.31560/pimentacultural/2023.97778.19 S U M Á R I O INTRODUÇÃO Nos últimos anos, discursos sobre uma suposta imoralidade no ambiente escolar intermediada pelos professores aos estudan- tes têm ganhado notoriedade nas falas de alguns representantes públicos do nosso país. Além disso, criou-se um movimento no Bra- sil por nome de “Escola Sem Partido” que foi inspirado em outros programas do mundo que centralizam suas discussões voltadas para combater a “doutrinação ideológica” de professores, da escola e do Estado, para com os estudantes. Em nosso trabalho, destacamos o artigo 4 do anteprojeto de Lei Federal elaborado pelo Movimento Escola Sem Partido e a seção “Educação Moral” que consta no sítio do programa. Nossa inquieta- ção surge ao ler o artigo 4 do anteprojeto de Lei, que destaca quais devem ser as funções do professor. Nessa seara, em sua redação, uma das recomendações se refere a educação moral, então, ao acessar a página virtual do movimento, deparamo-nos com a seção Educação Moral em que estabelece discursos, sobretudo, ao com- bate da discussão dos temas de gênero/sexualidades na escola. Dessa forma, nos propusemos a analisar os discursos refe- ridos acima com o objetivo de investigar a posição ideológica do “Escola Sem Partido” no que corresponde ao discurso da Educação Moral, isto é, analisar qual(is) sentido(s) de Educação Moral deve(m) ser mantido(s) e qual(is) deve(m) ser punido(s) na forma da lei. Na mesma direção, outros questionamentos surgem: Quais os efeitos de sentido são construídos sob a expressão “Educação Moral” no anteprojeto de lei federal do programa Escola Sem Par- tido? Por que esta expressão também remete às discussões sobre educação sexual e de gênero? Quais são os discursos válidos para reflexão da “Educação Moral”? Quais sujeitos e discursos são silen- ciados/invalidados neste anteprojeto de Lei? Qual gênero e sexuali- dade se torna inteligível? 378 Utilizamos o aporte teórico-metodológico da Análise de Dis- curso (AD), filiada à teoria do discurso de Michel Pêcheux, que tem o discurso como objeto de análise. Para a discussão do nosso tra- balho também nos respaldamos dos estudos de gênero e sexuali- dades, ancorados em Louro (1997), Wolff e Saldanha (2015), entre outros, bem como resgatamos o processo histórico da formação do Movimento Escola Sem Partido por intermédio de Algebaile (2017) e Silveira (2019). O Partido dos Trabalhadores é um partido político brasileiro. Fundado em 1980, integra um dos maiores e mais importantes movimentos de esquerda e de centro-esquerda da América Latina. Maiores informações em:  www.pt.org.br/. O PROGRAMA ESCOLA SEM PARTIDO: UM MOVIMENTO CONSERVADOR No ano de 2003 tem-se um importante marco histórico na sociedade brasileira, pois um ex-operário e militante do Partido dos Trabalhadores (PT)35, por nome de Luiz Inácio Lula da Silva, torna-se presidente da república brasileira. Com a ascensão de uma repre- sentação político-partidária de esquerda no Brasil, foram criados alguns programas sociais que possibilitaram uma melhor qualidade de vida para muitos trabalhadores. A vitória de Lula nas eleições brasileiras trouxe esperança e resoluções parciais para uma nação que estava subjugada a grandes dívidas e pouca visibilidade no mercado internacional. Mencionar estas conquistas de um governo brasileiro que se posicionou com políticas de esquerda para a transformação do país recupera, em certa medida, a memória de outros líderes de mesma orientação política. 35 379 S U M Á R I O No mesmo ano da posse de Lula, um professor de História, ao ministrar sua aula na educação básica em uma escola privada, resgata esta memória política de um representante de esquerda, o Che Guevara36, e provoca a insatisfação por parte do pai de uma estudante que tem por nome: Miguel Nagib. O desconforto de Nagib ocorre quando o professor “[...] teria comparado Che Guevara a São Francisco de Assis com o intuito de fazer a turma, na qual sua filha estava matriculada, crer que, ambos, Che Guevara e São Francisco, foram abnegados em nome de sua ideologia” (SILVEIRA, 2019, p. 24). De acordo com Silveira (2019), a fala deste professor inco- moda Nagib ao ponto de ele redigir uma carta aberta direcionada ao professor de História, acusando-o de “doutrinação de ideologia política”, todavia, não obteve sucesso com à direção da instituição de ensino, pais e demais membros da comunidade escolar. Não satisfeito com o resultado, Miguel Nagib37 inspira-se em outros movimentos fora do Brasil, que tangenciam nas mesmas ideias apresentadas na carta, para fundar o Movimento Escola Sem Partido (MESP) juntamente com outras pessoas que partilham das mesmas convicções. Dessa forma, o fato ocorrido na escola torna-se um precedente para a emergência deste movimento que começa a criar suas raízes por meio de uma situação subjetiva, mas que, ao mesmo tempo, está associado a um posicionamento político-reli- gioso (SILVEIRA, 2019). Ernesto Guevara, conhecido como “Che” Guevara, foi um guerrilheiro, político, jornalista, escritor e médico argentino-cubano. Foi um dos ideólogos e comandantes que lideraram a Revolução Cubana que levou a um novo regime político em Cuba. Possui a ocupação social de Procurador da Justiça e professa sua crença e espiritualidade no catolicismo. 38 Disponível em: http://www.escolasempartido.org/. 39 Trataremos deste site que contém os anteprojetos mais adiante. O PROGRAMA ESCOLA SEM PARTIDO: UM MOVIMENTO CONSERVADOR Para os fundadores do MESP: [...] a maioria dos professores, livros e programas curricu- lares, desconsideram  a diferença entre o ato de educar e o ato de doutrinar, por esta razão o sítio torna-se ins- trumento de denúncia anônima, por parte dos estudan- tes, ex-estudantes, pais e responsáveis, sobre o conteúdo 36 36 37 380 programático, o  material pedagógico e as atitudes e com- portamento dos professores na transmissão do conheci- mento (ESCOLA SEM PARTIDO, s/d). S U M Á R I O Os fundadores do movimento no Brasil, liderados por Miguel Nagib, criam um site38 para a sistematização das suas ideias, denún- cias, práticas de vigilância, controle e criminalização as práticas de “doutrinação”. “A partir de seu site e de suas ramificações, o Escola sem Partido vem realizando, desde sua criação, ações sistematica- mente orientadas para coibir a abordagem de determinados temas no processo formativo escolar” (ALGEBAILE, 2017, p. 66-67). O acesso ao site do MESP abre um outro link, na página inicial, que possibilita o acesso a um site paralelo que contém os anteprojetos de lei municipal, estadual e federal para que os parla- mentares possam utilizar como modelo e levar para discussão nos seus locais de atuação política39. No site do MESP, há algumas abas que contêm informações sobre a história do movimento, seus funda- dores, objetivos e um espaço para denúncias de estudantes, pais e demais cidadãos que sentirem-se coagidos/lesados com possíveis “doutrinações ideológicas” de professores nas escolas. No site do MESP, coloca-se como um dos objetivos centrais a exigência do discurso do professor e dos materiais utilizados em sala de aula ser apresentados aos estudantes de forma “neutra/impar- cial”, para evitar práticas de “doutrinação ideológica” que venham ferir as convicções religiosas, políticas, morais etc. já estabeleci- das pelas famílias destes estudantes. Todavia, os organizadores do MESP, ao assumirem este posicionamento, também acionam alguns sentidos: o primeiro, refere-se à desqualificação do trabalho docente, pois compreende a atividade como mera “transmissão do saber”, não oportunizando ao estudante possíveis questionamentos ou críticas sobre a conjuntura social na qual está inserido na sociedade. 39 381 Um outro sentido refere-se ao desejo de uma “neutralidade” no dizer do professor em sala de aula, entretanto, essa exigência depara-se apenas com temas que vão de encontro com as convic- ções religiosas, políticas, morais, etc. O PROGRAMA ESCOLA SEM PARTIDO: UM MOVIMENTO CONSERVADOR defendidas pelo MESP, isto é, “o movimento encontra-se em contradição consigo mesmo no que preconiza: contrariamente ao seu programa de neutralidade ético- -política” (SILVEIRA, 2019, p. 26). Além disso, o MESP revela seu comprometimento social e político com “o direitismo político, o con- servadorismo comportamental, bem como o reacionarismo cultural” (DEMIER, 2017, p. 90). A discussão política no MESP é fomentada por meio da ade- são dos parlamentares de diferentes partidos que se assemelham às ideias inscritas numa perspectiva ideológica de políticas de direita que sustentam uma visão de sociedade mantenedora dos interesses conservadores de sua época em todas as esferas sociais. Nesse ínterim, Algebaile (2017) nos diz que o MESP é responsável por esta- belecer procedimentos de vigilância com a criação de anteprojetos de lei para monitorar as atividades e materiais escolares e acadêmi- cos discutidos pelo professor na sala de aula. A finalidade destes anteprojetos de lei funciona “tanto como um instrumento estratégico de mobilização e propaganda, quanto como um instrumento jurídico-político de controle da escola” (ALGE- BAILE, 2017, p. 70). Os parlamentares que se identificam com o MESP utilizam tais anteprojetos para sustentar suas campanhas políticas e reforçam a legitimação das ideias conservadoras sobre o que é a escola, a atividade do professor e cristaliza uma possível homoge- neização de sentido sobre o “verdadeiro” processo de ensino-apren- dizagem do estudante. Para o MESP, a participação dos parlamentares é fundamen- tal, pois, com a aprovação destes anteprojetos de lei, permite-se “[...] o controle prévio da atividade escolar, da atividade docente e da discus- são educacional, a partir da disseminação da ameaça de exposição 382 pública e de criminalização” (ALGEIBAILE, 2017, p. 71). Somado ao fator político, Algebaile (2017, p. 70) afirma que: [...] o teor do modelo de Projeto de Lei e sua forma de divulgação difundem, por si, um clima de vigilância sus- peição, denúncia e punição, não é preciso haver um pro- jeto aprovado para que se cumpra parte dos efeitos espe- rados, que não são necessariamente a efetiva responsa- bilização criminal e a aplicação jurídica de punição, mas a autocensura, o constrangimento e a coibição de compor- tamentos e práticas que possam, mesmo remotamente, ser identificados como “doutrinação” ou “desrespeito às convicções morais da família”. O PROGRAMA ESCOLA SEM PARTIDO: UM MOVIMENTO CONSERVADOR S U M Á R I O Com a falta de informação adequada, formação educacional, acompanhamento nas diversas atividades escolares dos seus filhos/ as e a situação político-social do Brasil, instala-se um clima de tensão entre pais/responsáveis e demais membros da comunidade escolar, especialmente os professores, que são submetidos à censura sobre o que podem e devem dizer/fazer em suas práticas profissionais. Sendo assim, boa parte destes pais/responsáveis, estudan- tes e outros cidadãos, que se alinham ao pensamento do MESP, encontram subsídios no site deste movimento que possam ser uti- lizados para denunciar professores que utilizem metodologias de ensino plural em sala de aula e envolve os diversos saberes sobre um mesmo tema. Dessa forma, o MESP, por meio do seu site, passa a impressão de um movimento coletivo, no entanto, idealiza: [...] o propósito subjacente da suposta interatividade, por meio da qual também as pessoas atendem ao chamado do Escola sem Partido, reforçando suas ideias e ações, não é ampliar uma associação de pessoas que, coletiva- mente e de forma horizontal e solidária, conduzem uma luta relacionada a objetivos comuns. É incorporar pes- soas de forma utilitária, seletiva e subordinada, de modo que elas possam, por meio de sua participação fragmen- tária, referendar posições que já estão definidas e deci- sões que são tomadas por um rol bem mais restrito de participantes (ALGEBAILE, 2017, p. 69). 383 Com isso, o MESP se organiza com caráter personalista, cen- tralizado e partidário em sua coordenação (ALGEBALILE, 2017) ao advogar discursos que estão devidamente orientados a práticas polí- ticas, religiosas e ideias morais específicas, persuadindo assim pes- soas que ocupam diferentes cargos na sociedade. Entretanto, estes sujeitos se filiam às ideias defendidas pelo movimento com objetivo de cercear e intimidar principalmente os professores que se posi- cionam a favor de uma transformação social também por meio de uma educação humanizada, pluralizada e descentralizada das ideias dominantes do sistema educacional vigente. S U M Á R I O Nesta seção, nossa intenção é de fazermos uma sucinta abordagem sobre o surgimento das cate- gorias de gênero e sexualidade no século XX.   O movimento pelo sufrágio feminino é um movimento social, político e econômico de reforma, com o objetivo de estender o sufrágio (direito de votar) às mulheres. Em 1893, a Nova Zelândia se tornou o primeiro país a garantir o sufrágio feminino, graças ao movimento liderado por Kate Sheppard. GÊNERO E SEXUALIDADES: UMA BREVE CARTOGRAFIA40 A década de 1960 é marcada por muitas revoluções sociais, históricas, culturais, linguísticas, etc. que movimentaram muitas dis- cussões na sociedade, sobretudo, ao romper com paradigmas hege- mônicos atribuídos a mulheres e homens em suas práticas cotidia- nas. Seguindo este percurso de revoluções, nesta época, temos a segunda onda do movimento feminista que perpassa países como Estados Unidos, França, Alemanha, Inglaterra, entre outros, com demandas que não haviam sido alcançadas no primeiro momento sufragista41. Dessa forma, O movimento feminista contemporâneo ressurge, expres- sando- senão apenas através de grupos de conscientiza- ção, marchas e protestos públicos, mas também através Nesta seção, nossa intenção é de fazermos uma sucinta abordagem sobre o surgimento das cate- gorias de gênero e sexualidade no século XX. 40 41 O movimento pelo sufrágio feminino é um movimento social, político e econômico de reforma, com o objetivo de estender o sufrágio (direito de votar) às mulheres. Em 1893, a Nova Zelândia se tornou o primeiro país a garantir o sufrágio feminino, graças ao movimento liderado por Kate Sheppard. 384 S U M Á R I O de livros, jornais e revistas. Algumas obras hoje clássi- cas — como, por exemplo, Le deuxième sexe, de Simone Beauvoir (1949), The feminine mystíque, de Betty Friedman (1963), Sexual politics, de Kate Millett (1969) — marcaram esse novo momento (LOURO, 1997, p. 16, grifos da autora). de livros, jornais e revistas. Algumas obras hoje clássi- cas — como, por exemplo, Le deuxième sexe, de Simone Beauvoir (1949), The feminine mystíque, de Betty Friedman (1963), Sexual politics, de Kate Millett (1969) — marcaram esse novo momento (LOURO, 1997, p. 16, grifos da autora). de livros, jornais e revistas. Algumas obras hoje clássi- cas — como, por exemplo, Le deuxième sexe, de Simone Beauvoir (1949), The feminine mystíque, de Betty Friedman (1963), Sexual politics, de Kate Millett (1969) — marcaram esse novo momento (LOURO, 1997, p. 16, grifos da autora). O movimento feminista emerge inspirado em mulheres con- temporâneas de sua época que militavam também através da escrita de seus livros. 42 Em 1949, uma obra de cunho filosófico marcou o cenário francês, e mundial: “O segundo sexo”, de Simone de Beauvoir, um livro que analisava, sob vários aspectos, as causas e as maneiras pelas quais as mulheres estavam historicamente subordinadas aos homens, na sociedade ocidental (CHAPERON, 1999). 43 Diz respeito ao órgão sexual que possuímos em nosso corpo. Todavia, este conceito também passa a ser entendido como constructo sociocultural a partir das discussões da teoria queer. 44 É um antropólogo cultural americano mais conhecido como ativista e teórico da política sexual e de gênero. 45 De acordo com Pedro (2005) o uso da nomeclatura gênero já havia sido utilizada para diferenciar às pessoas intersexuais das não intersexuais pelo psicólogo Robert Stoller, em 1968. Em 1949, uma obra de cunho filosófico marcou o cenário francês, e mundial: “O segundo sexo”, de Simone de Beauvoir, um livro que analisava, sob vários aspectos, as causas e as maneiras pelas quais as mulheres estavam historicamente subordinadas aos homens, na sociedade ocidental (CHAPERON, 1999). GÊNERO E SEXUALIDADES: UMA BREVE CARTOGRAFIA40 A publicação de obras famosas, como “Le deuxième sexe”, que contém a célebre frase de Beauvoir (1949) “[...] ninguém nasce mulher, torna-se mulher”42, provocam inquietações nas mulhe- res que acentuam cada vez mais em seus diálogos a necessidade de reivindicar a visibilidade da mulher nos diversos espaços sociais, bem como pensar as relações de homens e mulheres para além de uma distinção biológica orientada pelo sexo43. Neste momento, sociólogas, historiadoras e ativistas de alguns movimentos sociais marginalizados reúnem-se e passam a discutir sobre essa visibilidade das mulheres como fundamental no processo das relações sociais. Respaldado por estas reflexões, Gayle Rubin (2010)44 foi o responsável por trazer a categoria de gênero para a teoria feminista45 pela primeira vez em seu livro “The Traffic in Women”, no ano de 1975, ampliando assim o entendimento sobre as mulheres para além de um biologismo, de um órgão anatômico, mas ser mulher como uma construção social. 42 Diz respeito ao órgão sexual que possuímos em nosso corpo. Todavia, este conceito também passa a ser entendido como constructo sociocultural a partir das discussões da teoria queer. É um antropólogo cultural americano mais conhecido como ativista e teórico da política sexual e de gênero. De acordo com Pedro (2005) o uso da nomeclatura gênero já havia sido utilizada para diferenciar às pessoas intersexuais das não intersexuais pelo psicólogo Robert Stoller, em 1968. 385 Na mesma direção, há a publicação do artigo de Joan Scott, intitulado “Gênero: uma categoria de análise histórica”, trazendo a discussão do ser mulher para o campo do gênero, objetivando “rejei- tar um determinismo biológico implícito no uso de termos como sexo ou diferença sexual, elas desejam acentuar, através da linguagem, o caráter fundamentalmente social das distinções baseadas no sexo” (SCOTT, 1995, p. 72). Emergem-se os estudos no campo de gênero (social) que em um primeiro momento reúne as principais pesquisas etnográfi- cas que descrevem a vida e o trabalho das mais diferentes mulheres no dia a dia (LOURO, 1997). Todavia, este trabalho de recuperação da história e vida das mulheres não se torna suficiente, ao ser con- templado apenas descritivamente, há uma subversão dos paradig- mas teóricos vigentes (marxismo, psicanálise, etc.) que ampliaram a leitura de uma mulher universal, possibilitando a compreensão das subjetividades das mulheres, isto é, entende-se a mulher plura- lizada, com necessidades específicas a partir das divisões de clas- ses, etnias/raças, gerações, etc., consequentemente às questões de gênero também avançam. Wolff e Saldanha (2015, p. 38) afirmam que o termo ‘“Queer “pode ser traduzido por estranho, talvez ridículo, excêntrico, raro, extraordinário”, diz Louro (2004, p. 38), e era usado nas línguas anglo-sax- ônicas como um xingamento que denotava anormalidade, perversão e desvio. Queer era, então, usado como sinônimo de estranho, diferente dos demais. A escolha de queer para denominar uma nova proposta teórica servia para destacar o compromisso em desenvolver uma análise da normalização de identidades que, naquele momento, era focada na sexualidade”. GÊNERO E SEXUALIDADES: UMA BREVE CARTOGRAFIA40 Ao trazer a categoria de gênero “pretende-se, dessa forma, recolocar o debate no campo do social, pois é nele que se cons- troem e se reproduzem as relações (desiguais) entre os sujeitos. [...] é no âmbito das relações sociais que se constroem os gêne- ros” (LOURO, 1997, p. 22). Destarte, entende-se gênero entre duas proposições: “[...] o gênero é um elemento constitutivo de relações sociais fundadas sobre as diferenças percebidas entre os sexos e o gênero é um primeiro modo de dar significado às relações de poder” (SCOTT, 1990, p. 86). Com a reflexão sobre gênero enquanto um instrumento ana- lítico e político, surge uma alternativa possível para explicar o funcio- namento da desigualdade social das mulheres e dos homens com suas respectivas subjetividades, além disso, abre-se um retorno ao 386 S U M Á R I O diálogo sobre as sexualidades, uma vez que o movimento LGBT tam- bém se apropria destas discussões em anos posteriores. Assim, no fim da década de 1980 surge nos Estados Unidos a teoria queer46, que nasce “como forma de oposição e crítica aos estu- dos sociológicos sobre gênero e minorias sexuais, com o anseio de tentar entender a dinâmica da sexualidade e do desejo na organiza- ção das relações sociais” (WOLFF; SALDANHA, 2015, p. 37), sendo assim, a sexualidade passa a ser compreendida como um compo- nente dos estudos de gênero. Todavia, a teoria queer se diferencia dos estudos sociológicos vigentes na época, pois esta última ressaltava “um pressuposto de que a forma ‘normal’ de sexualidade eram as relações ‘heterosse- xuais’” (WOLFF; SALDANHA, 2015, p. 37). De acordo com Miskolci (2009), a norma da sexualidade nos estudos sociológicos era apenas compreendida sobre uma matriz heterossexual e as demais formas de aparição das sexualidades seriam desvios do padrão de sexualidade. A contribuição da teoria queer torna-se necessária, pois visi- biliza sujeitos insurgentes, desviantes e invalidados pelas normas de gênero e sexualidades estabelecidas socioculturalmente. Como afir- mam Wolff e Saldanha (2015, p. 37), “Apesar de tanto a teoria queer, quanto a sociologia (e a teoria social) compreenderem a sexualidade como uma construção social e histórica”, nos estudos queer a sexu- alidade passa a ser pluralizada e legitimada fora do eixo do “desvio”, da não “normalidade”,  integrando o campo do desejo. Somado a isto, Jeffrey Weeks (1993, p. GÊNERO E SEXUALIDADES: UMA BREVE CARTOGRAFIA40 6) afirma inúmeras vezes que “a sexualidade tem tanto a ver com as palavras, as imagens, 46 387 o ritual e a fantasia como com o corpo”, não podendo assim ser redu- zida apenas ao componente biológico, uma vez que ele fala da impos- sibilidade de se “compreender a sexualidade observando apenas seus componentes ‘naturais’[...], pois esses ganham sentido através de processos inconscientes e formas culturais” (WEEKS, 1993, p. 21). [...] a sexualidade, como foi construída ao longo dos últi- mos séculos, é composta por dois principais aspectos: o desejo (a atração) e a prática (prazeres). O desejo acaba por delimitar a construção de identificações sexuais, sobretudo com aspectos políticos muito claros, que lutam contra a heteronormatividade: são o que atualmente cha- mamos de LGBT: lésbicas, gays, bissexuais, transgêneros, ou seja, pessoas que não se reconhecem dentro de lógicas e práticas da heterossexualidade ea quem têm sido nega- dos muitos direitos (WOLFF; SALDANHA, 2015, p. 42). S U M Á R I O Portanto, abarcar os estudos queer dentro das discussões de gênero é também recuperar e visibilizar sujeitos que são anula- dos socialmente pelas diversas práticas discursivas, físicas, psico- lógicas, etc. Nessa esteira, Seidman (1996, p. 13) reforça ser queer o estudo “daqueles conhecimentos e daquelas práticas sociais que organizam a ‘sociedade’ como um todo, sexualizando – heterosse- xualizando ou homossexualizando – corpos, desejos, atos, identida- des, relações sociais, conhecimentos, cultura e instituições sociais” (SEIDMAN, 1996, p. 13). As falas de gênero/sexualidades em nosso cotidiano são importantes, pois também funcionam como mecanismos de compre- ensão da formação da sociedade, isto é, como as pessoas constroem suas experiências/narrativas de vida por meio do gênero e das sexua- lidades. Ao sair na rua, vestimos um gênero, este, por sua vez, pode dar (ou não!) certa aparência da sexualidade que constituí o sujeito, sendo assim, nosso gênero/sexualidades está em constante vigilância e, 388 caso fujam da norma cisgênero47 e heterossexual, podem sofrer puni- ções, consequências, uma vez que nosso gênero/sexualidades está em vários espaços da sociedade, seja de forma velada ou não. 47 Cisgênero é um termo utilizado para se referir às pessoas cujo gênero é o mesmo que o desig- nado em seu nascimento. Isto é, configura uma concordância entre a identidade de gênero de um indivíduo com o gênero associado ao seu sexo biológico e/ou designação social. Enquanto Heterossexual compreende a atração afetiva de pessoas de gêneros distintos. A ANÁLISE DE DISCURSO PÊCHEUXTIANA: CONSIDERAÇÕES TEÓRICAS INICIAIS Na Análise de Discurso não existe um sentido único, homo- gêneo, linear, afinal, os sentidos sempre podem ser outros a partir do contexto e lugar social em que o(s) sujeito(s) enuncia(m) o seu dizer. Sendo o discurso “[...] o índice potencial de uma agitação nas filia- ções sócio-históricas de identificação” (PÊCHEUX, 2006, p. 56), tam- bém entendido como práxis social, não pode ser confundido com fala ou texto, já que o discurso refere-se aos efeitos de sentidos existen- tes a partir das relações sociais, dessa forma os sentidos não estão fixados na palavra/língua/linguagem, mas encontram-se dispersos, em constante movimento no cotidiano das pessoas. Trata-se, portanto, de situar o discurso como exterior à língua e que se materializa na língua e produz sentidos (ORLANDI, 2003). Cabe então assinalar que “[...] todo discurso já é uma fala que fala com outras palavras, através de outras palavras (da perspectiva dis- cursiva, as palavras já são sempre discursos na sua relação com os sentidos)” (ORLANDI, 2007, p. 15). Como o discurso está filiado às condições sócio-históricas, é coerente tratar de uma categoria fundamental que são as con- dições de produção do discurso, pois a partir dela é possível fazer 47 389 a relação dos sentidos possibilitados e as condições sociais, his- tóricas e ideológicas para que o(s) discurso(s) possa(m) emergir, ou seja, “[...] um discurso é sempre pronunciado a partir de condi- ções de produção dadas” (PÊCHEUX, 1993, p. 77). Vasconcelos e Cavalcante (2013) reforçam essa noção das condições de produção apoiadas em Pêcheux: S U M Á R I O [Para Pêcheux (1993)] A noção de condições de pro- dução (CP) refere-se ao contexto ou às circunstâncias históricas – mediatas e imediatas – que permitem que um determinado discurso venha à tona, e não um outro qualquer. A expressão CP traz a implicação de que um discurso não pode ser analisado apenas como um texto, pois ele é um acontecimento dentro de um período his- tórico e produto de situações específicas que fizeram com que ele (e não outro) aflorasse (VASCONCELOS; CAVANCANTI, 2013, p. 78). De acordo com Orlandi (2003), as condições de produção podem ser divididas em dois sentidos: amplos e estritos. Quando em sentidos amplos, referem-se ao contexto sócio-histórico-ideoló- gico em que o discurso foi produzido, já no sentido estrito, por sua vez, são as condições imediatas do discurso, ou seja, as circuns- tâncias da enunciação. A ANÁLISE DE DISCURSO PÊCHEUXTIANA: CONSIDERAÇÕES TEÓRICAS INICIAIS Neste contexto, a categoria da formação ideológica também é fundamental no processo de análise do discurso(s), pois remete dire- tamente ao lugar social, político e ideológico ocupado pelo sujeito do discurso. Os sentidos existentes nas palavras estarão imbricados na posição sujeito assumidos pelo enunciador, logo a formação ideoló- gica é considerada práxis sociais, já que é representada na realidade por meio das formações discursivas. Sobre esta categoria, Pêcheux (1993, p. 166) argumenta: [...] as formações ideológicas de que acabamos de falar comportam necessariamente, como um dos seus compo- nentes, uma ou várias formações discursivas interligadas que determinam o que pode e deve ser dito [...] a partir de [...] as formações ideológicas de que acabamos de falar comportam necessariamente, como um dos seus compo- nentes, uma ou várias formações discursivas interligadas que determinam o que pode e deve ser dito [...] a partir de 390 uma posição dada numa conjuntura, isto é, numa certa relação de lugares no interior de um aparelho ideológico, e inscrita numa relação de classes. Para Pêcheux (1988), as palavras mudam de sentido segundo as posições sustentadas pelos enunciadores e estas posições estão inscritas em formações ideológicas que determinam os sentidos que podem ser produzidos. Enquanto a formação discursiva trata-se da “[...] manifestação, no discurso, de uma determinada formação ideo- lógica em uma situação de enunciação específica” (LEANDRO FER- REIRA, 2001, p. 15). Afirmamos também que o silêncio significa, portanto, “[...] quando dizemos que há silêncio nas palavras, estamos dizendo que: elas são atravessadas de silêncio; elas produzem silêncio; o silên- cio fala por elas” (ORLANDI, 2007, p. 11). Logo, podemos dizer que o silêncio surge para estabilizar os discursos constituídos, formulados e postos em circulação. Por sua vez, não se confunde com o ato de calar, mas nova forma de expressividade no meio social, tonificando, assim, múltiplos sentidos, pois, como assevera Orlandi (2007, p. 72), o sujeito passa “das palavras ao silêncio e do silêncio às palavras”. 48 Encontra-se em um site específico para aqueles que desejam apenas acessar esta informação: https://www.programaescolasempartido.org/pl-federal. 49 http://www.escolasempartido.org/. 49 http://www.escolasempartido.org/. 8 Encontra-se em um site específico para aqueles que desejam apenas acessar esta informação https://www.programaescolasempartido.org/pl-federal. Figura 1 – Página do site Escola Sem Partido Figura 1 – Página do site Escola Sem Partido Figura 1 – Página do site Escola Sem Partido Fonte: Escola sem Partido (2018). CORPUS O corpus de nossa pesquisa é constituído do anteprojeto de lei federal do MESP48 e da seção “Educação Moral” que se encontra em um segundo site49 do MESP, que fornece informações sobre o movimento apresentando seções que dialogam com a finalidade do anteprojeto de lei federal. 49 391 Ao nos depararmos com o anteprojeto de lei federal, que con- tém 11 artigos e um anexo com os deveres dos professores, obser- vamos que a Educação moral é expressa nos artigos: 1, 4 e 6. Neste momento, faremos um recorte do nosso corpus e analisaremos dis- cursivamente apenas o artigo 4 e os incisos I, II e V, que compreende às funções do professor associados ao discurso da moralidade, em paralelo com a seção “Educação Moral” do segundo site do MESP. S U M Á R I O Art. 4º. No exercício de suas funções, o professor: Art. 4º. No exercício de suas funções, o professor: I – não se aproveitará da audiência cativa dos alunos para promover os seus próprios interesses, opiniões, con- cepções ou preferências ideológicas, religiosas, morais, políticas e partidárias; I – não se aproveitará da audiência cativa dos alunos para promover os seus próprios interesses, opiniões, con- cepções ou preferências ideológicas, religiosas, morais, políticas e partidárias; II – não favorecerá nem prejudicará ou constrangerá os alunos em razão de suas convicções políticas, ideológi- cas, morais ou religiosas, ou da falta delas; V – respeitará o direito dos pais dos alunos a que seus filhos recebam a educação religiosa e moral que esteja de acordo com as suas próprias convicções (ESCOLA SEM PARTIDO, s/d). Fonte: Escola sem Partido (2018). 392 http://portal.mec.gov.br/component/tags/tag/35090-brasil-sem-homofobia  O primeiro PNE foi estabelecido entre os anos de 2001 a 2010 e não contemplava às discussões de gênero/sexualidades. Ver “Ideologia de gênero: uma falácia construída sobre os planos de educação brasileiros” (REIS; EGGERT, 2017). ANÁLISE DAS MATERIALIDADES DISCURSIVAS Ao fazer o confrontamento do discurso escrito no artigo 4 do anteprojeto de lei federal do MESP e o discurso da seção “Educa- ção Moral”, no site paralelo ao do anteprojeto, alguns questionamen- tos surgiram: Quais condições de produção permitem o surgimento deste discurso? Quais os efeitos de sentido são construídos sob a expressão “Educação Moral” no anteprojeto de lei federal do pro- grama Escola Sem Partido? Por que esta expressão também remete às discussões sobre educação sexual e de gênero? Quais são os dis- cursos válidos para reflexão da “Educação Moral”? Quais sujeitos e discursos são silenciados/invalidados neste anteprojeto de lei? Qual gênero e sexualidade se tornam inteligível? Este projeto tratou-se de uma iniciativa não governamen- tal proposta para compor o Programa Brasil sem Homofobia50 do Governo Federal Brasileiro. Dentre as atividades do programa estava a elaboração de materiais para o combate à homofobia nas escolas, como vídeos, cartilhas e sugestões de sequências didáti- cas para professores. Além disso, estava previsto a inserção das discussões de gênero e sexualidades na versão do segundo51 Plano Nacional de Educação (PNE), apresentada à câmara dos deputados em 20 de dezembro de 2010, com a finalidade de promover a equidade de gênero e o respeito à diversidade sexual. A discussão já vinha sendo implementada em anos anteriores ao fim do primeiro PNE, pelo setor da educação que realizou: 393 [...] a Conferência Nacional da Educação Básica – Coneb (2008) e as Conferências Nacionais de Educação – Conae (2010 e 2014), também com as respectivas etapas muni- cipais e estaduais. O Documento Final da Coneb registra que durante toda a Conferência e na plenária final houve destaque para “uma educação com qualidade social que [...] vise à superação das desigualdades sociais, raciais,de gênero, de idade e de orientação sexual” (BRASIL, 2008, p. 1). O Documento Final da Conae 2010 também se encontra permeado de referências a gênero, diversidade sexual, orientação sexual e identidade de gênero, inclu- sive com deliberações aprovadas sobre gênero e diversi- dade sexual (REIS; EGGERT, 2017, p. 13). Reis e Eggert (2017) ainda reforçam que as deliberações da Coneb de 2008 e da Conae de 2010 foram vistas, portanto, como se constituindo “em marco para a construção de um novo Plano Nacional de Educação com ampla participação das sociedades civil e política” (BRASIL, 2010, p. 14), uma vez que o primeiro PNE estava chegando ao fim na época. ANÁLISE DAS MATERIALIDADES DISCURSIVAS Dessa forma, foi inserido nas diretrizes do segundo PNE, no artigo 2º, inciso III, “- a superação das desigualdades educacionais, com ênfase na promoção da igualdade racial, regional, de gênero e de orientação sexual e na erradicação de todas as formas de discri- minação” (BRASIL, 2012). Todavia, “Em 17 de dezembro de 2013, o Plenário do Senado aprovou o Substitutivo ao Projeto de Lei, no qual retirou da reda- ção do inciso III do artigo 2º” (REIS; EGGERT, 2017, p. 15), além disso, “[...] Também suprimiu, em todo o texto, a flexão de gênero, adotando a forma genérica masculina [...] (BRASIL, 2013)” (REIS; EGGER, 2017, p. 15). Considerando as discussões acima sobre o Projeto Escola Sem Homofobia e o PNE, o MESP também se posiciona de forma direta (artigo 2º) e indireta (Educação Moral) na redação do seu ante- projeto de lei federal sustentado em um discurso que não priorize 394 S U M Á R I O as discussões de gênero/sexualidades no ambiente escolar. Sendo assim, o discurso do MESP surge “na relação com a exterioridade, nas condições em que eles são produzidos e que não dependem só das intenções dos sujeitos” (ORLANDI, 1999, p. 30). Todo discurso evoca outros discursos, outros dizeres que já foram ditos em outros espaços ou situações discursivas. Conse- quentemente, os efeitos de sentido que estão postos em circulação na cotidianidade também não são exclusivistas/único-literais, ao contrário, “os sentidos são abertos, incompletos, sujeitos a derivas, fazendo irromper outros sentidos” (SILVA SOBRINHO, 2007, p. 43). Sendo assim, especificamente no artigo 4 do anteprojeto de lei federal do MESP, os primeiros efeitos de sentidos do texto reme- tem à condição do professor e às suas funções na escola estabeleci- das apenas por uma relação de poder ativa/passiva, isto é, o sentido estabelecido sobre a educação/professor/estudante é compreen- dido dentro de uma visão positivista, na   qual, os estudantes têm apenas a função de absorver o conteúdo ministrado em sala de aula. Isto é expresso no inciso I quando diz que o professor (SD 1)52: “não se aproveitará da audiência cativa dos alunos para promover os seus próprios interesses, opiniões, concepções ou preferências ide- ológicas, religiosas, morais, políticas e partidárias” (PL 7180/2014), ou seja, aciona-se o efeito de sentido de que os estudantes [alunos] não são capazes de construir seus próprios saberes e, tampouco, podem interagir em sala de aula. SD refere-se à expressão Sequência Discursiva. ANÁLISE DAS MATERIALIDADES DISCURSIVAS Somado a esta situação, o professor não é visto como aquele que promove o conhecimento formativo/técnico/especializado/ associado com a realidade dos estudantes, mas como aquele que possivelmente pode articular apenas uma discussão de interesses puramente subjetivos, colocando o professor numa posição de apro- veitador, desprezando assim sua qualificação profissional. 52 395 No inciso II, o professor (SD 2): “não favorecerá nem prejudi- cará ou constragerá os alunos em razão de suas convicções políticas, ideológicas, morais ou religiosas, ou da falta delas” (PL 7180/2014). O professor continua sendo apresentado como o “vilão”, detentor de um poder avaliativo que tem por finalidade apenas favorecer, preju- dicar ou constranger o estudante, resumindo a atividade docente a interesses puramente subjetivos. Entretanto, silencia que o processo avaliativo educacional adotado pelo professor é respaldado pela Lei de Diretrizes e Bases da Educação Nacional (LDB/BRASIL, 1996), portanto, o professor ao submeter o estudante a qualquer atividade escolar e, por conse- guinte, avaliá-lo, deve seguir rigorosamente às orientações da LDB, não podendo assim “favorecer, prejudicar ou constranger” qualquer estudante. Ao utilizar o pronome possessivo “seus/suas” nos incisos I e II também reforça este sentido de posse, poder do professor, na ministração de suas aulas, associando as palavras “política, ideologia, religião e moral” como fundamentais para uma possível “doutrinação”. No entanto, questionamos: É possível qualquer discurso sem ideologia? A política, a religião, a moral, etc. são manifestações da ideologia, por que a ideologia aparece como algo paralelo a estas outras palavras? O que o MESP compreende por ideologia? Por que política, religião e moral são as palavras de ordem do anteprojeto de lei? Se a política, a religião e a moral, em seu sentido amplo, fazem parte da formação da humanidade, como o professor poderá minis- trar o conteúdo das suas aulas, nos momentos cabíveis, sem fazer a devida correlação com estas palavras? A própria redação do PL 7180/2014 possibilita outros gestos de interpretação que nos leva a estas e, possivelmente, outras indagações. Seguindo, temos o inciso V, o qual afirma que uma das fun- ções do professor (SD 3): “respeitará o direito dos pais dos alunos a que seus filhos recebam a educação religiosa e moral que esteja de acordo com suas próprias convicções”. ANÁLISE DAS MATERIALIDADES DISCURSIVAS Desta vez, a palavra moral 396 S U M Á R I O está associada aos pais dos estudantes, rompendo assim com o sen- tido de moral que é atribuído ao professor nos incisos I e II. Há, no mínimo, um duplo movimento de sentidos cristalizados sobre a “edu- cação moral” do ponto de vista familiar e da escola. No entanto, a LDB (BRASIL, 1996) orienta que cabe à escola o processo de educação escolar vinculada ao mundo do trabalho e à prática social, bem como o ensino também deve ser ministrado considerando o respeito à liberdade e apreço à tolerância. Não é tarefa do professor dar educação religiosa ou moral, todavia, é de sua responsabilidade e de sua formação preparar os estudantes, na educação escolar, para lidar com as práticas sociais, isto é, dialogar por meio dos conteúdos da disciplina e/ou temas transversais sobre o funcionamento da sociedade na qual estamos inseridos, provendo assim o respeito à liberdade e o apreço à tolerância religiosa, raça/ etnia, geração, gênero/sexualidades, etc. Discursivamente os sentidos atribuídos ao professor sobre a “Educação Moral” inclinam-se para uma preocupação no que está sendo falado em sala de aula, incitando aos pais dos estudan- tes estarem em constante vigilância da atividade docente e, caso fuja do propósito de educação moral que é esperado pelos pais, deve ser denunciado e o professor punido pelo Estado por subver- ter a ideia dos pais. Do ponto de vista dos dizeres analisados, enfatizamos como a língua funciona como o lugar da materialização do discurso, uma vez que “o discurso é o lugar em que se pode observar essa relação entre língua e ideologia, compreendendo-se como a língua produz sentidos por/para os sujeitos” (ORLANDI, 2015, p. 15). Compreende- mos, assim, que o discurso funciona como entremeio da língua e da ideologia, sendo produtor de efeitos de sentido entre locutores. Então, nos perguntamos: Qual a posição ideológica de Edu- cação Moral do MESP? Qual(is) discurso(s) dos professores deve(m) 397 ser punido(s) e os que podem ser mantidos? Neste momento somos convocados a ir para o site paralelo do MESP, que dispõe da seção “Educação Moral: Direito dos Pais”, para desvelar quais sentidos podem ser construídos em torno da “Educação Moral” defendida pelo MESP e como estes sentidos produzem efeitos no cotidiano escolar, especialmente, dos professores. ANÁLISE DAS MATERIALIDADES DISCURSIVAS Ao acessarmos no site a seção Educação Moral, nos deparamos com o texto abaixo (SD 4): Educação Moral: Direito dos Pais. Publicaremos nesta seção artigos, denúncias, depoimentos e reportagens relacionadas à usurpação, pelas escolas e pelo governo, do direito dos pais a que seus filhos recebam a educa- ção moral que esteja de acordo com suas próprias con- vicções, direito este assegurado pela Convenção Ameri- cana de Direitos Humanos. O ponto de vista que adota- mos está expresso no artigo DIREITO DOS PAIS OU DO ESTADO?, de autoria do Prof. Luiz Carlos Faria da Silva e do coordenador do ESP, Miguel Nagib (ESCOLA SEM PARTIDO, s/d, online, grifos no original). O título “Educação Moral: Direito dos Pais” já nos coloca diante de uma afirmativa, ou seja, segundo o MESP, a referida edu- cação não cabe à escola e, no intuito de convencer as pessoas que a escola é um espaço nocivo/prejudicial, elabora um texto curto para mostrar aos seus interlocutores os possíveis “danos morais” que os estudantes estão subjugados no ambiente escolar. A seção funciona como um mecanismo de investigação, de “provas”, por meio da publicação de artigos, denúncias, depoimentos e reporta- gens, como já enuncia. Ao acessar os 54 conteúdos publicados em 18 páginas virtu- ais da seção “Educação Moral”, entendemos que toda e qualquer dis- cussão que possibilite a incursão de gênero/sexualidades, religiões de matriz africana, educação que possibilite novas experiências do mundo individual para o coletivo, etc. são entendidas como usurpa- ção do direito dos pais a educação moral dos filhos. Outrossim, nos surpreende que 42 dos conteúdos publicados referem-se às ques- tões de gênero/sexualidades. 398 Os conteúdos de gênero/sexualidades denunciados pelo MESP referem-se à utilização de livros paradidáticos, pesquisas aca- dêmicas, aulas de educação sexual, entre outros mecanismos que são adotados por escolas e professores em aulas específicas para tratar do tema. Entretanto, nas redações destas supostas denúncias, mui- tas vezes, não possuem autoria e/ou são escritas vagamente, porém são enfáticas no discurso de marginalização do ambiente escolar, sobretudo, trazendo o discurso do medo por meio da ideologia. ANÁLISE DAS MATERIALIDADES DISCURSIVAS Ao compararmos o discurso das funções do professor corre- lacionados com a moralidade no anteprojeto de lei federal e o dis- curso da Educação Moral, ambos do MESP, percebe-se que o sen- tido construído em torno da educação moral dos pais se relaciona diretamente com um gênero/sexualidade inteligível (cisgênero-he- terossexualidade) de acordo com a norma biológica, religiosa e do direito da ideologia dominante vigente, corroborando assim com “as sociedades, frente aos seus projetos políticos de sistema sexo-gê- nero” (SANTOS FILHO, 2017, p. 121). Neste sentido, o discurso do MESP produz efeitos de que a escola é um espaço que é transgressor da norma “moral” estabele- cida socialmente, bem como o professor é o principal fomentador de supostas “imoralidades” ao estabelecer uma conexão entre o con- teúdo disciplinar e a realidade cotidiana dos estudantes acerca da discussão de gênero/sexualidades. Há um desejo de uma pseudopreservação do gênero e da sexualidade hegemônica em nome da “moral” para evitar uma pos- sível “contaminação” de gênero/sexualidades não inteligíveis por meio de um “determinismo linguístico”, ou seja, o discurso do MESP é orientado para “seguir a matriz de inteligibilidade de gênero, sendo regulado pela perspectiva da heterossexualidade como norma/regra para a prática sexual e social. (...) estabelece e regula a heteronorma- tividdade” (SANTOS FILHO, 2017, p. 122). 399 Na intenção de cristalizar um sentido de um gênero/sexuali- dade inteligível, o MESP assegura em seu discurso da Educação Moral “as descontinuidades e as incoerências [que devem ser] constante- mente proibidas, negadas, excluídas e fortemente rejeitadas” (SAN- TOS FILHO, 2017, p. 122, acréscimos nossos). A escola e o professor tornam-se alvos de vigilância da sociedade apenas no que se refere à discussão de Educação Sexual, esta por sua vez não se relaciona, sob nenhuma hipótese, com uma suposta “ideologia de gênero”. Na mesma direção, retomamos duas perguntas realizadas no início da nossa análise: Quais são os discursos válidos para reflexão da “Educação Moral”? Quais sujeitos e discursos são silenciados/invalidados neste anteprojeto de lei? O MESP, mesmo defendendo a neutralidade, entra em contradição, pois assume em seu discurso uma posição-sujeito associada a uma determinada “formação ideológica que está entrelaçada à realidade da socie- dade e com isso, a prática do funcionamento discursivo ganha novos efeitos de sentido, por causa da dinamicidade e das escolhas realizadas pelos sujeitos dentro das alternativas que estão postas no meio social” (BARBOSA SILVA, 2017, p. 39). ANÁLISE DAS MATERIALIDADES DISCURSIVAS Os discursos que se tornam válidos para uma educação moral autorizados para discutir sobre gênero/sexualidades, não existindo punição para a escola e/ou o professor, devem obedecer a orientação conservadora biológica, religiosa e do direito que entende o sexo, o gênero e a sexualidade como categorias homogêneas. O sentido estabelecido da educação moral do MESP, tanto no anteprojeto quanto no site, compreendido na lógica binária de se conceber o gênero enquanto a sexualidade, é apresentada de forma unitária, de acordo com a ideologia dominante, outras mani- festações/compreensões do gênero/sexualidades são entendidas como “desvios”, contribuindo, assim, para que sujeitos e discursos que estejam numa contranorma desta ideologia sejam severamente punidos, pela forma da lei, e ao mesmo tempo sejam silenciados e invalidados no espaço da educação escolar. 400 Assim, concordando com Santos Filho (2017, p. 122): É em decorrência desse ângulo de ver os gêneros e as práticas sexuais que nascem os diversos preconceitos contra os homossexuais, os bissexuais, os transexuais, os heterossexuais que fogem aos modos de gênero binário e tornam-se dissidentes etc., visto que estes não se enqua- dram nos moldes de um gênero inteligível. O MESP silencia em seu discurso que as práticas de “edu- cação moral” que atendem à perspectiva da ideologia domi- nante não serão investigadas e/ou punidas nem pelos pais e nem pela lei, logo, torna-se um discurso “natural”, isto é, possivelmente “fora” de uma perspectiva ideológica, corroborando com a falsa ideia de neutralidade. Ao mesmo tempo, estabelece o discurso da “proteção” dos estudantes em nome da “moral”, por meio do anteprojeto de lei e do acesso ao site, reforçando o preconceito e a discriminação dos sujeitos que não se enquadram nas normas de gênero/sexualidade dominantes e intimidando professores que abordem no conteúdo obrigatório escolar qualquer relação com a temática em questão. CONSIDERAÇÕES (NÃO) FINAIS Em nosso trabalho, analisamos o discurso da Educação Moral implementada pelo Movimento Escola Sem Partido (MESP) por meio do anteprojeto de lei federal e a seção “Educação Moral” disponível em sua página virtual. O MESP advoga que esta educação deve ser dada pelos pais e ao mesmo tempo solicita a neutralidade do pro- fessor em suas aulas nos conteúdos disciplinares, pois há uma ten- tativa de se apagar os efeitos da história e da ideologia, no entanto, nenhum discurso é neutro, destituído de ideologia (ORLANDI, 2015). 401 O MESP, ao se posicionar discursivamente tanto na defesa da neutralidade quanto no tema Educação Moral, já se inscreve em uma determinada formação ideológica. É pela via da análise de discurso que problematizamos as maneiras de se ler o discurso da Educação Moral do MESP, isto é, um discurso que está sujeito a equívocos, à opacidade e “não há neutralidade nem mesmo no uso mais aparen- temente cotidiano dos signos” (ORLANDI, 2015, p. 7). O discurso da Educação Moral do MESP vincula-se a produ- zir efeitos de sentido que negam/invisibilizam/anulam subjetivida- des com suas práticas discursivas no ambiente escolar que envolve temas como política, religião, gênero/sexualidades, etc. No site do movimento, dos 54 conteúdos que tratam sobre a Educação Moral, 42 se referem às discussões de gênero/sexualidades em sala de aula. Ao trazer a discussão de gênero/sexualidades com maior proporção, deseja-se legitimar um discurso de que o papel do pro- fessor em sala de aula é levar um conteúdo “imoral” para os estudan- tes por meio destas reflexões em conteúdos disciplinares obrigató- rios e/ou temas transversais. O MESP se posiciona ideologicamente ao naturalizar o discurso da heteronormatividade como evidente, o já lá, isto é, “naturaliza-se o que é produzido na relação do histórico com o simbólico” (ORLANDI, 2015, p. 43). Atribui-se o sentido da ideologia dominante sobre o que se pode e deve dizer acerca da Educação Moral e ao mesmo tempo produz efeitos de sentido sobre qual discurso pertence a uma “edu- cação imoral” (gênero/sexualidades) que possa ser propagada pelo professor em sala de aula. Este processo discursivo ocorre, pois, a “evidência do sentido, que, na realidade é um efeito ideológico, não nos deixa perceber seu caráter material, a historicidade de sua cons- trução” (ORLANDI, 2015, p. 43). CONSIDERAÇÕES (NÃO) FINAIS Com as contribuições proporcionadas pela base teórico-me- todológica da Análise de Discurso, foi possível mobilizar algumas 402 categorias a partir de pressupostos teóricos que auxiliaram no pro- cesso de descorporificação das materialidades discursivas. Por se tratar de um artigo, nos limitamos na análise com o objetivo de aten- der à proposta solicitada, mas com o desejo de alargar outras ques- tões requisitadas pelo objeto de pesquisa. REFERÊNCIAS ALGEBAILE, Eveline. 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Acesso em: 17 jan. 2023. 405 20 Maria Vanessa Monteiro das Chagas A LEITURA LITERÁRIA EM SALA DE AULA DE LÍNGUA PORTUGUESA: REFLEXÕES SOBRE O ESPAÇO DA AUTORIA FEMININA NO LIVRO DIDÁTICO DO ENSINO FUNDAMENTAL DOI:10.31560/pimentacultural/2023.97778.20 S U M Á R I O INTRODUÇÃO A leitura é uma atividade essencial no processo de ensino- -aprendizagem estabelecido na escola, uma vez que é através dela que as/os discentes têm acesso aos mais diversos conteúdos dos componentes curriculares. Porém, é necessário atentar que além de um meio de acesso ao conhecimento, esta atividade é, também, um objeto do conhecimento, ou seja, é passível de ser ensinada e apren- dida, pois não é inata ao ser humano, haja vista que ninguém nasce sabendo ler, mas aprende a ler (FLÔRES, 2001). Sendo essencial a todas as disciplinas, conforme sugere a Lei de Diretrizes e Bases da Educação – LDB (BRASIL, 1996) e rei- teram as Diretrizes Curriculares Nacionais – DCN (BRASIL, 2013), seu ensino é de incumbência de todos os componentes curriculares, uma vez que constitui um alicerce para a aprendizagem como um todo e favorece o desenvolvimento de letramentos múltiplos. Dentre estes, destacamos o letramento literário, que constitui um conjunto de práticas (comportamentos) e eventos (situações), que podem ser viabilizados pela leitura literária, seja de autoras/es canônicas/os ou não (COSSON, 2009). Em se tratando do letramento literário, a disciplina de Língua Portuguesa tende a ocupar o lugar de carro-chefe para o seu desen- volvimento, pois é de sua incumbência o ensino da literatura. Sobre ela, cabe refletir que, para além do entretenimento, um dos motivos pelos quais recorremos à leitura literária em nosso dia a dia, a literatura possui importante papel para a reflexão e o aprimoramento individual da vocação de ser humano (TODOROV, 2009). Assim, a literatura, concebida por Antonio Candido (2011) como um bem incompressí- vel, ou seja, um direito considerado básico, que deveria ser assegu- rado a todos os seres humanos, possui importante papel na cons- tituição destes como tal. Isso se dá porque “os valores que a socie- dade preconiza, ou os que considera prejudiciais, estão presentes 407 S U M Á R I O nas diversas manifestações da ficção, da poesia e da ação dramá- tica” (CANDIDO, 2011, p. 175), de modo que o contato com o texto literário proporciona (re)conhecer a sociedade e a nós mesmas/os. Dada a sua função humanizadora, faz-se necessário refle- tir sobre o que é ofertado à leitura em sala de aula. INTRODUÇÃO Assim, acerca da seleção de textos que serão lidos, nos interessa refletir sobre o acervo proposto pelos livros didáticos, haja vista que este material constitui a principal fonte de leitura de grande parte das/os alunas/ os, sendo, em alguns casos, o único recurso escrito a que se tem acesso, e ocupando um papel fundamental na formação de leitores (FERNANDES, 2010; JURADO; ROJO, 2006). Nesse sentido, neste capítulo, apresentamos um recorte dos resultados de uma pesquisa quali-quantitativa de análise documen- tal (SILVEIRA; CÓRDOVA, 2009) , que buscou investigar a represen- tatividade da literatura feminina na sala de aula de língua portuguesa no ensino fundamental através do livro didático. Em específico, foca- mos nos aspectos interseccionais que compõem o perfil-autor pre- sente nos livros didáticos Se liga na Língua: leitura, produção de texto e linguagem, de autoria de Wilton Ormundo e Cristiane Siniscalchi (2018), e Tecendo Linguagens, de autoria de Tania Oliveira e Lucy Araújo (2018), dirigidos ao 9º ano do Ensino Fundamental. Para tal, foram realizadas catalogações dos gêneros presen- tes nos livros didáticos analisados, com atenção especial aos textos literários de autoria feminina. Para análise dos dados, recorremos ao aporte teórico da Análise Crítica do Discurso (BEZERRA, 2016; FAIR- CLOUGH, 2012; 2016; MEURER, 2005), e discussões sobre intersec- cionalidade (CRENSHAW, 1991; 1989) e descolonialidade (SANTOS, 2007), a fim de verificar a presença da representatividade no LD, e dis- cutir sobre seus limites e implicações na formação leitora e pessoal. 408 ORIENTAÇÕES SOBRE A LEITURA NOS DOCUMENTOS OFICIAIS Quanto à seleção de textos, há em comum nos documen- tos oficiais a orientação de que devem ser privilegiados os gêneros frequentes na realidade social e universo escolar, tais quais notícias, artigos de opinião, contos, romances e gêneros comuns ao espaço digital, como o meme e o gif. Ademais, nos Parâmetros Curriculares para o Ensino Fundamental – PCN (BRASIL, 1998, p.25) afirma-se que Os textos a serem selecionados são aqueles que, por suas características e usos, podem favorecer a reflexão crítica, o exercício de formas de pensamento mais elaboradas e abstratas, bem como a fruição estética dos usos artísticos da linguagem, ou seja, os mais vitais para a plena partici- pação numa sociedade letrada. Assim, é perceptível o alinhamento do trabalho com a lei- tura com o objetivo de formar indivíduos para a vida em sociedade, visando proporcionar meios para o desenvolvimento da proficiên- cia leitora na perspectiva dos letramentos, visto que esta, enfatizada pelos Referenciais Curriculares para o Ensino Fundamental – RCEF (PARAÍBA, 2010), pode favorecer o contato, em sala de aula, com práticas de letramento dominantes e não dominantes (KLEIMAN, 1995), por exemplo, a leitura de autoras/es canônicas/os ou não, que publicam em livros ou redes sociais etc. No âmbito nacional, a BNCC (BRASIL, 2018) sugere um tra- balho com a leitura de diferentes textos, não só os canônicos, que dizem respeito “não somente ao texto escrito, mas também a ima- gens estáticas (foto, pintura, desenho, esquema, gráfico, diagrama) ou em movimento (filmes, vídeos etc.) e ao som (música), que acom- panha e cossignifica em muitos gêneros digitais” (BRASIL, 2018, p. 72). Tal concepção de leitura contribui para a ampliação dos letra- mentos, visto que possibilita a inserção das/os alunas/os em diversas 409 S U M Á R I O práticas sociais, o que constitui um importante passo para a des- privatização da leitura discutida por Guedes (2006), a partir da qual seriam ofertadas obras diversas, tanto aquelas consideradas clássi- cas, quanto as de autoras/es desconhecidas/os. Assim, em se tratando da abordagem da leitura no currículo, é possível compreender esta atividade enquanto importante ferra- menta não somente para o conhecimento de si e do local físico e sócio-histórico em que está inserido, mas também para cruzar fron- teiras. Deve-se, então, buscar favorecer a ampliação de horizontes, visando possibilitar que se vá além dos conhecimentos pessoais e locais, expandindo os horizontes intelectuais e superando as fron- teiras físicas. ORIENTAÇÕES SOBRE A LEITURA NOS DOCUMENTOS OFICIAIS Outrossim, o documento aponta que a partir de uma leitura crítica, desencadeadora de questionamentos e reflexões, tor- na-se possível a (re)produção da cultura. Essa leitura crítica relaciona-se intimamente com a proposta apresentada nas Diretrizes Nacionais para a Educação em Direitos Humanos que constam nas DCN (BRASIL, 2013), pois esta consi- dera que o currículo não é uma simples transmissão desinteressada do conhecimento social, mas uma arena política (ARROYO, 2011) e, portanto, reflete e pode contribuir para a perpetuação de relações de poder, que são reproduzidas em contexto escolar através da transmissão de determinada ideologia e cultura. Cabe ressaltar que, de acordo com as DCN (BRASIL, 2013), é imprescindível à escola possibilitar discussões sobre “os princípios e as práticas de um pro- cesso de inclusão social, que garanta o acesso e considere a diversi- dade humana, social, cultural, econômica dos grupos historicamente excluídos” (BRASIL, 2013, p. 16). Estes, de acordo com o documento, são atravessados pelas categorias classe, gênero, raça, etnia, gera- ção e compõem a diversidade social brasileira. Ao trazer para a discussão a situação enfrentada por diver- sos grupos, tais como negros, indígenas, mulheres, crianças e ado- lescentes, homossexuais, pessoas com deficiência, busca-se dar 410 S U M Á R I O visibilidade e voz a estes que foram e são silenciados e ocultados sob o véu da naturalização das desigualdades. Assim, abrir este espaço, é colocar em debate questões como a igualdade formal; compreen- são do background de preconceitos e discriminações que alimentam desigualdades e são naturalizados; processos de dominação e ques- tões outras que favorecem uma perspectiva multicultural que pode atuar no desvelamento das relações de poder. Compreendemos que a abordagem destas e outras questões pode possibilitar um ensino-aprendizagem que considera a relação entre a linguagem e o mundo social, o que, sob o viés da Linguística Aplicada, implica pensar a influência e a importância dos contextos sócio-históricos dos sujeitos sociais, que estão distantes de terem um perfil homogêneo, sendo, assim como o mundo social, hetero- gêneos, fragmentados e fluidos (MOITA LOPES, 2006). Desse modo, associando as documentações oficiais aos estudos desta área de pesquisa, consideramos que “envolver-se na construção do signifi- cado nos processos de letramentos é inseparável da construção de quem somos como seres sociais” (MOITA LOPES, 2013, p. 241). APORTE TEÓRICO-METODOLÓGICO: A ANÁLISE CRÍTICA DO DISCURSO E OS ESTUDOS INTERSECCIONAIS A Análise Crítica do Discurso foi definida por Norman Fair- clough, como “uma forma de ciência social crítica, projetada para mostrar problemas enfrentados pelas pessoas em razão das formas particulares de vida social, fornecendo recursos para que se chegue a uma solução” (FAIRCLOUGH, 2012 [2005], p. 312). Nesse sentido, a abordagem da ACD baseia-se em objetivos emancipatórios (FAIR- CLOUGH, 2012 [2005]), uma vez que busca colocar em evidência 411 os indivíduos marginalizados, ou seja, que são socialmente excluídos devido a relações opressivas de raça, gênero, classe social e demais marcadores. A correlação entre estes pode ser analisada sob a ótica da interseccionalidade. Cunhado por Kimberlé Crenshaw, o termo demarca o paradigma teórico e metodológico da tradi- ção feminista negra, promovendo intervenções políticas e letramentos jurídicos sobre quais condições estruturais o racismo, o sexismo e violências correlatas se sobrepõem, discriminam e criam encargos singulares às mulheres negras. (CRENSHAW, 1991, p. 54) S U M Á R I O A partir da proposição da autora, é possível representar a relação interseccional de alguns marcadores sociais a partir do seguinte diagrama de conjuntos: ura 1 – Representação da interseccionalidade em diagram Fonte: Elaboração própria com base em Crenshaw (1989). Figura 1 – Representação da interseccionalidade em diagrama Fonte: Elaboração própria com base em Crenshaw (1989). Estes são apenas alguns marcadores que podem ser consi- derados, de modo que podem somar-se a estes a orientação sexual, identidade de gênero, idade, deficiência, entre outros. A configuração da imagem que forma interseções entre os conjuntos demonstra que estes não devem ser dissociados, visto que todos contribuem em algum grau na constituição do todo. 412 Nesse sentido, considerando os aspectos interseccionais, é possível compreender as distintas vivências de diferentes grupos em sociedade, como a) mulheres, brancas, de classe baixa e sulistas; b) mulheres, negras, de classe baixa e sulistas; c) homens, brancos, de classe baixa e sulistas; d) homens, negros, de classe baixa e sulistas. Nesses exemplos, foram modificados apenas os marcadores gênero e raça, e assim formados quatro grupos distintos, cujas sobreposi- ções de marcadores tendem a direcionar e delimitar os espaços que podem frequentar e as experiências vivenciadas no dia a dia. APORTE TEÓRICO-METODOLÓGICO: A ANÁLISE CRÍTICA DO DISCURSO E OS ESTUDOS INTERSECCIONAIS Logo, a interseccionalidade estabelecida entre os marcadores compõe algo que vai além de uma lista de características, trata-se da considera- ção dos múltiplos aspectos que formam identidades e têm implica- ções na atuação em sociedade e concepção de si e do outro, com o qual compartilha ou não determinados marcadores. Crenshaw (1989) postula que a desconsideração da inter- seccionalidade pode acarretar duas situações problemáticas: a) a superinclusão, quando um único marcador, como o gênero, é tratado como suficiente para descrever e compreender toda a situação, não considerando as diferenças; b) a subinclusão, que ocorre quando questões enfrentadas por determinado grupo, como as mulheres negras, não são comuns àquelas do grupo hegemônico e passam a ser desconsideradas. Em resumo, “nas abordagens subinclusivas da discriminação, a diferença torna invisível um conjunto de problemas; enquanto que, em abordagens superinclusivas, a própria diferença é invisível” (CRENSHAW, 1989, p. 176, grifo da autora). Para compreender tais relações, é pertinente situar que a ACD concebe a linguagem como prática social. Dessa forma, “o dis- curso é visto como língua em uso, que precisa ser compreendido em seu nível textual, mas também em suas dimensões mais amplas de produção e consumo, e em suas implicações e marcas sociais” (BEZERRA, 2016, p. 193). A partir dessa concepção, a ACD de base faircloughiana analisa as conexões estabelecidas entre linguagem e poder, e, para tal, considera a relação dialética entre o discurso e a 413 estrutura social, visto que esta última é considerada tanto condição quanto efeito da primeira (FAIRCLOUGH, 2016 [1992]). Fairclough (2016 [1992]) propôs um modelo tridimensional, que, posteriormente, foi adaptado por Meurer (2005), que contem- pla texto, práticas discursivas e práticas sociais, conforme pode ser observado na Fig. 2. S U M Á R I O Figura 2 – Representação do modelo tridimensional de Fairclough, adaptado por Meurer (2005) Fonte: Meurer (2005). Figura 2 – Representação do modelo tridimensional de Fairclough, adaptado por Meurer (2005) 414 Sendo a ACD um aparato teórico metodológico, cada dimen- são implica uma etapa de análise, conforme explicitado por Chagas (2022), através do Quadro 1. Quadro 1 – Dimensões, categorias e etapas da ACD Dimensão Categoria Etapas da análise Texto Léxico Análise linguística realizada através da descrição do texto, considerando os elementos linguísticos/textuais que o compõem. APORTE TEÓRICO-METODOLÓGICO: A ANÁLISE CRÍTICA DO DISCURSO E OS ESTUDOS INTERSECCIONAIS Gramática Coesão Estrutura Práticas discursivas Força Análise intra e intertextual e interdiscursiva, que caracteriza o processo de interpretação, através do qual considera-se a relação dialética estabelecida entre o texto e as práticas sociais e são analisadas as possíveis intenções do autor, a compreensão do leitor e as relações estabelecidas com outros textos e discursos. Coerência Intertextualidade Interdiscursividade Práticas sociais Poder Análise social que busca explicar a correlação entre o texto e as práticas sociais, aprofundando as discussões sobre a relação entre a linguagem e a estrutura social, problematizando e refletindo sobre o background dos discursos e as possíveis implicações de sua manutenção ou mudança das relações de poder. Ideologia Hegemonia Fonte: Chagas (2022). Quadro 1 – Dimensões, categorias e etapas da ACD Fonte: Chagas (2022). Fonte: Chagas (2022). A partir deste modelo, podemos, por exemplo, tomar o LD como um texto (evento discursivo), na primeira dimensão, e des- crevê-lo a partir de seus elementos linguísticos, tais quais o léxico, a gramática, a coesão e a estrutura. Tal descrição, no entanto, não deve se dar de maneira isolada, mas contextualizada e aliada aos elementos implicados nas demais dimensões, pois, no nível micro (o contexto mais imediato do texto), há reflexos do macro (o con- texto cultural e institucional mais amplo) (BEZERRA, 2016), uma vez que os elementos co-textuais e contextuais se inter-relacionam na construção do todo. 415 S U M Á R I O 3 Quanto ao marcador “gênero”, foram utilizadas quatro categorias: Feminino (em azul), Masculino (em laranja), Não se aplica (em cinza) e Não identificado (em amarelo), de modo que as duas últimas referem-se, respectivamente, aos casos em que não é possível atribuir a autoria a uma pessoa em específico, pois é assinado por uma empresa ou algum órgão ou entidade; e aos casos em que não foi possível identificar a autoria devido à falta de acesso a imagens da/o autora/autor. 4 Sobre os números relacionados aos marcadores sociais, cabe esclarecer que a porcentagem foi cal- culada considerando o quantitativo de autoras/es, assim, aquelas/es que possuem mais de um texto ou fragmentos de um mesmo texto ao longo do livro foram contabilizadas/os apenas uma vez. Além de idealizadora editora da Escaleras, Débora Gil Pantaleão é escritora e professora licenciada em Letras-Inglês pela Universidade Federal da Paraíba (UFPB), onde cursou também seu mestrado e está cursando o doutorado. O PERFIL-AUTOR NOS LIVROS DIDÁTICOS: QUAL O ESPAÇO DA AUTORIA FEMININA? As catalogações dos gêneros nos dois LD demonstraram que há um vasto acervo disponível à leitura, de modo que, no total, foram catalogados 211 textos no LD Se Liga na Língua (2018) e 98 no Tecendo Linguagens (2018), pertencentes a gêneros diversos, que circulam nas mais variadas esferas, tais quais o conto, a entrevista, o anúncio e a tirinha. Os números revelam um primeiro dado que nos chama a atenção: no primeiro livro, há mais que o dobro de tex- tos que no segundo, mas, para além do quantitativo geral, cabe-nos analisar a autoria destes, considerando os aspectos interseccionais que compõem as identidades das/os autoras/es presentes. Nesse sentido, ao conferir as autorias dos textos, constata- mos uma evidente disparidade em relação a todos os marcadores sociais considerados, a saber: o gênero, a raça e a origem. Em rela- ção ao gênero social53, foi constatado que, em ambos os livros, o quantitativo de textos54 de autoria feminina está próximo dos 15%, enquanto a masculina figura mais de 60%, e os casos em que “Não se aplica” variam entre 20,7%, no Se liga na Língua (2018), e 22,2%, no Tecendo Linguagens (2018). Neste primeiro, no entanto, houve dois casos de autoria não identificada, que corresponde a 1,1% do total, conforme pode ser verificado no Gráfico 1. 53 54 416 Gráfico 1 – Percentual do marcador “gênero” nos LD Gráfico 1 – Percentual do marcador “gênero” nos LD Gráfico 1 – Percentual do marcador “gênero” nos LD Gráfico 1 – Percentual do marcador “gênero” nos LD Fonte: Chagas (2022). Fonte: Chagas (2022). Cabe refletir que os números estão em desalinho com o total de residentes no Brasil segundo dados do IBGE (BRASIL, 2020), que utiliza dados da Pesquisa Nacional por Amostra de Domicílios Con- tínua (PNAD Contínua) entre os anos 2012 e 2019 e aponta que a população do país é composta por 48,2% de homens e 51,8% de mulheres. Esses dados fazem-nos refletir sobre algumas questões, como o porquê de haver tanta disparidade entre a presença de autoras e autores nos livros se o quantitativo de indivíduos é pró- ximo, e por quais motivos sócio-históricos não haveria um maior número de mulheres que produza, publique e tenha seus textos em livros didáticos. Acerca disso, sabe-se que atualmente o mercado está con- sideravelmente mais aberto à publicação destas, o que pode ser constatado pela crescente participação feminina no mercado edi- torial, situação exemplificada por editoras paraibanas como a Esca- leras, que é comandada por Débora Gil Pantaleão55, e que tem foco 55 417 S U M Á R I O na edição de autoras, como Isabor Quintiere56, escritora de literatura fantástica paraibana premiada nacionalmente, e a editora Triluna, idealizada por Aline Cardoso57, que tem como foco a publicação de mulheres negras. Mas, olhando para sua estante, e as das escolas que frequenta/ou como docente ou estudante, pode-se considerar que há um público considerável consumindo tais publicações? Em relação ao marcador “raça”, os livros apresentaram um quantitativo de brancos similar entre si, sendo 62,7% no Se liga na Língua (2018) e 63,7% no Tecendo Linguagens (2018). Dessa forma, conforme pode ser observado no Gráfico 2, há maioria branca, seguida de um quantitativo de 7,9% de negros no primeiro livro, e 5,5% no segundo. Ademais, no Se liga na Língua (2018) há 2,1% de amarelos, e nenhum caso identificado no Tecendo Linguagens (2018). Quanto à raça indígena58, não foi identificada nenhuma ocorrência em nenhum dos livros. Sobre não identificações, as/os autoras/es cuja raça não foi identificada, quer seja por não ter fotografia aces- sível ou por estar em preto e branco, foram, respectivamente, 6,4% e 8,8%. Por fim, os casos em que não se aplica a raça, por não haver autoria em específico, variou em apenas -0,2% no segundo livro, em relação à categoria anterior. 56 Licenciada em Letras-Inglês pela UFPB e atualmente mestranda pela mesma instituição, Isabor Quintiere ganhou o prêmio Odisseia de Literatura Fantástica na categoria Narrativa Curta Horror por conto do livro “A Cor Humana”, seu livro de estreia, publicado pela Escaleras. 57 Professora de Linguagem, Mestra em análise do discurso pela UFPB, Aline Cardoso é também autora, tendo publicado, através de sua própria editora, livros de poesia: A proporção Áurea do Caos, Harpia e o seu primeiro livro de ficção: Ritos Encantarórios & Outras Ladainhas. 58 Para a identificação das/os autoras/es indígenas, foram consideradas não somente os traços fenotípicos, como também a presença da etnia em seus nomes, que consistem em uma impor- tante forma de marcação de pertencimento. Licenciada em Letras-Inglês pela UFPB e atualmente mestranda pela mesma instituição, Isabor Quintiere ganhou o prêmio Odisseia de Literatura Fantástica na categoria Narrativa Curta Horror por conto do livro “A Cor Humana”, seu livro de estreia, publicado pela Escaleras. Sobre esta categoria, Osorio (2003) aponta que a agregação de “pardos” e “pretos” no grupo “ne- gro” justifica-se por estes partilharem características socioeconômicas e vivenciarem situações similares de discriminações e desigualdades em relação aos brancos. Gráfico 1 – Percentual do marcador “gênero” nos LD 56 Licenciada em Letras-Inglês pela UFPB e atualmente mestranda pela mesma instituição, Isabor Quintiere ganhou o prêmio Odisseia de Literatura Fantástica na categoria Narrativa Curta Horror por conto do livro “A Cor Humana”, seu livro de estreia, publicado pela Escaleras. 57 Professora de Linguagem, Mestra em análise do discurso pela UFPB, Aline Cardoso é também autora, tendo publicado, através de sua própria editora, livros de poesia: A proporção Áurea do Caos, Harpia e o seu primeiro livro de ficção: Ritos Encantarórios & Outras Ladainhas. 58 Para a identificação das/os autoras/es indígenas, foram consideradas não somente os traços fenotípicos, como também a presença da etnia em seus nomes, que consistem em uma impor- tante forma de marcação de pertencimento. Licenciada em Letras-Inglês pela UFPB e atualmente mestranda pela mesma instituição, Isabor Quintiere ganhou o prêmio Odisseia de Literatura Fantástica na categoria Narrativa Curta Horror por conto do livro “A Cor Humana”, seu livro de estreia, publicado pela Escaleras. 56 57 Professora de Linguagem, Mestra em análise do discurso pela UFPB, Aline Cardoso é também autora, tendo publicado, através de sua própria editora, livros de poesia: A proporção Áurea do Caos, Harpia e o seu primeiro livro de ficção: Ritos Encantarórios & Outras Ladainhas. 58 Para a identificação das/os autoras/es indígenas, foram consideradas não somente os traços fenotípicos, como também a presença da etnia em seus nomes, que consistem em uma impor- tante forma de marcação de pertencimento. 418 Gráfico 2 – Percentual do marcador “raça” nos LD Gráfico 2 – Percentual do marcador “raça” nos LD Fonte: Chagas (2022). Fonte: Chagas (2022). Fonte: Chagas (2022). Mais uma vez observamos disparidade entre dados estatís- ticos sobre a população brasileira e as representações nos LD, haja vista que, de acordo com o PNAD (BRASIL, 2019), esta é composta por 42,7% brancos, 46,8% pardos, 9,4% pretos e 1,1% como amarelos ou indígenas. Optamos, neste trabalho, por agrupar os grupos “par- dos” e “pretos”, considerados como distintos pelo IBGE, na categoria “negro”59, que configura uma categoria política que tem, no campo dos movimentos sociais, importante impacto no combate ao racismo. Com isso, intentamos evitar equívocos na classificação, que é feita por meio da autodeclaração no Brasil, e obtemos um percentual de 56,2% de negros, ou seja, observamos que a maior parte da popu- lação é negra, porém, este grupo representa menos de 10% das/os autoras/es nos dois LD analisados. No que tange a origem, o Gráfico 3 demonstra que a região Sudeste do país lidera em quantidade de autoras/es em ambos os LD, de modo que são 34% no Se liga na Língua (2018) e 42,2% 59 419 no Tecendo Linguagens (2018). Em seguida, temos as/os de origem estrangeira, com 22,9% e 15,6%, seguidas/os das/os sulistas, que são 9,6% e 5,6%. Por fim, a região Nordeste representa 4,8% e 3,3%, enquanto o Norte tem o menor percentual no primeiro LD (1%), não tendo qualquer representante no segundo. S U M Á R I O Gráfico 3 – Quantitativo do marcador “origem” nos LD Fonte: Chagas (2022). Gráfico 3 – Quantitativo do marcador “origem” nos LD Fonte: Chagas (2022). Fonte: Chagas (2022). Analisando a situação por estado, obtemos as seguintes visualizações das autorias por estado brasileiro: Figura 3 – Representação da autoria por estado brasileiro nos LD Figura 3 – Representação da autoria por estado brasileiro nos LD Fonte: Chagas (2022). Fonte: Chagas (2022). Fonte: Chagas (2022). 420 Com esta representação, constatamos que há maior cober- tura dos estados das regiões média e inferior do mapa, que corres- pondem às regiões Centro-oeste, Sul e Sudeste, enquanto as regi- ões Norte e Nordeste, que, somadas, contemplam mais da metade dos estados brasileiros, têm baixa representatividade. Além disso, há de se refletir sobre o fato de haver maior percentual de autoras/es estrangeiras/os que nordestinas/os e nortistas, sugerindo uma maior valorização da produção internacional do que destas regiões que são historicamente subjugadas, uma vez que não compõem o centro hegemônico do país e situam-se do outro lado da linha abissal que separa não só geográfica, mas também ideologicamente as diferen- tes regiões do país (SANTOS, 2007). Acerca da autoria estrangeira, cabe destacar que há maioria estadunidense no LD Se liga na Língua (2018), pois das/os quarenta e três (43) autoras/es estrangeiras/os, doze (12) são estaduniden- ses, e dentre estes apenas duas são mulheres. Em segundo lugar está a França, com cinco (5) autores, seguida da Alemanha, Portugal e Reino Unido, que têm três (3) cada. Por fim, há países com dois (2) (Irlanda e República Tcheca) ou um (1) (Angola, Áustria, Bulgária, Chile, Coreia do Sul, Hungria, Itália, México, Noruega, Polônia, Suécia, Ucrânia, Venezuela) representantes. Já no LD Tecendo Linguagens (2018) os números são mais próximos, de modo que das/os quatorze (14) autoras/es, dois (2) são estadunidenses, dois (2) portugueses e os demais países contem- plados (Argentina, Austrália, Áustria, Bélgica, Chile, Espanha, França, Inglaterra, Países Baixos, Rússia) têm uma/um (1) representante cada. Unindo os dados com quantitativo mais expressivo, compre- endemos que o perfil geral da autoria nos dois livros é o mesmo: o homem branco sudestino. Dessa forma, as/os alunas/os cujo repertório de leitura é, em grande medida, formado em sala de aula, podem conceber este perfil-autor como o padrão, o que pode auxiliar inconscientemente na perpetuação desse status, uma vez que são 421 S U M Á R I O às produções de homens, brancos e sudestinos que se tem acesso em sala de aula. Figura 3 – Representação da autoria por estado brasileiro nos LD Assim, a ausência da literatura de autoria feminina, em especial as do Norte e Nordeste, a coloca em um lugar de não- -existência (SANTOS, 2007), uma vez que vêm sendo invisibiliza- das, haja vista que os holofotes do cânone se voltam às produções masculinas, o que contribuiu para o estabelecimento da primazia de obras masculinas em detrimento das femininas, alimentando, assim, uma lógica capitalista patriarcal. Tal situação tem implicações na for- mação leitora das/os alunas/os, pois a escassez de outras referên- cias tende a restringir os horizontes de leituras e, por conseguinte, limitar a variedade de eventos de letramento literário. Após esse primeiro levantamento, foi realizada uma catalo- gação secundária, cuja finalidade foi quantificar os textos literários de autoria feminina. A partir desta, foi constatado o seguinte: Gráfico 4 – Gêneros literários nos LD Fonte: Chagas (2022). Podemos observar que há entre cinco (5) e seis (6) gêneros literários nos LD, de modo que, no total, são trinta e um (31) textos no Se liga na Língua (2018), o que corresponde a 14,6% do total, e vinte e dois (22) textos no Tecendo Linguagens (2018), ou seja, 22,4% do total. Destes, são, respectivamente, 25,8% e 4,5% de autoria feminina. Gráfico 4 – Gêneros literários nos LD Fonte: Chagas (2022). Gráfico 4 – Gêneros literários nos LD Fonte: Chagas (2022). Podemos observar que há entre cinco (5) e seis (6) gêneros literários nos LD, de modo que, no total, são trinta e um (31) textos no Se liga na Língua (2018), o que corresponde a 14,6% do total, e vinte e dois (22) textos no Tecendo Linguagens (2018), ou seja, 22,4% do total. Destes, são, respectivamente, 25,8% e 4,5% de autoria feminina. 422 Em ambos os livros, há uma evidente disparidade no quantitativo, todavia, o livro Tecendo Linguagens (2018) apresenta uma situação problemática: a presença de um único texto literário de autoria femi- nina. Foi justamente este cenário que nos levou a eleger o gênero conto para análise, pois é o único que tornaria possível a análise comparativa das atividades de leitura de um mesmo gênero nos dois LD. No entanto, mesmo este caminho apresentou intempéries, pois este único texto está presente apenas no Manual do Professor. Figura 3 – Representação da autoria por estado brasileiro nos LD Em relação aos textos literários de autoria feminina e o perfil específico de suas autoras, obtivemos o seguinte quadro: Quadro 2 – Textos literários de autoria feminina no LD Se liga na Língua (2018) g g TEXTOS LITERÁRIOS DE AUTORIA FEMININA DO LD SE LIGA NA LÍNGUA (2018) Gênero Título Autor(a) Raça Origem Página Canção Efêmera Tulipa Ruiz Branca SP 16-17 Conto O primeiro beijo Clarice Lispector Branca Ucrânia– PE - RJ 180-181 Conto (fragmento) Pomba enamorada ou uma história de amor Lygia Fagundes Telles Branca SP 221-222 Poema Intuições Ryane Leão Negra MT 191 Poema - Haicai - Alice Ruiz Branca PR 25 Poema visual - Regina Célia Barbosa Branca AL 268 Romance (fragmento) A máquina Adriana Falcão Branca RJ 84-86 Romance (fragmento) PS beijei Adriana Falcão e Mariana Veríssimo Branca e Branca RJ e RS 185 Fonte: Chagas (2022). TEXTOS LITERÁRIOS DE AUTORIA FEMININA DO LD SE LIGA NA LÍNGUA (2018) Ao observar os títulos dos textos, é possível perceber que a temática do amor romântico é predominante nos textos em prosa, 423 S U M Á R I O podendo favorecer a formação de um imaginário que concebe que este é o tema de interesse feminino, o que pode contribuir para a perpetuação de um estereótipo. Tal situação é problemática uma vez que invisibiliza as demais produções dessas autoras, pois não estão acessíveis no livro, podendo levar a crer que produções femininas tendem a seguir certa linearidade. Haja vista que as/os alunas/os estão no processo de formação de gostos literários, seria interes- sante que as temáticas dos textos lidos fossem mais diversas, a fim de possibilitar experiências leitoras variadas. Quanto aos marcadores de “raça” e de “origem”, o quadro demonstra que a maior parte das autoras de literatura é branca e sudestina, havendo apenas duas deste primeiro grupo que são nor- destinas, e uma negra do Centro-oeste. Quanto à origem de Cla- rice Lispector, cabe esclarecer que a autora nasceu na Ucrânia, mas cresceu no Brasil, país onde refugiou-se com sua família judia, tendo passado a infância e parte da adolescência em Pernambuco, no Nordeste, e grande parte da vida no estado do Rio de Janeiro, situ- ado na região Sudeste. Dessa forma, podemos considerar que há apenas três autoras que não são sudestinas: Mariana Veríssimo, do Sul; Regina Célia Barbosa, do Nordeste; Ryane Leão, do Centro-O- este. Figura 3 – Representação da autoria por estado brasileiro nos LD Traçando um perfil geral, temos a autoria feminina de literatura neste LD como sendo branca e sudestina, situação similar ao qua- dro geral discutido no item 6.1.1 e ao livro Tecendo linguagens (2018), que tem como única autora de literatura apresentada a paulista Lygia Fagundes Telles, com o conto “A disciplina do amor”, que consta no Manual do Professor. Assim, o perfil branco e sudestino que compõe o quadro geral dos LD se mantem preponderante neste recorte. Ademais, chama a atenção o fato de haver apenas um texto literário de autoria feminina justamente no LD Tecendo Linguagens (2018), cujas auto- ras são mulheres. Outro fato curioso é a forma como a Apresenta- ção do MP é assinada, pois, apesar de a autoria e a maior parte da equipe de editoração ser feminina, na assinatura consta “Os autores”, 424 com artigo definido e desinências correspondentes ao gênero mas- culino em língua portuguesa, aspecto linguístico que faz-nos refletir sobre o que pode ter motivado tal escolha. Compreendemos, pois, conforme proposto por Fairclough (2016 [1992]), que tal escolha lexical, “acidental” ou não, relaciona-se e reflete ideologias que con- tribuem para uma hegemonia patriarcal que omite o indivíduo e as produções femininas, delegando-as o não-lugar e a invisibilidade. CONSIDERAÇÕES FINAIS Através da investigação, foi constatado que o perfil-autor predominante é o do homem branco sudestino, e mesmo realizando um recorte com enfoque no gênero feminino, estes dois últimos mar- cadores sociais da diferença seguem predominantes. Assim, con- sideramos que há baixa representatividade da autoria feminina em ambos os livros analisados, pois não apenas há poucos textos escri- tos por mulheres, como o perfil presente é restrito. Ao fornecer mais espaço físico nas páginas do LD, colocando em evidência a/o autora/autor branca/o sudestina/o, pode-se con- tribuir com a perpetuação da hegemonia deste grupo e naturali- zação do apagamento das produções de outras regiões e grupos raciais, o que é substancialmente incoerente com as orientações das documentações oficiais, que preconizam a discussão e valorização da diversidade. É certo que seria inviável que um único LD con- templasse autoras/es de todos os estados brasileiros e países do mundo, porém é necessária a atenção para a configuração despro- porcional dos acervos, que tendem a priorizar determinado perfil em detrimento de outros. Assim, constatada a baixa representatividade das produções nortista e nordestina, cabe, especialmente nos estados situados dessas regiões, um movimento similar à perspectiva do suleamento 425 adotada pela Linguística Aplicada no que concerne a produção e disseminação do conhecimento. Ou seja, impõe-se como urgente que as/os docentes, inseridas/os em seus contextos, adquiram fami- liaridade com as produções de suas regiões para que seu acesso possa ser viabilizado no contexto de ensino-aprendizagem uma vez que estas não são contempladas a contento no LD adotado. REFERÊNCIAS ARROYO, Miguel G. Currículo: Território em Disputa. Petrópolis, RJ: Vozes, 2011. ARROYO, Miguel G. Currículo: Território em Disputa. Petrópolis, RJ: Vozes, 2011. BEZERRA, Fábio. A análise crítica do discurso e os multiletramentos: o papel da linguagem no fazer docente contemporâneo. In: NÓBREGA, Carmem; ARCOVERDE, BEZERRA, Fábio. A análise crítica do discurso e os multiletramentos: o papel da linguagem no fazer docente contemporâneo. In: NÓBREGA, Carmem; ARCOVERDE, Rossana; BRANCO, Sinara; FARIAS, Washington (Orgs.). Educação linguística e literária: discursos políticas e práticas Campina Grande: UFCG 2016 p 189-204 linguagem no fazer docente contemporâneo. In: NÓBREGA, Carmem; ARCOVERDE, Rossana; BRANCO, Sinara; FARIAS, Washington (Orgs.). Educação linguística e literária: discursos, políticas e práticas. Campina Grande: UFCG, 2016, p. 189-204. BRASIL. Base Nacional Comum Curricular. Brasília: MEC. 2018. A leitura no livro didático de Língua Portuguesa de Ensino Médio. Campinas, SP: [s.n.] 2010. FLÔRES, Onici. Eu leio, tu lês, eles deveriam ter acesso à leitura. In: FLÔRES, Onici. Ensino de Língua e Literatura. Canoas: Ed. ULBRA, 2001. GUEDES, Paulo Coimbra. A formação do professor de português: que língua vamos ensinar? São Paulo: Parábola, 2006. BRASIL. Base Nacional Comum Curricular. Brasília: MEC. 2018. BRASIL. Base Nacional Comum Curricular. Brasília: MEC. 2018. BRASIL. Instituto Brasileiro de Geografia e Estatística. Conheça o Brasil – População. 2020. Disponível em: < http://bit.ly/3zcdOOU>. Acesso em: 21 jul. 2021. BRASIL. Lei de Diretrizes e Bases da Educação Nacional – LDB nº 9394/96. Disponível em: <https://bit.ly/3TLB3Je>.htm Acesso em: 14 abril. 2021. BRASIL. Ministério da Educação. Secretária de Educação Básica. Diretoria de Currículos e Educação Integral. Diretrizes Curriculares Nacionais Gerais da Educação Básica. Brasília: MEC, SEB, DICEI, 2013. Brasília: MEC, SEB, DICEI, 2013. BRASIL. Secretaria de Educação Fundamental. Parâmetros curriculares nacionais: terceiro e quarto ciclos do ensino fundamental: língua portuguesa. Brasília: MEC/SEF, 1998. CANDIDO, Antônio. Vários escritos. 5. ed. Rio de Janeiro: Ouro sobre Azul, 2011. CHAGAS, Maria Vanessa Monteiro das. Leitura literária no ensino fundamental e a representatividade da literatura de autoria feminina no livro didático de língua portuguesa: um estudo à luz da análise crítica do discurso. 148 f. Dissertação (Mestrado) – UFPB/CCHLA, João Pessoa, 2022. BRASIL. Secretaria de Educação Fundamental. Parâmetros curriculares nacionais: terceiro e quarto ciclos do ensino fundamental: língua portuguesa. Brasília: MEC/SEF, 1998. BRASIL. Secretaria de Educação Fundamental. Parâmetros curriculares nacionais: terceiro e quarto ciclos do ensino fundamental: língua portuguesa. Brasília: MEC/SEF, 1998. CANDIDO, Antônio. Vários escritos. 5. ed. Rio de Janeiro: Ouro sobre Azul, 2011. CHAGAS, Maria Vanessa Monteiro das. Leitura literária no ensino fundamental e a representatividade da literatura de autoria feminina no livro didático de língua portuguesa: um estudo à luz da análise crítica do discurso. 148 f. Dissertação (Mestrado) – UFPB/CCHLA, João Pessoa, 2022. CHAGAS, Maria Vanessa Monteiro das. Leitura literária no ensino fundamental e a representatividade da literatura de autoria feminina no livro didático de língua portuguesa: um estudo à luz da análise crítica do discurso. 148 f. Dissertação (Mestrado) – UFPB/CCHLA, João Pessoa, 2022. 426 COSSON, Rildo. Letramento literário: teoria e prática. São Paulo: Editora Contexto, 2009 CRENSHAW, Kimberle. Demarginalizing the intersection of race and sex: a black feminist critique of antidiscrimination doctrine, feminist theory and antiracist politics. 1989. CRENSHAW, Kimberle. Mapeando as margens: interseccionalidade, políticas de identidade e violência contra mulheres não-brancas. 1991. Tradução de Carol Correia. Disponível em: <https://bit.ly/3TNNiF2>. Acesso em: 31 jun. 2021. FAIRCLOUGH, Norman. Análise Crítica Do Discurso como método em Pesquisa Social Científica. Tradução de Iran Ferreira de Melo. Língua d’Água, v. 25, n. 2, 2012, p. 307-329. FAIRCLOUGH, Norman. Discurso e mudança social. 2 ed. Brasília: Editora Universidade de Brasília. 2016. FERNANDES, Marly Aparecida. KLEIMAN, Angela B. Os significados do letramento: uma nova perspectiva sobre a prática social da escrita. Campinas: Mercado das Letras, 1995. MEURER, José Luiz. Gêneros textuais na análise crítica de Fairclough. In: MEURER, José Luiz; BONINI, Adair; MOTTA-ROTH, Désirée (Orgs.). Gêneros: teorias, métodos, debates. São Paulo: Parábola Editorial, 2005, p. 81-106. GUEDES, Paulo Coimbra. A formação do professor de português: que língua vamos ensinar? São Paulo: Parábola, 2006. JURADO, Shirley; ROJO, Roxane. A leitura no ensino médio: o que dizem os documentos oficiais e o que se faz? In: BUNZEN, Clecio; MENDONÇA, Márcia; et al. (Orgs.). Português no ensino médio e formação do professor. São Paulo: Parábola Editorial, 2006. KLEIMAN, Angela B. Os significados do letramento: uma nova perspectiva sobre a prática social da escrita. Campinas: Mercado das Letras, 1995. MOITA-LOPES, Luiz Paulo da (Org.). Por uma linguística aplicada indisciplinar. São Paulo: Parábola Editorial, 2006. MOITA-LOPES, Luiz Paulo da (Org.). Por uma linguística aplicada indisciplinar. São Paulo: Parábola Editorial, 2006. MOITA-LOPES, Luiz Paulo da. Gênero, sexualidade, raça em contextos de letramentos escolares. In: MOITA-LOPES, Luiz Paulo da (Org.). Linguística aplicada na modernidade recente: Festschrift para Antonieta Celani. São Paulo: Parábola Editorial, 2013, p. 227-247. MOITA-LOPES, Luiz Paulo da. Gênero, sexualidade, raça em contextos de letramentos escolares. In: MOITA-LOPES, Luiz Paulo da (Org.). Linguística aplicada na modernidade recente: Festschrift para Antonieta Celani. São Paulo: Parábola Editorial, 2013, p. 227-247. 427 OLIVEIRA, Tânia Amaral; ARAÚJO, Lucy Aparecida Melo. Tecendo linguagens: língua portuguesa. 5. Ed. Barueri [SP]: IBEP, 2018. ORMUNDO, Wilton; SINISCALCHI, Cristiane. Se liga na língua: leitura, produção e linguagem. 1. Ed. São Paulo: Moderna, 2018. S U M Á R I O PARAÍBA. Referenciais Curriculares do Ensino Fundamental do Estado da Paraíba. Paraíba. 2010. SANTOS, Boaventura de Souza. Para além do pensamento abissal: das linhas globais a uma ecologia de saberes. Novos Estudos, 79, nov. 2007. SILVEIRA, Denise Tolfo; CÓRDOVA, Fernanda Peixoto. A pesquisa científica. In: GERHARDT, Tatiana Engel; SILVEIRA, Denise Tolfo. (Orgs.). Métodos de pesquisa. Porto Alegre: Editora da UFRGS, 2009. TODOROV, Tzvetan. Literatura em perigo. Rio de Janeiro: Difel, 2009. 428 Quando citamos a proposição de gênero, consideramos o que os autores Rodrigues e Pereira (2021, p.169) dizem: “Os gêneros são considerados modos específicos de visualizar uma dada re- alidade. Nós pensamos e conceituamos o mundo na forma concreta de enunciados; e os enun- ciados são constituídos e funcionam segundo princípios genéricos (de gênero). Esses princípios estabelecem maneiras de ver e conceitualizar a realidade. Os gêneros, portanto, são formas rel- ativamente estáveis de conceitualizar as experiências humanas. Com isso, a prática de análise linguística na perspectiva dos gêneros do discurso, não apenas reitera a posição da língua como/ para interação social, como ratifica a ancoragem do estudo dos gêneros do discurso no âmbito das atividades humanas mediadas pela linguagem.” INTRODUÇÃO S U M Á R I O Vivemos em um país cujo cenário do poder executivo encon- trou-se em desarmonia, carregado de opiniões contrárias à da popu- lação que clama por saúde. A pandemia da COVID-19, que surgiu no início do ano de 2020, foi tratada por outros países (incluindo aqueles menos favorecidos que o Brasil) de forma mais sensata em relação ao uso e à compra das vacinas, o que diferiu do cenário político bra- sileiro no período pandêmico que, sem bases científicas, defendeu o uso de medicamentos não relacionados à doença viral e foi investi- gado por rejeitar a oferta de vacinas que, possivelmente, salvariam a vida da população. Nesse contexto, surgiu a Comissão Parlamentar de Inquérito (CPI da COVID) que investigou as fraudes realizadas pelo poder executivo. A CPI foi idealizada em abril de 2021 pelo senador Randolfe Rodrigues, do Partido REDE (AP), como tendo motivação inicial a crise sanitária no estado do Amazonas: fato revelador das omissões do Governo Federal, sob a Presidência de Jair Messias Bolsonaro, no âmbito administrativo da área da saúde. As investigações foram finalizadas em outubro de 2021, apon- tando o ex-presidente junto a alguns de seus ministros aos crimes de homicídio qualificado, genocídio e crimes contra a humanidade. A proposta deste capítulo consiste em analisar relações dia- lógicas de sentidos no gênero60 artigo de opinião a partir de deter- minadas construções sintáticas adjetivas sob uma perspectiva 60 430 de enunciados concretos (abrangidos pelos estudos funcionalis- tas), uma vez que “as análises linguísticas de orientação funciona- lista trabalham diretamente sobre o postulado básico – a língua é uma estrutura maleável, sujeita às pressões do uso e constituída de um código não totalmente arbitrário.” (FURTADO DA CUNHA, 2001, p. 02), como também afirmam Pereira e Costa-Hubes, (2021, p. 118) sobre o que pensam Bakhtin e Volochínov: Ambos concordam com a explicação de que a análise da língua não pode ser baseada apenas no estudo das formas puramente gramaticais, pois elas, por si mesmas, não respondem às questões de uso. O uso de formas gra- maticais, em situações reais e concretas de interação, só podem ser entendidas quando as formas gramaticais se colorem de perspectiva estilística. INTRODUÇÃO Diante dessa situação, utilizamos como abordagem espe- cífica a investigação sobre a produtividade discursiva dada pelas orações subordinadas adjetivas, fazendo-se necessário descrever e classificar tais ocorrências, analisando-as como valorações presen- tes nos enunciados, e lendo-as como estratégias do discurso em marcar e demarcar pontos de vista. Então foi necessário validar uma análise enunciativa de des- crição de língua, especificamente da construção sintática oracional subordinativa adjetiva, que contemple a vida verbo-ideológica pre- sente no gênero artigo de opinião que trata da CPI da COVID-19. Deste pressuposto, elegemos como necessário abordar a seguinte questão-problema: Como construções sintáticas adjetivas consti- tuem enunciados concretos no gênero artigo de opinião? Vale desta- car que a discussão presente neste capítulo se refere a um recorte de uma pesquisa acadêmica maior, cuja finalidade incidiu em investigar a produtividade de orações adjetivas no funcionamento dialógico- -discursivo do gênero artigo de opinião. Nesse sentido, cabe ao presente manuscrito, como objetivo específico, analisar as relações dialógicas de sentidos e a construção 431 de pontos de vista no gênero artigo de opinião a partir da abordagem de construções sintáticas adjetivas como enunciados concretos. Essa delimitação de estudo faz jus à nossa justificativa, que se dá por meio de duas frentes: a primeira, por estabelecer relações entre estudos de descrição linguística (especificamente sintaxe) e estudos de enunciados concretos em um campo da comunica- ção discursiva (o jornalístico); e a segunda, por contribuir com a área de estudos de natureza dialógica (Bakhtin e o Círculo) atra- vés de investigações que situam a COVID-19 inserida em um olhar histórico-discursivo. A pesquisa possui em destaque as fontes (textos do gênero jornalístico artigo de opinião), sendo colhidas através de meios digitais e advindas de arquivos contidos em portais informativos de cunho jornalístico. O critério temático para a seleção dos artigos que compõem cada artigo de opinião desta pesquisa é a CPI da COVID-19, inicial- mente ocorrida em abril de 2021, e que repercutiu enunciações carre- gadas de valorações proferidas pelos sujeitos produtores de discurso. A organização dos dados se deu através da seleção de infor- mações digitais no âmbito midiático, sendo as fontes os portais de notícia: o jornal eletrônico Brasil de Fato/RS; e o site eletrônico R7 - Notícias da Record, onde cada portal possui filiações ideológicas distintas, totalizando em um número de dois artigos de opinião. INTRODUÇÃO Para iniciar nossa fundamentação teórica, trazemos alguns autores reconhecidos pela Teoria Dialógica da Linguagem, sendo eles: Bakhtin (2016); Sériot (2015), Leite e Barbosa (2014); e trazemos também a noção de Gramática Funcional, represen- tada por Neves (1997). 432 A TEORIA DIALÓGICA DA LINGUAGEM E A GRAMÁTICA FUNCIONAL: UM ENCONTRO POSSÍVEL “Função” nos estudos de dialogismo diz respeito, sobretudo, a estudar os enunciados dentro dos vínculos sociais que lhe deram origem. Como a Gramática Funcional analisa a função de determina- dos elementos linguísticos presentes em uma língua, significa dizer que estes fatores possuem a múltipla capacidade de produzir senti- dos, sendo estes frutos de um número infinito de capacidades cons- trutivas da linguagem. Segundo Sériot (2015, p. 90, grifos do autor), podemos encon- trar as funções da linguagem sendo usadas [...] através das “trocas sociais”, que são os diversos usos da língua sob diversos contextos socialmente distintos. Uma troca social pode se dar dentro de uma organiza- ção social (como uma instituição administrativa ou de ensino), no local de produção do enunciado (nos locais de trabalho – onde se encontram as determinadas classes sociais), no nosso cotidiano (em casa, em meio às ruas, nos mercados etc.) e na divulgação de propagandas com fins ideológicos. (SÉRIOT, 2015, p. 90). Para podermos fazer um encaixe entre a Teoria Dialógica da Linguagem e a Gramática Funcional, precisamos compreender os meios de circulação em que a língua tramita. Como os estudos dialógicos também analisam o uso pelos sujeitos – e são estes que dão vida à própria linguagem –, a gramática funcional vem com o apoio de compreender as possíveis interpretações discursivas sob a experiência concreta da representação social dos sujeitos, como, por exemplo, os gêneros do discurso que possuem, por função comuni- cativa, representar os diversos setores de atuação humana. 433 Assim, enxergamos como necessário abordar a Gramática Funcional pelo motivo que ela tende a ser um laço entre a língua, o texto verbal e o uso, estando estes fatores a disposição dos sujeitos sociais. Para Neves (1997, p. 03, grifos da autora), “A principal tarefa de uma gramática funcional, como acentua de Beaugrande (1993, cap .III), é ‘fazer correlações ricas entre forma e significado dentro do contexto global que se encontra presente no discurso’”. Assegurando isto, Castilho (1994, p. 76) em seu texto diz o seguinte: A gramática funcional a que me refiro postula a língua como uma atividade social. [...] a gramática funcional concentra a atenção nos usuários e nos usos da língua, mediante uma valorização do receptor, do emissor e da variação linguística no quadro da reflexão gramatical. A gramática funcional a que me refiro postula a língua como uma atividade social. A TEORIA DIALÓGICA DA LINGUAGEM E A GRAMÁTICA FUNCIONAL: UM ENCONTRO POSSÍVEL [...] a gramática funcional concentra a atenção nos usuários e nos usos da língua, mediante uma valorização do receptor, do emissor e da variação linguística no quadro da reflexão gramatical. No presente estudo, daremos enfoque às funções internas da linguagem (que se referem ao funcionamento oracional-discur- sivo); às externas (que dizem respeito à aplicação da escolha ao dis- curso); e à semântica no que diz respeito amplo às interpretações dos sentidos. A mesma autora vem acentuar o que tange às funções da linguagem, onde, em sua perspectiva, Neves (1997, p. 07), diz: “[...] o termo função pode designar as seguintes relações: i) Entre uma forma e outra (função interna); ii) Entre uma forma e seu significado (função semântica); e iii) Entre o sistema de formas e seu contexto (função externa)” – o que acaba se referindo ao papel pragmático desempenhado no uso efetivo da linguagem. Destacamos que a função sempre será dada pelo fator social- -comunicativo, sendo reconhecido através da necessidade interativa e escolha argumentativa entre os sujeitos do discurso. Então percebemos que a linguagem é, sobretudo, utilizada para atender às necessidades humanas; e essas necessidades estão vinculadas à circulação de gêneros do discurso. Os gêneros represen- tam a atividade humana e não excluem a presença da gramática. A função de um gênero (como veremos mais adiante) é a de representar 434 S U M Á R I O as situações humanas, fazendo uso das possíveis inclinações grama- ticais. Portanto, a Gramática Funcional atua na dimensão dos gêne- ros do discurso, conforme os enunciados trabalham para os gêne- ros. Assim, em termos funcionais, tudo se resume numa questão de adequação de natureza semântica destinada ao uso e à escolha, a depender do contexto no qual a gramática é empregada. O termo “função”, quando buscado no dicionário, possui várias definições. Na linguagem, também funciona da mesma forma. Como neste estudo nos atentamos aos eventos de expressividade que demarcaram escolha gramatical, nos referimos à função externa da linguagem, descrita por Neves (1997, p. 10) da seguinte maneira: “A função externa da linguagem apontada como básica é a comu- nicativa, à qual se segue (e com a qual se mescla), como secun- dária; e a expressiva, que se refere à manifestação espontânea das emoções do falante”. A TEORIA DIALÓGICA DA LINGUAGEM E A GRAMÁTICA FUNCIONAL: UM ENCONTRO POSSÍVEL Quando nos referimos à manifestação espontânea das emo- ções dos falantes, não queremos dizer que essa manifestação se trata de um trecho comunicativo desordenado e carregado de emoções, mas sim de uma fala pré-elaborada dotada de escolha e adequação às linguagens demandadas pelo ato discursivo. Dada a associação entre escolha e uso discursivo, é oportuno destacarmos o que Neves (1997, p. 15) afirma sobre a gramática funcional: Por gramática funcional entende-se, em geral, uma teoria da organização gramatical das línguas naturais que pro- cura integrar-se em uma teoria global da interação social. Trata-se de uma teoria que assenta que as relações entre as unidades e as funções das unidades têm prioridade sobre seus limites e sua posição, e que entende a gramá- tica como acessível às pressões do uso. É com base nessa definição que compreendemos a Gramá- tica Funcional como uma teoria que abarca as interações humanas – estabelecendo, nesses termos – relações de parceria com o dialo- gismo estudado pelo Círculo de Bakhtin. 435 Partindo da compreensão discorrida sobre a Gramática Fun- cional e os gêneros do discurso, consideramos necessário, de modo pragmático, trazer à tona a noção de enunciados concretos. Segundo a Teoria Dialógica da Linguagem (Círculo de Bakh- tin), o que prediz um enunciado é a relação presente no peso sócio hierárquico existente entre os interlocutores. Assim, no modo em que fazemos construções dialógicas a cada momento, devemos saber que os enunciados (sendo a língua sob uso real) são únicos e irre- petíveis – justificativas estas que se dão em prol do evento comuni- cativo, que aborda sujeitos e valorações únicas; um local e tempo corrido definidos; uma variação linguística utilizada e determinada situação de interação – sob determinados contextos históricos. Pelas relações dialógicas serem estritamente de natureza humana, temos sempre a noção de ação e resposta a enunciados anteriores; o que gera fenômenos de responsividade e interação entre os sujeitos presentes no discurso. Com isso, cremos como necessário trazer a noção de enunciado para o Círculo de Bakhtin a partir do que nos apresentam Leite e Barbosa (2014, p. A TEORIA DIALÓGICA DA LINGUAGEM E A GRAMÁTICA FUNCIONAL: UM ENCONTRO POSSÍVEL 49-50): Sendo assim, o enunciado é um todo constituído pela parte material (linguagem verbal e/ou visual) e pela reali- dade concreta, isto é, os contextos de produção, circula- ção e recepção, bem como os conhecimentos produzidos pelos interlocutores na interação verbal, o juízo aprecia- tivo dos sujeitos, os interlocutores envolvidos e os pró- prios enunciados que o precedem ou são presumidos. Um dos objetivos que alicerça esta pesquisa é a possibili- dade de poder entender a realidade concreta de enunciados – o que implica dizer que os aspectos dialógicos a serem trabalhados são aqueles que atuam diretamente com o contexto em que os enuncia- dos em estudo foram proferidos, assim como os possíveis meios de circulação em que há diversas ações de responsividade e valorações que são sempre ligados à atividade humana e a recepção intera- tiva, como sendo representações de um sujeito em relação a outros; o que em outras palavras se resume à significação (re)produzida e compreendida pelos sujeitos. 436 Essa atividade humana em utilização da linguagem presume certa relação inerente aos enunciados: a presença do falante e sua plateia. Dentro dessa relação, podemos encontrar determinada orga- nização denominada de enunciados. Essa organização diz respeito aos aspectos temáticos, formais, ideológicos e estilísticos, dos quais em fase posterior, darão origem aos gêneros discursivos. Assim, tam- bém podemos reconhecer um enunciado como concreto, quando identificamos os posicionamentos tomados pelos sujeitos atuantes no campo da enunciação. Portanto, a função maior de um enunciado é a de situar. Essa primordial característica faz com que todo enun- ciado seja irrepetível61 sob determinado contexto histórico-social, que pressupõe a presença de características enunciativas já supracitadas. Diante destas definições, trazemos à tona o capítulo Cons- truções sintáticas adjetivas no funcionamento discursivo do artigo de opinião, contendo os dois corpora que subsidiam nossa pesquisa. Os enunciados são irrepetíveis pelo fato de que Bakhtin entende o evento enunciativo como singular. CONSTRUÇÕES SINTÁTICAS ADJETIVAS NO FUNCIONAMENTO DISCURSIVO DO ARTIGO DE OPINIÃO Para atingir a proposta desta pesquisa, é necessário analisar a função que a língua exerce dentro dos enunciados concretos, par- tindo dos níveis de escrita ou de reproduções da fala. Quando fazemos uma análise das orações subordinadas adje- tivas sob a perspectiva do discurso, percebemos que elas tendem a passar de um grau interpretativo mais simples, para outro de comple- xidade maior, visto que os sentidos de tais orações possuem discur- sivamente fortes naturezas tendenciosas ideologicamente situadas. 61 437 Associando o conceito de sujeito, podemos perceber – e também possivelmente interpretar – o que determinados dizeres querem dizer. Assim, o reconhecimento dos pronomes relativos den- tro das orações subordinadas adjetivas auxiliará, semanticamente, os possíveis posicionamentos do enunciador no ato do dizer. Diante dessas colocações, podemos fazer diversas pergun- tas: Até onde o reconhecimento de um “que” dentro de uma sentença pode ser necessariamente impositivo? E qual seria a relação dessa semântica impositiva com as orações restritivas ou explicativas? Ou mesmo, até onde uma sentença declarativa pode ser interpretada – visto que – discursivamente, as partículas relativas aparentam ser cada vez mais tendenciosas? Como podemos enxergar em um texto, se na presença ou ausência de vírgulas, tanto as adjetivas restritivas quanto explicativas possuem o mesmo sentido? Essas discussões são necessárias para podermos dar início às possíveis interpreta- ções analíticas sob um olhar discursivo. Sem almejar adiantar análises, enfatizamos que o gênero jornalístico artigo de opinião, por possuir natureza argumentativa e sociopolítica (se atendo às diversas camadas e classes sociais que redigem os textos), possibilita a presença de entonações semânti- cas sobre o discurso. Fonte: CONTREIRA, Thiago. Opinião: senadores desrespeitam Luciano Hang. Disponível em: <https:// bit.ly/3HDgvgi>. Acesso em 26 out. 2022. 62 O canal de notícias R7 é conhecido por ser um canal de filiação ideológica da direita. 63 O segundo artigo analisado possui representação valorativa da esquerda. AS ORAÇÕES SUBORDINADAS ADJETIVAS COMO ENUNCIADOS CONCRETOS NO GÊNERO ARTIGO DE OPINIÃO COM TEMÁTICA SOBRE A CPI DA COVID-19 Para essa análise, trouxemos dois artigos de opiniões distin- tas: o primeiro, redigido por um sujeito atuante em canal noticiário 438 de direita62; o segundo por um sujeito filiado a um canal de notí- cias de esquerda63. Partindo destas considerações, fazemos, nesse momento, a chamada do primeiro artigo selecionado como Corpus desta pesquisa. Convocamos no quadro a seguir, o texto intitulado de “Opinião: senadores desrespeitam Luciano Hang”, assinado pelo jornalista Thiago Contreira. S U M Á R I O Quadro 01 – Corpus 1 - Artigo de opinião assinado por Thiago Contreira Opinião: senadores desrespeitam Luciano Hang A CPI virou um circo e a culpa é dos senadores que comandam a CPI da Covid Thiago Contreira O que o Brasil está vendo no dia de hoje é lamentável, uma vergonha para o Senado. O depoimento do empresário Luciano Hang, proprietário da rede de lojas Havan, foi transformado em um circo, e a culpa é toda dos senadores que comandam a CPI da Covid. O senador Renan Calheiros foi desrespeitoso. Mesmo sem citar nomes, não se pode chamar um depoente de “bobo da corte”, seja ele quem for, tenha o depoente a posição política que tenha. Do mesmo modo, o presidente da CPI, Omar Aziz, não pode se transformar em um fiscal das virtudes, uma espécie tosca de comentarista de respostas do inquirido. Já o senador Randolfe Rodrigues, contrariando qualquer prática democrática, chegou ao absurdo de proibir o depoente de usar adjetivos nas respostas. Os senadores precisam ser lembrados de que estão na CPI para investigar o combate à Covid e, para isso, devem fazer perguntas objetivas, sérias e, é claro, deixar que os depoentes falem. Também é preciso exigir isonomia por parte dos senadores. É absurda a diferença de tratamento que os senadores dispensam a Luciano Hang em relação aos depoentes “alinhados” com a oposição ao presidente Bolsonaro. Basta rever o depoimento da advogada dos ex-médicos da Prevent Senior ontem. Para esta CPI ser levada a sério pela população, os senadores que a compõem devem esquecer os holofotes e lembrar que são funcionários do Brasil. Fonte: CONTREIRA, Thiago. Opinião: senadores desrespeitam Luciano Hang. Disponível em: <https:// bit.ly/3HDgvgi>. Acesso em 26 out. 2022. AS ORAÇÕES SUBORDINADAS ADJETIVAS COMO ENUNCIADOS CONCRETOS NO GÊNERO ARTIGO DE OPINIÃO COM TEMÁTICA SOBRE A CPI DA COVID-19 Quadro 01 – Corpus 1 - Artigo de opinião assinado por Thiago Contreira Opinião: senadores desrespeitam Luciano Hang A CPI virou um circo e a culpa é dos senadores que comandam a CPI da Covid Thiago Contreira O que o Brasil está vendo no dia de hoje é lamentável, uma vergonha para o Senado. O depoimento do empresário Luciano Hang, proprietário da rede de lojas Havan, foi transformado em um circo, e a culpa é toda dos senadores que comandam a CPI da Covid. O senador Renan Calheiros foi desrespeitoso. Mesmo sem citar nomes, não se pode chamar um depoente de “bobo da corte”, seja ele quem for, tenha o depoente a posição política que tenha. Do mesmo modo, o presidente da CPI, Omar Aziz, não pode se transformar em um fiscal das virtudes, uma espécie tosca de comentarista de respostas do inquirido. Já o senador Randolfe Rodrigues, contrariando qualquer prática democrática, chegou ao absurdo de proibir o depoente de usar adjetivos nas respostas. Os senadores precisam ser lembrados de que estão na CPI para investigar o combate à Covid e, para isso, devem fazer perguntas objetivas, sérias e, é claro, deixar que os depoentes falem. Também é preciso exigir isonomia por parte dos senadores. É absurda a diferença de tratamento que os senadores dispensam a Luciano Hang em relação aos depoentes “alinhados” com a oposição ao presidente Bolsonaro. Basta rever o depoimento da advogada dos ex-médicos da Prevent Senior ontem. Para esta CPI ser levada a sério pela população, os senadores que a compõem devem esquecer os holofotes e lembrar que são funcionários do Brasil. Quadro 01 – Corpus 1 - Artigo de opinião assinado por Thiago Contreira Thiago Contreira O que o Brasil está vendo no dia de hoje é lamentável, uma vergonha para o Senado. O depoimento do empresário Luciano Hang, proprietário da rede de lojas Havan, foi transformado em um circo, e a culpa é toda dos senadores que comandam a CPI da Covid. O senador Renan Calheiros foi desrespeitoso. Mesmo sem citar nomes, não se pode chamar um depoente de “bobo da corte”, seja ele quem for, tenha o depoente a posição política que tenha. Do mesmo modo, o presidente da CPI, Omar Aziz, não pode se transformar em um fiscal das virtudes, uma espécie tosca de comentarista de respostas do inquirido. Já o senador Randolfe Rodrigues, contrariando qualquer prática democrática, chegou ao absurdo de proibir o depoente de usar adjetivos nas respostas. Os senadores precisam ser lembrados de que estão na CPI para investigar o combate à Covid e, para isso, devem fazer perguntas objetivas, sérias e, é claro, deixar que os depoentes falem. Os senadores precisam ser lembrados de que estão na CPI para investigar o combate à Covid e, para isso, devem fazer perguntas objetivas, sérias e, é claro, deixar que os depoentes falem. Também é preciso exigir isonomia por parte dos senadores. É absurda a diferença de tratamento que os senadores dispensam a Luciano Hang em relação aos depoentes “alinhados” com a oposição ao presidente Bolsonaro. Basta rever o depoimento da advogada dos ex-médicos da Prevent Senior ontem. Para esta CPI ser levada a sério pela população, os senadores que a compõem devem esquecer os holofotes e lembrar que são funcionários do Brasil. 63 439 O Corpus 1 provém do canal de notícias R7. Trata-se do artigo de opinião assinado por Thiago Contreira, em 29 de setembro de 2021. O autor deste artigo atua como diretor de jornalismo na Record TV e demais áreas prioritárias, como: o Jornal da Record, a editoria de política e a de opinião da rede de comunicação. Vejamos, a seguir, as ocorrências das orações adjetivas no texto em análise. O critério inicial da geração de dados foi o reconhe- cimento da construção sintática, para, então, partir para o funciona- mento da organização discursiva do gênero, planejada e elaborada pelo articulista do artigo. Thiago Contreira Eis os dados presentes na Tabela 01: Tabela 01 – Apresentação dos dados reconhecidos no Corpus 1 Orações subordinadas adjetivas Construção sintática adjetiva 1: que comandam a CPI da Covid Construção sintática adjetiva 2: quem for Construção sintática adjetiva 3: que tenha Construção sintática adjetiva 4: que os senadores dispensam a Luciano Hang em relação aos depoentes “alinhados” com a oposição ao presidente Bolsonaro Construção sintática adjetiva 5: que a compõem Fonte: Autoria própria. Tabela 01 – Apresentação dos dados reconhecidos no Corpus 1 A partir do dado apresentado, verificamos que a oração adjetiva “que comandam a CPI da Covid” indica que, para o autor do artigo, existe certa pressão que faz com que a CPI, de fato, seja um espetáculo; mais do que uma apuração. Percebemos que, pela construção da mesma oração, conseguimos compreender um posi- cionamento do autor contrário à CPI. A oração “quem for” vem complementar a oração princi- pal “[...] não se pode chamar um depoente de “bobo da corte, [...]” 440 e se refere, primeiramente, ao que se diz sobre o depoente; para depois se referir a sua posição política. Dentro do possível modo de ver do articulista, a escolha do verbo tenha inserido na oração “que tenha”, indica necessi- dade de neutralidade por parte dos senadores sobre o inquérito, se relacionando sobre as posições político-partidárias dos sujei- tos autuados pela CPI. A mesma ideia de neutralidade é retomada pela quarta oração em destaque, onde o articulista expõe a necessidade dos senadores exercerem isonomia, ao afirmar sobre a diferença de tratamento “que os senadores dispensam a Luciano Hang em relação aos depoentes “alinhados” com a oposição ao presidente Bolsonaro”. A escolha do verbo dispensam mostra um possível tom de pessoalidade, sendo marcada no discurso do articulista que, talvez, seja desfavorável à comissão de inquérito. A última oração adjetiva “que a compõem” se refere aos sena- dores da CPI e vem expor o posicionamento de Contreira sobre o comportamento dos membros integrantes da CPI. Esse posiciona- mento é marcado pelos verbos impositivos devem e esquecer, que finalizam o artigo de opinião com tons de patriotismo, sendo estes demarcados pelo trecho “[...] devem esquecer os holofotes e lembrar que são funcionários do Brasil”. Pensando na estrutura composicional do artigo, que segundo Bakhtin (2016), é “um elemento inerente aos gêneros do discurso”, percebemos que o articulista demarca maiores valorações no desen- volvimento do texto em questão. Fonte: MARCON, Dionilso. CPI DA covid sim! Precisamos salvar o Brasil do genocídio. Disponível em: <http://bit.ly/3YffnGC>. Acesso em 26 out. 2022. Thiago Contreira Sua política genocida conduziu o país ao caos social.” Dionilso Marcon Thiago Contreira No que toca à ocorrência das construções sintáticas adjeti- vas presentes no Corpus 1, convocamos a tabela a seguir: 441 Tabela 02 – Construção oracional reconhecida no Corpus 1 Orações adjetivas explicativas Orações adjetivas restritivas quem for que comandam a CPI da Covid que tenha que os senadores dispensam a Luciano Hang em relação aos depoentes “alinhados” com a oposição ao presidente Bolsonaro que a compõem Fonte: Autoria própria. Tabela 02 – Construção oracional reconhecida no Corpus 1 A respeito do uso não aleatório das orações adjetivas (ou seja, a escolha destas orações), percebemos que o discurso do arti- culista tende a se inclinar em desfavor dos demais investigadores e depoentes contra Bolsonaro. Esse fator é lido pelo peso semântico que as orações adjetivas – previamente escolhidas – carregam no decorrer do artigo de opinião em análise. Podemos, ainda, destacar que, para o articulista, a CPI da COVID-19 é um enunciado conhecido, pois ele possui tematização delimitada; sujeitos definidos; discussões de naturezas política, his- tórica e social; local e tempo definidos; estrutura organizacional pré- -elaborada; e movimentos de interação ocorridos na relação entre o sujeito investigador e o sujeito investigado, tratando-se portanto de um conjunto de enunciados concretos. O uso consciente da gramática funcional pode ser atribuído ao articulista deste Corpus, no momento em que suas ocorrências adjetivais são usadas de maneira valorativa – visto que o articulista Contreira se encontra sob um contexto de interação social e represen- tação de filiação ideológica, fazendo jus a uma análise completa por incluir referência ao falante sob aspectos da enunciação discursiva. Dadas as identificações oracionais associadas às interpre- tações discursivas encontradas no Corpus 1, fazemos chamada ao nosso segundo Corpus utilizado para esta pesquisa. 442 O Corpus 2 provém do canal de notícias Brasil de Fato/RS. Trata-se de artigo de opinião assinado por Dionilso Marcon. O arti- culista em questão atua como deputado federal no Rio Grande do Sul pelo Partido dos Trabalhadores, desde 2011. O canal de notícias Brasil de Fato/RS também é proveniente do Rio Grande do Sul e se trata de um jornal lançado no ano de 2018. Vejamos, a seguir, no Quadro 02, o segundo artigo de opinião selecionado como dado desta investigação. Quadro 02 – Corpus 2 - Artigo de Opinião assinado por Dionilso Marcon CPI da covid sim! Precisamos salvar o Brasil do genocídio “Bolsonaro precisa ser investigado, julgado e responsabilizado. Dionilso Marcon Todo dia morrem milhares de brasileiros e brasileiras. Na semana passada, chegamos à triste marca de 4 mil mortes por covid-19 por dia. É como se todo o dia um município pequeno do nosso Interior desaparecesse: são 185 municípios gaúchos com menos de 4 mil habitantes. Até quando vamos ficar amarrados diante desta situ- ação, respeitando um calendário legal, enquanto a vida morre em todo o nosso país? Crimes de Responsabilidade não faltam para dar Impeachment em Bolsonaro e seu governo genocida. O mais re- cente é o conteúdo da conversa com o senador Kajuru (Cidadania - GO), que deixou nítida a intenção de interferir no Supremo Tribunal Federal, após a decisão do ministro Barroso de instalar a CPI da covid no Senado. A conversa de Bolsonaro com Kajuru é grave e apresenta a reação do presidente que teme ser responsabilizado pelos seus atos. Além de grave, é criminosa! Bolsonaro precisa ser investigado, julgado e responsabilizado. Sua política genocida conduziu o país ao caos social, sanitário e econômico. Nossa luta em Brasília continua sendo pela vida do povo, por vacina para todos os brasileiros, auxílio emergen- cial de R$ 600 até o fim da pandemia, para colocar comida na mesa e fomentar o comércio, pelos subsídios às micro e pequenas empresas e por uma investigação séria, que chegue na raiz do problema e considere o que vie- mos denunciando desde o início dessa pandemia. Bolsonaro não adotou medidas sanitárias, quebrou micro e pequenas empresas, beneficiou grandes empresári- os, trouxe a fome de volta para a mesa dos brasileiros e é responsável pelas mais de 350 mil mortes. O Impeachment de Bolsonaro é urgente! A mobilização popular é necessária e os parlamentares precisam honrar o compromisso com o povo brasileiro. Fora Bolsonaro e seu governo genocida! 443 No decorrer do texto, foram encontradas poucas ocorrên- cias de orações subordinadas adjetivas. Segue a Tabela 03 com as devidas identificações. Tabela 03 – Apresentação dos dados reconhecidos no Corpus 2 Orações subordinadas adjetivas Construção sintática adjetiva 1: que deixou nítida a intenção Construção sintática adjetiva 2: que teme ser responsabilizado pelos seus atos. Construção sintática adjetiva 3: que chegue na raiz do problema Construção sintática adjetiva 4: (que) considere Fonte: Autoria própria No desenvolvimento do artigo assinado por Dionilso Mar- con, encontramos a presença da oração adjetiva “que deixou nítida a intenção” se referindo à conversa realizada entre o senador Jorge Kajuru e o presidente Bolsonaro. A possível escolha desta oração se dá ao seu poder argu- mentativo sendo exercido em relação à intenção de intervenção sobre as decisões do Supremo Tribunal Federal, através das atitudes arbitrárias tomadas pelo então presidente. O mesmo fato é justificado através da segunda oração em relação à conversa em questão. A oração “que teme ser responsabili- zado pelos seus atos” mostra uma posição favorável às investigações contra o presidente Bolsonaro, visto que a formulação semântica da oração se originou a partir do verbo “teme”, que apresenta certo receio do presidente acerca das investigações realizadas. O articulista conclui em sua última oração acerca do inqué- rito “que chegue na raiz do problema” e “(que) considere”, que as investigações da CPI devem ser resolutivas e assertivas, no intuito de que comprovem as acusações feitas contra as atitudes do chefe do executivo. O segundo pronome relativo que vem acompanhado ocultamente, atribuindo sentido mais amplo à oração principal. 444 Percebemos que há maior incidência de orações adjetivas no movimento de desenvolvimento do artigo, o que justifica no texto a presença de certos tons de caracterização e delimitação escolhi- dos pelo articulista para a construção de seu discurso sobre a CPI. Acerca do uso não aleatório das orações adjetivas, encontramos estruturas discursivas semelhantes (com pontos de vista distintos) às encontradas no Corpus 1, visto que as orações adjetivas escolhi- das para compor o artigo possuem um tom de maior pessoalidade acerca das investigações contra o presidente Bolsonaro. Segue, então, a tabela com as construções sintáticas adjeti- vas reconhecidas no Corpus 2. Tabela 04 – Construção oracional reconhecida no Corpus 2 Orações adjetivas explicativas Orações adjetivas restritivas Que deixou nítida a intenção Que teme ser responsabilizado pelos seus atos Que chegue na raiz do problema (que) considere Fonte: Autoria própria. Conseguimos reconhecer que as orações encontradas no texto podem funcionar – aos olhos do discurso – com formas adjeti- vais diferentes, se valendo de interpretações mais ou menos articulá- veis, a depender do crítico ponto de vista dos articulistas. A linguística funcional abordada neste trabalho considera que determinados usos linguísticos são motivados por fatores extra- linguísticos. Tabela 03 – Apresentação dos dados reconhecidos no Corpus 2 Orações subordinadas adjetivas Construção sintática adjetiva 1: que deixou nítida a intenção Construção sintática adjetiva 2: que teme ser responsabilizado pelos seus atos. Construção sintática adjetiva 3: que chegue na raiz do problema Construção sintática adjetiva 4: (que) considere Fonte: Autoria própria Deste modo, concordamos e compreendemos que estes fatores são de natureza subjetiva, cultural e social, uma vez que temos nosso olhar voltado para o sujeito em sociedade. Em linhas gerais, a análise empreendida nesta investigação ergueu-se pela tentativa de compreender a presença das constru- ções sintáticas adjetivas como enunciados concretos que estão nos projetos de dizer dos articulistas implicados nos corpora da pesquisa. 445 Ao nosso ver, esse movimento permite-nos efetuar um pro- cesso analítico de descrição linguística à luz de um olhar mais fle- xível e contextual, como apregoado pelos estudos da Gramática Funcional, e de um modo de observar/ler fenômenos linguísticos a partir de uma perspectiva dialógica e discursiva, como são situ- adas as abordagens teórico-metodológicas da Teoria Dialógica da Linguagem. Com a finalização das etapas desta pesquisa, fazemos encaminhamento para as considerações finais. CONSIDERAÇÕES FINAIS Após observarmos a riqueza de nossa língua sob seus aspec- tos sintático-discursivos, obtivemos êxito ao responder a pergunta de nossa pesquisa, a saber: Como construções sintáticas adjetivas constituem enunciados concretos no gênero artigo de opinião? Durante nossa pesquisa, conseguimos responder que as construções sintáticas adjetivas só se realizam como fenômenos do discurso, através da escolha discursiva dentro dos enunciados concretos, que são o próprio palco para os gêneros do discurso; e acerca das entoações valorativas e expressivas, faz-se necessá- rio observar a posição social na qual os sujeitos da enunciação se encontram, para que estes se demonstrem favoráveis ou desfavorá- veis perante as situações que demandam posicionamentos (enun- ciados) e que, por sua vez, demandam da atividade interativa daque- les que usam a linguagem. Percebemos, portanto, que há uma relação direta entre o sujeito e a filiação ideológica a qual ele pertence – concordância esta que justifica a escolha de um conjunto de corpus de natureza estritamente política. 446 Vale também, retornar aos objetivos assumidos nesta pes- quisa. Em linhas gerais, acreditamos ter alcançado tais objetivos, uma vez que logramos êxito em compreender a construção dialógica de sentidos dentro da função dos discursos analisados, sob a ocorrência das orações adjetivas – visto que o discurso possui tendências valo- rativas ao assumir posicionamentos de naturezas verbo-ideológicas durante a interação entre os sujeitos participantes da enunciação. A pesquisa possui relevância na área da linguística porque abordou determinados fenômenos sintáticos – orações adjetivas – analisados em perspectiva dialógica e discursiva – o que nos faz enten- der a produtiva relação entre a concepção de gramática dos estudos funcionalistas, como os de Neves (1997), por exemplo, e a concepção de enunciado concreto oriunda dos estudos do Círculo de Bakhtin. Este trabalho proporcionou maior brilho à linguagem no momento em que ela é colocada em funcionamento sobre situações que deman- dam seu uso, oferecendo aos estudiosos um leque de possibilidades que a linguagem é capaz de assumir, a depender de seu contexto (como sendo os enunciados) e, sobretudo, acerca de seu objeto de estudo (os sujeitos que põem a língua/linguagem para funcionar). CONSIDERAÇÕES FINAIS Por fim, o que aprendemos como cientistas da linguagem nos estudos abordados nesta pesquisa diz respeito às capacidades produtivas naturais à língua, sendo estes fatores o que geram curio- sidades por parte dos estudantes da linguagem e também deter- minada necessidade social e humana em (re)analisar a linguística sob os vieses discursivos e estruturais que, normalmente, se trans- formam a partir da dependência usual da linguagem. Reconhecemos que outras discussões analíticas poderão ser realizadas sobre nosso estudo. De fato, as abordagens sempre dei- xam algo por dizer, o que legitima a própria visão de linguagem como inacabada para Bakhtin. Todavia, defendemos que, dentro do possí- vel em um trabalho de conclusão de curso de graduação, lemos que realizamos uma pesquisa que surta o efeito de servir como estímulo a outras investigações. 447 E que venham mais pesquisas que interconectem Gramática Funcional e Teoria Dialógica da Linguagem. S U M Á R I O BAKHTIN, Mikhail. Os gêneros do discurso. Tradução de Paulo Bezerra. São Paulo: 34, 2016. BAKHTIN, Mikhail. Os gêneros do discurso. Tradução de Paulo Bezerra. São Paulo: 34, 2016. CASTILHO, Ataliba Teixeira de. Um  ponto de  vista  funcional  sobre  a  predicação. Alfa,  São  Paulo,  n. 38, p. 75-95, 1994. FURTADO DA CUNHA, Maria Angélica. O modelo das motivações competidoras no domínio funcional da negação. Revista DELTA, São Paulo, v. 17, n. 1, p. 1-30, 2001. LEITE, Lucila Carvalho; BARBOSA, Tatyana Mabel Nobre. Linguagem e educação: diálogos entre Mikhail Bakhtin e Paulo Freire. In.: LEITE, Lucila Carvalho; BARBOSA, Tatyana Mabel Nobre (Orgs.). Cartografia da produção textual: livros didáticos, gênero do discurso, políticas e indicadores. Natal: EDUFRN, 2014, p. 49-70. NEVES, Maria Helena de Moura. A gramática funcional. São Paulo: Martins Fontes, 1997. PEREIRA, Rodrigo Acosta; COSTA-HÜBES, Terezinha da Conceição. Sobre a Análise da Língua: considerações em Bakhtin e Volochínov. In: PEREIRA, Rodrigo Acosta; COSTA- HÜBES, Terezinha da Conceição (Orgs.). Prática de análise linguística nas aulas de Língua Portuguesa. São Carlos: Pedro & João, 2021, p. 109-131. PEREIRA, Rodrigo Acosta; COSTA-HÜBES, Terezinha da Conceição. Sobre a Análise da Língua: considerações em Bakhtin e Volochínov. In: PEREIRA, Rodrigo Acosta; COSTA- Língua: considerações em Bakhtin e Volochínov. In: PEREIRA, Rodrigo Acosta; COSTA- HÜBES, Terezinha da Conceição (Orgs.). Prática de análise linguística nas aulas de Língua Portuguesa. São Carlos: Pedro & João, 2021, p. 109-131. HÜBES, Terezinha da Conceição (Orgs.). Prática de análise linguística nas aulas de Língua Portuguesa. São Carlos: Pedro & João, 2021, p. 109-131. HÜBES, Terezinha da Conceição (Orgs.). Prática de análise linguística nas aulas de Língua Portuguesa São Carlos: Pedro & João 2021 p 109-131 RODRIGUES, Rosângela Hammes; PEREIRA, Rodrigo Acosta;. Os Gêneros do Discurso como elementos integradores para/nas aulas de leitura, escuta, produção textual e análise linguística: subsídios teórico-metodológicos. In: PEREIRA, Rodrigo Acosta; COSTA-HÜBES, Terezinha da Conceição (Orgs.). Prática de análise linguística nas aulas de Língua Portuguesa. São Carlos: Pedro & João, 2021, p.157-181. SÉRIOT, Patrick. Volosinov e a filosofia da linguagem. Tradução de Marcos Bagno. São Paulo: Parábola, 2015. 448 S U M Á R I O INTRODUÇÃO A expressão “monstros à solta” diz respeito à ideia de que algo está fora de ordem e como “monstro” pode causar medo. Então, aqui, ao tratarmos de gramática como um componente instável, em que as categorias podem ser flutuantes, podendo ir do concreto ao abstrato, torna-se um receio para alguns professores, pois iremos demonstrar como o uso linguístico move categorias que compêndios normativos indicam como estáveis. Contudo, esperamos demonstrar que realinhar a análise para uma prática descritiva, em vez de pres- critiva, não é um “bicho de sete cabeças”. Então, ensinar não é uma tarefa simples, demanda tempo, ânimo e vontade eterna de aprender. Especialmente, no ensino de Língua Portuguesa (LP), uma vez que seu objeto se faz e refaz todos os dias, dentro e fora da escola. Nesse contexto, o professor de LP não pode nem deve estar “algemado” à gramática normativa e seus preceitos tradicionais como única ferramenta de ensino sobre a lín- gua. Afinal, “[...] há muito tempo a função do professor de português não é a de guardião daquela língua que ele não fala – nunca falou – e na qual raramente se atreve a escrever” (GUEDES, 2006, p. 13). Objetivamos, nesta proposta, analisar usos linguísticos que se distanciam daqueles que a gramática tradicional prescreve como “corretos”. Utilizaremos um corpus inédito, que é composto por trans- crição de entrevistas feitas com falantes do interior da PB, coletadas por discentes da Educação Básica na localidade. A escolha desse corpus está de acordo com a adoção do Linguística Funcional, uma vez que essa perspectiva teórica analisa dados reais de uso da lín- gua, retirados de situações concretas de comunicação (FURTADO DA CUNHA, 2017). Em termos de metodologia, temos uma natureza qualitativa de caráter propositivo, haja vista os interesses pedagógi- cos que atravessam esta discussão. 450 Nesse sentido, justificamos este trabalho a partir das inquie- tações enquanto professores de língua(s), estudantes da linguagem, bem como pela necessidade social de se estabelecer caminhos favoráveis e possíveis para uma (re)significação do ensino de LP, o qual, por vezes, ainda tem se limitado a postulações metalinguísti- cas. Nesse sentido, as contribuições aqui defendidas são de cará- ter pedagógico, guiadas pelo rigor científico e fundamentadas nos estudos funcionais da língua, com foco na gramaticalização, a fim de analisar a relação entre as estruturas gramaticais e os contextos efetivos de comunicação. Esse artigo estrutura-se em 4 seções. INTRODUÇÃO Depois desta introdu- ção, algumas colocações teóricas acerca da Linguística Funcional e conceitos de Gramática são discutidos; na seção seguinte refletimos sobre a emergência de uma gramática reflexiva para o ensino de língua, uma vez que os próprios documentos oficiais indicam a rele- vância da análise linguística. Adiante, na seção metodológica e ana- lítica, estabelecemos o contexto de produção do corpus; logo depois, as propostas de análise são evidenciadas. Por fim, apresentamos as considerações finais, seguidas das referências que ancoram as ideias aqui expostas. EM DEFESA DE UMA BASE FUNCIONALISTA PARA A GRAMÁTICA Uma teoria é chamada de funcionalista quando está oposta àquelas conhecidas como formalistas, como é o caso do Estrutu- ralismo e, atualmente, em domínio, o Gerativismo, instaurado por Noam Chomsky nos anos 60. Nessa direção, modelos pós-forma- listas são, por exemplo, a Sociolinguística, a Linguística Cognitiva, a Análise da Conversação e a Análise do Discurso. Além desses, há 451 também os estudos específicos da Linguística Funcional, a qual se interessa pela língua em uso como “uma base comunicativa que for- nece dados que regulam a interação entre falantes” (FURTADO DA CUNHA et al. 2017, p. 19). Pela necessidade de adotar uma concepção de língua no instante em que são propostos os objetivos de uma pesquisa, con- sideramos, para os desdobramentos do que se escreve neste traba- lho, o postulado de que língua é um instrumento da interação social. Assim, buscamos investigar os fatos linguísticos, concebendo os propósitos comunicativos dos interlocutores e o contexto em que se situa a comunicação e ultrapassando a estrutura gramatical, como expressa Furtado da Cunha (2017). Assim, a Linguística Funcional vislumbra o seguinte: [...] as estruturas linguísticas estão correlacionadas às circunstâncias discursivas nas quais são geradas, entre- laçando aspectos culturais e cognitivos na produção e veiculação de informações. O conteúdo semântico e os propósitos explícitos ou implícitos nos atos de fala são fatores relevantes, influenciando os estudos funcionalis- tas e redimensionando a preocupação com as situações concretas de uso [...] (SILVA, 2011, p. 71). Mediante as colocações de Silva (2011), estabelecemos que critérios puramente formais são insuficientes para a análise linguís- tica; não se trata de postular que práticas estruturalistas e gerati- vistas façam uma análise de menor valor, mas é uma análise que desconsidera o uso linguístico. Nesse sentido, a análise linguística de cunho funcionalista compreende, de acordo com Furtado da Cunha (2017), duas questões: i) a língua exerce funções que são externas ao sistema linguístico e ii) as ações externas influem na organização interna do sistema linguístico. Portanto, não se concebe a língua em si e por si, uma vez que ela está interligada ao comportamento social dos falantes, a partir das variadas práticas de comunicação. 452 S U M Á R I O Especificamente, a gramaticalização é a possibilidade que itens linguísticos têm de renovar suas funções gramaticais, devido a sua expressividade e grande frequência de uso. EM DEFESA DE UMA BASE FUNCIONALISTA PARA A GRAMÁTICA Portanto, trata-se da “[...] produção de novos recursos gramaticai já s a partir de (re) processamentos cognitivos, por parte dos falantes, impostos aos recursos gramaticais já existentes (BAGNO, 2013, p. 163). Nessa dire- ção, Gonçalves et al. (2007, p. 20) reforçam essa perspectiva sobre gramaticalização dizendo que “palavras de uma categoria lexical plena (nomes, verbos e adjetivos) podem passar a integrar a classe das categorias gramaticais (preposições, advérbios, auxiliares etc.), as quais, em momento posterior, podem vir até mesmo a se tornar afixes”. E, outra maneira de entendermos o processo de gramaticali- zação é pensarmos no contínuo apresentado por Givón (1979, p. 209 apud BYBEE, 2020, p. 283), que aponta a mudança linguística como “ondas cíclicas” que repetem o seguinte padrão: Discurso – Sintaxe – Morfologia – Morfofonêmica – Zero. Mediante o exposto, fica em evidência que a linguística de caráter funcional, especificamente, o Funcionalismo Linguístico, coloca em evidência o uso linguístico em uma análise que não retira o uso das instâncias discursivas, tampouco o coloca inacessível a questionamentos como: quem disse? Como disse? Por que disse? E para quê? Dito isso, o quadro a seguir expõe um resumo da visão funcionalista sobre a linguagem, com base em premissas aponta- das por Givón (1995): 453 Quadro 01 – Premissas funcionalistas ■ A linguagem é uma atividade sociocultural; ■ A estrutura serve a funções cognitivas e comunicativas; ■ A estrutura é não arbitrária, motivada, icônica; ■ Mudança e variação estão sempre presentes; ■ O sentido é contextualmente dependente e não atômico; ■ As categorias não são discretas; ■ A estrutura é maleável e não rígida; ■ As gramáticas são emergentes; ■ As regras gramaticais permitem algumas exceções. Fonte: apud Martelotta & Kenedy, 2015, p. 20. ■ A linguagem é uma atividade sociocultural; ■ A estrutura serve a funções cognitivas e comunicativas; ■ A estrutura é não arbitrária, motivada, icônica; ■ Mudança e variação estão sempre presentes; ■ O sentido é contextualmente dependente e não atômico; ■ As categorias não são discretas; ■ A estrutura é maleável e não rígida; ■ As gramáticas são emergentes; ■ As regras gramaticais permitem algumas exceções. Fonte: apud Martelotta & Kenedy, 2015, p. 20. Na direção dessas premissas, consideramos importante con- siderar um conceito de gramática a partir do qual possamos enten- der, mais à frente, o método pelo qual encaminhamos nossas análi- ses. EM DEFESA DE UMA BASE FUNCIONALISTA PARA A GRAMÁTICA Para tanto, faz-se importante compreender que a maneira como compreendemos os fenômenos relacionados à gramática da(s) lín- gua(s) se modificou no decorrer dos anos, isso desde a gramática grega até os estudos mais modernos da linguística, conforme Marte- lotta (2017). Desse modo, tinha-se uma concepção de base filosófica ancorada nas relações entre a linguagem e o pensamento lógico de viés aristotélico, então, com o advento da linguística moderna proce- dimentos científicos da chamada ciência moderna passaram a ser incorporados nos estudos da linguagem, abandonando uma visão sem comprovações empíricas, de acordo com o autor supracitado. O estudo voltado para a gramática percorreu grandes obs- táculos ao longo do tempo, visto que o uso do termo sobredito se tornou problemático dado o seu direcionamento unilateral. Dito isso, podemos destacar a gramática e suas multifaces por meio de estudos, a exemplo do esquematizado por Martelotta (2017), o qual desmistifica diferentes tipos de gramática, possibilitando uma maior compreensão sobre esse componente/elemento intrínseco à língua. 454 Desse modo, os sujeitos, de forma natural, fazem combinações das unidades classificadas por Martelotta (2017) como menores univer- sais, haja vista a existência nas mais diversas línguas, que compõem o discurso, tais como: morfemas, vocábulos, frases. Desse modo, os sujeitos, de forma natural, fazem combinações das unidades classificadas por Martelotta (2017) como menores univer- sais, haja vista a existência nas mais diversas línguas, que compõem o discurso, tais como: morfemas, vocábulos, frases. Isso posto, Martelotta (2017, p. 43) faz questionamentos acerca do processo elaborativo/combinatório dos elementos cita- dos acima: como se dá essa combinação? Os falantes combinam os elementos na frase do modo como bem entendem ou existem res- trições impostas pelas línguas no que diz respeito a esse processo? Se existem restrições, qual a sua natureza? Elas provêm dos padrões de correção de uso da língua impostos pela comunidade? São arbi- trárias? Refletem o funcionamento natural da mente humana, sendo, portanto, universais? O pesquisador tem a tarefa de evidenciar as numerosas maneiras de abordar temas que envolvem o funciona- mento da língua, assim como as diferentes propostas de sistemati- zação descritiva. À vista disso, “o conjunto dessas interpretações e descrições acerca do funcionamento da língua recebe o nome de gramática” (MARTELOTTA, 2017, p.44). EM DEFESA DE UMA BASE FUNCIONALISTA PARA A GRAMÁTICA É válido ressaltar a dicotomia presente na definição do termo gramática, posto que, prioritariamente, define-se gramática como um conjunto de regras pré-estabelecidas, as quais demarcam limi- tes entre os elementos do discurso, o que designa o funcionamento da língua e auxilia na construção do discurso em diversas situações de uso. No entanto, ainda podemos estabelecer que a gramática tem enfoque nos estudos que buscam explicar a natureza dos elementos discursivos, assim como suas respectivas características restritivas e combinatórias. Por esse viés, é plausível dizer que a gramática “se refere aos modelos teóricos criados pelos cientistas a fim de explicar o funcionamento da língua” (MARTELOTTA, 2017, p. 44). Ao observar a língua em sua imanência esbarramos em alguns conceitos expostos desde muito cedo por meio das metodologias e/ou abordagens que podem privilegiar determinada concepção. 455 O estudo acerca da gramática tradicional se debruça sobre os estu- dos filosóficos da Grécia antiga, Aristóteles evidencia a relação existente entre linguagem e lógica. Nesse sentido, Martelotta (2017, p. 45-46) advoga que: O estudo acerca da gramática tradicional se debruça sobre os estu- dos filosóficos da Grécia antiga, Aristóteles evidencia a relação existente entre linguagem e lógica. Nesse sentido, Martelotta (2017, p. 45-46) advoga que: A lógica seria o instrumento que precede o exercício do pensamento e da linguagem, oferecendo-lhes meios para realizar o conhecimento e o discurso. Assim, a lógica aristotélica buscava descrever a forma pura e geral do pensamento, não se preocupando com os conteú- dos por ela veiculados. Os ideais conceituais da gramática tradicional surgem com os interesses gregos em tornar taxativo o uso de alguns elementos da língua, ditando padrões e prescrevendo determinadas estruturas de uso, o que refletiu diretamente na língua latina, e consequente- mente, perdurou de forma “genética” a todas as línguas descenden- tes. Dito isto, pode-se perceber o caráter normativo adotado histori- camente, o que possibilita a criação de concepções específicas para esse tipo de gramática. O estudo da gramática percorreu variados conceitos ao longo do tempo, os quais visam analisar novas características sobre os elementos discursivos da língua. A gramática histórico-compara- tiva, por exemplo, tem como foco correlacionar elementos gramati- cais de uma determinada língua com a língua “original”. Além disso, é possível criar relação e evidenciar a pistas das proximidades entre as línguas por meio de algumas características extralinguísticas, como as oriundas de características fonético-fonológicas. Como exemplo, Martelotta (2017, p. EM DEFESA DE UMA BASE FUNCIONALISTA PARA A GRAMÁTICA 48) indica a palavra “cavalo” que em espanhol é “caballo”, em italiano como “cavallo”, em francês temos “cheval”, por fim, em latim “cāballus”, assim, identificamos um padrão entre línguas de uma mesma ramificação. Partindo desse viés comparativo, ocorre um marco no tocante ao estudo linguístico da época, o que impulsiona o interesse 456 S U M Á R I O dos estudiosos da área. A esse respeito, Martelotta (2017) indica que o interesse na análise da estrutura das diferentes línguas surgiu, de modo especial, com Gottfried Wilhelm von Leibniz. Em vista disso, alguns nomes surgiram como precursores dos estudos da gramática histórico-comparativa, tais como Franz Bopp e Jacob Grimm. Assim, os trabalhos advindos da gramática supracitada objetivaram a rup- tura da visão aristotélica/filosófica acerca da linguagem, delineando um processo empírico que considera algumas pistas extralinguísti- cas para facilitar a correlação entre as línguas. A língua é conceituada como “um produto social da facul- dade da linguagem”, um sistema homogêneo de signos distintos em oposição, depositados na mente dos falantes através de estímulos externos, para que seja possível que os indivíduos se compreendam. Já a fala é um ato individual, heterogêneo e acidental, que se uti- liza do código para exprimir o pensamento do indivíduo. Para Saus- sure (apud MARTELOTTA, 2017), portanto, o objeto da linguística é a langue, tendo em vista sua centralidade e sistematicidade, logo, língua é forma, e não substância, pois ela não está na substância/ matéria fônica, gráfica ou conceitual, e sim na forma, nas regras do sistema. Para exemplificar, o autor se utiliza de metáfora do jogo de xadrez. Para jogá-lo, não interessa se serão utilizadas peças de marfim ou de plástico, mas se são conhecidas as regras do jogo, as funções de cada peça. Nesse sentido, a gramática estrutural está ligada à normati- zação das estruturas do discurso de determinada língua. Com isso, as abordagens relacionadas a análise linguística associam-se a uma visão de língua autônoma, no que se refere ao seu sistema, reali- zando combinações seguindo suas “leis” internas. Ainda sobre o exposto, Martelotta (2017, p. EM DEFESA DE UMA BASE FUNCIONALISTA PARA A GRAMÁTICA 54) descreve alguns aspectos importan- tes para a gramática estruturalista: a) a existência de um conjunto de elementos; b) o fato de que cada elemento só tem valor em rela- ção a outros, organizando-se solidariamente em um todo, que deve sempre ter prioridade sobre as partes que contém; c) a existência 457 de um conjunto de regras que comanda a combinação dos elemen- tos para formar unidades maiores. A estruturalização gramatical deixa de lado os aspectos ligados a parole, o que focaliza na elucidação dos elementos que compõem a língua. A posteriori, com a evolução dos estudos sobre a linguagem, Chomsky surge com a teoria denominada de gerati- vismo, com isso, o autor relaciona a linguagem à natureza humana, ressaltando a criatividade como componente inerente no processo cognitivo humano. Nessa ótica, “a natureza da linguagem é, assim, relacionada à estrutura biológica humana, e a teoria linguística passa a ter o objetivo de explicar o funcionamento de um órgão mental particular responsável pelo funcionamento da linguagem humana” (MARTELOTTA, 2017, p. 58). Consoantes ao sobredito, o desenvolvimento da gramática gerativa tornou-se inerente dentro do campo de estudo, o qual tem a linguagem como reflexo do inatismo humano. Logo, Martelotta (2017, p. 58) advoga que “a gramática gerativa analisa a estrutura grama- tical das línguas, vendo-a como o reflexo de um modelo formal de linguagem preexistente às línguas naturais e faz desse modelo o pró- prio objeto de estudo da linguística”. Dito isso, línguas de diferentes origens podem ser analisadas de formas semelhantes, uma vez que obedecem de forma regular o princípio inatista que organiza o fun- cionamento gramatical das línguas. Durante todo o processo de gramatização gerativa, Chomsky ressalta dois fatores importantes para a construção do entendimento das relações entre os elementos do discurso, que é a dicotomia entre competência e desempenho. Por esse viés, assim como a teoria gra- matical saussuriana, a gramática gerativa isola a língua dos compo- nentes extralinguísticos, o que mantém a concepção de linguagem como um sistema autônomo/intrínseco. 458 Assim, a gramática, caracterizada por estudos da linguística moderna, também diz respeito “[...] ao conjunto e à natureza dos elementos que compõem uma língua e às restrições que coman- dam sua união para formar unidades maiores nos contextos reais de uso” (MARTELOTTA, 2017, p. 44). EM DEFESA DE UMA BASE FUNCIONALISTA PARA A GRAMÁTICA Essa posição possibilita-nos adotar uma gramática centrada no uso linguístico e que parte dos seguintes princípios: S U M Á R I O [...] não se pode analisar a competência como algo dis- tinto do desempenho, ou, nos termos funcionalistas, a gramática não pode ser vista como independente do uso concreto da língua, ou seja, do discurso. Quando falamos, valemo-nos de uma gramática, ou seja, de um conjunto de procedimentos necessários para, através da utiliza- ção de elementos linguísticos, produzirmos significados em situações reais de comunicação. Mas, ao adaptar- mos esses procedimentos aos diferentes contextos de comunicação, podemos remodelar essa gramática, que, na prática, seria o resultado de um conjunto de princípios dinâmicos que se associam a rotinas cognitivas e intera- tivas moldadas, mantidas e modificadas pelo uso (MAR- TELOTTA, 2017, p. 63). Nessa perspectiva, a gramática não é colocada como está- tica e finalizada em si. Portanto, é preciso ampliar o olhar sobre os estudos dos itens linguísticos em contexto de sala de aula, possibi- litando analisar os diferentes usos da linguagem entendendo que as categorias gramaticais, antes de tudo, são categorias linguísticas, sendo assim, devem estar associadas aos falantes, seus propósi- tos comunicativos etc. Dito isso, tecer-se-á considerações sobre a necessidade de um ensino de língua significativo, para a abordagem de categorias gramaticais, as quais são, por vezes, tratadas na escola em visão unívoca: a tradicional/normativa que, por sua vez, descon- sidera os usos reais da língua. 459 POR UMA GRAMÁTICA REFLEXIVA NA SALA DE AULA Nesta seção, iremos discutir a necessidade de um ensino de gramática de caráter reflexivo, que não pode nem deve anco- rar suas análises em postulados de cunho unicamente tradicional, considerando apenas visões normativas e estabelecendo regras. De acordo com Antunes (2014), um ensino produtivo de língua não pode desconsiderar a gramática, pois esta é um componente consti- tutivo da língua, logo, não há ação de linguagem sem gramática, bem como os itens lexicais e os fatores contextuais também são relevan- tes para a interação. Entretanto, por vezes, as salas de aula de LP da educação básica têm se baseado, exclusivamente, em compêndios normati- vos, os quais podem ser guiados por análises superficiais, bem como preconceituosas, desconsiderando a variação e a mudança linguís- ticas. Entendemos por gramática normativa aquela que “recomenda como se deve falar e escrever segundo o uso e a autoridade dos escritores corretos e dos gramáticos e dicionaristas esclarecidos” (BECHARA, 2011, p. 54). Identificamos que há uma hierarquia sobre quem deve deter- minar o bom uso da língua. Entretanto, concebemos, neste trabalho, uma premissa também importante: os melhores gramáticos de uma língua são seus falantes. Com isso, não se está ditando uma aboli- ção do ensino de gramática normativa, mas que ela deve ser traba- lhada em condições mais realistas (PERINI, 2007). Dito isso, concor- damos com o seguinte: [...] não se trata de, aqui, estarmos negando ou questio- nando a importância dessas “gramáticas”. Preocupa-nos é o fato de que, valorizar uma única variedade de fala como legítima e acentuar o estigma de que são vítimas fala e falantes das demais variedades, o ensino desperdiça 460 a oportunidade de estimular a discussão e reflexão sobre os usos linguísticos reais. Assim, despreza-se a observa- ção de suas singularidades, fugindo-se à análise de suas motivações e suas (in)adequações em relação à diversi- dade de contextos sociais em que os falantes se relacio- nam (MARTINS, 2013, p. 42). Nessa direção, compreendemos que não se trata efetiva- mente de discutir se se deve ou não ensinar gramática, mas o como ensinar, uma vez que as tendências pedagógicas funcionais estabe- lecem a necessidade de (re)significar o ensino de língua, buscando torná-lo prazeroso e eficiente, refletindo, assim, na postura assumida pelo professor, haja vista que a adoção e aplicação de aportes teóri- co-metodológicos incitam a apropriação de crenças e valores (MAR- TINS, 2013, OLIVEIRA & WILSON, 2017). POR UMA GRAMÁTICA REFLEXIVA NA SALA DE AULA Nesse sentido, concorda- mos com Perini (2007, p. 31) ao indicar os benefícios de se estudar gramática como instrumento para: [...] exercitar o raciocínio e a observação; pode dar a opor- tunidade de formular e testar hipóteses; e pode levar à descoberta de fatias dessa admirável e complexa estru- tura que é uma língua natural. O aluno pode sentir que está participando desse ato de descoberta, através de sua contribuição à discussão, ao argumento, à procura de novos exemplos e contra-exemplos (sic) cruciais para a testagem da hipótese dada. [...] exercitar o raciocínio e a observação; pode dar a opor- tunidade de formular e testar hipóteses; e pode levar à descoberta de fatias dessa admirável e complexa estru- tura que é uma língua natural. O aluno pode sentir que está participando desse ato de descoberta, através de sua contribuição à discussão, ao argumento, à procura de novos exemplos e contra-exemplos (sic) cruciais para a testagem da hipótese dada. A partir do exposto, compreendemos a relevância em trans- formar o discente em investigador da língua que ele já fala, mas sabendo que precisa aprender como a adaptá-la aos variados con- textos de uso, formais e informais; orais e escritos, por exemplo. Nesse sentido, os estudos funcionalistas também favorecem análi- ses da língua em uma abordagem reflexiva e centrada no uso. Por- tanto, essa abordagem tem como objetivo “desenvolver no aluno a reflexão sobre os efeitos de sentido provocados pelos usos dos dife- rentes recursos disponíveis na língua” (TRINDADE, 2011, p. 96). Para tal, documentos oficiais também fomentam refletir, analisar e pensar sobre os fatos da língua. O quadro a seguir resume esses pontos: 461 Quadro 02 – A análise linguística em documentos oficiais (01) Base Nacional Comum Curricular: [...] o eixo da análise linguística/semiótica, que envolve o conhecimento sobre a língua, sobre a norma-padrão e sobre as outras semioses, que se desenvolve transversalmente aos dois eixos – leitura/escuta e produção oral, escrita e multissemióticos – e que envolve análise textual, gramatical, lexical, fonológica e das materialidades das outras semioses (BRASIL, 2018, p. 80). (02) Orientações Curriculares para o Ensino Médio: construir habilidades e conhecimentos que o capacitem a refletir sobre os usos da língua(gem) nos textos e sobre fatores que concorrem para sua variação e variabilidade, seja a lingüística (sic), seja a textual, seja a pragmática (BRASIL, 2006, p. 32). POR UMA GRAMÁTICA REFLEXIVA NA SALA DE AULA (03) Proposta Curricular para a Educação de Jovens e Adultos: vista na perspectiva de atividade de reflexão sobre a língua, a análise lingüística (sic) ajuda a desenvolver habilidades intelectuais e a compreender aspectos do universo social (BRASIL, 2002, p. 17). (04) Parâmetros Curriculares Nacionais: aproprie-se dos instrumentos de natureza procedimental e conceitual necessários para a análise e reflexão lingüística (sic) (delimitação e identificação de unidades, compreensão das relações estabelecidas entre as unidades e das funções discursivas associadas a elas no contexto) (BRASIL, 1998, p. 52). Fonte: elaborado pelos autores. Quadro 02 – A análise linguística em documentos oficiais Fonte: elaborado pelos autores. Para 1, a análise não é apenas linguística, também é semió- tica, haja vista ser o documento mais recente encaminhando abor- dagens acerca do ensino de diferentes componentes curriculares, evidenciando as dinâmicas da vida moderna, em que, logicamente, os textos têm assumido novos designers, os quais requerem domí- nios de questões da multimodalidade. Além disso, o trecho 1 sinaliza para a análise gramatical, a qual deve ser trabalhada numa perspec- tiva que possibilite conhecimentos sobre a língua e sobre a norma padrão. Desse modo, “[...] a metalinguagem seria o meio, e não mais o fim, o que levaria à substituição de um modelo de avaliação que se restringe às atividades classificatórias” (TRINDADE, 2011, p. 94). No documento 2, destinado ao ensino médio, identificamos a busca por um “refinamento” de conhecimentos linguísticos que pos- sibilitem ao estudante refletir sobre os usos da linguagem e os fatores que favorecem a variabilidade linguística, textual e pragmática, favo- recendo uma abordagem ancorada em textos. Dito isso, é preciso 462 S U M Á R I O que os alunos aperfeiçoem a análise linguística para aplicarem nos textos presentes na sociedade, bem como em suas próprias produ- ções, o que lhes possibilita configuração na norma padrão, por exem- plo, estando conscientes de suas ações linguageiras. Logo, permite aos discentes “[...] na condição de autores, [que] pensem no efeito de sentido desejado, a fim de se buscar o recurso mais adequado à situação em que seu texto aparecerá” (TRINDADE, 2011, p. 95). Conforme Trindade (2011), é preciso que o professor de LP favoreça atividades de cunho epilinguístico em relação àquelas de cunho metalinguístico, fazendo isso, terá condições de ofertar em sala de aula o que o documento 3, da Educação de Jovens e Adul- tos, solicita. Documento esse que destaca a análise linguística como algo que oportuniza desenvolvimento intelectual e compreensão de questões sociais diante do uso da língua. Por último, temos o documento 4 – ao qual muitas publica- ções voltadas ao ensino fazem menção – que considera a análise e reflexão linguística, ambas a partir do uso, a partir de diferentes manifestações linguísticas no seio de diferentes gêneros textuais/ discursivos, considerando como necessárias a instrumentalização técnica e metodológica que possibilitarão a compreensão das rela- ções das unidades linguísticas, seus efeitos e suas funções discur- sivas. Fonte: elaborado pelos autores. Portanto, “trata-se de uma atividade que transcende a sim- ples classificação, categorização, enfim, memorização de regras [...]” (TRINDADE, 2011, p. 96). Exposto isso, identificamos que há mais de vinte anos, docu- mentos oficiais têm considerado a análise linguística como um eixo vertical no ensino de LP, uma vez que possibilita aos discentes refle- tirem e intervirem em suas próprias produções de linguagem, sejam orais ou escritas, bem como em suas percepções em atividades de leitura e escuta de textos. Logo, a análise linguística se faz válida no aperfeiçoamento da competência comunicativa dos alunos. A ado- ção dessas perspectivas, por parte dos professores de LP, requererá 463 S U M Á R I O aperfeiçoamento, modificação e/ou reconfiguração metodológica, uma vez que “[...] a metodologia da gramática reflexiva consiste em perguntar quais alternativas entre os recursos linguísticos dispo- níveis foram utilizadas na construção do enunciado e em compa- rar os efeitos de sentido nas situações de interação comunicativa” (TRINDADE, 2011, p. 125). Portanto, identificamos uma abordagem mais preocupada com a maneira que atuamos com a língua do que com a classificação e categorização dos itens linguísticos conforme ideais prescritivos e, mediante as colocações dos documentos oficiais, constitui-se a necessidade de um ensino de gramática que deve considerar aspec- tos discursivos e pragmáticos que são próprios da atividade verbal, refletindo uma gramática flexível, a qual não pode estar distante dos contextos da prática linguageira durante a análise linguística. Logo, a adoção do termo “Análise Linguística” pelos documentos oficiais não é mera remodelagem de nomenclatura, mas a emergência de um ensino de língua que busque analisar os usos linguísticos reais, conforme Martins (2013). METODOLOGIA E PROPOSTAS DE ANÁLISE Para este trabalho, adota-se não só uma concepção de lín- gua, mas também de profissional, assumindo-o como professor-pes- quisador. Nesse sentido, “[...] não se vê apenas como um usuário de conhecimento produzido por outros pesquisadores, mas se propõe também a produzir conhecimentos sobre seus problemas profissio- nais, de forma a melhorar sua prática” (BORTONI-RICARDO, 2008, p. 46). Logo, o professor é um agente que reflete sobre suas práticas de ensino e se interessa pelo aprimoramento de seus conhecimentos. 464 Com base em Paiva (2019), a pesquisa é qualitativa, uma vez que busca descrever e analisar um fenômeno específico. Isso favo- rece seu caráter interpretativo, pois se vale dos sentidos, com a fina- lidade de compreender os significados que as pessoas conferem às ações sociais (BORTONI-RICARDO, 2008). Dito isso, estabelece-se a natureza do corpus que irá compor as propostas de análise, o qual resultou de um projeto de letramento desenvolvido com alunos da educação básica da rede particular de ensino, no interior do estado da Paraíba, em quatro turmas dos anos finais do fundamental, tota- lizando 32 integrantes. Cada um deles aplicou e transcreveu uma entrevista, utilizando o recurso de áudio do WhatsApp para coleta. A ação de pesquisa ocorreu paralelamente aos conteúdos curriculares em 2017, durante os meses de outubro e novembro, quarto bimestre da escola-campo. Foi desenvolvido nesse período, na escola, com alunos do ensino fundamental, o projeto de letra- mento que teve como ponto de partida e chegada a análise e refle- xão dos usos concretos da língua, com o foco na variação. O intuito foi levar os alunos a considerarem alguns fenômenos que interferem nesse uso, e, para tanto, elencamos gêneros que pudessem suscitar as discussões acerca dos níveis: fonético-fonológico, morfossintá- tico e semântico-pragmático. Foram no total oito aulas (sendo quatro de base teórico-re- flexiva e quatro de base analítica). O projeto de letramento foi desen- volvido entre os referidos meses. Em artigo publicado, a partir dos resultados do projeto, os autores apontam “[...] a necessidade de se debater e refletir a respeito de práticas pedagógicas possíveis para adentrarem às salas de aula e também para as transpor” (SILVA & SILVA, 2019, p. 779). Esses apontamentos colaboram para a seção a seguir, na qual será apresentada uma seleção de exemplos de usos reais da língua, mediante a coleta de dados da fala de alguns cida- dãos do município supracitado. METODOLOGIA E PROPOSTAS DE ANÁLISE 465 Na direção do que se propõe aqui, evidenciamos que “[...] o grande mal não é o ensino de gramática em si, mas como os conte- údos são trabalhados” (MARTINS, 2013, p. 45). Nesse sentido, faz-se necessário ter em mente que a divisão das palavras em classes gra- máticas resulta de uma herança grega, concebida pelo pensamento de Aristóteles, as quais, segundo Martins (2012), são categorias que foram utilizadas pelo filósofo para organizar o mundo e foram trans- plantadas para a língua. Partindo dessa classificação tradicional, identificamos que as diferentes manifestações linguísticas são anali- sadas a partir de uma visão normativa e apresentadas aos estudan- tes de modo descontextualizado, logo, “[...] são analisadas em frases isoladas, não apenas do texto, mas de situações reais de comunica- ção. Este isolamento nega a mobilidade possível entre alguns meca- nismos [...]” (MARTINS, 2012, p. 200). Na sequência, as amostras consideram usos linguísticos que estão além do padrão tradicional e que são passíveis de uma análise por meio da linguística funcional: a. “[...] nunca aconteceu nada não que me fez mudar da minha cidade” b. “Então... falando a verdade, eu num gosto muito de João Pessoa não. Num tive uma boa experiência lá naquela cidade não [...]” Com base nesses enunciados, pode-se explicar aos alunos como o uso frequente de uma construção pode promover a existên- cia de uma nova forma de se negar algo, haja vista a necessidade de reforçar a negação. Assim, em (a), o advérbio “nunca” já indica a negação do acontecimento, mas o “não”, ao fim da oração principal, antecedido pelo pronome “nada”, fortalece a negativa. O falante usa três itens negativos para afirmar e reafirmar que em circunstância alguma houve motivações para que se mudasse daquela cidade. Já em (b), há uma outra forma de se enfatizar a construção negativa: de acordo com Furtado da Cunha et. el. (2017), o segundo “não” é utilizado para suprir o enfraquecimento do “num” pré-verbal, consti- tuindo o que os autores chamam de dupla negativa. 466 c. “e a parte da manhã que eu não estudava, brincava mais os meninos” c. “e a parte da manhã que eu não estudava, brincava mais os meninos” c. “e a parte da manhã que eu não estudava, brincava mais os meninos” d. METODOLOGIA E PROPOSTAS DE ANÁLISE “Trabalho com algo que gosto, agora eu gostaria de mudar de profissão” No excerto (c), é possível identificar o uso do item “mais”, o qual é definido pela gramática tradicional como adverbial intensifica- dor ou conjunção aditiva. Entretanto, no excerto, “mais” não é usado nessas funções, uma vez que remete ao fato de o falante estar brin- cando na companhia de amigos, o que canonicamente se faria pelo uso da preposição “com”. Esse uso indica uma função prepositiva para o item em análise. Adiante, em (d), o item “agora” está corre- lacionando as orações com os verbos trabalho e gostaria: esse item linguístico é um dêitico temporal, mas é classificado como advér- bio pela tradição gramatical. Em (d), passa a exercer uma função de conectivo adversativo, uma vez que, apesar de gostar do trabalho, o falante gostaria de mudar de profissão. E, por que esse uso ocorre? Vejamos o que indica Martins (2013, p. 47): [...] motivada por um conjunto de parâmetros por condi- cionamentos que favorecem ou inibem o emprego de um determinado item. Ou seja, as inovações gramaticais ou qualquer expressão linguística não podem ser analisadas sem que se tenha em mente que elas realizam funções não apenas das intenções e das informações transmitidas pelo falante, mas também das informações pragmáticas do destinatário e do seu conhecimento a respeito das intenções do emissor. Essa afirmação da autora corrobora a criação de novos usos, os quais são constantemente atualizados pelos falantes, uma vez que os falantes não interagem somente pela língua, mas com a lín- gua, desse modo, participam cooperativamente da construção dos referentes e dos diversos significados manifestados em diferentes contextos de uso, conforme aponta Martins (2012). Portanto, “[...] os significados são construídos por meio de escolhas que esses 467 falantes fazem durante a ‘negociação’ dos processos comunicati- vos” (MARTINS, 2012, p. 220). Essa questão continuará em evidência nas análises adiante. Furtado da Cunha et al. (2015) citam o estudo de Leonor Oli- veira, sobre a gramaticalização do onde, que aponta para um desli- zamento de sentido desse item linguístico. É possível constatar isso nas ocorrências abaixo: e. “Sempre gostei das escolas onde estudei, sempre fui muito chegado a meus professores [...]” f. “A minha infância foi uma infância muito boa onde eu pude brincar muito com os meus... METODOLOGIA E PROPOSTAS DE ANÁLISE com as minhas coleguinhas” Observe que em (e), o item “onde” remete à espacialidade, uma vez que retoma às escolas, ou seja, espaço físico. Este uso é canônico para a norma padrão, porém, distancia-se daquele em (f), tendo em vista que agora remete à infância do falante, que foi muito boa, constituindo um sentido mais temporal do que locativo. Temos, aqui, um processo de gramaticalização lato sensu, pois ocorre, inter- namente, na própria gramática (FURTADO DA CUNHA, et. al, 2015). Em vista de casos como este, Silva (2011, p. 76) adverte: A gramática, numa perspectiva funcional, não pode ser idealizada como um conjunto de categorias inertes, pre- viamente estruturadas e imune a mudanças. As palavras não estão acondicionadas em arquivos de onde são reti- radas para a produção de enunciados, retornando após o uso, como se voltassem para um castelo indevassável. Essa afirmação esclarece o fato de a gramática se fazer no discurso, não anterior a ele, afinal, é ingenuidade estabelecer usos linguísticos como corretos antes mesmo de eles serem realizados. Ressaltamos que não se trata da negação do uso da norma padrão, apenas está se constatando que a gramática não vem antes do dis- curso, está no próprio discurso. Esse aspecto também pode ser iden- tificado, a seguir, nos últimos itens de análise deste trabalho: 468 g. “Eu gosto de morar aqui em Belém porque foi a cidade que minh’acolheu, me recebeu, vivo e sobrevivo até hoje aqui [...]” g. “Eu gosto de morar aqui em Belém porque foi a cidade que minh’acolheu, me recebeu, vivo e sobrevivo até hoje aqui [...]” g. “Eu gosto de morar aqui em Belém porque foi a cidade que minh’acolheu, me recebeu, vivo e sobrevivo até hoje aqui [...]” h. “Gostava sim da escola, até porque minha professora de primá- rio até a quarta série foi a mesma [...]” h. “Gostava sim da escola, até porque minha professora de primá- rio até a quarta série foi a mesma [...]” O item “até”, na perspectiva da gramática tradicional, é uma preposição utilizada para indicar espacialidade, ou seja, aponta a existência de um espaço entre dois pontos, um lugar concreto. Em (g), esse uso se expande, pois o item é utilizado para indicar tempo- ralidade, o que se confirma com o emprego do advérbio “hoje”, logo após a preposição. METODOLOGIA E PROPOSTAS DE ANÁLISE Já em (h), identifica-se uma função argumenta- tiva, uma vez que o item “até” é usado junto ao “porque” explicativo. Nesse sentido, seria possível a substituição da expressão “até por- que” por “inclusive”, indicando a introdução de um argumento sobre o motivo de gostar da escola, funcionando como operador argumen- tativo (MARTINS & SILVA, 2018). Esse uso indica como a operacio- nalização desse uso vai do mais concreto para o mais abstrato. A adoção de uma postura teórico-metodológica como essa, por parte dos professores de LP, possibilita cientificidade na aula de gramática, conforme defende Perini (2014, p. 58), ao estabelecer que “a gramática é uma disciplina científica, pois tem como finalidade o estudo, a descrição e a explicação de fenômenos do mundo real”. Nessa direção, esse autor defende que se o estudo de gramática não for parte da formação científica dos alunos, não terá muita utilidade, contribuindo para a analfabetização científica. Logo, deve levar em conta os resultados das Ciências da Linguagem como faz a Matemá- tica, a Biologia e a Química. Por isso, concordamos com Furtado da Cunha (2017, p. 174), ao esclarecer que “o funcionalismo admite que um grande conjunto de fenômenos linguísticos é o resultado da adaptação da estrutura gramatical às necessidades comunicativas do usuário da língua”. Assim, a gramática adapta-se à língua em uso e coloca em ação questões sociopragmáticas que indicam a habilidade dos falan- tes numa comunicação efetiva por meio da língua, uma vez que, 469 S U M Á R I O conforme Martins (2013), as estruturas linguísticas estão a serviço do sentido, sendo o conteúdo determinante no formato final que se materializa linguisticamente nos textos. Desse modo, é possível praticar com os alunos análises epi- linguísticas, as quais intensificam o exercício com textos escritos ou orais (impressos ou digitais). Assim, a análise com excertos de fala serve para potencializar o trato também dos textos orais, lembrando que não é adequado estabelecer uma hierarquia entre fala e escrita, o que deve ser dito aos alunos. Os discentes devem entender que são habilidades diferentes e que demandam registros linguísticos também diferentes. Portanto, não há uma melhor ou pior, ambas devem ser promovidas com intenção de desenvolver a competência comunicativa dos educandos. Na direção das questões que refletimos aqui, acreditamos na evidência de um convite aos professores, uma proposta de ação que reconfigure o ensino de língua. METODOLOGIA E PROPOSTAS DE ANÁLISE A partir dessa postura, os professores de LP podem passar a transformar as relações entre língua e socie- dade e junto aos seus discentes, conforme Bagno (2014), construir um novo senso comum sobre a língua e suas mudanças. Desse modo, ao legitimarmos novas visões sobre a língua, possibilitaremos que “[...] o Brasil exorcize de vez o fantasma colonial que ainda assombra nossas concepções de língua e de ensino de língua” (BAGNO, 2014, p. 111), instaurando um ensino de gramática centrado nos usos do português brasileiro a partir de um viés reflexivo que considera a gra- mática como emergente em foco semântico-pragmático-discursivo. REFERÊNCIAS ANTUNES, Irandé. Gramática contextualizada: limpando “o pó das ideias simples”. São Paulo: Parábola Editorial, 2014. CONSIDERAÇÕES FINAIS Diante do que já se expôs, é válido ressaltar que há uma rela- ção entre língua e sociedade que é indissociável. Desse modo, um ensino de língua que desconsidere as ações de mudança e varia- ção da língua é um ensino desprovido de vida e que caminhará em 470 S U M Á R I O práticas obscurantistas, desconsiderando os próprios sujeitos que se prestam a analisá-la. Como visto, os usos linguísticos examinados nas propostas de estudo se mostraram inovadores, rompem com os postulados da gramática tradicional e evidenciam o dinamismo den- tro e fora do sistema linguístico. Possibilitar essas análises questio- nadoras com os alunos oportuniza um trabalho de ordem cognitiva, cultural e prática. Portanto, esperamos que as proposições apresen- tadas sejam uma alternativa para a (re)significação do ensino de LP, bem como mobilize questionamentos pelos docentes que realizam papel fundamental na arte de aprender. Ademais, as reflexões aqui expostas não finalizam as discus- sões, mas são a busca por alternativas que contemplem a adoção das ciências da linguagem no espaço escolar. Assim, ficou evidente que é possível alcançar proposições estabelecidas nos documentos oficiais, visto que se demonstrou que classes e funções variam. Essa variação coloca o ensino de língua em lugar de interação, na qual os falantes estão sempre em processos de “negociação” na comu- nicação e potencializando formas inovadoras no uso da língua. Por fim, não esqueçamos do que afirma Antunes (2014, p. 145, grifo da autora): “toda língua é, portanto, uma realidade altamente complexa, que foge inevitavelmente a qualquer pretensão de confinamento, de redução, de univocidade, de simplificação”. Por último, evidenciamos que a análise linguística proposta aqui auxilia o trabalho do professor no que se refere ao uso das cate- gorias gramaticais, contribuindo para a ampliação do conhecimento dos docentes acerca das “palavras” como elementos constituintes do discurso de modo móvel. 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João Pessoa: UFPB, 2011, 65-87. SILVA, Camilo Rosa. Processos combinatórios: coordenação e subordinação. In: SILVA, Camilo. Rosa; MATOS, Denilson Pereira de (Org.). Sintaxe do Português: abordagens funcionalistas, v. 2. João Pessoa: UFPB, 2011, 65-87. S U M Á R I O TRINDADE, Mônica Mano. Sobre o ensino de gramática: uso e reflexão nas aulas de língua portuguesa. In: FRANCELINO, Pedro Farias (Org.). Linguística Aplicada à Língua Portuguesa no ensino médio: reflexões teórico-metodológicas. João Pessoa: UFPB, 2011, p. 89-128. TRINDADE, Mônica Mano. Sobre o ensino de gramática: uso e reflexão nas aulas de língua portuguesa. In: FRANCELINO, Pedro Farias (Org.). Linguística Aplicada à Língua Portuguesa no ensino médio: reflexões teórico-metodológicas. João Pessoa: UFPB, 2011, p. 89-128. TRINDADE, Mônica Mano. Sobre o ensino de gramática: uso e reflexão nas aulas de língua portuguesa. In: FRANCELINO, Pedro Farias (Org.). Linguística Aplicada à Língua Portuguesa no ensino médio: reflexões teórico-metodológicas. João Pessoa: UFPB, 2011, p. 89-128. 474 23 Natássia Thais do Nascimento Ribeiro VARIAÇÃO E MUDANÇA LINGUÍSTICAS EM GRAMÁTICAS ESCOLARES DE LÍNGUA PORTUGUESA DA DÉCADA DE 2000 DOI:10.31560/pimentacultural/2023.97778.23 S U M Á R I O INTRODUÇÃO Este capítulo traz os resultados de uma pesquisa que teve como principal objeto investigativo gramáticas escolares de língua portuguesa da década de 2000 (cf. RIBEIRO, 2020). O trabalho se inscreve no campo teórico-metodológico da Historiografia Linguís- tica (doravante, HL), entendida como área interdisciplinar dentro da história da ciência e das ideias (SWIGGERS, 2013). Além disso, enquadra-se no âmbito investigativo da Linguística Aplicada e da Sociolinguística variacionista, haja vista os objetivos aqui pretendi- dos, bem como o tratamento dado ao objeto de análise. Tendo como princípio norteador a reconstrução do contexto (social, ideológico, intelectual) no qual esteve inserido determinado objeto de pesquisa, o fazer historiográfico na linguística deve repou- sar na tentativa de refazer determinados caminhos, buscando res- ponder a questionamentos que envolvem, por vezes, teorias e práti- cas já instituídas no âmbito da linguística ou, no que compete a este trabalho, dentro do ensino de gramática. Em se tratando dos estudos gramaticais, é visível a contribui- ção deixada pela tradição greco-romana para o ensino de gramática, que pode ser atestada, por exemplo, pelo fato de a autoria da pri- meira gramática ser atribuída ao estudioso alexandrino Dionísio Trá- cio, do século 2 a.C. Pode-se dizer que foi a partir desses estudos que se constituiu todo o lineamento taxonômico, conceitual, metodoló- gico e ideológico de nossas gramáticas normativas contemporâneas, afirmação que perpassa a noção de Paradigma Tradicional de Gra- matização (PTG), termo cunhado por  Vieira (2018) para se referir ao modelo milenar utilizado na elaboração de gramáticas no ocidente. As gramáticas elaboradas sob o crivo do PTG apresentam um padrão de construção e tratamento dos conteúdos linguístico- -gramaticais que lida com a ideia de língua homogênea e estática, 476 S U M Á R I O e com a prescrição de regras, a partir do que Vieira (2020) denomi- nou de diretrizes epistemológicas da gramática tradicional. Os compêndios de Gramática Tradicional (doravante, GT), foram, durante muito tempo, o principal suporte pedagógico utilizado nas salas de aula da educação básica. Com o tempo, esses compên- dios foram sendo gradativamente substituídos por gramáticas que adquiriram nova configuração, de caráter eminentemente didático. Tal configuração incluiu reformulações desde o design de apresen- tação à forma de abordar os conteúdos, características que levaram esses instrumentos a ser conhecidos como gramáticas escolares, como as conhecemos hoje. INTRODUÇÃO Esse movimento de ruptura com o PTG adveio em grande parte do que Gueiros (2019) denomina Tradição Sociodiscursiva (TSD), que consiste, grosso modo, em uma virada na reflexão sobre o ensino de língua materna provocada pelo advento das teorias linguísticas no Brasil, e que teria atravessado três fases: emergên- cia, desenvolvimento e consolidação, nas décadas de 1970, 1980 e 1990, respectivamente. Dentre as frentes teóricas que constituem essa tradição de estudos sociodiscursivos, a Sociolinguística, sobretudo a variacio- nista, emerge como área que se ocupa em estudar os fenômenos de variação da língua e a maneira como ela se modifica paralelamente às mudanças sociais. A partir de tal percepção, a Sociolinguística foi responsável por trazer à tona, entre outros aspectos, trabalhos relacionados à variação e à mudança na língua, o que foi de grande importância no âmbito das discussões sobre o ensino de língua por- tuguesa protagonizadas no bojo da TSD. Diante disso, o estudo objetivou analisar os textos de apre- sentação de gramáticas escolares da década de 2000, época em que a TSD já estaria consolidada há pelo menos uma década, a fim de per- ceber se, atravessadas pela atmosfera intelectual acerca do trabalho 477 S U M Á R I O com conteúdos linguístico-gramaticais à época de suas publica- ções, essas gramáticas trazem, por meio dos conceitos de variação e mudança linguística, uma retórica que demonstra intenção de romper com os dogmas da tradição gramatical, alinhando-se, assim, ao movimento da TSD. A metodologia aqui empreendida pode ser classificada como quali-quantitativa, uma vez que os resultados foram interpretados tanto com base no discurso das apresentações das gramáticas esco- lares analisadas, quanto por meio da quantidade de realizações dos termos pertencentes ao arcabouço teórico da sociolinguística. No tangente à seleção e recorte das fontes, considerou-se as obras publicadas entre 2001 e 2010, tendo em vista que o interstício de tempo entre a emergência e a consolidação da TSD foi de 1970 a 1990 e, por isso, inferimos haver uma presença mais acentuada dos pressupostos dessa tradição de estudos uma década após sua con- solidação, nos termos de Gueiros (2019). O levantamento, feito em sebos físicos e online, constatou a existência de 17 compêndios de gramáticas escolares no período de tempo pesquisado. APORTE TEÓRICO O aporte teórico que serviu de norte para a condução desta pesquisa partiu sobretudo das contribuições da Sociolinguística de cunho variacionista, através dos escritos de Labov (2008), Tarallo (1994), entre outros. Além disso, utilizamos conceitos e catego- rias da Historiografia da Linguística, por meio das contribuições de Batista (2013), Koerner (1996) e Murray (1994). Aparecem ainda os trabalhos de Vieira (2018, 2020), sobre as diretrizes que consti- tuem a gramática tradicional, e de Gueiros (2019), acerca das fases que desaguaram na TSD. INTRODUÇÃO Após esse levantamento, procedemos à leitura e análise dos textos de apresentação das 17 obras, buscando observar neles i) se há uma retórica de ruptura com as diretrizes da gramática tradicio- nal; ii) se os compêndios que trazem essa retórica de ruptura apre- sentam, implícita ou explicitamente, conceitos da Sociolinguística, a exemplo de variação e mudança linguística. O tratamento do material de análise está ancorado em dois princípios de Koerner (1996): contextualização e imanência, que se encontram explicitados no aporte teórico. O princípio da contextu- alização diz respeito aqui às fases de constituição da TSD e a ima- nência constitui a análise propriamente dita dos textos de apresen- tação dessas gramáticas. A análise desses dois princípios permite entender de que maneira o contexto de produção das gramáticas escolares analisadas influenciou a retórica dos autores nas apresen- tações de seus manuais. 478 HISTORIOGRAFIA DA LINGUÍSTICA A HL não deve ser tomada por uma prática historiográfica que pretende apenas construir crônicas cronologicamente lineares acerca de eventos ligados à língua e à linguagem (ALTMAN, 2003). O que se pretende em HL é antes de tudo a reconstrução e interpre- tação, tendo como base critérios e categorias específicas, de fatos históricos considerados relevantes para determinado estudo. Nesse sentido, o trabalho em HL deve estar comprometido em desenvolver uma narrativa que perceba a história a ser contada como uma versão possível, que pode ser revista a partir de outros prismas, a depender do recorte feito acerca do objeto. Sobre isso, Coelho e Hackerott (2012, p. 406) afirmam: ao considerarmos a diversidade de perspectivas [...] entendemos prontamente que a história da produção do conhecimento linguístico pode ser reconstruída de dife- rentes modos [...] Mais do que isso, entendemos que há mais de uma versão possível para a história e que o valor 479 daquela que se advoga para um autor, período, problema, tradição ou escola depende da boa costura dos elemen- tos acionados para compô-la. Partindo da percepção acerca da importância de se obser- var as relações entre as dimensões internas e externas envolvendo o objeto de estudo, Koerner (1996) propôs realizar a investigação em HL a partir de três princípios, a saber: contextualização, imanência e adequação. Interessa-nos, aqui, apenas os dois primeiros princípios. O princípio da contextualização consiste no resgate do clima de opinião (Koerner, 1996), entendido como o contexto intelectual, cultural e político em que as teorias se desenvolveram. A reconstru- ção desse contexto tem como intuito permitir ao historiógrafo esta- belecer relações, diretas ou indiretas, entre as ideias que circulavam à época e o modo como surgiu ou se desenvolveu seu objeto de estudo. Essa tentativa de resgate do clima de opinião pelo histori- ógrafo em linguística deve se dar também no sentido de perceber as estratégias de convencimento utilizadas na tentativa de legiti- mar determinada teoria, denominadas por Murray (1994) de retórica revolucionária, a qual pode ser percebida no modo como o discurso científico é colocado acerca de determinada teoria ou paradigma, fazendo com que sejam aceitos e, consequentemente, com que os anteriores sejam de certo modo rejeitados. Após tais considerações acerca da observação de aspectos externos ao objeto de pesquisa, passemos ao princípio da imanên- cia. HISTORIOGRAFIA DA LINGUÍSTICA Consoante Koerner (1996) a análise interna do objeto de pes- quisa (paradigma, teoria, obra) deve ser feita respeitando o recorte histórico do seu momento de produção. Desse modo, o pesquisador deve evitar realizar aproximações com a epistemologia utilizada atu- almente ou leituras críticas envolvendo teorias e conceitos emprega- dos no material observado, sob o risco de não ser fiel ao que, de fato, configuram as ideias expressas no texto em análise. 480 GRAMÁTICA TRADICIONAL VS. TRADIÇÃO SOCIODISCURSIVA: BREVES CONSIDERAÇÕES A despeito de toda a reflexão que perpassou o cenário de dis- cussões no âmbito do ensino de língua materna a partir da década de 1960, no Brasil, pautadas principalmente nas contribuições das teorias linguísticas e que culminaram no que alguns estudiosos cha- mam virada linguística, a força exercida pela tradição gramatical parece ser, nos dias atuais, ainda muito evidente. Vieira (2018) defende que há uma espécie de paradigma que perpassa a produção de instrumentos gramaticais desde a antigui- dade até os dias atuais. Tal padrão de construção desses instrumen- tos foi denominado pelo autor de Paradigma Tradicional de Grama- tização . Esse paradigma apresenta cinco diretrizes epistemológicas responsáveis por balizar a produção desses instrumentos grama- ticais. Para este trabalho, interessam-nos, sobretudo, as duas pri- meiras diretrizes, que parecem ir de encontro aos pressupostos da Sociolinguística de cunho variacionista. A primeira delas, a partir da qual o autor afirma que os instru- mentos produzidos sob o crivo do PTG “buscam construir e ensinar um padrão linguístico ideal a partir da prescrição de supostas for- mas corretas legítimas” (VIEIRA, 2018, p. 10), , diz respeito ao caráter pedagógico e prescritivo desde sempre inerente aos compêndios de gramática tradicional. Esses instrumentos surgem já na antiguidade grega com o intuito de ensinar um padrão de língua ideal, que serviu também e sobretudo a fins políticos, uma vez que a língua padroni- zada era imposta aos territórios conquistados, desde o período hele- nístico, como forma de difundir a civilização grega e aumentar seu poderio ante as demais culturas. 481 A segunda diretriz traz a ideia de que os compêndios de GT “veem as variedades linguísticas dominantes na sociedade como superiores às de menor prestígio [...] entendendo a língua como objeto autônomo, homogêneo e estático, independente de seus usu- ários e a serviço da expressão do pensamento” (VIEIRA, 2018, p. 10). Sobre isso, Vieira (2020) afirma que essa diretriz está intimamente ligada à primeira, uma vez que a elaboração de um padrão idealizado ensinável acaba por promover a ideia de que as variações existentes na língua devem ser rechaçadas por serem tidas como responsáveis pelo suposto caos linguístico. GRAMÁTICA TRADICIONAL VS. TRADIÇÃO SOCIODISCURSIVA: BREVES CONSIDERAÇÕES Gueiros (2019) afirma que, apesar de já na década de 1960 ter-se iniciado uma atmosfera de reflexão pautada nas contribuições das teorias linguísticas, é só a partir da década de 1970 que se pode falar, de fato, em um início de mudança na reflexão sobre o ensino de língua materna no Brasil, que teria se consolidado anos mais tarde dando corpo ao que esse autor denomina de Tradição Sociodiscur- siva (TSD). Sobre isso, o autor defende a tese de que a TSD passou por três fases: emergência, desenvolvimento e consolidação, nas décadas de 1970, 1980 e 1990, respectivamente. SOCIOLINGUÍSTICA VARIACIONISTA Dentro do amplo contexto que abrigou as discussões res- ponsáveis por delinear e dar forma à TSD, a Sociolinguística, ao lado da Linguística textual, da Análise do Discurso e da Linguística Fun- cionalista, podem ser tomadas, consoante Gueiros (2019) como as principais áreas que contribuíram para balizar e dar corpo às dis- cussões no âmbito do ensino de língua materna. Interessa-nos aqui, sobretudo, a primeira, haja vista os objetivos pretendidos. Desse modo, faz-se necessário apresentar seus principais pressupostos, bem como os conceitos de variação e mudança, a partir dos quais desenvolveu-se a linha analítica deste trabalho. 482 A Sociolinguística pode ser definida, de modo sucinto, como “una ciencia interdisciplinar que se ocupa de las relaciones existentes entre el linguaje e la sociedade” (HERNANDEZ CAMPOY; ALMEIDA, 2005). Dito isto, é possível inferir que ela surgiu como uma área que se contrapõe totalmente ao que preconizam as teorias formalistas acerca da língua, haja vista ser o estudo imanente da língua, a partir do contato com falantes ideais, um dos principais postulados dessas frentes teóricas. Sobre isso, Labov (2008, p. 217) afirma que [...] de modo bastante curioso, os lingüistas que traba- lham dentro da tradição Saussuriana [...] não levam em conta de modo nenhum a vida social: trabalham com um ou dois informantes em seus escritórios, ou exami- nam seu próprio conhecimento da langue. Além disso, insistem em que as explicações dos fatos lingüísticos sejam derivadas de outros fatos linguísticos, não de quaisquer dados externos. Tal percepção esteve presente nos famosos estudos desen- volvidos por Labov acerca da realização dos ditongos na ilha de Marthas’s Vineyard e sobre a estratificação do R em lojas de depar- tamento de Nova York, os quais foram responsáveis pelo surgimento da Sociolinguística variacionista à década de 1960 nos EUA. Vertente teórica que se ocupa do estudo das variações e mudança linguística, tomando fatores linguísticos e sociais como principais condicionan- tes, a partir de uma abordagem quantitativa dos dados. Neste cenário, a variação, tida como responsável pelo “caos” linguístico pelo estruturalismo saussuriano, figura agora como aspecto central de estudos, por ser considerada algo inerente à lín- gua. Nas palavras de Tarallo (1994, p. 7), “[...] a cada situação de fala em que nos inserimos e da qual participamos, notamos que a língua falada é, a um só tempo, heterogênea e diversificada. E é precisa- mente essa situação de heterogeneidade que deve ser sistematizada”. SOCIOLINGUÍSTICA VARIACIONISTA As variações ocorrem na língua através de fatores inter- nos e externos, e entendê-los é essencial para que se perceba tais variações como ocorrências legítimas da língua e não como meros 483 S U M Á R I O desvios, como quer fazer crer a tradição gramatical. Nesse sentido, Hernandez Campoy e Almeida (2005) trazem quatro principais tipos de variação linguística, que serviram também de norte para análise dos objetos de estudo deste trabalho. São quatro os principais tipos de variação, quais sejam: diacrônica, que diz respeito às variações que ocorrem na língua e que ao longo do tempo; diatópica (ou geo- gráfica), que diz respeito às diferenças que ocorrem entre os fala- res de pessoas por pertencerem a regiões diferentes; diastrática, ligada exclusivamente ao fator social e ocorre justamente atrelado aos estratos sociais, servindo tanto para marcar a identidade de um grupo e diafásica, que ocorre respeitando o contexto situacional. Atrelado ao conceito de variação, está o de mudança linguís- tica, haja vista ser esta consequência direta daquela. Esses conceitos vão de encontro ao que preconiza a gramática tradicional e adentra- ram na cena dos estudos linguísticos por meio de desdobramentos e ampliações da ideia, já postulada pelo estruturalismo, de que nenhuma forma ou variedade linguística seria inferior à outra dentro do sistema linguístico. Nesse sentido, Lyons (1987 apud CAMACHO, 2013, p. 30) afirma que “Levados ao extremo, esses postulados abriram caminho para novos enfoques do objeto da linguística, um dos quais, a Sociolin- guística variacionista, impôs o postulado de que o binômio variação-e- -mudança é uma propriedade constitutiva da linguagem [...]”. VARIAÇÃO E MUDANÇA EM GRAMÁTICAS ESCOLARES: UMA ANÁLISE À LUZ DA RETÓRICA DE RUPTURA (MURRAY, 1994) Essa breve investigação teve como objetivos já mencionados perceber a retórica de ruptura presente nos textos de apresentação das gramáticas a serem investigadas, bem como observar se dentro 484 dessas retóricas esses manuais trazem, de modo explícito ou não, conceitos próprios da sociolinguística de cunho variacionista. Após a análise das 17 obras selecionadas, chegamos à divisão de quatro tipos de gramáticas: as que não trazem retórica de ruptura, as que apresentam retórica de ruptura e, dentro desse último tipo, as que trazem conceitos da sociolinguística e as que não o fazem. A partir disso, optamos por seguir essa divisão na exposição e discus- são dos resultados aqui apresentados. S U M Á R I O Gráfico 01 – Retórica de ruptura Fonte: Ribeiro (2020). Gráfico 01 – Retórica de ruptura Fonte: Ribeiro (2020). O gráfico 01 representa a primeira subdivisão das gramáticas escolares analisadas, entre as que apresentam retórica de ruptura em sua apresentação (ARR) e as que não o fazem (NRR). A partir dos resultados observados, percebemos que a maioria desses manuais já apresentavam, à década de 2000, uma retórica que se propunha romper com a gramática tradicional e se filiar aos pressupostos da VSD. Isso mostra que a atmosfera intelectual à época de produção desses materiais e uma década depois da consolidação da VSD, já conseguia influenciar boa parte dos autores dessas gramáticas e 485 que, apesar de ainda circularem materiais que seguem a doutrina da tradição gramatical, uma mudança clara, pelo menos a nível de retórica, já podia ser notada. Após esse primeiro recorte, das 17 gramáticas analisadas, 13 apresentaram esse tipo de retórica revolucionária, representando, assim, o segundo tipo do qual se falou anteriormente. A partir disso, procedemos à análise, observando agora se esses compêndios que trazem em suas apresentações uma retórica de ruptura com a GT, apresentam também elementos da sociolinguística variacionista. Os resultados obtidos por meio dessa segunda leitura podem ser observados no gráfico 02. S U M Á R I O Gráfico 02 – Variação e mudança linguística Fonte: Ribeiro (2020). Gráfico 02 – Variação e mudança linguística Gráfico 02 – Variação e mudança linguística Fonte: Ribeiro (2020). O gráfico 02 traz os dois outros tipos de manuais, inseridos entre os que apresentam a retórica de ruptura, que são as gramá- ticas que trazem termos relacionados aos conceitos de variação e mudança linguística, da sociolinguística variacionista e as que não trazem esses termos. Com base no que constatamos, a partir do que foi dito pelos autores nas apresentações desses manuais, quase metade deles fazem menção, implícita ou explicitamente, aos con- ceitos de variação e mudança linguística. Colocando em números, 486 S U M Á R I O do total de 13, observou-se que 4 abordam esses aspectos, enquanto os outros 9 não o fazem, priorizando, em seu discurso, o trabalho com gêneros textuais. Tal resultado permite entender que a Socio- linguística, à época de produção desses materiais, já era uma área profícua no que diz respeito à possibilidade de abordagens em sala de aula, haja vista o número considerável de materiais que promete- ram trabalhar a partir de seus pressupostos. Diante disso, e tendo em vista o objetivo central deste traba- lho, cujo intuito é analisar a retórica nas apresentações das gramáti- cas escolares selecionadas, a fim de perceber se estas já objetivavam trabalhar com conceitos da sociolinguística, faz-se válido mostrar de modo mais detalhado como isso se deu, à nível de retórica, nesses textos de apresentação. Para fins de didatização, o quadro 01 apre- senta as gramáticas que se enquadram nesse recorte. Quadro 01 – Gramáticas que apresentam os conceitos de variação e mudança linguística CÓDIGO OBRA G1 CEREJA, W. R.; MAGALHÃES, T. C. Gramática reflexiva: texto, semântica e interação. 3. ed. São Paulo: Atual, 2009. G2 D’ÁVILA, S. Gramática em prática: textos e exercícios. 2. ed. São Paulo: Editora do Brasil, 2009. G3 NICOLA, J. de. Gramática: palavra, frase, texto. 1. ed. São Paulo: Scipione, 2004. G4 TERRA, E. Gramática de hoje. 8. ed. São Paulo: Scipione, 2010. Fonte: Ribeiro (2020). Quadro 01 – Gramáticas que apresentam os conceitos de variação e mudança linguística Fonte: Ribeiro (2020). As 4 gramáticas listadas no quadro 01 terão aqui o papel de ilustrar de que modo os autores destes manuais utilizavam uma retó- rica que prometia trabalhar com os conceitos de variação e mudança linguística. Dito isto, optamos por destacar alguns fragmentos dos textos de apresentação desses manuais, para tornar esse discurso mais visível. Passemos então à essa explanação. Gráfico 02 – Variação e mudança linguística 487 A G1, que já em seu título demonstra filiação com os pressu- postos da VSD, traz em sua apresentação o seguinte trecho S U M Á R I O [...] em situações, como numa entrevista, num discurso público, numa carta a um jornal ou numa dissertação escolar, é indispensável o uso da variedade padrão e for- mal da língua. Já numa conversa com amigos ou familia- res, a língua padrão seria formal demais e poderia con- tribuir para o distanciamento entre as pessoas (CEREJA; MAGALHÃES, 2009, p. 3). Tendo como base esse fragmento, é possível inferir que ele traz uma clara alusão à variação diafásica, mencionada por Her- nandez Campoy e Almeida (2005), uma vez que parte da ideia de que situações formais de uso da língua exigem uma varie- dade padrão, enquanto em situações formais essa variedade se torna desnecessária. Já a G2, que também possui um título sugestivo de filiação à VSD, traz em sua apresentação o seguinte excerto As diferentes formas de usar o português são capazes de caracterizar que as está usando. Você reconhece um gaúcho (ou um carioca, ou um nordestino etc.) pela sua pronúncia; você sabe quando está falando ao telefone com uma criança por causa de seu vocabulário; você tem dificuldade de entender uma notícia sobre uma descoberta científica, a não ser que esteja familiarizado com os assun- tos ali tratados. (D’AVILLA, 2009, p. 2, grifos nossos). O trecho citado revela, assim como na G1, referência ao aspecto variacionista da língua, agora tendo como norte dois tipos de variação, apresentados em Hernandez Campoy e Almeida (2005): a variação diatópica e a variação diastrática. A primeira é percebida logo no início da citação, quando a autora afirma ser a língua, em suas diferentes realizações de acordo com a região em que se vive, um instrumento de identidade. A segunda pode ser notada já no final do trecho destacado, quando a autora faz menção à dificuldade que muitos podem enfrentar para entender uma notícia científica, prova- velmente pelos termos técnicos da área. 488 A G3 segue a mesma linha das duas primeiras, no que diz respeito ao título, o qual sugere uma ruptura com a GT, haja vista prometer não se limitar ao estudo da frase como unidade máxima, indo para o domínio do texto. Gráfico 02 – Variação e mudança linguística No que tange aos conceitos da socio- linguística, destacou-se o seguinte excerto [...] saber expressar-se numa língua não é simplesmente dominar o modo de estruturação de suas frases, mas é saber combinar essas unidades sintáticas em peças comunicativas eficientes, o que envolve a capacidade de adequar os enunciados às situações, aos objetivos da comunicação e às condições de interlocução [...] (NICOLA, 2004, p. 3, grifos nossos). S U M Á R I O Apesar de apresentar mais elementos próprios da linguística de texto e da pragmática, o trecho em destaque também revela uma alusão à capacidade de adequação dos enunciados às situações de comunicação, o que pode ser claramente entendido como uma refe- rência à variação diafásica, que apareceu também na G1. Na G4, última gramática da lista, tem-se um título bem suges- tivo, haja vista ser possível inferir a partir dele que se trata de um manual que se dispõe a trabalhar a partir não mais da tradição gra- matical, mas sim de uma abordagem que considere todas a mudan- ças ocorridas tanto na língua, como no modo de refletir sobre ela. O texto de apresentação desse compêndio traz logo de início uma citação de Celso Pedro Luft que parece ilustrar o foco principal a ser considerado ao longo dessa gramática. Segundo esse autor, “A língua é o que é, e não o que poderia ou deveria ser: ela é como a fizeram e fazem os que a falaram e falam”. O trecho mostra como os autores desse manual percebem a língua como um organismo vivo, que pode ser e é modificado pelo seu falante, não devendo permanecer preso às regras impostas pela tradição gramatical. Tal percepção faz parte dos pressupostos da sociolinguística no que tange à mudança linguística, a qual ocorre justamente por ação dos falantes de uma língua. 489 Em outro fragmento, encontrado mais adiante no texto de apresentação da G4, os autores afirmam que “[...] como referência para estabelecer o que é correto, utilizamos a língua efetivamente cultivada no Brasil de hoje, em suas diversas manifestações [...]” (TERRA, 2010, p. 3). Percebe-se aqui, além da alusão à aceitação da mudança linguística, uma referência aos diferentes falares do país, que servem como forma de marcar a nossa identidade. CONSIDERAÇÕES FINAIS Este estudo teve como objetivo analisar gramáticas escolares de língua portuguesa da década de 2000, a fim de perceber se estas trazem uma retórica de ruptura (MURRAY, 1994) com a tradição gra- matical, buscando se filiar aos pressupostos da TSD. Objetivou-se, ainda, investigar se os manuais que apresentam essa retórica, fazem também alusão aos conceitos de variação e mudança, pertencentes à sociolinguística variacionista. A análise mostrou que, das 17 obras catalogadas, apenas 4 não apresentaram um discurso de mudança com relação à tradição gramatical, enquanto as demais traziam em suas apresentações uma clara filiação à TSD, trazendo aspectos das principais teorias que pro- tagonizaram essa virada na reflexão sobre o ensino de língua materna. Após esse primeiro recorte, procedeu-se a análise dos textos de apresentação das 13 gramáticas que apresentaram retórica de ruptura, a fim de observar se elas continham elementos ligados aos conceitos de variação e mudança linguística. Essa segunda parte da análise mostrou que, desse total, 4 compêndios apresentam, implí- cita ou explicitamente, esses conceitos. Diante da análise empreendida, percebeu-se uma clara filia- ção das gramáticas escolares analisadas com a tradição sociodis- cursiva e uma consequente desvinculação aos dogmas da gramática 490 S U M Á R I O tradicional. No entanto, como mostrou a análise, ainda coexistiam, à época, manuais que propunham um trabalho totalmente voltado à tradição gramatical, a despeito de toda a discussão que vinha sendo empreendida nesse sentido. Apesar de o ensino de gramática se configurar como um tema amplamente discutido na esfera acadêmica (cf. ANTUNES 2007; CASTILHO, 2008; PRUDENTE, 2008; FARACO, 2017; entre outros), trabalhos que versam especificamente sobre o objeto gra- mática escolar não são numerosos e os estudos encontrados, em sua maioria, repousam sobre questões atuais envolvendo aspectos discursivos dessas obras, bem como sua adequação com as atuais discussões acerca do ensino de gramática. Aliada a isso está a necessidade de proporcionar ao professor de língua portuguesa maior conhecimento acerca do delineamento que constitui os materiais pedagógicos por ele utilizados, o que se faz importante, se concordamos com Koerner (1999 apud BATISTA, 2013), dado o fato de ser imprescindível na formação do cientista e do intelectual entender acerca do passado responsável pelos sabe- res constituídos no seu campo de atuação, podendo, assim, posicio- nar-se criticamente sobre estes. CONSIDERAÇÕES FINAIS Por fim, cabe dizer também que o fato de a maioria das gra- máticas selecionadas apresentarem uma retórica que prometia tra- balhar a partir do que preconizavam as teorias linguísticas, sobretudo a sociolinguística variacionista, não significa que esses materiais de fato traziam essa proposta ao longo de seus capítulos, o que sugere que pesquisas futuras nesse sentido são extremamente relevantes. REFERÊNCIAS ALTMAN, Cristina. A pesquisa linguística no Brasil (1968-1988). 2.ed. São Paulo: Humanitas, 2003. 491 ANTUNES, Irandé. Gramática contextualizada: limpando “o pó” das ideias simples. São Paulo: Parábola editorial, 2014. ANTUNES, Irandé. Gramática contextualizada: limpando “o pó” das ideias simples. São Paulo: Parábola editorial, 2014. BATISTA, Ronaldo de Oliveira. Introdução à Historiografia da Linguística. São Paulo: Cortez, 2013. CAMACHO, Roberto Gomes. Da linguística formal à linguística social. São Paulo: Parábola, 2013. CAMACHO, Roberto Gomes. Da linguística formal à linguística social. São Paulo: Parábola, 2013. CASTILHO, Luciana Tomé de Souza. A abordagem da gramática em material didático do ensino Médio. Dissertação (Mestrado em Linguística) – Universidade de Taubaté, Centro de Ciências Sociais e Letras, 2008. CEREJA, William Roberto; MAGALHÃES, Thereza Cochar. Gramática reflexiva: texto, semântica e interação. 3 ed. São Paulo: Atual, 2009. COELHO, Olga; HACKEROTT, Maria Mercedes Saraiva. Historiografia Linguística. In: GONÇALVES, Adair V.; GÓIS, Marcos Lúcio S. (Orgs.). Ciências da linguagem: o fazer científico. Campinas, SP: Mercado das Letras, 2012, p. 381-407. D’ÁVILA, Suzana. Gramática em prática: textos e exercícios. 2. ed. São Paulo: Editora do Brasil, 2009. FARACO, Carlos. Gramática e Ensino. Diadorim. Rio de Janeiro, v. 19, n. 2, p. 11-26, jul./dez. 2017. GUEIROS, Leonardo. Da emergência à consolidação da tradição sociodiscursiva na pesquisa linguística brasileira e suas implicações para a reflexão sobre ensino de língua portuguesa. 251 f. Tese (Doutorado em Letras) – Programa de Pós-Graduação em Letras, Universidade Federal de Pernambuco, Recife, 2019. HERNÁNDEZ-CAMPOY, Juan Manuel; ALMEIDA, Manuel. Metodología de la Investigación Sociolingüística. Granada: Comares, 2005. KOERNER, Korad. Questões que persistem em historiografia linguística. Revista da ANPOLL, n. 2, p. 45-70, 1996. LABOV, William. Padrões Sociolinguísticos. São Paulo: Parábola, 2008. TERRA, Ernani. Gramática de hoje. 8 ed. São Paulo: Scipione, 2010. VIEIRA, Francisco Eduardo. A gramática tradicional: história crítica. São Paulo: Parábola Editorial, 2018. TARALLO, Fernando. A pesquisa sociolinguística. São Paulo: Ática, 2005. TERRA, Ernani. Gramática de hoje. 8 ed. São Paulo: Scipione, 2010. LABOV, William. Padrões Sociolinguísticos. São Paulo: Parábola, 2008. MURRAY, Stephen O. Theory Groups and the Study of Language in North America: a Social History. Amsterdã: John Benjamins, 1994. NICOLA, José de. Gramática: palavra, frase, texto. São Paulo: Scipione, 2004. 492 PRUDENTE, Virginia Mary Mendonça. A gramática no banco dos réus: culpada ou inocente? Dissertação (Mestrado em Linguística) – Universidade Federal de Minas Gerais, 2008. PRUDENTE, Virginia Mary Mendonça. A gramática no banco dos réus: culpada ou inocente? Dissertação (Mestrado em Linguística) – Universidade Federal de Minas Gerais, 2008. SWIGGERS, Pierre. A historiografia da linguística: objeto, objetivos, organização. Confluência, Rio de Janeiro, n. 44-45, p. 39-59, 2013. S U M Á R I O VIEIRA, Francisco Eduardo. A gramática tradicional: história crítica. São Paulo: Parábola Editorial, 2018. VIEIRA, Francisco Eduardo. Tradição gramatical: história, epistemologia e ensino. In: VIEIRA, Francisco Eduardo. Tradição gramatical: história, epistemologia e ensino. In: VIEIRA, Francisco Eduardo; BAGNO, Marcos (Orgs.). História das línguas, histórias da Linguística: homenagem a Carlos Alberto Faraco, v. 1. São Paulo: Parábola Editorial, 2020 p 85 125 , ç g , p g VIEIRA, Francisco Eduardo; BAGNO, Marcos (Orgs.). História das línguas, histórias da Linguística: homenagem a Carlos Alberto Faraco v 1 São Paulo: Parábola Editorial VIEIRA, Francisco Eduardo; BAGNO, Marcos (Orgs.). História das línguas, histórias g g da Linguística: homenagem a Carlos Alberto Faraco, v. 1. São Paulo: Parábola Editorial, 2020, p. 85-125. da Linguística: homenagem a Carlos Alberto Faraco, v. 1. São Paulo: Parábola Editorial, 2020, p. 85-125. 493 24 Emily Gonçalves de Medeiros Ferreira AS REGRAS DA LINGUA PORTUGUEZA, ESPELHO DA LINGUA LATINA, DE JERÓNIMO CONTADOR DE ARGOTE (1725), E A GRAMATIZAÇÃO DA COLOCAÇÃO PRONOMINAL: APONTAMENTOS HISTORIOGRÁFICOS DOI:10.31560/pimentacultural/2023.97778.24 S U M Á R I O INTRODUÇÃO Este capítulo se propõe a refletir sobre a abordagem da colocação pronominal nas Regras da Lingua Portugueza, espe- lho da lingua latina, de Jerónimo Contador de Argote (1725), e suas motivações e implicações no processo de gramatização do por- tuguês no século 18. O conceito de gramatização, proposto por Auroux (2014, p. 65, grifos do autor), diz respeito ao “processo que conduz a descrever e a instrumentar uma língua na base de duas tecnologias, que são ainda hoje os pilares de nosso saber metalinguístico: a gramática e o dicionário”. No que se refere ao instrumento gramática, em particu- lar, o autor apresenta três aspectos básicos que caracterizam esse processo: a categorização das unidades, que supõe a fragmentação da cadeia falada e termos teóricos (as partes do discurso, com suas definições e propriedades); os exemplos, que se configuram como o cerne da língua normatizada e manifestam a existência de uma realidade linguística em particular; e as regras mais ou menos explí- citas para construir enunciados, cujo lugar pode ser tomado pelos exemplos e que podem ser encaradas por um viés prescritivo (como deve ou não ser dito) ou descritivo (como é dito). Desse modo, enten- demos que o processo de gramatização de um dado fenômeno da língua se concretiza quando o tema é abordado em conformidade a esse conjunto de aspectos. No processo de constituição de uma nação, diversas políti- cas são estabelecidas, nas quais a língua tem um papel fundamental e desempenha uma função prática e uma função simbólica (THIE- SSE, 1999 apud FIORIN, 2008). É prática, na medida em que se torna uma ferramenta essencial à administração, à economia, ao poder legislativo e judiciário, ao ensino, ao desenvolvimento das ciências, enfim, àquilo que rege e constitui a nação. É simbólica, na medida em que se torna a encarnação da nação, “a expressão viva, orgânica, 495 S U M Á R I O do espírito do povo” (HERDER apud FIORIN, 2008, p. 59). Nessa pers- pectiva, a cultura, os princípios, os valores, tudo aquilo que molda a nação como tal é expresso e apreendido a partir de sua língua. A elaboração de instrumentos de gramatização, então, per- mite que se estabeleçam no imaginário dos sujeitos normas e refe- rências àquilo que constitui a unidade de sua língua e, consequen- temente, à unidade linguística de sua nação. Como afirma Auroux (2014, p. INTRODUÇÃO 70), “do mesmo modo que um martelo prolonga o gesto da mão, transformando-o, uma gramática prolonga a fala natural e dá acesso a um corpo de regras e de formas que não figuram juntas na competência de um mesmo locutor”. Esse movimento modifica os espaços de comunicação da sociedade, ampliando e alterando seus conhecimentos e sua capacidade linguística (AQUINO, 2012). Nessa medida, transforma o fazer linguístico de uma sociedade e as relações estabelecidas entre os indivíduos que a constituem, solidificando e enriquecendo o construto que os coloca como membros de uma unidade histó- rica, cultural e social. A produção de gramáticas e sua utilização por parte da população em geral tomou maiores proporções especialmente após o surgimento da imprensa, no século 15. Esse instrumento de gra- matização fixou-se como um gênero e suporte específico de refe- rência na compreensão e caracterização das línguas. As gramáticas de língua portuguesa, em particular, passaram a ser produzidas a partir do século 16. Quanto à posição dos pronomes oblíquos átonos na língua portuguesa, este tema gramatical passou a ser discutido particu- larmente a partir do final do século 19, quando particularidades do português brasileiro como língua materna passaram a ser incorpora- das à literatura brasileira, o que desencadeou uma série de polêmi- cas em que se contrapunham esta variedade da língua e a lusitana. 496 À época, estudos da filologia evidenciaram diferenças linguísticas entre Brasil e Portugal e a problemática da colocação pronominal foi um tópico de grande importância nesse debate, especialmente a partir das últimas décadas do século 19, dadas as diferenças linguís- ticas apontadas em relação a cada nação (cf. GURGEL, 2008). De fato, tal período corresponde também ao momento da consolidação do processo de gramatização desse fenômeno linguístico na grama- ticografia brasileira (cf. FERREIRA, 2021). Apesar de ter sido publicada um século antes da querela em torno desse tema gramatical, a obra Regras da Lingua Portugueza, espelho da lingua latina, de Contador de Argote (1725), foi apon- tada por diferentes estudos como sendo a primeira a sistematizar o fenômeno da ênclise na língua portuguesa (cf. LEITE, 2011; 2013; KEMMLER, 2012; 2013; MARQUES, 2016). Dito isso, o objetivo deste   é discutir a abordagem da colocação pronominal nessa obra, tanto no que se refere à ênclise quanto a outros aspectos relacionados ao tema. INTRODUÇÃO Nossa análise se insere no horizonte de reflexões desenvolvi- das no campo de estudo da Historiografia da Linguística, nos termos de Koerner (1996, 2014) e Swiggers (2013, 2019), que consiste em uma disciplina cientificamente fundamentada que busca descrever e interpretar a compreensão sobre a linguagem situada num dado recorte temporal e contextual. Além desta introdução, o capítulo é organizado em outras quatro seções. Na seção a seguir, procuramos contextualizar a atmos- fera intelectual do século 18, período em que a gramática de Conta- dor de Argote (1725) veio a lume. Na sequência, apresentamos autor e obra. Mais adiante, analisamos a abordagem da ênclise, em parti- cular, e de aspectos relativos à colocação pronominal na gramática em questão. Finalmente, seguem-se nossas últimas considerações. 497 O conceito de clima de opinião que utilizamos deriva das concepções adotadas em Koerner (1996), e remete à atmosfera intelectual historicamente situada, em que as ideias linguísticas são gera- das, circuladas, propagadas ou esquecidas. SOBRE AS IDEIAS LINGUÍSTICAS DO SÉCULO 18 Para compreendermos a conjuntura portuguesa do século 18, é necessário aludirmos aos movimentos intelectuais de ruptura então correntes no clima de opinião64 europeu. Esse período foi mar- cado por transformações de ordem social, política, econômica e edu- cacional, aliadas a uma crítica profunda às instituições e aos princí- pios até então dominantes, principalmente à supremacia da fé e da tradição sobre a razão, o espírito crítico e o progresso. Tal momento da história é denominado Iluminismo. De acordo com Miranda (2011), o ideário dos filósofos ilu- ministas, moderados ou radicais, acreditava no progresso material humano e no progresso moral através da educação e da leitura. A educação era defendida por estes como “a única e legítima realidade reformadora da sociedade, da política, da religião e dos valores de uma forma geral” (MIRANDA, 2011, p. 81). Ainda segundo o autor, ao longo do século 18, a educação se desloca de um modelo secular clerical para um modelo de laicização progressiva, e o livro, que tem sua importância ampliada já na Modernidade, alcança novas instân- cias e passa a carregar também as aspirações de uma geração de pensadores que compreendiam o conhecimento como fundamento de uma nova organização social. É nesse contexto, em que a noção de valor individual do homem e de sua capacidade de produzir coisas novas modifica os espaços sociais, que as discussões acerca dos direitos naturais e universais do homem tomam destaque, e este novo homem, o 64 498 homem das letras, baseado na razão e na experiência, recebe o papel de esclarecer as massas e conduzi-las a uma cidadania mais ampla. Apesar das transformações desencadeadas pelo movi- mento iluminista europeu alcançarem o pensamento português desde o início do século 18, Moura (2012) nos conta que esse reflexo destinava-se a círculos eruditos, permanecendo ignorado pela maioria da população: A elite aristocrática portuguesa, a partir dos finais do século XVII e início do século XVIII, começou a evidenciar um crescente interesse intelectual, ao mesmo tempo que procurou ostentar um padrão de vida moderno, levando-a à criação de determinados círculos particulares onde se debatiam, essencialmente, as preocupações literárias barrocas, constituindo sobretudo um espaço de entrete- nimento cultural. SOBRE AS IDEIAS LINGUÍSTICAS DO SÉCULO 18 Nestes locais, que entretanto abriram as suas portas a alguns eruditos nacionais e estrangeiros fora da esfera aristocrática, debateram-se cada vez mais os problemas da atualidade, mormente as novas orien- tações científicas, históricas e filosóficas, sendo que as questões literárias barrocas iam sendo relegadas para segundo plano (MOURA, 2012, p. 116). Essas reuniões desempenhavam um papel importante na disseminação do pensamento iluminista, ao mesmo tempo em que restringiam o conhecimento a uma parcela mínima da população. Assim, a evolução do pensamento iluminista em Portugal se deu de forma muito lenta, no decorrer do século 18. No que diz respeito às ideias linguísticas, o racionalismo é abraçado pelo iluminismo, partindo-se, então, do pressuposto de que as línguas são regidas por princípios universais da razão. Nesse esteio, passou-se a defender a existência de uma gramática geral, ou universal, cujas categorias lógicas e universais se desdobravam de diferentes modos em cada língua. Por esta razão, “à medida que o influxo racionalista, logicista ou mentalista ia ganhando ter- reno, assim a gramática portuguesa conferia mais ênfase à sintaxe 499 em vez de privilegiar apenas o tratamento das partes do discurso” (GONÇALVES, mimeo, p. 5). A mudança paradigmática alcançou também a educação e, consequentemente, o fazer gramatical. O século 18 vê, por quase toda a Europa, a consolidação das ideias da Grammaire générale et raisonnée65, elaborada por Antoine Arnauld e Claude Lancelot, que foi publicada em 1660; portanto, a consolidação de um fazer gramatical parcialmente distinto da tradição greco-latina. No entanto, conforme aponta Borges Neto (2018, p. 183), em Portugal, este período pouco acrescenta aos estudos gramaticais do século anterior, “embora os gramáticos passem a citar Port-Royal e a se dizerem influenciados por suas ideias (influência que, de modo geral, não é óbvia)”. Nesse contexto, desencadeou-se o processo de gramatiza- ção da língua portuguesa. A produção de instrumentos gramaticais foi impulsionada no correr do século 18, apesar de ter tomado maio- res proporções apenas a partir do século seguinte. Esse fato está intimamente ligado à importância concedida à língua portuguesa, cujo ensino precedente ao de qualquer outra língua passou a ser defendido por gramáticos e pedagogos, o que levou a um aumento significativo na produção de dicionários, tratados de ortografia e gra- máticas (MOURA, 2012). Traduzida como Gramática geral e razoada, também costuma ser referida simplesmente como Gramática de Port-Royal. SOBRE AS IDEIAS LINGUÍSTICAS DO SÉCULO 18 Jerónimo Contador de Argote (1676-1749) se destaca entre os gramáticos do período, sendo sua obra a primeira gramática de língua portuguesa a ser publicada no século 18, “depois de um longo hiato de produção metagramatical em língua portuguesa” (KEMMLER, 2014, p. 289), além da primeira a ter mais de uma edição em vida ela- borada pelo autor, a ser estruturada segundo o método dialógico e a ter a primeira edição publicada sob um pseudônimo. Destrinchamos estas e outras questões relacionadas à obra e ao autor a seguir. Traduzida como Gramática geral e razoada, também costuma ser referida simplesmente como Gramática de Port-Royal. 65 500 SOBRE O AUTOR E SUA OBRA Conforme testemunha o historiador setecentista Diogo Bar- bosa Machado (1747, Tomo II, p. 493), Jerónimo Contador de Argote nasceu na vila de Colares, em 8 de julho de 1676, e faleceu em 1749, no Convento dos Caetanos, em Lisboa. Era filho de Luiz Contador de Argote, desembargador da Relação do Porto e da Casa da Suplicação, e de D. Maria Josefa Lobo da Gama, cuja família advinha da nobreza. Sua formação teve início na cidade do Porto, mas foi em Lisboa, no colégio de São Francisco Xavier, com os padres jesuítas Álvaro Machado e Antônio Vieira, que Contador de Argote aprendeu latim. No entanto, conforme aponta Marques (2016), àquela altura, o padre Antonio Vieira se encontrava no Brasil, pois em 1681, após um tempo em Lisboa, havia retornado à Bahia, lá permanecendo até sua morte em 1697 (cf. MACHADO, 1741, Tomo I, p. 418-419). “Assim sendo, Machado utiliza a vinculação da formação latina de Contador de Argote a Vieira como um recurso de autoridade, a fim de con- firmar o conhecimento de Contador de Argote a respeito da língua latina” (MARQUES, 2016, p. 16). Em 1688, Contador de Argote ingressou na Ordem dos Clé- rigos Regulares Teatinos, na Casa de Nossa Senhora da Divina Pro- vidência, de Lisboa, onde estudou Gramática, Latim, Retórica e Filo- sofia. Em 1708, ocupou-se do ensino de Retórica e Latim, na mesma Casa, ao mesmo tempo iniciando a leitura da Filosofia, seguindo a razão e a experiência de filósofos modernos (cf. BEM, 1794). Tendo problemas de saúde, deixou o magistério e se mudou para Minho, onde ficou até 1715. Ali, dedicou-se ao “exame de antiguidades Romanas” e ao “estudo das letras humanas”, ensinou Filosofia e Matemática a algumas pessoas em particular, e auxiliou nas funções eclesiásticas daquela província (MARQUES, 2016). 501 Além de latim e filosofia, Contador de Argote desenvolveu seus conhecimentos em grego, francês, italiano e história sagrada e profana, especialmente em matéria de cronologia e geografia. Regis- tra-se sua presença como um dos membros fundadores da Acade- mia Real de História Portuguesa, nascida do movimento iluminista português, que tinha como objetivo escrever a história de Portugal e de seus domínios. Varia a natureza das obras escritas por Contador de Argote, entre as quais se destacam Memórias Históricas do Arce- bispado de Braga, publicada em 1744, dividida em quatro volumes e dedicada ao rei D. SOBRE O AUTOR E SUA OBRA João V; e Regras da Lingua Portugueza, espelho da lingua latina (doravante Regras), que teve sua primeira edição publi- cada em 1721, sob o pseudônimo Pe. Caetano Maldonado da Gama, e uma segunda edição em 1725, agora sob o nome do autor. De acordo com Marques (2016, p. 17), o período em que Contador de Argote escreveu as duas edições da gramática era, em termos político-sociais e culturais, ainda conturbado: Portu- gal permanecia “social e economicamente comprometido, mesmo com as riquezas provindas do ouro do Brasil, e com a predominân- cia da cultura escolástica e religiosa, apesar da emergência do ilu- minismo europeu”. Examinando as duas edições da obra, a autora identifica que Contador de Argote, preocupado com o uso da língua, tratou tanto de regras gerais quanto de regras particulares, dando ênfase às regras gerais. As Regras foram concebidas com interesse didático, para servir às aulas de gramática nas Casas de Nossa Senhora da Divina Providência. Segundo Ceia (2011, p. 35-42 apud MARQUES, 2016, p. 19), a rotina no Convento dos Caetanos era centrada não apenas no desenvolvimento da vida espiritual, mas também no desenvol- vimento intelectual, pois os teatinos também se tornariam “profes- sores, Padres, Mestres, confessores, pregadores, académicos”, e, para isso deviam destacar-se “no interior da Casa por seu talento, disciplina e produtividade intelectual”. A obra de Contador de Argote, assim, tem como objetivo fundamental descrever as normas 502 da língua portuguesa de modo a identificá-la completamente com a língua latina, favorecendo o ensino-aprendizagem desta última66. De fato, Leite (2011, p. 666) nos diz: S U M Á R I O O mestre deveria negar-se a ensinar aos alunos as estru- turas desviantes, os idiotismos, pois o foco do ensino era a busca pelas regras coincidentes com as do latim. Era a busca pelos universais linguísticos. Essas duas gramá- ticas constroem-se sobre dados linguísticos observados pelos autores na prática da língua e, portanto, não trazem exemplos literários67. Sobre a escolha do nome “espelho”, utilizada por Contador de Argote para demonstrar a conformi- dade das regras do português ao latim, Moura (2008, p. 112 apud MARQUES, 2016, p. 16) lembra que “os gramáticos especulativos da Idade Média consideravam a linguagem como um espelho que reflectia a verdade das coisas”, e que essa ideia remonta a Platão. À exceção disto, Leite (2011, p. 666) destaca, em nota de rodapé, que, quando o autor trata do “dialeto poético”, há alguns exemplos da linguagem literária. SOBRE O AUTOR E SUA OBRA Nesse sentido, características da língua portuguesa desvian- tes do padrão latino eram, em sua maioria, deixadas de lado, pois não serviam ao propósito para o qual a gramática fora produzida, sendo, por esta razão, as regras gerais enfatizadas, priorizadas, conforme podemos ver no Prólogo da edição de 1725: A presente Grammatica he Portugueza no nome, nas palavras, e nas regras; porém no intento, e effeyto, para que se compoz, he Latina; por isso a mayor parte das regras, que contèm, guardão ou total, ou parcial harmonia com as Latinas, e as demais, em que a Grammatica Por- tugueza discorda inteyramente da Latina, as reputa como Idiotismo, e assim as deyxa para aquelles, que houverem de compor da Grammatica Portugueza em toda a sua extensaõ. [...] (CONTADOR DE ARGOTE, 1725, “Prólogo”). A obra, segundo Moura (2012), manifesta a ideia de que a gra- mática é essencialmente normativa, apresentando exemplos forja- dos, o que aponta à tendência do autor em seguir o paradigma racio- nalista dos gramáticos franceses. Desvalorizando o conhecimento 66 67 503 S U M Á R I O fundamentado num critério de autoridade e conferindo uma impor- tância fundamental à razão, valorizavam, sobretudo, o uso. No entanto, continua a autora, na tentativa de encontrar semelhanças entre a língua portuguesa e a latina, Contador de Argote não ana- lisa as minúcias de alguns usos então contemporâneos do portu- guês, alguns modos de falar do momento, e por isso, no conjunto de prescrições que estabelece, identifica alguns usos como incorretos, impróprios, e proíbe-os68. Podemos então dizer que, apesar de reconhecerem impor- tantes diferenças entre o português e o latim, as Regras de Contador de Argote “seguem o projeto pedagógico inaugurado em Portugal por João de Barros: trata-se de uma gramática do português que se pretende meio para se chegar ao latim, a língua que, ao fim e ao cabo, é a meta da educação portuguesa (ainda no século XVIII)” (BORGES NETO, 2018, p. 185), por razões que se justificam na seção de introdução da obra: A Lingua Latina he universal em toda a Europa, e neces- saria para as occupaçoens da Republica, por isso muy- tos a aprendem, mas poucos a sabem suficientemente, e raros com perfeyçaõ. Em a aprender gastaõ os meni- nos a mayor parte da Puericia, e ainda da Adolescen- cia. Por exemplo, o autor considera que que a gramática portuguesa dos “verbos reciprocados” é “mui- to embaraçada”, discorda muito da latina e não deveria ser ensinada (cf. ARGOTE, 1725, p. 264). SOBRE O AUTOR E SUA OBRA Para evitar estas demoras, de que procedem graves danos, se tem proposto por alguns Varoens sabios diver- sos arbitrios. Entre estes o que se tem achado ser mais facil, util, e seguro, (ao menos para as Naçoens, cujas linguas vulgares saõ filhas da Latina, assim como a Por- tugueza, Castelhana, Italiana, e Franceza) he ensinar aos rapazes primeyro a Grammatica da sua lingua vulgar, e depois ensinar-lhes a Grammatica Latina: porque assim viraõ a aprendella fácil, e brevemente, segundo mos- tra a experiencia, e a razaõ (CONTADOR DE ARGOTE, 1725, “Introducçam”). 68 504 Leite (2011) nos diz que a obra de Contador de Argote foi a única gramática portuguesa a ser publicada, no século 18, anterior- mente à institucionalização da Arte de Grammatica Portugueza, de António José dos Reis Lobato (1770), como a primeira gramática ofi- cial da língua portuguesa do sistema de ensino em Portugal. À época de sua publicação, as Regras já gozavam certo destaque, o que se evidencia, ainda conforme Leite (2011, p. 668), pelo fato da obra ser retomada por Reis Lobato (1770) e, embora o autor teça severas crí- ticas a alguns “erros” de Contador de Argote, também afirma “não obstante ser huma das melhores, entre as que se tem escrito de lin- guas vulgares; he diminuta e contem muitas regras falsas”. S U M Á R I O De acordo com Borges Neto (2018, p. 188-189), as Regras de Contador de Argote exemplificam, no século 18, um tipo de manual de gramática muito frequente no período medieval: as regulae, “gra- máticas destinadas a estudantes que não tinham o latim como língua materna e assumiam o caráter de obras de consulta”, em oposição às gramáticas de escola (WEEDWOOD, 2002, p. 39-42 apud BORGES NETO, 2018, p. 189), que continham “uma exposição sistemática das categorias gramaticais, exemplificadas por meio do latim”. Conforme já dissemos, há duas edições das Regras: a pri- meira, de 1721, e a segunda, de 1725. A primeira edição, publicada sob o pseudônimo Pe. Caetano Maldonado da Gama69, consiste em uma gramática estruturada num livro de três partes: a primeira, destinada à morfologia, é composta por 11 capítulos; a segunda, sobre a sintaxe, compreende oito capítulos; e a terceira parte, da sintaxe figurada, divide-se em sete capítulos. A segunda edição veio a lume em 1725, “muito acrescentada e correta”, como informa o próprio autor no frontispício da gramática. Segundo Kemmler (2012), a escolha do pseudônimo advém da ordem dos Clérigos Regulares de São Caetano de Thiene, a que o autor pertencia, donde escolheu “Caetano” para identificar-se como teatino; e os sobrenomes pertencem à família da mãe do gramático. Um quadro comparativo entre os capítulos que compõem cada uma das duas edições pode ser encontrado em Kemmler (2012). SOBRE O AUTOR E SUA OBRA Apresenta algumas novidades em relação à primeira, a começar pela 69 505 S U M Á R I O publicação com o nome real de seu autor, que se identifica como clérigo regular e acadêmico da Academia Real de Historia Portu- gueza. Em se tratando de aspectos estruturais70, a obra comporta dois capítulos a mais na terceira parte (capítulos VI e VII, respecti- vamente, “Das figuras, da Dicçaõ” e “Das palavras Enclíticas”), além de uma quarta parte, voltada aos estudos da variação linguística (cf. KEMMLER, 2012, 2013; LEITE, 2011; SANTOS, 2014) e de um exer- cício elaborado pelo Pe. Antônio Vieira. Marques (2016) afirma que, enquanto na primeira edição Contador de Argote apresenta concisa- mente as características do português, na segunda, o autor chega a explorar com mais clareza os idiotismos, tratando também da morfo- logia, da sintaxe, e da semântica da língua portuguesa. As duas edições das Regras foram publicadas utilizando como recurso o discurso direto, à moda dos socráticos (FÁVERO, 1994/1995), por meio de um diálogo conduzido entre o Mes- tre (M.) e seu Discípulo (D.), em que o primeiro leva o segundo a refletir e encontrar respostas sobre os temas gramaticais a res- peito dos quais discutem. Na primeira edição, Contador de Argote chega a admitir a existência de erros em sua obra, e deixa claro não se importar com as críticas; na segunda edição, por sua vez, o autor não apenas admite a existência de tais erros, mas também exorta o leitor a repará-los. Tais questões nos permitem levantar a hipótese de que, como afir- mou Marques (2016), a primeira edição seria um esboço desta que viria quatro anos depois. Entendemos, em consonância a Marques (2017), que a aná- lise de mais de uma edição da mesma obra nos permite observar as (des)continuidades nas ideias linguísticas manifestadas pelo autor diante de um fato linguístico. Entretanto, para esta análise, 70 Um quadro comparativo entre os capítulos que compõem cada uma das duas edições pode ser encontrado em Kemmler (2012). 506 S U M Á R I O foi preciso nos limitarmos à segunda edição da obra devido à extrema raridade dos exemplares da primeira edição das Regras, de 1721 (KEMMLER, 2012; 2014). Além disso, a colocação pronominal, tema gramatical de nosso interesse, se encontra especificamente acrescida à edição de 1725, no capítulo VII da terceira seção, intitu- lado “Das palavras Enclíticas”. SOBRE O AUTOR E SUA OBRA Tratemos, enfim, de algumas questões relativas ao arcabouço categorial e conceitual e à abordagem descritivo-normativa desse fenômeno linguístico. SOBRE A COLOCAÇÃO PRONOMINAL NAS REGRAS (1725) A primeira questão a que chamamos a atenção é que o autor restringiu o conceito de ênclise ao problema da colocação prono- minal, apesar de não utilizar claramente este metatermo para se referir a ele. Esse conceito não se faz pela colocação da partícula depois do verbo; antes, o que define uma partícula enquanto enclí- tica ou não é a mudança de “tom”, de forte para fraco, como podemos ver no trecho a seguir: Mestre. Que cousa he palavra, ou dicçaõ Enclitica? D. Dicçaõ Enclitica he aquella particula, ou palavra, que perde o seu tom, e o poem na ultima syllaba da palavra antecedente, se he capaz dele. M. E que cousa he tom? M. E que cousa he tom? Em terceiro, o fato da gramática francesa dis- correr acerca de mudanças no latim decorrentes do acréscimo de uma partícula enclítica tornaria necessária sua abordagem também na obra de Contador de Argote, tendo em vista o seu propósito didá- tico de servir ao aprendizado da língua latina apoiado na portuguesa, a partir de que se tem a necessidade de espelhar, efetivamente, os aspectos linguísticos característicos do latim no português, apesar das diferenças estruturais entre uma língua e outra. Além disso, é preciso considerar, como vimos, que a língua latina exercia poder nos âmbitos educacional, social, político, cultural e religioso sobre a sociedade portuguesa. Na categorização das “dicções enclíticas” da língua portu- guesa, figuram “partículas” (nomenclatura semelhante à de Port- -Royal): os oblíquos me, te, se, lhe, nos, vos, lhes; e os relativos o, a, os, as. As regras que definem a ênclise desses elementos são: M. E quaes saõ as regras dos Encliticos? D. São estas. Todas as vezes que estas particulas, ou pro- nomes Me, Te, Se, Lhe, Nos, Vos, Lhes, se poem logo depois do Verbo, se fazem Encliticas. Isto he mudaõ o seu tom. [...] M. E quando he que saõ Encliticos os relativos O, Os, A, As? D. Quando se ajuntaõ aos pronomes Me, Te, &c. que vem logo depois dos Verbos (CONTADOR DE ARGOTE, 1725, p. 288-289, grifos do autor). M. E quaes saõ as regras dos Encliticos? M. E quaes saõ as regras dos Encliticos? D. São estas. Todas as vezes que estas particulas, ou pro- nomes Me, Te, Se, Lhe, Nos, Vos, Lhes, se poem logo depois do Verbo, se fazem Encliticas. Isto he mudaõ o seu tom. [...] D. São estas. Todas as vezes que estas particulas, ou pro- nomes Me, Te, Se, Lhe, Nos, Vos, Lhes, se poem logo depois do Verbo, se fazem Encliticas. Isto he mudaõ o seu tom. [...] M. E quando he que saõ Encliticos os relativos O, Os, A, As? M. E quando he que saõ Encliticos os relativos O, Os, A, As? D. Quando se ajuntaõ aos pronomes Me, Te, &c. que vem logo depois dos Verbos (CONTADOR DE ARGOTE, 1725, p. 288-289, grifos do autor). Estas regras ainda podem ser encontradas nas gramáti- cas brasileiras e portuguesas que se filiam à tradição, mas em sua gênese se viam livres de engessamentos. M. E que cousa he tom? D. He hum certo geyto, ou diversidade de som, com que pronunciamos a mesma palavra, ou particula (CONTA- DOR DE ARGOTE, 1725, p. 287, grifos nossos). 507 Sendo assim, o deslocamento é consequência, e não ênclise. O enfraquecimento do tom é que torna a partícula enclítica; logo, o que define a ênclise da língua portuguesa, em sua primeira acepção, é seu aspecto fonético – perspectiva que foi retomada, posteriormente, pelo gramático e filólogo brasileiro Manuel Said Ali Ida (1861-1953). Conforme percebemos, o capítulo é estruturado, assim como os demais da gramática, conforme os diálogos socráticos, entre mestre (M.) e discípulo (D.). Referências a outras obras ou outros autores não são explicitadas ao longo do capítulo. Entretanto, como demonstra Kemmler (2014), pode-se perceber uma possível influên- cia da obra de Claude Lancelot, gramático de Port-Royal, sobre sua abordagem desse fenômeno linguístico: Uma vez que não consta que a gramaticografia portu- guesa ou latino-portuguesa anteriormente a Contador de Argote se tenha servido do conceito da ‘partícula enclí- tica’ para a realidade portuguesa, justifica-se um olhar para a Nouvelle methode de Claude Lancelot. E real- mente, a obra do gramático de Port-Royal (baseando-se, entre outros, em Despautério) não somente se refere aos clíticos como partículas, mas faz questão de pronunciar- -se sobre a mudança na acentuação que o acréscimo de uma partícula enclítica traz consigo no latim (KEMMLER, 2014, p. 292-293, grifos nossos). Isto nos parece justificar ou no mínimo trazer alguma luz sobre o que levou à introdução desta seção, pela primeira vez, na história da gramaticografia do português; podemos pensar em pelo menos três razões. Em primeiro lugar, temos a forte disseminação das ideias de Port-Royal sobre as gramáticas na Europa setecentista, ainda que em Portugal essa influência tenha se dado muito mais a nível retórico do que prático, na abordagem efetiva dos aspectos linguísticos do português (cf. BORGES NETO, 2018). Em segundo lugar, temos o fato de que, nesta segunda edi- ção, Contador de Argote aponta às obras de Port-Royal e do Padre Lamy como as que melhor representavam o então clima de opinião 508 (cf. MARQUES, 2016). M. E que cousa he tom? De fato, como já apontou Leite (2011; 2013), o registro de Contador de Argote demonstra que, até então, a posição da partícula enclítica era variável no português europeu, podendo vir antes ou depois do verbo: “M. E esses prono- mes, ou particulas, podem-se por antes, ou depois do Verbo? / D. Commummente ou se podem por antes, ou depois” (CONTADOR DE ARGOTE, 1725, p. 289, grifos nossos). 509 É interessante observarmos a naturalidade com que a ausên- cia de regras de colocação ou conceitos que estejam engessados no que diz respeito ao posicionamento dos pronomes é retratada na obra: “M. Isto he huma cousa, que mal se percebe, ou conhece. / D. Assim he, e na verdade em algumas palavras se percebe mais, em outras menos” (CONTADOR DE ARGOTE, 1725, p. 289, grifos nossos). Outro fator importante é que, apesar do capítulo retratar explicitamente apenas a ênclise dos pronomes e de não estabele- cer terminologicamente a próclise ou as circunstâncias nas quais esta se dá, a percepção desse aspecto linguístico se faz presente no capítulo, como podemos ver no seguinte trecho: “M. E quando essas particulas, ou pronomes se põem antes do Verbo, saõ Encliticas? D. Naõ.” (CONTADOR DE ARGOTE, 1725, p. 289, grifos nossos). Já a mesóclise não figura em momento algum do capítulo, restringindo- -se a colocação pronominal, nesse caso, à ênclise e à próclise. Todos os exemplos apresentados pelo discípulo partem da intuição do autor, mas representam usos possíveis na estrutura do português, corroborando o que foi dito, em uma abordagem raciona- lista desse tema gramatical. CONSIDERAÇÕES FINAIS Neste capítulo, nos propusemos a analisar a abordagem da colocação pronominal nas Regras da Lingua Portugueza, espelho da lingua latina, de J. Contador de Argote (1725), e suas motivações e implicações no processo de gramatização do português no século 18. Dentre as razões pelas quais optamos por nos debruçar sobre tal instrumento gramatical, destacamos a importância da obra na história da gramaticografia da língua portuguesa, especialmente por ser considerada pioneira no processo de gramatização da colocação pronominal no português. 510 Como resultados, ressaltamos, a partir da Nouvelle methode, de Claude Lancelot, o vislumbre de algumas motivações que levaram à gramatização da colocação pronominal na língua portuguesa: a forte influência das ideias de Port-Royal sobre a gramatização euro- peia do século 18; a influência explícita da gramática de Port-Royal e, particularmente, sobre a edição de 1725 das Regras, onde encontra- mos o acréscimo do capítulo sobre o tema gramatical da colocação pronominal; e a associação desse fenômeno, na gramática francesa, à língua latina, que à época ainda exercia domínio político-social e cultural sobre a sociedade portuguesa. A obra de Contador de Argote sofreu influências teóricas do Iluminismo, que caracterizava o clima de opinião da Europa do século 18, mas não se pode considerar que haja um rompimento absoluto com a tradição, tendo em vista que abarca em si características de obras anteriores a esse período. A exemplo disso, podemos apontar o próprio propósito de ser um instrumento de consulta e um meio para se compreender a língua latina, e não como um fim em si mesma na aplicação dos princípios gerais de gramática ao português. Por sua vez, não se restringindo à abordagem da colocação pronominal, as Regras como um todo influenciaram o fazer gramati- cal posterior à sua própria produção. No século 18, a obra teve uma forte repercussão, tendo suas concepções retomadas por gramáti- cas de prestígio escritas ao longo do século. A colocação pronomi- nal ainda hoje se configura como um tema gramatical estudado por linguistas, gramáticos, acadêmicos, estudantes, e pela população em geral. A percepção desse fenômeno também teve grande importân- cia, como vimos, no debate sobre a língua brasileira e a portuguesa, exercendo funções política e simbólica sobre essas nações e influen- ciando o prisma sob o qual cada uma formou sua própria identidade. CONSIDERAÇÕES FINAIS Por fim, apontamos a necessidade de se estudar de modo mais profundo a abordagem desse fenômeno linguístico nas gra- máticas de Port-Royal e do Pe. Lamy, tecendo comparações entre 511 si e em relação às obras setecentistas do português, em busca de maior elucidação acerca de suas influências sobre a história da gra- maticografia portuguesa. REFERÊNCIAS AQUINO, José Edicarlos de. O que há de materno na língua? Considerações sobre os sentidos de língua materna no processo de gramatização brasileira nos séculos XIX e XX. Dissertação (Mestrado em Linguística) – Instituto de Estudos da Linguagem, Universidade Estadual de Campinas. Campinas: UNICAMP, 2012. 204 p. ARGOTE, Jerónimo Contador de. Regras da lingua portugueza, espelho da lingua latina. Lisboa: Officina da musica, 1725. ARGOTE, Jerónimo Contador de. Regras da lingua portugueza, espelho da lingua latina. Lisboa: Officina da musica, 1725. AUROUX, Sylvain. A revolução tecnológica da gramatização. Tradução de Eni Puccinelli Orlandi. 3. ed. Campinas: UNICAMP, 2014. BEM, Thomas Caetano de. Memorias chronologicas da sagrada religiaõ dos clerigos reguares em Portugal, e suas conquistas na India Oriental. Tomo II. Lisboa: Regia officina typografica, 1794. clerigos reguares em Portugal, e suas conquistas na India Oriental. Tomo II. Lisboa: Regia officina typografica, 1794. BORGES NETO, José. A gramática no século XVIII. In: BORGES NETO, José. História da Gramática. Curitiba, 2018. p. 183-199. (mimeo). FÁVERO, Leonor Lopes. Sentido e gramáticas no século XVIII. Língua e Literatura, n. 21, p. 109-130, 1994/1995. FERREIRA, Emily Gonçalves de Medeiros. Uma historiografia do processo brasileiro de gramatização da colocação pronominal em gramáticas oitocentistas FERREIRA, Emily Gonçalves de Medeiros. Uma historiografia do processo brasileiro FERREIRA, Emily Gonçalves de Medeiros. Uma historiografia do processo brasileiro de gramatização da colocação pronominal em gramáticas oitocentistas. Dissertação (Mestrado em Linguística) – Programa de Pós-Graduação em Linguística, Universidade Federal da Paraíba. João Pessoa, 2021. 232 p. FIORIN, José Luís. Língua portuguesa, identidade nacional e lusofonia. Confluência. Rio de Janeiro, n. 33/34, p. 53-68, 2008. GONÇALVES, Maria Filomena. Iluminismo e pensamento linguístico em Portugal: o exemplo das gramáticas filosóficas. (mimeo). Disponível em: <https://bit.ly/3XoY2dT>. Acesso em: 13 jan. 2023. 512 GURGEL, Silvana. O período dos estudos lingüísticos brasileiros dito científico na questão da colocação pronominal (1880-1920). Dissertação (Mestrado em Semiótica e Linguística Geral) – Programa de Pós-Graduação em Linguística, Universidade Federal de São Paulo. 2008. 142 p. KEMMLER, Rolf. Caetano Maldonado da Gama, D. Jerónimo Contador de Argote e as duas edições das Regras da lingua portugueza, espelho da lingua latina (1721, 1725). Limite, n. 6, p. 75-101, 2012. KEMMLER, Rolf. O gramático Jerónimo Contador de Argote e as duas edições das KEMMLER, Rolf. O gramático Jerónimo Contador de Argote e as duas edições das Regras da lingua portugueza, espelho da lingua latina (1721, 1725). In: MORENO, António et. al. XXIX Encontro Nacional da Associação Portuguesa de Linguística: Textos selecionados 2013. Porto: Associação Portuguesa de Linguística. 2014. p. 289-300. et. al. XXIX Encontro Nacional da Associação Portuguesa de Linguística: Textos selecionados 2013. Porto: Associação Portuguesa de Linguística. 2014. p. 289-300. KEMMLER, Rolf. Para uma melhor compreensão da história da gramática em Portugal: a gramaticografia portuguesa à luz da gramaticografia latino-portuguesa nos séculos XV a XIX. ARGOTE, Jerónimo Contador de. Regras da lingua portugueza, espelho da lingua latina. Lisboa: Officina da musica, 1725. Veredas, Santiago de Compostela, v. 19, p. 145-176, 2013. KOERNER, Konrad. Historiografia Linguística. In: KOERNER, Konrad. Quatro décadas de historiografia linguística: estudos selecionados. Seleção e edição de textos de Rolf Kemmler e Cristina Altman. Trás-os-Montes e Alto Douro: Centro de Estudos em Letras, Universidade de Trás-os-Montes e Alto Douro, 2014. p. 17-28. KOERNER, Konrad. Questões que persistem em historiografia linguística. Revista da ANPOLL, n. 2, p. 45-70, 1996. LEITE, Marli Quadros. A construção da norma linguística na gramática do século XVIII. Alfa, São Paulo, v. 55, n. 2, p. 665-684, 2011. LEITE, Marli Quadros. A gramatização da colocação dos pronomes átonos em gramáticas portuguesas e brasileiras. Todas as Letras V, v. 15, n. 2, p. 127-140, 2013. MACHADO, Diogo Barbosa. Bibliotheca lusitana historica, critica, e chronologica: na qual se comprehende a noticia dos authores Portuguezes, e das Obras, que compuzeraõ desde o tempo da promulgaçaõ da Ley da Graça até o tempo prezente. Lisboa: Officina de Ignacio Rodrigues, 1741-1759. Disponível em: <http://bit.ly/3HjXqRn>. Acesso em: 13 jan. 2023. MARQUES, Raquel do Nascimento. A configuração do português na gramática de D. Jeronymo Contador de Argote, Regras da lingua Portugueza, espelho da lingua latina. Dissertação (Mestrado em Filologia e Língua Portuguesa) – Faculdade de Filosofia, Letras e Ciências Humanas, Universidade de São Paulo. 2016. 143 p. 513 MARQUES, Raquel do Nascimento. As duas edições da gramática de Contador de Argote (1721, 1725). Revista da ABRALIN, v. 16, n. 1, p. 205-222, 2017. MIRANDA, Paulo André Batista. A cultura histórica iluminista: entre o projeto político e o livro didático. Dissertação (Mestrado em História) – Programa de Pós-Graduação em História, Universidade Federal da Paraíba. 2011. 172 p. MOURA, Tereza Maria Teixeira de. As ideias linguísticas portuguesas no século XVIII. Vila Real: Centro de Estudos em Letras, 2012. REIS LOBATO,  A. J. dos.  Arte  da  grammatica  da  lingua  portugueza.  Lisboa:  Regia  Officina  Typografica, 1770. REIS LOBATO,  A. J. dos. Arte  da  grammatica  da  lingua  portugueza. Lisboa:  Regia  Officina  Typografica, 1770. SANTOS, Tiago Scolari Chab dos. Artigo: da Tékhne Grammatiké aos dias de hoje. Dissertação (Mestrado em Letras) – Universidade Federal do Paraná. 2014. 108f. SWIGGERS, Pierre. A historiografia da linguística: objeto, objetivos, organização. Confluência, Rio de Janeiro, RJ, n. 44-45, p. 39-59, 2013. SWIGGERS, Pierre. Historiografia da Linguística: princípios, perspectivas, problemas. In: BATISTA, Ronaldo de Oliveira. Historiografia da Linguística. São Paulo: Contexto, 2019. p. 45-80. 514 25 Willian Ferreira Furtado de Lacerda A REALIZAÇÃO DO FONEMA LATERAL /L/ EM INGLÊS COMO L2 POR FALANTES BRASILEIROS DOI:10.31560/pimentacultural/2023.97778.25 Pensando nas diferentes formas com que uma comunidade se distingue de outra, a língua é postulada como fator identitário de um povo, apresentando variações tanto em nível macro (como por exemplo entre nações), como em níveis mais micro (se pensarmos em uma única nação, estado, cidade etc.). É por isso que muitas vezes encontramos, no Brasil, formas de falar em uma região que podem não ser de fácil reconhecimento por outras regiões. Neste estudo, abordaremos a variação no nível fonético-fo- nológico, investigando a variação do fonema lateral /l/ em posição de coda silábica no inglês como língua estrangeira (doravante L2) por falantes brasileiros. Ressaltamos que tal variação possui funções diferentes no português brasileiro (doravante PB) e no inglês. De modo geral, quando o /l/ ocupa a posição de coda silábica no PB, a variante que o falante utilizar não afetará o significado da palavra. Em con- trapartida, no inglês, a variante da lateral utilizada na coda silábica poderá ser determinante para o entendimento da mensagem que se deseja transmitir, tendo em vista que palavras parônimas, com significados distintos, poderão diferenciar-se exatamente pela pro- núncia do /l/ em coda. O objetivo do nosso estudo é investigar o comportamento do segmento /l/ em posição de coda silábica no inglês em con- texto de L2, de acordo com pesquisas anteriores realizadas sobre o tema, buscando identificar pontos de convergência nos resulta- dos de tais pesquisas. Para entendermos o que é a coda silábica, entramos no campo da estrutura silábica, a qual é referenciada por Selkirk (1982 apud HORA, PEDROSA e CARDOSO 2010) como composta por dois níveis. No primeiro nível, estão o onset e a rima, e, no segundo nível, a rima se subdivide em núcleo e coda. O onset corresponde à parte inicial da sílaba, e pode não ser preenchido foneticamente. 516 O núcleo refere-se à parte mais sonora da sílaba, e sempre é consti- tuído por uma vogal ou glide. Já a coda silábica, por sua vez, corres- ponde à parte final da sílaba e não tem a necessidade de ser foneti- camente preenchida, tornando-se a posição mais débil da sílaba em termos de sonoridade. Observe que, conforme definições acima, a coda silábica não precisa ser foneticamente preenchida e, mesmo quando o é, refere- -se a uma estrutura que favorece uma reestruturação silábica. 25 Willian Ferreira Furtado de Lacerda A REALIZAÇÃO DO FONEMA LATERAL /L/ EM INGLÊS COMO L2 POR FALANTES BRASILEIROS DOI:10.31560/pimentacultural/2023.97778.25 Essa “vulnerabilidade”, de certa forma já nos justifica, pelas próprias carac- terísticas da estrutura silábica, uma possível tendência a transformar a lateral pós-silábica em uma semivogal, ou até mesmo uma possível tendência ao apagamento do segmento. As considerações acima são importantes porque, conforme se verá a seguir, o PB admite apenas as consoantes [s], [r] e [l] em posição de coda silábica, enquanto no inglês há bem menos res- trições quanto a consoantes que pode ocupar essa posição. Dessa forma, se uma palavra em inglês terminar com uma sílaba fechada (ou travada) por outra consoante, um falante brasileiro de inglês como L2 poderá compensar a ausência do segmento vocálico inse- rindo uma vogal que não existe naquela sílaba. Silva (2012, p. 26, grifo da autora) afirma que “esse fenômeno – de inserção de vogal para evitar uma sílaba travada – é denominado epêntese”. Conforme mencionamos acima, uma diferença que se pode verificar entre o português e o inglês em relação à coda silábica diz respeito às consoantes que podem ocupar essa posição e, de acordo com Câmara Jr. (2004), as únicas consoantes possíveis de serem encontradas em posição de coda silábica na língua portu- guesa são a vibrante /r/, a lateral /l/, o arquifonema fricativo labial /S/ e o arquifonema nasal /N/. Interessante notar que o som nasal não é considerado por alguns autores como possível de ocorrer em coda silábica, como se pode verificar em Silva (2012, p. 26, grifos da autora), ao afirmar que 517 No PB, somente as consoantes ‘s, r, l’ ocorrem em final de sílaba. As consoantes nasais não são pronuncia- das em final de sílaba – embora ortograficamente pala- vras do português terminem em ‘m’ (‘sim, batom’) e as letras ‘m, n’ possam ocorrer em final de sílaba em meio de palavra (‘ponto, pombo’), sem ‘m, n’, nestes casos, serem pronunciados. Aquino (2013, p. 9) acentua as diferenças quanto à posição de coda no português e no inglês, deixando clara uma menor limi- tação das consoantes que podem ocupar esta posição silábica em língua inglesa. A autora afirma que Na posição de coda, em especial, são significativas as diferenças entre português e inglês. No português são permitidas apenas codas simples e duplas compostas por qualquer soante e/ou /S/. 25 Willian Ferreira Furtado de Lacerda A REALIZAÇÃO DO FONEMA LATERAL /L/ EM INGLÊS COMO L2 POR FALANTES BRASILEIROS DOI:10.31560/pimentacultural/2023.97778.25 No inglês, com a possibilidade de codas com até três elementos (as não sufixadas), as combinações são bastante variadas, permitindo a pre- sença de nasais, obstruintes e plosivas nessa posição. As ideias apresentadas até então permitem-nos entender que a posição de coda silábica, por suas características estruturais, está de certa forma mais propensa a sofrer alterações sonoras do que as demais partes constituintes da palavra e esse entendimento, por ora, entendemos como suficiente para a viabilização da análise a que esta pesquisa se propõe, tendo em vista que a vocalização da lateral pós-vocálica está contemplada em tais justificativas. Em relação ao fonema lateral, Baratieri (2006) afirma que estes sons fazem parte da classe de fonemas líquidos, que perten- cem ao grupo dos sons aproximantes. A explicação para o termo “líquido” é apresentada com base em Câmara Jr. (1977), segundo o qual o termo foi cunhado pelos gregos, levando em considera- ção que o ar, sempre que encontra uma obstrução, atua como um líquido, que consegue mudar de direção de modo a manter seu fluxo. Já a explicação para o termo “lateral” deve-se ao fato de o ar fluir para fora da boca livremente pelas laterais da língua, ou seja, o topo 518 da língua provoca uma obstrução central e faz com que o ar flua pelas laterais da língua. Em língua inglesa, esse fonema possui as variantes clara e escura, conforme apontado por Johnson e Britain (2003), cuja dife- rença estaria na posição que a consoante ocupa na palavra. Nesse sentido, Kelly (2000, p. 52, tradução nossa) assevera que O fechamento alveolar com a ponta da língua resulta em um l claro, como no segmento live. Isso ocorre antes de sons vocálicos. Depois de sons vocálicos, como no vocá- bulo pool), antes de consoantes (como no vocábulo help), a parte de trás da língua é elevada em direção ao palato mole, resultando no l escuro (um alofone). As definições acima convergem com o que preconiza Silva (2012, p. 155), quando esta autora afirma que “o l-claro ocorre em inglês: no início de palavra [...], seguindo s em início de palavra [...], no meio de palavra entre vogais [...] ou no meio de palavra precedido de outra consoante na sílaba anterior [...]”. No que diz respeito ao l-escuro /ɫ/, de acordo com a mesma autora, este alofone do fonema lateral “ocorre, tipicamente, em posição final de sílaba em inglês”. 25 Willian Ferreira Furtado de Lacerda A REALIZAÇÃO DO FONEMA LATERAL /L/ EM INGLÊS COMO L2 POR FALANTES BRASILEIROS DOI:10.31560/pimentacultural/2023.97778.25 A autora assevera que Em algumas variedades do inglês britânico, americano e australiano, o l-escuro, quando ocorre em final de sílaba, é pronunciado com w. Esse tipo de fenômeno é deno- minado de processo de vocalização do “l”. Assim, uma palavra que é tipicamente pronunciada em inglês com um l-escuro em final de sílaba – como feel fi:l –, é pronun- ciada como fi:w nos dialetos que têm a vocalização do “l” (SILVA, 2012, p. 157, grifos da autora). Conforme visto no excerto acima, as ocorrências do l escuro têm apontado para um processo de vocalização da lateral em posi- ção de coda silábica em língua inglesa, embora haja forte predomi- nância da realização do fonema como consoante lateral, conforme se verá na análise dos dados deste estudo. 519 No PB, essa vocalização se apresenta como uma variante do fonema lateral em posição de coda silábica, havendo predominância desta forma vocalizada em detrimento das realizações da variante consonantal. Não se pode afirmar, entretanto, que essa predominân- cia ocorre na mesma proporção entre as diversas regiões do Brasil. Além dos alofones claro e escuro, no PB também encontra- mos a lateral palatal [ʎ], que é pronunciada basicamente em seg- mentos grafados com o dígrafo lh. Seara, Nunes e Lazzarotto-Volcão (2011) nos fornecem os seguintes exemplos para diferenciação dos três tipos de lateral encontrados no PB: nas palavras lata, sal e telha, são encontradas, respectivamente: a) a lateral alveolar vozeada ([l] ata), a qual é produzida por meio de uma obstrução realizada com a ponta da língua no centro dos alvéolos; b) a lateral velar (vozeada) (sa[ɫ]), que é realizada através do bloqueio com o dorso da língua na região central do palato mole (produzida principalmente em algumas regiões do Rio Grande do Sul em posição de coda silábica) e, por fim, c) a lateral palatal (te[ʎ]a) (também vozeada), produzida com a parte anterior da língua tocando no centro do palato duro. Muitas reflexões podem ser feitas acerca das variações do fonema lateral, inclusive apontando outras variações que ocorrem em diversas outras línguas pelo mundo. Porém, aqui, vale lembrar que este estudo irá investigar as variações do fonema em posição de coda silábica no inglês na perspectiva de L2. 25 Willian Ferreira Furtado de Lacerda A REALIZAÇÃO DO FONEMA LATERAL /L/ EM INGLÊS COMO L2 POR FALANTES BRASILEIROS DOI:10.31560/pimentacultural/2023.97778.25 Seja em língua materna, seja em L2, entendemos a variação como um fenômeno que sofre influência de forças diversas, tanto internas como externas à língua e, assim, inevitavelmente entramos no campo da Sociolinguística Variacionista, cuja presença no pro- cesso de aquisição de L2 abordaremos a partir de agora. De acordo com Silva (2016, p. 47), “a Sociolinguística é a ciência que analisa o comportamento linguístico desde um ponto de vista sociológico. Deste modo, estudos variacionistas têm como 520 premissa básica os fatores sociais e linguísticos [...]”. Os estudos ante- riores ao advento da Sociolinguística, no século XX, através das cor- rentes estruturalista e gerativista, cujos principais expoentes foram Saussure e Chomsky, respectivamente, concebiam a língua como uma estrutura alheia aos fatores sociais, considerando-a um sistema homogêneo e independente do meio externo. Em consonância com as ideias acima, Coelho et al (2010, p. 14) complementam que o estruturalismo e o gerativismo considera- vam a língua como uma realidade abstrata, desvinculada de fatores históricos e sociais. É como uma reação a essas duas correntes que a Sociolinguística desponta nos Estados Unidos na década de 1960, tendo como um de seus maiores expoentes William Labov”. Esclarecemos, aqui, que as definições trazidas até então refe- rem-se à Sociolinguística em seu sentido mais amplo. Porém, a área possui algumas ramificações, conforme aponta Fragozo (2010, p. 45) [A Sociolinguística] se subdivide em três áreas: a Etno- grafia da Comunicação, a Teoria da Variação e a Socio- linguística Interacional. A Etnografia da Comunicação, liderada por Hymes, tem caráter qualitativo e apresenta uma intersecção com a antropologia. A Teoria da Varia- ção foi iniciada por Labov nos anos 60 e possui caráter quantitativo, por lidar com números e análise estatística dos dados coletados [...]. A Sociolinguística Interacional, liderada por Gumperz, estuda o comportamento do indi- víduo em situações de comunicação face a face. Ressaltamos que, neste estudo, tomaremos como base a Sociolinguística em seu viés variacionista e, a esse propósito, Lacerda, Cavalcante e Lucena (2020, p. 440) asseveram que “o estudo da variação como característica inerente à natureza da linguagem é a proposta central de Labov”, cuja abordagem ficou conhecida como Sociolinguística Variacionista, ou Sociolinguística Laboviana ou, ainda, Sociolinguística Quantitativa, tendo em vista o fato de operar com números e de fazer tratamento estatístico dos dados coletados. 25 Willian Ferreira Furtado de Lacerda A REALIZAÇÃO DO FONEMA LATERAL /L/ EM INGLÊS COMO L2 POR FALANTES BRASILEIROS DOI:10.31560/pimentacultural/2023.97778.25 521 Nessa perspectiva, Tarallo (1990) define a variação linguística como a possibilidade de haver mais de uma maneira de se dizer a mesma coisa, com o mesmo grau de verdade. Dessa forma, durante a fala, o indivíduo faz escolhas, e tais escolhas são baseadas em cri- térios (TARALLO, 1990). A essas maneiras diversas, dá-se o nome de variantes. O conjunto de variantes compõem a variável. Outro conceito importante no estudo da variação linguística diz respeito à variedade, que corresponde ao que conhecemos como dialeto, ou a forma de falar de determinada comunidade de fala. Ainda no tocante à variação, Tarallo (1990) considera que as variantes envolvidas estão “em batalha” e dispõem de armas para que possam ser escolhidas pelo indivíduo no momento da fala. O autor complementa que os contextos que favorecem uma variante ou outra são chamados fatores condicionadores, os quais fornecerão respostas ao pesquisador sobre quais as variantes mais prováveis de acontecer em certos contextos. Tais condicionadores podem tanto ser internos à língua (linguísticos) como externos à língua (extralin- guísticos), tais como classe social, sexo, faixa etária etc. A variação linguística pode se dar nos níveis fonético-fonoló- gico, morfológico e sintático. No caso de Labov, seu estudo inicial tra- tou de analisar uma variação no nível fonológico na ilha de Martha’s Vineyard, nos Estados Unidos. A variação consistia nas diferentes produções possíveis dos ditongos /ay/ e /aw/, para os quais o autor verificou a existência de três variantes: /ay/ era pronunciado ora como /ay/, ora como /ey/ e ora como /əy/ e, de forma semelhante, / aw/ apresentava as variantes /aw/, /ew/ e /əw/ (LABOV, 2008). Os resultados do estudo de Labov demonstraram que a varia- ção linguística estava acontecendo por influência de fatores externos à língua, ou seja, fatores extralinguísticos: os falantes optam por uma variante ou por outra de acordo com o seu sentimento de perten- cimento àquela comunidade de fala, a ilha de Martha’s Vineyard. 522 Quando este sentimento era positivo para o falante, este mantinha o seu estilo linguístico local, divergindo dos sotaques dos turistas. Para Fragozo (2010), os estudos sobre a Sociolinguística e a aquisição de L2 vêm ganhando espaço por conta da importân- cia verificada dos aspectos sociais na aprendizagem de uma língua estrangeira. 25 Willian Ferreira Furtado de Lacerda A REALIZAÇÃO DO FONEMA LATERAL /L/ EM INGLÊS COMO L2 POR FALANTES BRASILEIROS DOI:10.31560/pimentacultural/2023.97778.25 Sob esta perspectiva, entende-se que a variação linguís- tica ocorre pelos fatores mencionados (etnia, automonitoramento etc.), mas não isoladamente, pois há inúmeras forças atuando sobre o indivíduo no momento da produção da fala. Se tomarmos por base os “fatores isolados” mencionados acima, podemos concluir, por exemplo, que a quantidade de tempo dedicado ao planejamento do discurso pode exercer influência na fala do indivíduo, ao mesmo tempo que o tópico do discurso também o faz, bem como o monito- ramento do indivíduo sobre a sua própria fala etc. Assim sendo, ao pesquisador da área da Sociolinguística compete a missão de investigar não o fator único, mas quais os dife- rentes fatores que exercem influência no aprendiz e condicionam a variação na sua fala (BAYLEY, 2007). Nesse sentido, e com foco no aprendiz de L2, Fragozo (2010, p. 48) considera que “além de observarem-se os fatores linguís- ticos e psicolinguísticos comuns à pesquisa em aquisição de LE, incluem-se também fatores extralinguísticos que podem influenciar o processo de aprendizagem, como idade, sexo e vivência em país falante da língua alvo”. Bayley (2007) aponta, ainda, algumas contribuições da pes- quisa sociolinguística para os estudos em aquisição de L2, dentre as quais destacamos: o fornecimento de uma visão mais realista de como funciona a língua-alvo; a oferta de uma maneira empírica de estudar os efeitos das transferências linguísticas frente a uma alta gama de variáveis; a visão de que a forma como o aprendiz usa os processos de variação pode revelar uma identidade linguística etc. 523 Feitas tais considerações a respeito da interface Sociolin- guística – aquisição de L2, passaremos à análise dos dois estudos selecionados, destacando os seguintes aspectos de cada uma das pesquisas: objetivo, corpora e variáveis. Os resultados de cada uma delas serão oportunamente discutidos. Iniciemos pelo estudo de Baratieri (2006), cuja pesquisa cor- responde a uma dissertação de mestrado em que o autor buscou analisar a produção, por falantes do PB, do fonema /l/ em posição de coda silábica no inglês como L2, com fins de investigar o efeito do contexto fonológico seguinte na produção da lateral. O autor buscou, também, investigar propriedades acústicas e articulatórias desse fonema, além do pico silábico, de modo a verificar se ele mantém alguma relação com as realizações articulatórias da lateral. A amostra do autor foi composta por 20 estudantes brasi- leiros de inglês como L2. 25 Willian Ferreira Furtado de Lacerda A REALIZAÇÃO DO FONEMA LATERAL /L/ EM INGLÊS COMO L2 POR FALANTES BRASILEIROS DOI:10.31560/pimentacultural/2023.97778.25 Destes, 15 eram do sexo feminino e 5 do sexo masculino, e as idades variaram entre 14 e 22 anos. O autor afirma que os participantes da pesquisa nunca viajaram para outro país. Dentre os estudantes, 13 estavam matriculados no terceiro nível do curso de inglês “To the Top” (TT-3) e 7 tinham concluído este nível recentemente. O curso, de acordo com o autor, possui 3 níveis de inglês avançado e consiste em 57 horas de instrução por nível. Após completar o terceiro nível, os estudantes são instruídos a reali- zarem o teste TOEFL ITP. As variáveis controladas pelo autor foram puramente linguísti- cas: foi fixada a vogal média-baixa anterior /ɛ/ como contexto prece- dente para, a partir daí, analisar o comportamento da lateral de acordo com o contexto fonológico seguinte, ponto de articulação e modo de articulação. Embora o autor reconheça que pode haver variação de acordo com a variável sexo, esta não foi considerada no estudo. Baratieri (2006) sintetiza as características do /l/ no inglês britânico, no americano e no PB afirmando que, de um lado, essas 524 S U M Á R I O três variedades realizam o /l/ de forma semelhante quando este está em posição de onset (início de sílabas), apresentando características fonéticas do l claro. Por outro lado, quando o /l/ ocupa a rima silábica, é predominantemente realizado como l escuro tanto no inglês britâ- nico como no americano, enquanto no PB é realizado geralmente com nenhuma ou com poucas características do gesto consonantal. Os dados coletados foram processados pelo programa Praat, versão 4.4.12, no qual se buscou verificar a presença ou ausência de três aspectos, quais sejam: a) gesto consonantal, que denotaria que o fonema compartilha traços característicos das consoantes laterais; b) arredondamento labial, característico de produções vocálicas pos- teriores, e c) formantes nasais, de sons característicos. Baratieri (2006) sinaliza que seu estudo não buscou investi- gar a precisão da realização do /l/, mas sim a sua vocalização consi- derando o efeito do contexto fonológico seguinte e, por isso, agrupou as produções dos participantes de acordo com o grau de vocaliza- ção da lateral. 25 Willian Ferreira Furtado de Lacerda A REALIZAÇÃO DO FONEMA LATERAL /L/ EM INGLÊS COMO L2 POR FALANTES BRASILEIROS DOI:10.31560/pimentacultural/2023.97778.25 No total, foram verificadas cinco produções de /l/ dis- tintas, que chamaremos de variantes e que ele agrupou em três: a) as produções nas quais se verificasse apenas arredondamento labial, sem gesto consonantal (codificadas como “W” ou “Wo”) foram con- sideradas como totalmente vocalizadas, e a elas era atribuído o peso 10; b) as produções em cuja análise se verificasse tanto o gesto con- sonantal como o arredondamento labial (codificadas como “Lw” ou “Lwo”) foram classificadas como parcialmente vocalizadas, de peso 5; c) as produções cuja análise apontasse apenas o gesto consonan- tal (codificadas como “L”) foram classificadas como não-vocalizadas, peso zero. Todas as demais produções foram consideradas como valores nulos. No total, foram registradas 2.134 produções válidas. O tratamento estatístico dos dados foi realizado através do programa SPSS para Windows 10.0. Os resultados e discussão deno- tam que: a) o menor percentual de frequência  foi apresentado pela produção não-vocalizada (“L”), que totalizou 2,7% das ocorrências, 525 o que corresponde a 57 produções com movimentos exclusivamente consonantais dentre as 2.134 válidas; b) a produção parcialmente vocalizada (“Lw”) foi a que apresentou o maior percentual de frequ- ência, à razão de 61,8%, o que indica que 1.319 produções do /l/ foram realizadas com movimentos tanto vocálicos como consonantais; c) a produção vocalizada (“W”) – apresentou frequência de 35,5%, ou seja, 758 das 2.134 produções válidas apresentaram movimentos exclusivamente vocálicos. Baratieri (2006) considera que se poderia ser afirmado que os participantes da pesquisa transferiram o /l/ do PB para o /l/ em coda silábica no inglês, visto que ambas as variantes vocalizada e parcialmente vocalizada ocorrem no PB, e menciona que parece haver uma mudança na direção do menos marcado, ou seja, o /l/ claro [l] evolui para o escuro [ɫ], que evolui para o parcialmente voca- lizado [ɫw], que, finalmente, evolui para a forma vocalizada [w]. Já em sua pesquisa, os resultados parecem indicar que os participantes da pesquisa estão trilhando a direção oposta, partindo do menos marcado ([w]) para o mais marcado ([ɫ]), pois mais da metade das produções foi parcialmente vocalizada ([ɫw]). Isso, na visão do autor, pode indicar mais desenvolvimento interlinguístico do que a trans- ferência da língua materna, pois esta aumentaria a produção da variante vocalizada ([w]). 25 Willian Ferreira Furtado de Lacerda A REALIZAÇÃO DO FONEMA LATERAL /L/ EM INGLÊS COMO L2 POR FALANTES BRASILEIROS DOI:10.31560/pimentacultural/2023.97778.25 Nestes termos, o maior número de ocorrências da variante parcialmente vocalizada ([ɫw]) pode ser devido ao esforço dos parti- cipantes para produzir a lateral em coda silábica da forma mais pare- cida possível com a produção que fariam os nativos do inglês. Os efeitos do contexto fonológico analisados pelo autor per- mitiram-no fazer as seguintes inferências: a. os contextos fonológicos de pausa e consoante na pala- vra seguinte favorecem significativamente mais a vocaliza- ção do que consoante na mesma palavra e a diferença entre a interferência da pausa e da consoante na palavra seguinte não foi significativa; 526 b. quando o contexto fonológico seguinte é de consoantes desvo- zeadas, o fenômeno da vocalização é significativamente mais favorecido do que pelo contexto das consoantes vozeadas, seja na mesma palavra ou na palavra seguinte; c. quanto ao ponto de articulação, independentemente da posição da consoante (se na mesma palavra ou na palavra seguinte), a vocalização da lateral em coda silábica ocorreu mais frequen- temente (por ordem decrescente de favorecimento) antes de bilabiais, labiodentais, velares, pós-alveolares e, finalmente, antes de alveolares; d. a forma de articulação da consoante seguinte ao /l/ não é fator decisivo que causa vocalização; e. o ponto de articulação é fator decisivo na vocalização do /l/ em coda silábica no inglês. A segunda pesquisa selecionada que abordou o inglês como L2 por falantes brasileiros foi a de Hahn (2010), cuja proposta era a de investigar a influência do PB na aquisição da lateral /l/ em posição de coda silábica no inglês como segunda língua por falantes da região sul do Brasil, mais especificamente do estado do Rio Grande do Sul. O corpus analisado pela autora advinha dos dados coleta- dos de 25 estudantes brasileiros de inglês como L2. A análise foi feita auditivamente via programa Praat e, estatisticamente, utilizando o software Goldvarb X. Seus objetivos foram: observar as taxas de vocalização por falantes de inglês como L2 do Rio Grande do Sul e a relação do fenômeno com variáveis linguísticas e extralinguísticas; verificar se o comportamento de tais falantes em L2 se relaciona com características da variedade do PB da região e com características observadas na realização do /l/ em variedades do inglês. 25 Willian Ferreira Furtado de Lacerda A REALIZAÇÃO DO FONEMA LATERAL /L/ EM INGLÊS COMO L2 POR FALANTES BRASILEIROS DOI:10.31560/pimentacultural/2023.97778.25 As variáveis consideradas no estudo de Hahn foram divididas em linguísticas e sociais, conforme segue: 1) variáveis linguísticas: a) variável dependente: aplicação da regra da vocalização, transfor- mando o l escuro [ɫ] na semivogal [w]; b) variáveis independentes: 527 S U M Á R I O contexto anterior; contexto posterior; 2) variáveis sociais: a) sexo; b) nível de inglês; 3) informante (cada informante também foi codificado). Os resultados apontam, em relação à variável dependente, um equilíbrio entre as variantes. Do total de 1.377 dados válidos, a vocalização ocorreu em 49,2%, fazendo a autora inferir que houve um comportamento distinto entre a realização do /l/ no inglês e a realização desse fonema em língua materna, pois a literatura aponta predominância elevada da vocalização do /l/ em posição final no PB na capital gaúcha e região metropolitana, de onde é a maioria dos participantes da pesquisa. Esse percentual considerável de vocaliza- ção vai de encontro à hipótese da autora, de que haveria muito mais ocorrências do l escuro [ɫ]. Também foram frustradas as hipóteses da autora de que os contextos fonológicos anteriores (tanto o vocálico como o consonan- tal) exerceriam influência no processo de vocalização. O programa de tratamento estatístico Goldvarb X não selecionou nenhuma des- sas variáveis como relevantes para o fenômeno sob investigação. Quanto à hipótese de ressilabação resultante o /l/ em coda seguido de palavra iniciado com vogal, em que o /l/ tomaria a posi- ção de onset da palavra seguinte, inibindo a vocalização, a autora também se surpreendeu ao verificar que a ressilabação parece não estar atuando nos dados considerados, pois esta variável não foi selecionada pelo programa como sendo relevante. Os resultados mostraram que os percentuais de ocorrências de vocalização com a palavra seguinte iniciando com vogal atingiram o patamar de 49,1%, contra 49,2% de vocalização quando à coda silábica seguia uma pausa ou uma palavra iniciada por consoante. Em outras palavras, todos esses fatores das variáveis independentes permearam o ponto neutro, não sendo relevantes, de acordo com o software, para o fenô- meno da vocalização, cenário que a autora interpreta como distinto do que o que aconteceria em língua materna. Por isso, ela alega que os resultados sugerem uma manipulação da ressilabação com certo nível de consciência do falante. 528 Também houve a hipótese de que o sexo exerceria certa influ- ência no processo de vocalização. 25 Willian Ferreira Furtado de Lacerda A REALIZAÇÃO DO FONEMA LATERAL /L/ EM INGLÊS COMO L2 POR FALANTES BRASILEIROS DOI:10.31560/pimentacultural/2023.97778.25 A autora afirma que não foi pos- sível verificar qual seria a forma prestigiada pelos falantes no fenô- meno em análise e se esse eventual prestígio estaria relacionado ao sexo. Os resultados da pesquisa, segundo a autora, apenas indicam a proporção de vocalização por mulheres e homens do PB, que indi- cam 50,6% de vocalização pelas mulheres, perante 45,7% de voca- lização pelos homens. Observamos, aqui, que os dados poderiam indicar uma precisão maior, ou seja, poderiam indicar se a variável sexo exerce influência no fenômeno da vocalização, se a amostra da pesquisa contivesse representantes de cada sexo de forma ortogo- nal, apresentando a mesma quantidade de informantes para ambos os sexos (conforme delimitação da amostra, esta foi composta por 7 homens e 18 mulheres, havendo desequilíbrio significativo). Mesmo assim, a autora considera que os seus procedimentos metodológicos atenderam aos objetivos iniciais. A autora também apresentou a hipótese de que o grau de proficiência dos aprendizes exerce alguma influência na vocaliza- ção da lateral. Em relação a essa hipótese, a autora considera que suas expectativas foram superadas, pois esperava que a vocaliza- ção ocorresse com maior frequência no nível G (que, em sua amos- tra, corresponde ao nível de inglês elementar, representado por um único sujeito). O maior peso relativo foi apresentado pelo nível E, cujos representantes, de acordo com a autora, são considerados “usuários competentes” na língua. Ela interpreta tais dados pela ver- tente dos chamados erros de desenvolvimento, buscando respaldo em Archibald (1998), o que quer dizer que o aprendiz em nível inicial de proficiência (como o falante de nível G em seu estudo) apresenta baixo índice de erros de desenvolvimento, índice este que aumenta no nível intermediário e diminui ao passo que o nível de proficiência aumenta. Novamente, acreditamos que um equilíbrio na quantidade de informantes de cada nível poderia expressar resultados mais pre- cisos, embora a autora considere que o teste escolhido na meto- dologia, por ser bastante reconhecido, dá a devida sustentação aos resultados apresentados. 529 Quanto ao fato de as variáveis linguísticas não terem sido consideradas como relevantes, por não terem sido selecionadas nas rodadas do programa Goldvarb X, a autora sintetiza uma nova hipótese: a de que a vocalização não possui caráter assimilatório, mas que seja um fenômeno de lenição, explicável foneticamente, por propriedades inerentes à articulação do /l/ que se manifestam mais fortemente no contexto de coda. 25 Willian Ferreira Furtado de Lacerda A REALIZAÇÃO DO FONEMA LATERAL /L/ EM INGLÊS COMO L2 POR FALANTES BRASILEIROS DOI:10.31560/pimentacultural/2023.97778.25 Também é interpretado pela autora que a variação entre as formas vocalizada ([w]) e velarizada ([ɫ]) no mesmo falante sugere que este seja um processo fonológico mais simplificado do que aquele expresso pela regra telescópica (que se inicia nas grandes cidades), apresentado por Quednau (1993). Hahn entende que há uma naturalidade na passagem de /l/ para [w]. A autora destaca que, em relação aos informantes, existe uma grande variação entre os pesos relativos dentro de um mesmo nível de inglês. Tais variações, afirma, poderiam, talvez, ser atribuídas a fatores como sexo, idade, escolaridade, região geográfica e classe social, fatores que não foram controlados no estudo. Outros fatores de ordem social também poderiam exercer influência nessa varia- ção, tais como: relações sociais mais imediatas, atividades de lazer com a língua-alvo, experiência no exterior etc. A autora julga seus resultados como compatíveis com aque- les apresentados por Baratieri (2006), sugerindo que há um processo de desenvolvimento interlinguístico operando na aquisição de /l/ no inglês como L2 pelos participantes da sua pesquisa. De modo a tornar mais didática a interpretação dos resul- tados das pesquisas apresentadas, representaremos graficamente as variáveis controladas por ambas, que seguem no Quadro 2. Mas, antes se faz necessário identificar os códigos através dos quais os dados serão representados, e isso fazemos no Quadro 1, a seguir: 530 Quadro 1 – Legenda para a interpretação do próximo quadro Dado Significado P Variável controlada no estudo X Variável considerada relevante após análise dos dados pelos autores - (hífen), quando no lugar de P Variável não controlada no estudo - (hífen), quando no lugar de X Variável controlada no estudo, porém não julgada relevante após análise dos autores Fonte: adaptado de Lacerda (2021). Quadro 1 – Legenda para a interpretação do próximo quadro Dado Significado P Variável controlada no estudo X Variável considerada relevante após análise dos dados pelos autores - (hífen), quando no lugar de P Variável não controlada no estudo - (hífen), quando no lugar de X Variável controlada no estudo, porém não julgada relevante após análise dos autores Fonte: adaptado de Lacerda (2021). No Quadro 2, a seguir, estão dispostas as variáveis indepen- dentes controladas nos dois estudos. 25 Willian Ferreira Furtado de Lacerda A REALIZAÇÃO DO FONEMA LATERAL /L/ EM INGLÊS COMO L2 POR FALANTES BRASILEIROS DOI:10.31560/pimentacultural/2023.97778.25 Quadro 2 – Variáveis independentes nas pesquisas que abordam o inglês como L2 Pesquisa Variáveis independentes Baratieri (2006) Hahn (2010) Contexto fonológico anterior P X P - Contexto fonológico seguinte P X P - Ponto de articulação P X - - Modo de articulação P - - - Sexo - - P - Nível de inglês - - P X Informante - - P X Fonte: Lacerda (2021). Quadro 2 – Variáveis independentes nas pesquisas que abordam o inglês como L2 O Quadro 2, acima, nos permite visualizar um fato curioso: não houve sequer uma variável independente que tenha sido contro- lada pelas duas pesquisas é considerada como relevante por ambas. Enquanto a pesquisa de Baratieri (2006) apontou que as variáveis relevantes para o fenômeno da vocalização são o contexto fonológico 531 anterior, o contexto fonológico seguinte e o ponto de articulação, Hahn (2010) apontou que as variáveis relevantes são o nível de inglês e o informante. Apresentaremos, agora, a nossa interpretação do que pude- mos observar do Quadro 2 no sentido de identificar o quão relevan- tes foram as variáveis nas pesquisas analisadas. S U M Á R I O a. Contexto fonológico precedente: presente em ambas as pes- quisas, não considerado relevante na pesquisa de Hahn (2010): b. Gênero: controlada apenas por Hahn (2010), cujo resultado não apontou a variável como relevante para o fenômeno pesquisado; c. Contexto fonológico seguinte: variável controlada por ambas as pesquisas, mas julgada como relevante apenas no estudo de Baratieri (2006); d. Ponto de articulação: considerada apenas por Baratieri (2006), que a descobriu relevante para o fenômeno; e. Nível de inglês e informante: controladas apenas por Hahn (2010) e consideradas relevantes em suas respectivas pesquisas; f. Modo de articulação: controlada apenas por Baratieri (2006) e, mesmo assim, não foi considerada relevante para o fenômeno. f. Modo de articulação: controlada apenas por Baratieri (2006) e, mesmo assim, não foi considerada relevante para o fenômeno. A diversidade de variáveis controladas por ambas as pesqui- sas nos mostra um diversificado leque de possibilidades de investi- gação dos fenômenos linguísticos e interpretando-os à luz da influ- ência que inclusive fatores extralinguísticos (ou sociais) interferem no comportamento linguístico do falante. 25 Willian Ferreira Furtado de Lacerda A REALIZAÇÃO DO FONEMA LATERAL /L/ EM INGLÊS COMO L2 POR FALANTES BRASILEIROS DOI:10.31560/pimentacultural/2023.97778.25 Conforme foi possível verificar ao longo deste trabalho, a consoante lateral /l/ em posição de coda silábica possui algumas variantes tanto em língua portuguesa como em língua inglesa e pudemos observar, mediante análise de pesquisas anteriores sobre o tema, que fatores diversos, tanto linguísticos como extralinguísti- cos, atuam na decisão (consciente ou não) do falante por eleger uma variante ou outra no momento de sua fala. 532 Interessante notar que, como nosso objetivo era encontrar pontos de convergência entre as pesquisas, não houve qualquer variável que tenha sido controlada por ambas e descoberta como relevante por ambas. 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Fonética e fonologia do português: roteiro de estudos e guia de exercícios. 6ª ed. São Paulo: Contexto, 2002. 534 SILVA, Thaïs Cristófaro. Pronúncia do inglês: para falantes do português brasileiro. São Paulo: Contexto, 2012. SILVA, Thaïs Cristófaro. Pronúncia do inglês: para falantes do português brasileiro. São Paulo: Contexto, 2012. TARALLO, Fernando. A pesquisa sociolinguística. 3 ed. São Paulo: Ática, 1990. TARALLO, Fernando. A pesquisa sociolinguística. 3 ed. São Paulo: Ática, 1990. S U M Á R I O 535 INTRODUÇÃO A definição aristotélica de verdade é um maravilhoso quebra- -cabeças semântico e pragmático perfeito para iniciarmos as refle- xões que queremos trazer neste texto. Para Aristóteles: De fato, dizer que aquilo que é não é, ou que aquilo que não é é, é falso; por outro lado, dizer que aquilo que é é, ou que aquilo que não é não é, é verdadeiro. Por conse- guinte, quem pretende afirmar que algo é ou não é poderá estar dizendo algo verdadeiro ou algo falso. No entanto, não pretende afirmar que é ou que não é nem aquilo que é, nem aquilo que não é. (ARISTÓTELES, Metafísica, livro Γ (IV), 1011b23). Começamos, assim, com dois problemas principais: o pri- meiro, e mais complicado, definir o que “é” e o que “não é”. O segundo, imediatamente decorrente do primeiro, definir o que seja e o que não seja a partir do “dizer”, como numa teoria de verdade-como-corres- pondência, mas que inclua os usuários da língua. Neste sentido, dentro dos Estudos Linguísticos, muitas ver- tentes teóricas têm algo a acrescentar sobre como o “dizer” refere e descreve o que “é” e “não é” ou como, em tantos outros níveis, “dizer” cria de modo discursivo, enunciativo e performativo o que é e o que não é. Em termos ainda mais contemporâneos, vivemos em uma época em que vemos, cotidianamente, verdades sendo criadas por meio do que é “dito que é”, ainda que não seja, em termos objeti- vos, verificável no mundo real. Ainda além: muitas vezes o que é dito é verificável em termos de valor de verdade, mas os termos utiliza- dos para tanto não são capazes de referir de maneira inequívoca e indiscutível que aquele fato é daquele jeito. Este trabalho, assim, não se dedicará a falar sobre a verdade, mas sobre a sua falta: o que – e como – um falante faz quando, por decisão própria, opta por não dizer a verdade (ou por dizer o que é falso), com a intenção de enga- nar o seu interlocutor. 537 Caracterizar a mentira como um ato linguístico é algo relati- vamente óbvio, uma vez que não há como mentir sem que isso seja feito verbalmente. Evidentemente, estamos falando sobre “mentir” e não sobre “enganar”, “induzir ao erro”, “confundir”, “trapacear”, “trair”, “iludir”, “causar equívoco” ou demais tipos de atos que, ainda que passem por uma ação linguística, não se encerram em si. INTRODUÇÃO A mentira, da forma como iremos abordar neste trabalho, é única e exclusiva- mente feita por meio do uso linguístico e gera ações condizentes com esses proferimentos a partir do momento em que são feitos. Ou seja, a ação dos indivíduos, no mundo, passará a ser pautada pelos proferimentos feitos dentro do ato de fala. Evidentemente, isto não é verdade apenas para a mentira, mas para qualquer outro ato de fala, conforme demonstraram Aus- tin (1962a) e Searle (1979[1984]). Para o primeiro, existem sentenças cujo proferimento “não é descrever o ato que estaria praticando ao dizer o que disse, nem declarar o que estou praticando: é fazê-lo. Nenhum dos proferimentos citados é verdadeiro ou falso; consi- dero isso tão óbvio que sequer pretendo justificar.” (AUSTIN, 1991, p. 24-25). Já para o segundo, o ato de fala é a unidade mínima da comunicação humana (SEARLE, 1984, p. 26) e “realizar atos ilocucio- nários é adotar um comportamento governado por regras [...]”, o que ele pretende provar explicando a noção de um ato e locucionário estabelecendo um con- junto de condições suficientes e necessárias para realizar um tipo particular de ato ilocucionário, e extrair dele um conjunto de regras semânticas (ou o mecanismo sintático) para o uso da expressão que marca que a proposição é um ato ilocucionário daquele tipo. (SEARLE, 1964, p. 254) a noção de um ato e locucionário estabelecendo um con- junto de condições suficientes e necessárias para realizar um tipo particular de ato ilocucionário, e extrair dele um conjunto de regras semânticas (ou o mecanismo sintático) para o uso da expressão que marca que a proposição é um ato ilocucionário daquele tipo. (SEARLE, 1964, p. 254) Este trabalho, portanto, pretende caracterizar a mentira den- tro deste conjunto de características, extraindo suas condições sufi- cientes e necessárias para entendê-la como um fenômeno linguís- tico autônomo, pensando em suas condições de realização, em quais são suas regras definidoras e nos atos locucionário, ilocucionário 538 e perlocucionário envolvidos na sua realização. Entendemos que a mentira tenha semelhanças com outros atos de fala, da mesma forma que os demais atos listados por Searle (1969)71 também se inter-relacionam, o que não significa que ela seja uma excrescência de outro ato de fala. INTRODUÇÃO Assim, também podemos ver que a tipologia proposta por este autor não necessariamente se encerra nos atos por ele descritos, mas que continua válida e produtiva para descre- ver outros atos que podem ser encontrados e caracterizados a partir da relação com os primeiros. Para tanto, utilizaremos a terminologia clássica cunhada por Austin (1962a), reformulada por Searle (1969 [1984]), que entende que um ato de fala está dividido em três partes: (i) um ato locutório, que é o material linguístico efetivamente proferido em um enunciado e possui sentido e referência; (ii) um ato ilocutório, que é ação reali- zada pelo falante ao proferir o enunciado (por exemplo: ordem, pro- messa, conselho, pergunta, pedido de desculpa, aposta,...); (iii) um ato perlocutório, (acrescentado por Searle), que é o efeito causado pelo enunciado, tendo sido pretendido ou não (por exemplo: ao fazer uma aposta, posso deixar meu interlocutor animado, ou intimidado, ou irritado, ou sensibilizado, e isso pode acontecer por força do ato ilocutório ter conseguido seu objetivo ou à revelia do que o falante pretendia no início, porque os efeitos são impossíveis de controlar). Dessa forma, iniciaremos caracterizando a mentira, seguire- mos abordando aspectos relevantes para distinguir a mentira dentro da teoria e, por fim, apresentaremos a nossa própria proposta de análise dentro da taxonomia proposta por Searle (1969). Citado aqui a partir da tradução portuguesa, publicada em 1984. 71 71 539 S U M Á R I O A MENTIRA COMO ATO DE FALA Em um trabalho intitulado “The Description of Lies in Speech Acts Theory”, Anne Reboul (1994) faz um percurso que também que- remos fazer aqui, trazendo a ideia de que a grande questão da men- tira reside na distinção entre o ato locionário e ato perlocucionário. Para ela, é no ato perlocucionário, que é o efeito ao agir no mundo, onde reside a questão da mentira porque, se o efeito da mentira for obtido, o ato de fala assertivo falhou. Para esta autora, portanto, a mentira é um efeito resultante de um desvio em um ato assertivo, em que seu perlocucionário (efeito) resulta diferente, pois, ao invés de informar algo verdadeiro, informa algo falso e, assim, engana. Assim como Searle (1969), Reboul (1994) levanta a hipótese de que um ato de fala mal-sucedido seja entendido como um ato falso. Neste sentido, um falante que realizasse um ato de fala sem a intenção de efetivar o que tenha sido verbalmente acertado por meio daquele proferimento poderia estar mentindo. Seria o caso, por exem- plo, de uma promessa ou de uma aposta feitas sem a intenção de serem cumpridas. Neste sentido, ela apresenta uma distinção entre o ato feito e o compromisso do falante em realizar o ato, mais ou menos nos termos das condições de felicidade de Austin (1962a [1990]), que está também presente no texto de Searle (1969 [1984], p. 74): Em alguns casos, uma condição pode ser de fato intrín- seca à noção do ato em questão e não satisfeita em um dado caso, e ainda assim, o ato terá sido realizado. Em tais casos, dizemos que o ato foi “defeituoso”. A nossa noção de defeito no ato ilocucional está estritamente relacio- nada com a noção de “insucesso” de Austin. Estas noções são formuladas, por Austin, da seguinte forma: (A.1) Deve existir um procedimento convencionalmente aceito, que apresente um determinado efeito convencio- nal e que inclua o proferimento de certas palavras, por cer- tas pessoas, e em certas circunstâncias; e além disso, que 540 (A.2) as pessoas e circunstâncias particulares, em cada caso, devem ser adequadas ao procedimento específico invocado. (B.1) O procedimento tem de ser executado, por todos os participantes, de modo correto e (B.2) completo. (B.2) completo. (Γ.1) Nos casos em que, como ocorre com frequência, o procedimento visa às pessoas com seus pensamentos e sentimentos, ou visa à instauração de uma conduta cor- respondente por parte de alguns dos participantes, então aquele que participa do procedimento, e o invoca deve de fato ter tais pensamentos ou sentimentos, e os participan- tes devem ter a intenção de se conduzirem de maneira adequada, e, além disso, (Γ.2) devem realmente conduzir-se dessa maneira subse- quente (AUSTIN, 1990, p. 31). (Γ.2) devem realmente conduzir-se dessa maneira subse- quente (AUSTIN, 1990, p. 31). O que estamos tentando dizer aqui é que, nem para Aus- tin, nem para Searle, um ato de fala defeituoso é entendido como mentiroso, uma vez que qualquer problema na sua realização com- promete o ato, bem como as condutas sociais subsequentes a ele, mas não são mentiras expressamente realizadas (e nem serão assim julgadas ou entendidas pelos interlocutores) porque são atos reali- zados dentro das suas próprias regras, com defeitos que se referem às próprias regras, e é assim que serão interpretados. Além disso, estes defeitos, ainda que possam se dar pela falsa intenção do falante em se comprometer, também poderiam acontecer por razões externas a ele: quer porque a ação subsequente não possa ter sido realizada por condições quaisquer (as circunstâncias), quer porque o interlocutor não tenha vindo cobrar o que foi proferido (os partici- pantes), por exemplo. Como Searle (1969) ilustra com a análise da estrutura do ato de “prometer”, é necessário que haja um enunciado no qual a promessa esteja explícita verbalmente (“eu prometo”, “eu me comprometo a” etc), 541 a intenção do falante em se comprometer a realizar algo no futuro, uma situação contextual na qual não fosse óbvio para o interlocu- tor que o falante fosse realizar este ato sem um ato verbal que o colocasse diante deste compromisso e demais fatores relacionados. O próprio autor, porém, considera que promessas insinceras conti- nuam sendo promessas: S U M Á R I O [...] ao fazer uma promessa insincera, o falante não tem todas as intenções que teria se fizesse uma promessa sin- cera; em particular, falta-lhe a intenção de desempenhar o ato prometido. Entretanto, ele dá a entender que tem essa intenção. De fato, é justamente porque ele dá a entender que tem intenções que não tem, que descrevemos o seu ato como insincero. (B.2) completo. [...] Portanto, para explicar promessas insinceras, precisamos apenas rever as nossas condições para afirmar que o falante assume a responsabilidade por ter a intenção, em vez de afirmar que ele realmente a tem (SEARLE, 1984, p. 82). Ou seja, no entendimento de Searle (1969 [1984]), basta que o falante “assuma a responsabilidade” pela sinceridade do ato, ou seja, não há necessidade de que se prove verdadeiro ou falso o seu comprometimento em executar o que é prometido para que a ação de prometer seja desempenhada e para que seja assim entendido pelo seu interlocutor. Além disso, nem haveria, pela própria defini- ção do que sejam atos de fala, como se certificar da veracidade da proposição. A veracidade, a sinceridade e o compromisso com a rea- lização do ato e das condições subsequentes são assumidos pelo locutor, e tacitamente assumidos e esperados pelo interlocutor. A reflexão que pode se seguir daqui é que a sanção que o locutor não comprometido com a realização do que foi posto pelo seu ato de fala terá não é feita em relação ao ato de fala em si, que é realizado, mas em relação à sua atitude concreta que deixou de ser realizada no mundo. É por esta ação, e não pelo seu ato de fala, que ele será interpelado. Em outras palavras, o ato de fala continua valendo e é só por isso que o interlocutor pode cobrar do locutor 542 S U M Á R I O que ele tenha a atitude que foi enunciada pelo ato de fala. A conse- quência do não cumprimento do acordo é social: este falante pode passar a ser visto como alguém que não cumpre suas promessas, alguém não confiável, (o que, não necessariamente, significa que seja entendido como um mentiroso). Voltando ao raciocínio da autora, Reboul (1994) argumenta que a intenção do mentiroso é a de que “seu interlocutor acredite que ele (o falante) acredita em P, quando, de fato, ele não acredita.” (REBOUL, 1994, p. 294, tradução nossa) e, ainda, que “o falante acre- dita que p é falso e tem a intenção de comunicar o que ele pensa que é falso e tem uma intenção de persuadir o ouvinte de que ele (o falante) acredita que p é verdadeiro” (REBOUL, 1994, p. 293). (B.2) completo. Ela assim o faz assumindo as condições encontradas por Searle (1969 [1984]) para o ato assertivo: Para as asserções, as condições preparatórias incluem o fato de que o ouvinte deve ter alguma base para supor que a proposição asseverada é verdadeira, a condição de sinceridade consiste em ele acreditar que ela é verdadeira e a condição essencial tem a ver com o fato de a propo- sição ser apresentada como representando um estado de coisas real (SEARLE, 1984, p. 86). Para as asserções, as condições preparatórias incluem o fato de que o ouvinte deve ter alguma base para supor que a proposição asseverada é verdadeira, a condição de sinceridade consiste em ele acreditar que ela é verdadeira e a condição essencial tem a ver com o fato de a propo- sição ser apresentada como representando um estado de coisas real (SEARLE, 1984, p. 86). Desta forma, a autora elabora uma primeira definição para a mentira: “uma mentira é uma proposição correspondente à rea- lização de um ato ilocucionário assertivo em que: a) a condição de sinceridade não foi cumprida; b) o falante tem a intenção de que seu interlocutor acredite que o falante respeitou a condição de sinceri- dade” (REBOUL, 1994, p. 293). Contudo, no nosso entendimento, ainda que a crença na veracidade da proposição seja suficiente para a realização de um ato assertivo (ou seja, o falante que faz a asserção não precisa ter verificado a verdade da proposição e ter certeza de que se trata de uma proposição verdadeira para enunciá-la, mas o faz assumindo 543 a responsabilidade sobre a informação veiculada), para a mentira, não nos parece ser uma questão de crença na veracidade da pro- posição. Antes, nos parece que a mentira projeta uma realidade R na qual a proposição p é verdadeira e assume que essa realidade é a mesma realidade material M na qual elas estão. O mentiroso tem que saber que a proposição é falsa, então ele não “acredita” que o que está falando é verdade. Se ele acredita que é verdade, então ele não está dizendo uma mentira, e sim uma falsidade. (Este tipo de dis- tinção será discutida posteriormente.) Em termos formulaicos, então, F(P) → R = M ou seja, se o falante profere p, então R é igual a M. (B.2) completo. Além disso, a autora entende que os atos de fala, de maneira geral, são definidos por duas características principais: serem inten- cionais e serem convencionais (como vimos com Austin, e, poste- riormente, com Searle): São intencionais no sentido em que o falante que está realizando um ato ilocucionário deve ter, pelo menos, a intenção de realizá-lo. São convencionais no sentido que o reconhecimento pelo ouvinte da intenção do falante emerge da semântica da sentença. Além disso, o reco- nhecimento da intenção do ouvinte é vital para o sucesso do ato ilocucionário (SEARLE, 1994, p. 293). Para que mentir seja um ato bem sucedido, portanto, diferen- temente de um ato assertivo ou de uma promessa, o “que é crucial para satisfazer esta intenção, e para o sucesso da mentira, é que o ouvinte não deve estar ciente da sua intenção.” (REBOUL, 1994, p. 294) Não muito diferente do que propõe Reboul, Silva-Joaquim (1991), citando Falkenberg (1982), apresenta cinco aspectos defi- nidores da mentira elencados por este autor. Também para Falke- nberg (1982), um falante intencionalmente profere, a alguém, um ato verbal destinado a enganar. Contudo, nada é dito sobre o papel deste interlocutor aqui: 544 a) a mentira é pessoal: isto é, há sempre um autor para cada mentira. É esta a característica que distingue a men- tira do boato; b) A mentira é intencional: depende exclu- sivamente da vontade do autor. Ninguém mente sem querer; pode enganar-se, não saber ou confundir, mas isso não é mentir. Há mentiras bem intencionadas e mal intencionadas, mas são sempre um resultado da vontade ou, melhor, da intenção; c) A mentira é verbal: consiste essencialmente na enunciação de expressões linguísti- cas. Não há mentira se não houver exercício verbal, oral ou escrito, e há muitas maneiras de conduzir ao engano que não são propriamente mentiras. A mentira realiza-se na enunciação linguística e a sua manifestação máxima de afastamento em relação à situação canônica ocorre na enunciação corporal-linguística, não verbalizada, mas em que o gesto substitui principalmente enunciados do tipo “sim”/”não”; d) A mentira é temporal: pode ser sempre datada; e) A mentira é social: dirige-se sempre a outrem (SILVA-JOAQUIM, 1992, p. 121-122). A discussão principal que propomos com relação a Reboul (1994), no entanto, diz respeito ao seu entendimento de que a men- tira não pode ser caracterizada como um ato de fala pela impossibi- lidade de defini-la nos termos da teoria. (B.2) completo. Segundo ela, tanto atos ilocucionários em geral e mentiras são inten- cionais; assim, pelo menos neste tópico, não existe razão para que as mentiras não constituam um tipo particular de ato ilocucionário. O problema surge com o outro lado do ato ilocucionário, que é a convencionalidade: ainda que seja essencial para o sucesso do ato ilocucionário que a intenção na sua base seja reconhecida e, assim ele mesmo seja reconhecido como tal, por essa razão, a intenção é convencionalmente representada na própria proposição, é tão essencial para o sucesso da mentira que a intenção que a motiva não seja reconhecida e, é claro, não seja representada, convencionalmente ou de qualquer outra forma, no proferimento. Assim, ainda que os atos ilocucionários e as mentiras sejam intencionais, as mentiras não são atos ilocucionários porque não são convencionais. (REBOUL, 1994, p. 294, grifos nossos.) tanto atos ilocucionários em geral e mentiras são inten- cionais; assim, pelo menos neste tópico, não existe razão para que as mentiras não constituam um tipo particular de ato ilocucionário. O problema surge com o outro lado do ato ilocucionário, que é a convencionalidade: ainda que seja essencial para o sucesso do ato ilocucionário que a intenção na sua base seja reconhecida e, assim ele mesmo seja reconhecido como tal, por essa razão, a intenção é convencionalmente representada na própria proposição, é tão essencial para o sucesso da mentira que a intenção que a motiva não seja reconhecida e, é claro, não seja representada, convencionalmente ou de qualquer outra forma, no proferimento. Assim, ainda que os atos ilocucionários e as mentiras sejam intencionais, as mentiras não são atos ilocucionários porque não são convencionais. (REBOUL, 1994, p. 294, grifos nossos.) 545 Ou seja, para Reboul (1994), a mentira não possuiria conven- cionalidade na medida em que não pode ser percebida pelo interlo- cutor como uma mentira, porque assim, enquanto ato de fala, fracas- saria. No entanto, aplicando a mesma lógica que Searle (1969 [1984]) aplica às promessas insinceras, é possível que entendamos a men- tira como um ato em si (ou seja, o ato de mentir) cujo efeito pode ou não ser atingido, a depender de as condições de sucesso terem sido obtidas. Em outras palavras, uma mentira bem sucedida, que engana o interlocutor, não é percebida como uma mentira. (B.2) completo. O que escapa a Reboul (1994) é que uma mentira mal sucedida é percebida como tal, ainda que seu objetivo de enganar não seja alcançado. Qualquer interlocutor que seja capaz de perceber que foi enganado (ou per- ceber, ao menos, a tentativa de engano) elucida, imediatamente, as condições de realização do ato mentiroso. É por esta razão, então, que entendemos que podemos descrever a mentira enquanto um ato de fala que tem uma estrutura diversa da estrutura da asserção, como faremos a seguir. A ESTRUTURA LOCUCIONÁRIA DA MENTIRA Neste sentido, podemos afirmar que a mentira tem uma estru- tura de ato de fala semelhante a de um ato assertivo, com algumas diferenças cruciais que o levam a ser um ato mentiroso. Alguns des- tes critérios, como discutimos até aqui, residem na intenção de enga- nar do falante e no fato de existir um conteúdo proposicional falso. Assim, o enunciador que profere uma mentira e tem a intenção de que ela seja bem sucedida precisa, em primeiro lugar, escamotear a informação mentirosa em um ato de fala assertivo. Isso parece ponto pacífico entre os autores que problematizam a mentira, como vimos. 546 Em termos austinianos, o ato de fala da mentira pode ser entendido como qualquer enunciado falso dito com a intenção de enganar, o que necessariamente engendra o ato locucionário (o con- teúdo proferido, ou seja, uma proposição não verdadeira), o ato ilo- cucionário (ou seja, a intenção de enganar) e o perlocucionário (o efeito de informar de maneira enganosa sem que o interlocutor per- ceba o engano) em um jogo praticamente simultâneo. Porém, para entender melhor como esses atos se articulam, é preciso entender separadamente cada peça. S U M Á R I O Dados retirados do corpus sick.br Esta escolha se deu em razão da anotação de acarretamento feita pelos autores em uma extensão grande de dados. SOBRE A FALSIDADE DO CONTEÚDO PROPOSICIONAL A falsidade semântica de uma proposição certamente é o ponto de partida para a avaliação de qualquer mentira, tanto do ponto de vista do locutor quanto do ponto de vista do interlocutor (que venha a descobrir que o enunciado era mentiroso). A verifi- cação do valor de verdade, contudo, pode ser feita em relação ao mundo real (sobre o que falarei a seguir), aquele mesmo no qual a proposição é enunciada, ou ainda sobre qualquer mundo M sobre o qual a proposição se refere. É possível que se afirme algo falso, na tentativa de enganar um ouvinte sobre o enredo de algum filme ou livro, por exemplo (ainda que essa seja uma mentira facilmente des- mentível, mas isto não vem ao caso neste momento). Reboul (1994) coloca isso da seguinte forma: Se um enunciado ficcional é um enunciado que aparece na ficção e está falando sobre um objeto não existente, então, em um enunciado ficcional, assim como na men- tira, o falante que diz p não acredita que p é verdadeiro: ele acredita que p é falso. Ainda, na ficção, ele pretende comunicar p mas não persuadir seu interlocutor de que ele (o falante) acredita que p é verdadeiro. Em outras 547 palavras, o interlocutor não tem que acreditar que o falante acredita que p é verdadeiro para que o ato seja bem sucedido. Em uma mentira, por outro lado, o falante não acredita que p é verdadeiro, mas pretende comunicar p que pretende que seu interlocutor acredite que ele (o falante) acredita que p. (REBOUL, 1994, p. 294). Por outro lado, ser uma sentença com um valor de verdade falso para qualquer dos mundos considerados para a proposição não é uma condição suficiente para que a proposição, uma vez enunciada, se torne uma mentira. As verificações de valor de verdade possíveis de serem feitas, quando a sentença é tomada sem estar na situação de proferimento, são menos sensíveis a alguns fatores, como hipo- nímias, hiperonínimas, implicaturas escalares e outros fenômenos do tipo, que resultam em acarretamentos verdadeiros, por exemplo. Poderiam ser consideradas omissões, caso fossem intencionais, ou imprecisões, caso não intencionais. As situações abaixo pretendem exemplificar o que quero dizer: (01)  (a) Um menino está segurando uma pistola de água. (mais informação)72 (b) Uma criança está segurando uma pistola de água. SOBRE A FALSIDADE DO CONTEÚDO PROPOSICIONAL (02)  (a) A matéria de jornal foi publicada na China (menos informação) (b) A matéria de jornal foi publicada na Ásia. (03)   (a) João ganha 5 mil reais por mês (quando na ver- dade seu salário total é 10 mil). (menos informação, via implicatura escalar) (03) 72 548 Este raciocínio vem, é claro, tanto do cálculo de acarreta- mento, que é semântico, quanto do cálculo de implicaturas grice- ano (feito pela violação da máxima de quantidade), mas o ponto em questão aqui é que a informação veiculada não é falsa nem em ter- mos semânticos nem em termos pragmáticos; o que não impede que a intenção do falante ao proferir (b) ao invés de (a) em (01) e (02) (ou de proferir (a) em (03) optando por omitir a informação completa) possa ser a de enganar seu interlocutor. Sobre a intenção de enga- nar, porém, falaremos no tópico a seguir. A partir daqui, começamos a entender que existe, na enun- ciação de uma proposição qualquer, a presunção da intencionali- dade do falante na escolha lexical ou, até mesmo, do recorte infor- macional sobre a cena que ele pretende criar ao enunciar. Com isso, o conteúdo do ato locucionário passa a ser avaliado (tanto pelo locu- tor quanto pelo interlocutor) como sendo “verdadeiro” ou “falso” na medida em que seja mais ou menos adequado para se referir ao estado de coisas que o falante pretende descrever. Em outras pala- vras, é o conteúdo linguístico proferido que será avaliado como “ade- quado” ou “inadequado” para descrever o estado de coisas que fará, na troca verbal, a avaliação de “falsidade” ou “veracidade” do conte- údo enunciado. A comprovação do valor de verdade, portanto, vem do confronto com o mundo real (ou com o mundo referido pela fala) feito pelo interlocutor. É neste sentido que, adiante, afirmaremos que uma mentira pode ser instantaneamente descoberta pelo interlocu- tor independente da intenção do locutor. Outro caso completamente diferente é o do falante que pro- fere uma informação falsa sem o conhecimento de que esta informa- ção seja falsa. Neste caso, ele não poderia ter a intenção de enganar porque, entendendo que a informação é verdadeira, seu ato de fala é outro: ele realiza um ato de fala informativo/assertivo. 73 Esta é uma discussão particularmente relevante para o momento em que vivemos quando se pensa, por exemplo, na disseminação de fake news. Contudo, por razões de espaço, este não é um assunto que poderemos abordar longamente aqui. Sugiro, para tanto, o trabalho de TANDOC JR., E.; LIM, Z.W., LING, R. 2017. Defining “Fake News”: A Typology of Scholarly Definitions. SOBRE A FALSIDADE DO CONTEÚDO PROPOSICIONAL Evidente- mente, caso o interlocutor seja capaz de identificar que a informa- ção é falsa, ele poderá alertar o falante sobre a falsidade da propo- sição enunciada, mas não poderá cobrar o locutor por ter proferido 549 voluntariamente uma mentira. Contudo, esta é uma discussão que pode, por razões éticas, se estender além disso, justamente pelo pacto estabelecido pelo ato assertivo que está sendo realizado: ao assumir a veracidade da informação, é possível que se cobre do falante a responsabilidade sobre a enunciação do conteúdo falso como se fosse verdadeiro, ou seja, existe um ato social sendo reali- zado com o qual o falante está se comprometendo e, ao proferir algo falso, assumindo que é verdadeiro, este falante passa a ter alguma responsabilidade pela disseminação deste conteúdo falso73. 74 Fala de Jair Bolsonaro no debate promovido pela Rede Globo no dia 28/10/2022. Transcrição dis- ponível em <https://bit.ly/3XNSKZx>. 75 Manchete publicada no site de fofocas “TV foco”, na seção “Deu o que falar”. Disponível em <http:// bit.ly/3kpkCo9>. SOBRE A INTENÇÃO DE ENGANAR Como vimos, é difícil separar a intenção de enganar do con- teúdo proferido, como o último parágrafo do item acima demonstrou. Por isso, é necessário dizer que o enunciador precisa ter ciência da falsidade do enunciado e ter a intenção de transmitir este enunciado falso com a intenção de enganar o interlocutor para que possamos considerar que estamos diante de uma mentira. Caso não seja dessa forma, muitas outras coisas podem acontecer. Se o enunciador sou- ber da falsidade da proposição, mas a compartilhar sem a intenção de enganar, é possível que esteja, por exemplo, transmitindo um boato com a intenção de informar que existe o boato; ou, ainda, que explicite a intenção de criar um cenário falso, por exemplo, ficcional. Com a intenção de enganar, porém, um falante pode inclu- sive se utilizar de um enunciado verdadeiro, como vimos acima, para não contar toda a verdade a um interlocutor. Isso pode se dar de várias formas: não dando toda a informação por omissão 73 550 de partes dela; fazendo escolhas lexicais que induzem ao erro, enun- ciando sentenças que geram implicaturas não condizentes com a realidade e assim por diante. Abaixo, temos alguns exemplos disso: S U M Á R I O (04)  “E dizer também que segundo o Ipea, extrema pobreza é quem ganha US$ 1,9 por dia. Ou seja, R$ 10 por dia. O Auxílio Brasil paga R$ 20.”74 (04)  “E dizer também que segundo o Ipea, extrema pobreza é quem ganha US$ 1,9 por dia. Ou seja, R$ 10 por dia. O Auxílio Brasil paga R$ 20.”74 Este é um enunciado verdadeiro utilizado para enganar. O Auxílio Brasil era, no momento em que a sentença foi proferida, R$ 600 por mês, ou seja, 20 reais por dia, em média. Acontece que este dado se refere a um cálculo por pessoa, não por família. Em uma família composta por 5 pessoas, por exemplo, o valor de R$ 600 deve ser dividido por todos os membros da família, totalizando R$ 4 /dia/ pessoa (muito abaixo US$ 1,9, que, na data, chegava próximo de R$ 10). A informação aqui não é totalmente veiculada, dando o interlo- cutor a entender que cada pessoa cadastrada no Auxílio Brasil virá a receber R$ 20 por dia, o que não é o caso. SOBRE A INTENÇÃO DE ENGANAR (05)  Leonardo foi sincero sobre o que pensa da sogra, mãe de Poliana Rocha: “juro por Deus, viu?”75 (05)  Leonardo foi sincero sobre o que pensa da sogra, mãe de Poliana Rocha: “juro por Deus, viu?”75 Este é mais um enunciado que tem uma intenção que pode, talvez, não ser interpretada como a de enganar, mas certamente levará o interlocutor a um cálculo de implicatura diferente do que a matéria, posteriormente, informará. O título da matéria (já conhecido como “caça-clique” justamente pelo teor apelativo) induz, pela utili- zação de termos como “foi sincero”, “o que pensa da sogra”, “juro por Deus”, a um raciocínio, utilizado pelo senso comum, de que pessoas pensam coisas ruins da sogra, que se ele foi sincero (e jura por Deus) deve ter falado coisas ruins, por exemplo. A matéria, por outro lado, 551 apenas informa que o cantor Leonardo gosta muito da sogra e que se dão muito bem. Novamente, não é mentira, mas é uma informação verdadeira utilizada com uma intenção não transparente. Por fim, com a intenção explícita de enganar, resta ao locutor, então, emular um ato de fala afirmativo (declarativo), como temos visto até aqui. Ou, em outros termos, performar um ato de fala que leve o interlocutor a entender que se trata de um ato de fala decla- rativo. Assim, as regras de um ato de fala mentiroso são, em alguns aspectos, semelhantes às regras de um ato assertivo (declarativo). Cabe, então, retomar as regras deste ato de fala: S U M Á R I O afirmar, declarar Regra de conteúdo proposicional: Qualquer proposição p Regra de conteúdo proposicional: Qualquer proposição p Regras preparatórias: Regra de conteúdo proposicional: Qualquer proposição p Regras preparatórias: Regras preparatórias: 1. F tem evidências (razões, etc) para a verdade de p. 2. Não é óbvio nem para F nem para O que O saiba (não precise ser lembrado de, etc) p. Regra de sinceridade: F acredita em p Regra de sinceridade: F acredita em p Regra de sinceridade: F acredita em p Regra essencial: Equivale a afirmar que p repre- senta uma situação real. Comentário: Contrariamente a provar, estes não parecem estar essencialmente ligados à tentativa de convencer. Assim “eu estou simplesmente a declarar que p e não a tentar convencê-lo” é aceitável, mas “Eu estou a provar que p e não a tentar convencê-lo” parece contraditório (SEARLE, 1984, p. 88). Por outro lado, como podemos ver, o ato de fala declarativo tem a intenção de falar sobre uma situação real e, portanto, é com base neste tipo de compromisso assumido entre locutor e interlocu- tor que um ato mentiroso irá se basear. A ação do falante, aqui, pre- cisa ser a de fazer o ouvinte acreditar que se trata de um ato asser- tivo, ainda que as regras colocadas em jogo não sejam as mesmas. 552 Para Reboul (1994), neste ponto, reside o paradoxo sobre a mentira: “Se um falante produz um enunciado que é uma mentira, é necessário para o sucesso da mentira que o ato ilocucionário asser- tivo seja bem sucedido. Mas se o ato perlocucionário da mentira é bem sucedido, então o ato ilocucionário da asserção não é bem sucedido.” (REBOUL, 1994, p. 295) Em outros termos, o que a autora diz é que o ato assertivo precisa passar como sendo verdadeiro, mas não é. E se não for, seu efeito não será o efeito do ato assertivo. Con- tudo, entendemos que o problema é um pouco mais complicado do que isso, como veremos a seguir. SOBRE O QUE É ENGANAR Se o ato da mentira for bem sucedido, então, entendemos que é porque ele conseguiu surtir o efeito de enganar o interlocutor, mas ainda sobre isso é necessário discutir algumas questões. Parece que, no caso de um ato mentiroso bem sucedido, “enganar” aqui se trata de passar a ilusão de que o ato proferido era um ato asser- tivo e que, portanto, a informação veiculada era verdadeira e que o falante estava comprometido com a sua veracidade. Isto não é tudo em “enganar”, porém. Se, no minuto seguinte após a enunciação, o falante proferir “ah, te enganei”, o ato assertivo será cancelado, a afir- mação sobre o estado de coisas passará a ser interpretada com o valor de verdade invertido e a mentira emerge, revelada como ato de fala, que faz o interlocutor reinterpretar, retroativamente, o ato pro- ferido anteriormente. Além disso, existe um papel muito importante que não é levado em conta nem por Reboul (1994) na descrição da mentira, nem por Searle (1969) na do ato assertivo. Estes autores minoram a participação do interlocutor no ato de fala, não colocando em cena sua atuação para que o ato tenha o efeito pretendido pelo falante, 553 S U M Á R I O uma vez que este não tem o controle completo do que está reali- zando. Dito de outra forma, um locutor pode pretender enganar, mas não será possível efetivar a sua intenção se o interlocutor não “esti- ver apto” a ser enganado. Caso o interlocutor tenha a informação de que o conteúdo proposicional veiculado é falso, como dissemos anteriormente, inde- pendente da intenção de enganar do falante, o ato perlocucionário (ou seja, o engano) não será efetivado. Se o interlocutor tiver qual- quer razão para desconfiar do falante, o ato de fala ficará em sus- penso, sem que o interlocutor seja capaz de julgar se a informação veiculada pelo ato de fala é verdadeira ou falsa e, assim, sem que seja capaz de classificar se o ato proferido foi assertivo ou mentiroso. Se o interlocutor for capaz de perceber qualquer sinal na força ilocucioná- ria (hesitações, titubeações, contradições) que seja indicativo de que o ato não é assertivo, pode descobrir a mentira na hora. Se o interlo- cutor conseguir contrastar rapidamente a informação veiculada com o estado de coisas que esta informação pretende descrever, ele pode argumentar que o ato de fala foi mentiroso. SOBRE O QUE É ENGANAR Outra questão sobre o ato de enganar diz respeito ao com- portamento dos envolvidos na cena com relação ao que foi proferido. É por isso que a visão de Reboul (1994), que entende que o falante não acredita na veracidade da proposição que enuncia, ou que o faz sem compromisso com a sua veracidade, nos parece uma versão fraca do que acontece: o falante, ao proferir a mentira, se compromete com a criação de um cenário no qual aquele enunciado é verdadeiro, e precisa agir como se aquele cenário fosse verdadeiro para que o engano performado seja minimamente consistente. Caso contrário, a mentira pode ser facilmente descoberta e o efeito de “engano” não é realizado. É claro, podemos estar diante de um mentiroso convicto, que mente tentando angariar pessoas que acreditem em sua mentira mesmo sabendo que i) ele é considerado mentiroso por boa parte das pessoas do grupo e ii) que o que ele está dizendo é contrafac- tual (um efeito cognitivo que atualmente já é conhecido apesar de 554 ainda não completamente explicado pela psicologia e pela socio- logia), mas podemos estar diante, também, de alguém que queira fazer seu enunciado passar como sendo crível para que não sofra as consequências por qualquer atitude que tenha tido ou deixado de ter. Neste segundo caso, acreditamos que o efeito performativo do ato passa, inclusive, a ir para além do nível linguístico. O falante vai precisar, em determinados casos,  agir de forma a fazer ser crível a realidade R projetada pelo enunciado falso que proferiu. O exemplo abaixo pretende ilustrar o que queremos dizer: (06)  Catarina pediu à Simone uma mala emprestada, dizendo que iria fazer uma viagem. Quando a via- gem acabou, Simone solicitou à Catarina que a mala fosse devolvida. (C) A mala foi roubada dentro do hotel, durante a viagem. Simone ligou para o hotel, que informou que não havia tido nenhuma ocorrência de roubo nem nenhuma notificação a respeito disso no período em que Catarina esteve lá. Catarina parou de respon- der as mensagens de Simone e seguiu não tendo devolvido a mala.76 O que estamos colocando em questão aqui é o fato de que, uma vez que o enunciado proferido ao interlocutor diga que deter- minado objeto está perdido, este objeto não pode voltar às vistas de seu dono, sob pena de a mentira ser descoberta. 6 História adaptada do podcast Não inviabilize. Disponível em <http://bit.ly/41fVMaL>. SOBRE O QUE É ENGANAR Neste sentido, o trabalho do locutor mentiroso (e, agora, um possível ladrão) será o de criar uma realidade na qual o objeto tenha, de fato, desaparecido para o seu dono. O falante tanto está agindo no mundo após ter enunciado a fala que está, neste momento, para esta história, incor- rendo em consequências que excedem as linguísticas, tais como incorrer em processos judiciais. De qualquer forma, o perlocucioná- rio de “enganar” parece ter um escopo bastante amplo inclusive no que diz respeito ao tipo de atitude que pode vir a suceder a ele. 76 555 PROPOSTA DE REGRAS DE ATO ILOCUCIONAL DA MENTIRA Antes de apresentarmos a nossa proposta de descrição da mentira enquanto ato de fala, apresentaremos, brevemente, cada uma das condições utilizadas por Searle (1969 [1984]) para des- crever cada um dos atos de fala elencados por este autor. Assim, ele define cada um dos atos a partir de quatro condições, listadas e minimamente descritas a seguir: (i) condição de conteúdo proposicional: características rela- cionadas ao material linguístico efetivamente enunciado, para que o que foi proferido tenha sentido (ii) condições preparatórias: o que é necessário haver para que se possa realizar o ato de fala (iii) condição de sinceridade: o comprometimento assumido pelo falante com relação ao que está sendo veiculado. (iv) condição essencial: como o ato se realiza. A nossa proposta de enquadramento da mentira enquanto ato de fala é, portanto, a seguinte: A nossa proposta de enquadramento da mentira enquanto ato de fala é, portanto, a seguinte: (i) condição de conteúdo proposicional: Uma informação dis- sonante da verdade em alguma medida; (ii) condições preparatórias a) F sabe que P é falso; b) F sabe ou ao menos supõe que O não sabe que P é falso; c) F tem a intenção de enganar O ao proferir P. (iii) condição de sinceridade: (iii) condição de sinceridade: 556 a) F simula que está proferindo um ato declarativo e se com- promete com as regras deste ato; b) O assume que se trata de um ato declarativo e espera do falante a sinceridade tácita envolvida em atos deste tipo; c) Não há sinceridade. (iv) condição essencial: a) A informação dissonante da verdade; b) A intenção de F de enganar. c) O desconhece a veracidade da informação e a intenção de F. COMENTÁRIOS O falante projeta uma realidade R na qual a proposição P é verdadeira e, pela enunciação, projeta que essa realidade R é a mesma realidade material na qual elas estão. O mentiroso sabe que a proposição que enuncia é falsa, então ele não acredita que o que está falando é verdade. Se o falante acreditar que a proposição é verdade, então ele não está dizendo uma mentira, e sim uma falsidade. O falante passa a agir no mundo como se a informação veiculada por ele fosse verdade. O efeito causado no interlocutor é o de enganar, mas por meio do convencimento. Uma mentira bem sucedida é a que con- vence o interlocutor de que não é uma mentira, ou seja, seu efeito não é o de enganar, mas de informar. Percebendo o engano, o inter- locutor cancela a mentira imediatamente. O ouvinte passa a agir no mundo como se a informação veiculada fosse verdade. 557 Nossa insistência em enquadrar a mentira como um ato de fala separadamente de um ato informativo mal sucedido ou des- viante vem, portanto, dos argumentos que apresentamos: o entendi- mento, pelo interlocutor, de que o ato realizado pelo locutor não foi assertivo, mas mentiroso, apenas nos mostra como o ato de mentir tem a sua própria existência projetada. Como nos mostra Silva-Jo- aquim (1992, p. 122): A identificação da mentira não se faz simplesmente pela distinção entre o verdadeiro e o falso. Se na realidade uma coisa é falsa, não é obrigatoriamente mentira considerá-la verdadeira. E, por exemplo, dissimular é diferente de men- tir. A ignorância, a indecisão ou a indiferença, podem levar a posições não-verdadeiras, o que não deve ser confun- dido com mentiras. O contrário de “ser mentiroso” é “ser sincero”, e não “ser verdadeiro”, embora o uso linguístico cotidiano utilize muitas vezes as duas expressões como sinônimos, especialmente quando se trata de situações e de contatos em que o contraste entre elas não é pertinente. A identificação da mentira não se faz simplesmente pela distinção entre o verdadeiro e o falso. Se na realidade uma coisa é falsa, não é obrigatoriamente mentira considerá-la verdadeira. E, por exemplo, dissimular é diferente de men- tir. A ignorância, a indecisão ou a indiferença, podem levar a posições não-verdadeiras, o que não deve ser confun- dido com mentiras. COMENTÁRIOS O contrário de “ser mentiroso” é “ser sincero”, e não “ser verdadeiro”, embora o uso linguístico cotidiano utilize muitas vezes as duas expressões como sinônimos, especialmente quando se trata de situações e de contatos em que o contraste entre elas não é pertinente. Conforme nota Silva-Joaquim, “ser sincero” é o contrário de “ser mentiroso” e isso se deve ao fato de que ambas as coisas são atitudes do falante em relação ao conteúdo que será proferido. “Ser verdadeiro” ou “ser falso”, assim, pode se referir ao conteúdo da afirmação, e sobre a avaliação que o falante e o interlocutor podem ter sobre o estado de coisas e sobre como (ou quanto) a afirmação reflete este estado de coisas, mas não sobre a atitude do falante em relação ao interlocutor e sobre o efeito que ele pretende causar. Além disso, nos parece evidente que quando a mentira é percebida, anunciada, confrontada com a verdade de maneira que resulta muito óbvia etc, e o interlocutor entende que o ato enunciado ali foi uma mentira, ela deixa de ter seu efeito perlocucionário, ou seja, deixa (ou pelo menos, deveria deixar) de enganar, que era seu objetivo principal, mas não deixa de ser entendida como uma men- tira. Explicitamente, é nesse momento que passa a ser percebida como uma mentira pelo interlocutor, que sua configuração como ato mentiroso passa a ser revelada. 558 Há diversos outros pontos que podem e devem ser consi- derados em relação à mentira como fenômeno linguístico dentro do campo da pragmática, que deixaremos para trabalhos posterio- res, como, por exemplo, o que está em jogo com relação à face do mentiroso quando ele opta (ou precisa) proferir uma mentira, ou em que medida este falante está sendo (não) cooperativo. Este trabalho, porém, optou por mapear os traços de ato de fala e minimamente descrever que tipo de ação no mundo uma mentira evoca, uma vez que, para a contemporaneidade, este tipo de discussão é cada vez mais relevante pelo tipo de implicação prática e ética que evoca. REFERÊNCIAS ARISTÓTELES. Metafísica. Tradução: Lucas Angioni. Campinas: Edipro, 2007. ARISTÓTELES. Metafísica. Tradução: Lucas Angioni. Campinas: Edipro, 2007. AUSTIN, John Langshaw. Quando dizer é fazer. Porto Alegre: Artes Médicas, 1990. AUSTIN, John Langshaw. Quando dizer é fazer. Porto Alegre: Artes Médicas, 1990. LEGROSKI, Marina, GEREMIAS, Fernanda de Fátima; GODOY, Fernanda Ferreira. Revisitando um experimento sobre a mentira. Muitas Vozes, Ponta Grossa, v. 10, p. 1-15, 2022. REAL, Livy; RODRIGUES, V. et al. SICK-BR: a Portuguese corpus for inference. PROPOR2018 (International Conference on the Computational Processing of Portuguese), 26 September 2018. Canela, Brazil. Disponível em <http://bit.ly/3UlGQFP>. Acesso em: 12 out. 2022. REBOUL, Anne. The description of lies in speech act theory. In: PARRET, Herman (Org.). Pretending to communicate. v. 292. W. de Gruyter, 1994, p. 291-298. SEARLE, John Rogers. Os actos de fala: um ensaio de filosofia da linguagem. Coimbra. 1984. SEARLE, John Rogers. Os actos de fala: um ensaio de filosofia da linguagem. Coimbra. 1984 SEARLE, John Rogers. What is a speech act. In: STAINTON, Robert (Org.). Perspectives in the philosophy of language: a concise anthology. Toronto. 1964, p. 258-272. SILVA JOAQUIM, Cândida. Mentiras. Sociologia. Problemas e Práticas, n. 9, p. 121-126, 1991. TANDOC JR., Edson; LIM, Zheng Wei, LING, Richard. Defining “Fake News”: A Typology of Scholarly Definitions. Digital Journalism, London, 6(2):1-17, ago. 2017. 559 S U M Á R I O Héliton Diego Lau É graduado em Letras Inglês pela UNICENTRO (2013), especialista em Educação Especial com Ênfase em Libras pelo ISAM (2015), mestre em Linguagem, Identidade e Subjetividade pela UEPG (2016) e doutor em Letras pela UFPR (2021). Foi professor substituto da PUC/PR do curso de Letras Português/Inglês (2022). Atualmente é pesquisador do grupo interinstitucional de pesquisas “Estudos do texto e do discurso: entrelaçamentos teóricos e analíticos” (GPTD/UNI- CENTRO-UFPR/CNPq), integrante do Núcleo de Relações Étnico-Raciais, de Gênero e Sexualidade (NUREGS/UEPG) e do Núcleo de Estudos e Pesquisas sobre Diversidade Sexual (NUDISEX/UEM/CNPq). Tem experiência na área de Letras, com ênfase em Linguística, atuando principalmente nos seguintes temas: análise de discurso, estudos de gênero, estudos culturais, teoria queer, linguística queer e Libras. E-mail: [email protected] Lattes: http://lattes.cnpq.br/9091109424675179 Lattes: http://lattes.cnpq.br/9091109424675179 André Luiz Souza-Silva Professor de Língua Portuguesa com doutorado em andamento pela Universidade Federal da Paraíba (PROLING/ UFPB), sendo Bolsista do Programa de Excelência Acadêmica (PROEX/CAPES), Mestre em Linguística (UFPB) com especialização em Língua, Linguística e Literatura pelo Centro Universitário de Patos (UNIFIP) e em Ensino de Lín- guas e Literaturas pela Universidade Estadual da Paraíba (UEPB). Também tem graduação em Letras pela UEPB e é integrante do Grupo de Pesquisa em Contato Linguístico (UFPB/CNPq). Tem interesse na área da Teoria e Análise Linguística, com ênfase na Diversidade e Identidade Linguísticas, Tabus Linguísticos e Ensino de Língua(s), atuando com base na Sociolinguística e suas interfaces. E-mail: [email protected] Lattes: http://lattes.cnpq.br/0875968155387666 E-mail: [email protected] Lattes: http://lattes.cnpq.br/0875968155387666 Zuleica Aparecida Michalkiewicz Tem graduação em licenciatura em letras Português/Inglês pela Faculdade Estadual de Filosofia, Ciências e Letras de União da Vitória (1998). Especialização em Magistério da Educação Básica com concentração em Língua Portuguesa. Mestra em Linguagem, Identidade e Subjetividade pela Universidade Estadual de Ponta Grossa. Doutora em Letras pela Universidade Federal do Paraná. Atua como pesquisadora da Linguística Aplicada, principalmente nos seguintes temas: formação docente, ideologias linguísticas, políticas linguísticas, letramento digital. Desenvolveu o projeto “Olhares reflexivos acerca do letramento digital”, no qual coordenou o GETEC - grupos de estudos sobre tecnologia, 560 com graduandos dos cursos de Letras na UNESPAR. Foi professora da educação básica no setor público e privado e na Educação superior como professora de Linguística no departamento de Letras/Espanhol na UNESPAR- campus de União da Vitória. Fez parte da UAB como membro da equipe multidisciplinar na Universidade Federal de Santa Maria. Professora na Especialização da Faculdades Integradas Santa Cruz e FIC-PR. Tem experiência na produção de conteúdo e material didático para EAD, elaboração de questões para avaliação de larga escala, coordenação de projetos educacionais, trabalha com assessoria na Intensifique assessoria acadêmica. Atualmente é pós-doutoranda naCátedradeEducaçãoBásicaAlfredoBosinaUniversidadedeSãoPaulo-IEA-USP. S U M Á R I O E-mail: [email protected] Lattes: https://lattes.cnpq.br/6104287697533395 561 S U M Á R I O Caroline Soder Especialista em Libras (Língua Brasileira de Sinais); Educação Especial; Alfabetização e Letramento, graduada pela Universidade Estadual do Oeste do Paraná (UNIOESTE) em Letras Libras Bacharelado e Pedagogia, possui mais de 14 anos de experiência na área da Surdez. Trabalha em escolas municipais e estaduais como professora e Intérprete de Libras, ministra cursos de Libras em empresas e cooperativas e cursos particulares. E-mail: [email protected] L tt htt //l tt b /8016400614724731 Lattes: http://lattes.cnpq.br/8016400614724731 Lattes: http://lattes.cnpq.br/8016400614724731 Cristiane Malinoski Pianaro Angelo É Doutora em Letras pela Universidade Estadual de Maringá. Docente do Departamento de Letras e do Programa de Pós-Graduação em Letras da Universidade Estadual do Centro-Oeste - UNICENTRO. E-mail: [email protected] Lattes:https://lattes.cnpq.br/3063731236262817 É Doutora em Letras pela Universidade Estadual de Maringá. Docente do Departamento de Letras e do Programa de Pós-Graduação em Letras da Universidade Estadual do Centro-Oeste - UNICENTRO. Lattes: https://lattes.cnpq.br/3063731236262817 Caroline Kretzmann Graduada em Letras Português/Inglês (PUCPR, 2004). Especialista em Língua Portuguesa e Literatura Brasileira (PUCPR, 2006). Mestre em Educação (PUCPR, 2008). Tem experiência como docente de Língua Portuguesa e Produção de Textos no Ensino Fundamental e Médio. Atualmente é professora assistente na PUCPR e doutoranda do Programa de Pós-Graduação em Letras da UFPR. E-mail: [email protected] E-mail: [email protected] Lattes: http://lattes.cnpq.br/7628756114891520 Lattes: http://lattes.cnpq.br/7628756114891520 Emily Gonçalves de Medeiros Ferreira Doutoranda e mestra em Linguística pelo Programa de Pós-Graduação em Linguística (PROLING), da Universidade Federal da Paraíba (UFPB). Bolsista do Programa de Excelência Acadêmica (PROEX/CAPES). Integrante do grupo de pesquisa “Historiografia, Gramática e Ensino de Línguas” (HGEL). E-mail: [email protected] Lattes: http://lattes.cnpq.br/0468181162952625 Eliziane Manosso Streiechen É Doutora em Educação pela Universidade Estadual de Ponta Grossa/PR. Docente do Departamento de Letras e do Programa de Pós-Graduação em Educação da Universidade Estadual do Centro-Oeste - UNICENTRO. Possui alguns livros publicados, entre eles: “Língua Brasileira de Sinais - Libras”, pela Editora UNICENTRO de Guarapuava/PR, 2012; “Libras: aprender está em suas mãos”, pela Editora CRV de Curitiba/PR, 2013 (2ª. ed. 2017), “A aquisição da Libras em um contexto multilinguístico”, pela Editora UNICENTRO, 2017. “Pedro vai à escola: uma história de silenciamentos e superações”, Editora UNICENTRO, 2023. É fundadora da ONG: ‘Amigo Fiel Ivaí’, onde atua fervorosamente na defesa e proteção dos animais de rua. Eliziane é Mãe do Eric e a Pamela, com imenso orgulho! Lattes: http://lattes.cnpq.br/1062660746215044 É Graduada em Letras-Inglês pela Universidade Estadual do Centro-Oeste (UNICENTRO). E-mail: [email protected] Lattes: http://lattes.cnpq.br/5706023568538367 É Graduada em Letras-Inglês pela Universidade Estadual do Centro-Oeste (UNICENTRO). E-mail: [email protected] Lattes: http://lattes.cnpq.br/5706023568538367 É Graduada em Letras-Inglês pela Universidade Estadual do Centro-Oeste (UNICENTRO). E-mail: [email protected] Lattes: http://lattes.cnpq.br/5706023568538367 Danielle dos Santos Mendes Coppi Possui graduação em Letras - Língua Portuguesa (2011), especialização em Língua e Linguística (2014), especial- ização em Fundamentos da Educação: Práticas Pedagógicas Interdisciplinares (2014), mestrado em Letras - PROF- LETRAS - (2016), ambos pela Universidade Estadual da Paraíba (UEPB), doutoranda em Linguística pela Universidade Federal de Pernambuco (PPGL - UFPE). Atuou como docente nos cursos de Letras e Pedagogia na UEPB (2018-2019), assim como na Especialização em Ensino de Línguas e Literaturas na Educação Básica nessa mesma instituição (2019) e, como supervisora do Subprojeto: PIBID/Letras-Português da UEPB (2020-2022). Atualmente, é professora - Educação Básica 3 D II - do Governo do Estado da Paraíba. Atua, também, no curso de Especialização em Língua 562 S U M Á R I O Linguística e Literatura da Faculdade Integrada de Patos-FIP. Integra o Grupo de Pesquisa Estudos de Linguagens e Ensino (GELIE) da Universidade Estadual da Paraíba e o Grupo de Estudos Saberes e Práticas do Professor (GESPP) da Universidade Federal da Paraíba. Tem experiência na área de Letras, com ênfase em Língua Portuguesa, atuando, principalmente, nos seguintes temas: Ensino, Língua Portuguesa, Letramento. Denise Gabriel de Oliveira É Graduada em Letras-Inglês pela Universidade Estadual do Centro-Oeste (UNICENTRO). E-mail: [email protected] Lattes: http://lattes.cnpq.br/5706023568538367 Lattes: http://lattes.cnpq.br/1989289031368586 Lattes: http://lattes.cnpq.br/1989289031368586 Lattes: http://lattes.cnpq.br/1989289031368586 Jaqueline Ângelo dos Santos Denardin Doutoranda em Estudos da Linguagem no Programa de Pós-Graduacão em Estudos da Linguagem da Universidade Federal do Mato Grosso, mestre em Letras pela Universidade Estadual do Oeste do Paraná (Unioeste), graduada em Pedagogia, Ciências Sociais e Letras, especialista em Educação Especial: Deficiências Múltiplas, Educação do Campo, Docência no Ensino Superior e Neuropsicopedagogia Clínica e Institucional. Professora e Pedagoga do NEa- DUNI (Núcleo de Educação a distância da UNIOESTE) Unioeste – campus Cascavel/PR. Docente de AEE (Atendimento Educacional Especializado) no PEE (Programa Institucional de Ações Relativas às Pessoas com Necessidades Espe- ciais) campus Toledo. E-mail: [email protected] E-mail: [email protected] Lattes: http://lattes.cnpq.br/4355836698232493 Lattes: http://lattes.cnpq.br/4355836698232493 Gabriel Fernandes de Oliveira Especialista em Língua, Linguística e Literatura pelo Centro Universitário de Patos (UNIFIP). Graduado em Letras/ Português pela Universidade Estadual da Paraíba (UEPB); foi integrante bolsista do Subprojeto de Língua Portu- guesa do Programa de Residência Pedagógica (PRP/CAPES/CNPq/2018-2020) pela mesma instituição. Ainda, atuou 563 S U M Á R I O como mediador de Língua Portuguesa do Programa Novo Mais Educação (PNME/2017-2018) na E.E.E.F.M. Engª Márcia Guedes A. de Carvalho em Belém-PB. Possui interesse em pesquisas vinculadas à Linguística Aplicada (LA) com ênfase em: Formação e Trabalho Docente, Ensino de Língua Portuguesa, Letramentos, Leitura e Produção de Textos, Gêneros Textuais e Ensino. Atualmente, é professor de Língua Portuguesa dos anos finais na E.M.E.F. Anita de Melo Barbosa Lima em Belém-PB. E-mail: [email protected] Lattes: http://lattes.cnpq.br/8453269886689356 Lattes: http://lattes.cnpq.br/8453269886689356 Jamil Cabral Sierra Jamil Cabral Sierra é Licenciado em Letras pela Universidade Estadual do Oeste do Paraná - UNIOESTE (1999), Espe- cialista em Linguística Aplicada pela Universidade Estadual do Oeste do Paraná - UNIOESTE (2002), Mestre em Letras pela Universidade Estadual de Maringá - UEM (2004), Doutor em Educação pela Universidade Federal do Paraná - UFPR (2013), Pós-Doutor em Educação pela Universidade Federal do Rio Grande do Sul - UFRGS (2017). Foi bolsista de estágio doutoral no Instituto de Educação, da Universidade de Lisboa - UL (2012). Atualmente é professor asso- ciado no Departamento de Planejamento e Administração Escolar - DEPLAE e professor permanente no Programa de Pós-graduação em Educação - PPGE, do Setor de Educação, da Universidade Federal do Paraná - UFPR. É coordenador e pesquisador do GILDA - Grupo Interdisciplinar em Linguagem, Diferença e Subjetivação (UFPR/CNPq). E-mail: [email protected] José Walbérico da Silva WCosta Doutorando em Estudos de Linguagem (Posling/UFF), Mestre em Linguística (Proling/UFPB), especialista em Língua Portuguesa (FUNESO/UFPB) e licenciado em Língua Portuguesa pela Universidade Estadual Vale do Acaraú (UVA). Atualmente é professor substituto na Unidade Acadêmica de Letras da Universidade Federal de Campina Grande Doutorando em Estudos de Linguagem (Posling/UFF), Mestre em Linguística (Proling/UFPB), especialista em Língua Portuguesa (FUNESO/UFPB) e licenciado em Língua Portuguesa pela Universidade Estadual Vale do Acaraú (UVA). Atualmente é professor substituto na Unidade Acadêmica de Letras da Universidade Federal de Campina Grande - UFCG. Tem experiência no Ensino Superior, Técnico, Básico e EJA. Atuou como tutor do curso Lato Sensu CLELP/ UFPBVirtual (2015-2017). É membro do Grupo de Pesquisa Teorias Linguísticas de Base – (TLB/UFPB/CNPq), bem como doGrupodePesquisaConectivoseConexõesdeOrações-CCO/UFF/CNPq EstudaepesquisanaáreadaLinguística - UFCG. Tem experiência no Ensino Superior, Técnico, Básico e EJA. Atuou como tutor do curso Lato Sensu CLELP/ UFPBVirtual (2015-2017). É membro do Grupo de Pesquisa Teorias Linguísticas de Base – (TLB/UFPB/CNPq), bem como do Grupo de Pesquisa Conectivos e Conexões de Orações - CCO/UFF/CNPq. Estuda e pesquisa na área da Linguística Funcional Clássica (LFC), na Linguística Funcional Centrada no Uso (LFCU) e Gramática de Construções (GC). E-mail: [email protected] Lattes: http://lattes.cnpq.br/8854550133914938 José Cristovão Maia Lucena Marreiro Graduado em Letras – Língua Inglesa pela Universidade Estadual da Paraíba (UEPB), com especialização em Línguas Estrangeiras Modernas pelo Instituto Federal de Ciência e Tecnologia da Paraíba (IFPB), graduando em Pedagogia pela Universidade de Santo Amaro (UNISA) e Pós-graduando em Gestão de Ensino a Distância, pelo Instituto Federal 564 S U M Á R I O de Ciência e Tecnologia de Rondônia (IFRO). Atualmente atua em escola pública e privada nas séries do ensino funda- mental – anos finais, 6º ao 9º ano –, nas cidades de Pirpirituba – PB e Belém – PB; é também Tutor Externo do curso de graduação em Letras – Inglês EAD na faculdade Uniasselvi. E-mail: [email protected] Lattes: http://lattes.cnpq.br/7339903480478766 de Ciência e Tecnologia de Rondônia (IFRO). Atualmente atua em escola pública e privada nas séries do ensino funda- mental – anos finais, 6º ao 9º ano –, nas cidades de Pirpirituba – PB e Belém – PB; é também Tutor Externo do curso de graduação em Letras – Inglês EAD na faculdade Uniasselvi. E-mail: [email protected] Lattes: http://lattes.cnpq.br/7339903480478766 E-mail: [email protected] Lattes: http://lattes.cnpq.br/7339903480478766 Keila Gentil Neves de Lima Graduada em Pedagogia e Letras/Libras – Bacharelado. Pós-graduada em Libras – Educação Especial com Ênfase no Intelectual – Educação Profissional e  Língua Inglesa nas séries iniciais. Atua na rede municipal de ensino como intérprete de libras desde 2018 até o presente ano – Docente AEE (Atendimento Educacional Especializado) Unioeste – Universidade do Oeste do Paraná na rede estadual desde 2022 até a presente data. Atuou por 4 anos como intérprete de Libras Unioeste. E-mail: [email protected] Lattes: http://lattes.cnpq.br/1182440998110488 Lattes: http://lattes.cnpq.br/1182440998110488 Juliana Ferreira Benke Graduada em Letras Português/Inglês (PUCPR, 2021). Foi professora de inglês como segunda língua por quatro anos (2018-2022) e atualmente atua na área da educação para uma empresa de Israel. E-mail: [email protected] Lattes: http://lattes.cnpq.br/9081006409541883 Graduada em Letras Português/Inglês (PUCPR, 2021). Foi professora de inglês como segunda língua por quatro anos (2018-2022) e atualmente atua na área da educação para uma empresa de Israel. E-mail: [email protected] Lattes: http://lattes.cnpq.br/9081006409541883 E-mail: [email protected] Lattes: http://lattes.cnpq.br/9081006409541883 Klecio de Assis Raimundo Mestre em Linguística e Ensino pela UFPB e graduado em Letras, com habilitação em Língua Inglesa pela UEPB. Espe- cializando-se em Língua Inglesa pelo UNIFIP. Atualmente, atua como professor da rede estadual de ensino da Paraíba. E-mail: [email protected] Lattes: http://lattes.cnpq.br/2501933788228978 Joyce da Silva Cruz de Mendonça Especialista em Língua, Linguística e Literatura pelo Centro Universitário de Patos (UNIFIP) e em Ensino de Línguas e Literaturas na Educação Básica pela Universidade Estadual da Paraíba (UEPB). Graduanda em Pedagogia (UEPB/CH) e Pós-graduanda em Intervenção ABA aplicada ao transtorno do espectro autista -TEA pela  Escola de Ensino Superior do Agreste Paraibano (EESAP). Graduada em Letras com habilitação em Língua Portuguesa (UEPB/CH). Atualmente faz parte do Programa Institucional de Bolsas de Iniciação à Docência (Pibid) na UEPB/CH como bolsista (CAPES). Foi monitora da disciplina de Latim I e atuou como monitora bolsista da disciplina de Prática Pedagógica I (UEPB/ CH), como também foi integrante bolsista do Subprojeto de Língua Portuguesa do Programa Residência Pedagógica (CAPES/CNPq). Integrou como bolsista (CNPq) no projeto intitulado “Identificação de Micro-Organismos Decomposi- tores de Serrapilheira e Solo em Áreas Degradadas no Seridó Oriental Paraibano” (2015-2016) pelo Instituto Federal da Paraíba (IFPB), é discente no curso subsequente em Meio Ambiente na referida instituição. Estudou Libras no Centro de Línguas Estrangeiras (CELEST/PB), cursou Libras em contexto na Fundação Centro Integrado de Apoio ao Portador de Deficiência (FUNAD/PB). Formou-se no Curso para Cuidadores na Educação Inclusiva no Centro Universitário de Patos (UNIFIP), como também se formou pela Clínica Entre Afetos e pelo Instituto Neuro como uma profissional de Acompanhamento Terapêutico (AT). Possui interesse nas seguintes temáticas: Educação Especial na perspectiva Inclusiva; Educação dos Surdos; Libras; Formação Docente; Letramento, Linguagem & ensino. E-mail: [email protected] Lattes: http://lattes.cnpq.br/7757800213663845 565 Karina Pacheco dos Santos Vander Broock Graduada em Letras Português (PUCPR, 2013). Especialista em Desenvolvimento Editorial com Ênfase em Materiais Didáticos (PUCPR, 2015). Mestre em Estudos da Linguagem (UTFPR, 2019). Atualmente é professora do curso de Letras Português/Inglês da PUCPR e atua em um projeto voltado à produção do letramento acadêmicos dos estudantes da universidade. E-mail: [email protected] Lattes: http://lattes.cnpq.br/4445812284172031 Leonardo Kominek Barrentin Especialista em Produção e Revisão Textual pela FAE Business School. Bacharel em Direito pelo Centro Universitário Curitiba (Unicuritiba). Acadêmico do curso de Letras Português/Inglês da Pontifícia Universidade Católica do Paraná (PUC/PR). Atualmente é integrante do Programa Institucional de Bolsas de Iniciação à Pesquisa em literatura, coor- denado pelo Prof. Dr. Otto Leopoldo Winck. E-mail: [email protected] Lattes: http://lattes.cnpq.br/7073428636750543 Leonardo Carvalho de Oliveira Bacharel e Licenciatura em Psicologia - Universidade Estadual da Paraíba (UEPB), Graduando em Letras/Português - Universidade Santo Amaro (UNISA). Formação em Logoterapia e Análise Existencial - Núcleo Viktor Frankl (UEPB), Pós-graduação em nível lato sensu em Terapia Cognitivo Comportamental - UNICORP. Psicólogo Comunitário e Social no Centro de Referência da Assistência Social (Pilõezinhos/PB) Psicólogo da Saúde no Centro de Atenção Psicossocial (Mulungu/PB) Professor dos cursos técnicos de Enfermagem e Radiologia na Escola Técnica São Vicente de Paula - Polo Guarabira/PB. E-mail: [email protected] Lattes: http://lattes.cnpq.br/8991338931466060 Letícia Petel de Sousa Estudante do quinto período de Letras Português-Inglês pela PUC-PR (Pontifícia Universidade Católica do Paraná). E-mail: [email protected] Lattes: http://lattes.cnpq.br/0818076885666151 Lattes: http://lattes.cnpq.br/0818076885666151 Lattes: http://lattes.cnpq.br/0818076885666151 Larissa da Silva Duarte Acadêmica do curso de Letras Português-Inglês - PUCPR (Pontifícia Universidade Católica do Paraná). E-mail: [email protected] Lattes: http://lattes.cnpq.br/7895189401053866 566 Lucas Possatti de Oliveira Formado em Letras - Inglês. Mestre e doutorando em linguística, atuando principalmente nos seguintes temas: Aco- modação Linguística, Atitudes Linguísticas, Identidade. E-mail: [email protected] Lattes: http://lattes.cnpq.br/1805996944632329 E-mail: [email protected] Lattes: http://lattes.cnpq.br/1805996944632329 Maísa Cardoso Possui graduação em Letras - Português pela Universidade Estadual de Maringá (2001) e mestrado em Letras pela Universidade Estadual de Maringá (2009). Seus estudos e pesquisas localizam-se nos Estudos Linguísticos, especifi- camente, na área da Linguística Aplicada, atuando principalmente nos seguintes temas: formação de professores, ensino-aprendizagem de língua portuguesa, educação para relações étnico-raciais, análise do discurso bakhtiniana, epistemologias do Sul, gêneros textuais, gêneros discursivos, letramentos, ensino-aprendizagem de produção tex- tual e formação de leitor. É doutora em Letras pela Universidade Federal do Paraná. Atualmente, é docente perten- cente ao quadro da Secretaria do Estado do Paraná. E-mail: [email protected] Lattes: http://lattes.cnpq.br/2909642428993166 E-mail: [email protected] Lattes: http://lattes.cnpq.br/2909642428993166 Mábia Camargo Professora Adjunta no Departamento de Secretariado Executivo da Universidade Estadual do Centro-Oeste (UNICEN- TRO). Doutorado em Linguística Aplicada (UFRJ). Mestrado em Linguagem, Identidade e Subjetividade (UEPG). Bacha- relado em Secretariado Executivo (UNICENTRO). Graduação em andamento em Letras Português-Inglês (PUCPR). E-mail: [email protected] Lattes: http://lattes.cnpq.br/6010379968530768 567 Manassés Morais Xavier Doutor em Linguística pela Universidade Federal da Paraíba. Mestre em Linguagem e Ensino pela Universidade Federal de Campina Grande. Especialista em Tecnologias Digitais na Educação, Bacharel em Comunicação Social - Jornalismo e Licenciado em Letras - Língua Portuguesa pela Universidade Estadual da Paraíba. Realizou Estágio Pós-Doutoral em Linguística na Universidade Federal da Paraíba. Professor Adjunto III de Língua Portuguesa e Lin- guística na Unidade Acadêmica de Letras, Centro de Humanidades, da Universidade Federal de Campina Grande (UAL/ CH/UFCG) e Professor Permanente no Programa de Pós-Graduação em Linguagem e Ensino da Universidade Federal de Campina Grande (PPGLE/UFCG). Membro dos Grupos de Pesquisa: Linguagem, Interação e Cultura (GELInC/UFCG); Teorias da Linguagem e Ensino (CNPq/UFCG); Linguagem, Enunciação e Interação (GPLEI/CNPq/UFPB); e O Círculo de Bakhtin em Diálogo (CNPq/UEPB). Desenvolve pesquisas tendo como referências teórico-metodológicas estudos da Teoria Dialógica da Linguagem, da Linguística Aplicada, da Educomunicação e das Teorias da Comunicação e do Jornalismo. Tem interesse por temas como formação de professores e de comunicadores sociais, leitura, análise linguística, gêneros jornalísticos e midiáticos, práticas educomunicativas e redes sociais digitais. E-mail: [email protected] Lattes: http://lattes.cnpq.br/7230669265797896 Lattes: http://lattes.cnpq.br/7230669265797896 Merylin Ricieli dos Santos Licenciada em História pela Universidade Estadual de Ponta Grossa (UEPG), Mestra em Linguagem, Identidade e Subjetividade pela mesma Intituição. Doutora em História pela Universidade do Estado de Santa Catarina (UDESC). Especialista em História, Arte e Cultura pela Universidade Estadual de Ponta Grossa (UEPG), Especialista em Gênero e Diversidade na Escola pela Universidade Federal do Paraná (UFPR), Especialista em Gestão Educacional: Organização Escolar e Trabalho Pedagógico (UEPG). Graduada em Pedagogia pelo Centro Universitário Internacional (UNINTER). E-mail: [email protected] Lattes: http://lattes.cnpq.br/4389814022309360 Maria Vanessa Monteiro das Chagas É graduada em Letras - Licenciatura plena em Língua Portuguesa, pela Universidade Federal da Paraíba (UFPB - CAM- PUS IV), Mestre em Linguística pelo Programa de Pós-Graduação em Linguística (PROLING), pela Universidade Federal da Paraíba (UFPB - CAMPUS I), e atualmente, é doutoranda em Linguística pelo mesmo programa, com pesquisa na área de Linguística Aplicada. Desde 2020 é membro do Grupo de Estudos e Pesquisa em Linguística Sistêmico-Fun- cional, Análise Crítica do Discurso e Multimodalidade/Multiletramentos (GEPLAM) e docente de Língua Portuguesa e Produção Textual na Educação Básica, na rede estadual da Paraíba. E-mail: [email protected] Lattes: https://lattes.cnpq.br/9790043060830056 568 Lattes: http://lattes.cnpq.br/9583480590467208 Lattes: http://lattes.cnpq.br/9583480590467208 Natássia Thais do Nascimento Ribeiro Mestre em Linguística pelo Programa de Pós-Graduação em Linguística (PROLING), da Universidade Federal da Paraíba (UFPB). Professora efetiva de língua portuguesa da rede estadual da Paraíba, desde 2013. E-mail: [email protected] Lattes: http://lattes.cnpq.br/5561460505626050 Marina Chiara Legroski Doutora em Letras pela UFPR. Professora adjunta da Universidade Estadual de Ponta Grossa desde 2015. Trabalho com semântica, pragmática e leciono disciplinas de linguística. Apaixonada por fenômenos da linguagem. E-mail: [email protected] Lattes: http://lattes.cnpq.br/4208603228366576 Lattes: http://lattes.cnpq.br/4208603228366576 Ronna Freitas de Oliveira Licenciada em Letras Português/Inglês (UEPG, 2019), mestra em Estudos da Linguagem (PPGEL/UEPG, 2022) e graduanda em Geografia (UEPG). Ativista e pesquisadora transfeminista, com atuação nos Estudos de Gênero e na Linguística Aplicada. E-mail: [email protected] Lattes: http://lattes.cnpq.br/9583480590467208 Silvely Brandes Mestre em Linguagem, Identidade e Subjetividade pela Universidade Estadual de Ponta Grossa e doutora em Estudos Linguísticos pela Universidade Federal do Paraná. E-mail: [email protected] Lattes: http://lattes.cnpq.br/7700433462005191 Mestre em Linguagem, Identidade e Subjetividade pela Universidade Estadual de Ponta Grossa e doutora em Estudos Linguísticos pela Universidade Federal do Paraná. E-mail: [email protected] Lattes: http://lattes.cnpq.br/7700433462005191 Lattes: http://lattes.cnpq.br/7700433462005191 Samuel Barbosa Silva Doutor (2021) e Mestre em Linguística pela Universidade Federal de Alagoas (2017), Especialista em Língua Portuguesa e suas Literaturas pela Universidade de Pernambuco - campus Garanhuns (2014), Especialista em Gestão Escolar pela Faculdade de Teologia Integrada (Fatin). Possui Graduação em Letras (Língua Portuguesa e suas Literaturas) pela Uni- versidade de Pernambuco (2012). Suas pesquisas e produções acadêmicas são desenvolvidas na área de Linguística na linha de pesquisa em Análise de Discurso Pêcheutiana, tendo ênfase nos estudos sobre gênero/sexualidades, 569 raça, cultura, subjetividades e política. Se interessa também nas discussões dos seguintes temas: religião, mídias sociais, educação e diversidade, decolonialidade e quaisquer outros debates dos estudos linguísticos. E-mail: [email protected] Lattes: http://lattes.cnpq.br/0543187841218105 raça, cultura, subjetividades e política. Se interessa também nas discussões dos seguintes temas: religião, mídias sociais, educação e diversidade, decolonialidade e quaisquer outros debates dos estudos linguísticos. Susana de Freitas Vaz Graduada em Letras/Libras Bacharelado pela Universidade Estadual do Oeste do Paraná. E-mail: [email protected] E-mail: [email protected] E-mail: [email protected] Lattes: http://lattes.cnpq.br/0153520162103636 Samuel Filipe Guedes do Nascimento Graduado em Letras – Língua Portuguesa (UFCG). Possui experiência nos níveis Fundamental e Médio das redes estadual e municipal de ensino. Participou de publicações no campo da literatura nacional e internacional, pela editora Chiado e pelo editorial colombiano La Semilla Amarilla. Atualmente faz estudos independentes na área de Linguística Funcional e Análise discursiva. Venan Lucas de Oliveira Alencar Professor no Departamento de Secretariado Executivo da Universidade Estadual do Centro-Oeste (Unicentro). Dou- tor e Mestre em Estudos Linguísticos (UFMG). Bacharel em Letras Português (Unesa) e em Secretariado Executivo Trilíngue (UFV). E-mail: [email protected] Lattes: http://lattes.cnpq.br/9908168525607718 E-mail: [email protected] Tamires Tolomeotti Pereira Doutora em Educação pela Universidade Federal do Paraná - UFPR. Atualmente é professora substituta no Departa- mento de Planejamento e Administração Escolar da Universidade Federal do Paraná. Pesquisadora do GILDA - Grupo Interdisciplinar em Linguagem, Diferença e Subjetivação (UFPR/CNPq). E-mail: [email protected] Lattes: http://lattes.cnpq.br/7956987169844661 570 Willian Ferreira Furtado de Lacerda Possui graduação em Administração pelo Instituto de Educação Superior da Paraíba (2010), graduação em Letras - Inglês pela Universidade Federal da Paraíba (2018), pós-Graduação Lato Sensu em Língua, Linguagem e Literatura pelo Centro Integrado de Tecnologia e Pesquisa (2019), pós-Graduação Lato Sensu Master Business Administration em Gestão de Instituições Públicas pelo Instituto Federal de Rondônia (2021). É mestre e doutorando em Linguística pela Universidade Federal da Paraíba (2021). Atualmente é Assistente de Negócios - BANCO DO BRASIL S/A. Tem experiência na área de administração, com ênfase em instituições financeiras bancárias, e na área de Letras, com ênfase em língua Inglesa. Integrante do Grupo de Estudo Contato Linguístico na Universidade Federal da Paraíba, voltado para a pesquisa sociolinguística, contato dialetal, atitudes e crenças linguísticas, coordenado pelo professor Dr. Rubens Marques de Lucena. E-mail: [email protected] Lattes: http://lattes.cnpq.br/117748119788942 571 gays 18, 29, 40, 43, 55, 62, 100, 103, 127, 348, 355, 356, 388 gênero 10, 11, 12, 15, 16, 19, 21, 29, 32, 39, 42, 44, 47, 48, 52, 55, 56, 57, 59, 60, 61, 65, 69, 96, 97, 100, 101, 103, 106, 107, 109, 111, 112, 113, 114, 115, 118, 119, 120, 124, 126, 128, 129, 130, 131, 141, 153, 185, 192, 222, 227, 228, 229, 233, 235, 236, 241, 242, 243, 247, 248, 249, 252, 253, 255, 256, 258, 259, 260, 267, 269, 314, 359, 363, 364, 377, 378, 379, 384, 385, 386, 387, 388, 389, 393, 394, 395, 397, 398, 399, 400, 401, 402, 404, 410, 412, 413, 416, 417, 423, 425, 429, 430, 431, 432, 434, 438, 440, 446, 448, 496, 560, 570 gênero 10, 11, 12, 15, 16, 19, 21, 29, 32, 39, 42, 44, 47, 48, 52, 55, 56, 57, 59, 60, 61, 65, 69, 96, 97, 100, 101, 103, 106, 107, 109, 111, 112, 113, 114, 115, 118, 119, 120, 124, 126, 128, 129, 130, 131, 141, 153, 185, 192, 222, 227, 228, 229, 233, 235, 236, 241, 242, 243, 247, 248, 249, 252, 253, 255, 256, 258, 259, 260, 267, 269, 314, 359, 363, 364, 377, 378, 379, 384, 385, 386, 387, 388, 389, 393, 394, 395, 397, 398, 399, 400, 401, 402, 404, 410, 412, 413, 416, 417, 423, 425, 429, 430, 431, 432, 434, 438, 440, 446, 448, 496, 560, 570 identidade 19, 42, 44, 52, 57, 64, 69, 85, 91, 96, 101, 103, 104, 105, 107, 109, 130, 134, 135, 137, 138, 139, 140, 141, 142, 146, 147, 148, 149, 162, 168, 221, 226, 299, 302, 303, 343, 348, 389, 394, 412, 427, 484, 488, 490, 511, 512, 523 identidade 19, 42, 44, 52, 57, 64, 69, 85, 91, 96, 101, 103, 104, 105, 107, 109, 130, 134, 135, 137, 138, 139, 140, 141, 142, 146, 147, 148, 149, 162, 168, 221, 226, 299, 302, 303, 343, 348, 389, 394, 412, 427, 484, 488, 490, 511, 512, 523 gênero artigo de opinião 12, 247, 429, 431, 432, 446 gênero artigo de opinião 12, 247, 429, 431, 432, 446 gênero dissertativo-argumentativo 21, 241, 247 ÍNDICE REMISSIVO A acomodação dialetal 20, 132, 133, 134, 149 AD 343, 349, 359, 360, 362, 374, 379 agenda pedagógica 47 agentes do campo 14 agonística 14 alfabetização 41, 42, 88, 153, 283, 332, 341, 471 alfabetização de pequenos agricultores 153 aluno surdo 166, 167, 169, 170, 171, 172, 173, 174, 175, 176, 187, 188, 190, 191 Análise de Discurso 24, 343, 359, 375, 379, 389, 402, 404, 569 análise linguística 13, 24, 47, 53, 248, 259, 261, 430, 448, 449, 451, 452, 457, 462, 463, 464, 471, 568 antirracista 22, 281, 282, 284, 290, 298 Antologia Nacional 81, 93 antropologia 30, 67, 105, 521 aprendizagem da Língua Portuguesa 10, 20, 166, 167, 173, 174, 175, 176 aquisição/aprendizagem 21, 180, 181, 182, 183, 185, 197, 198 assexuais 29, 348 assujeitamento 19, 96, 345, 346 atitudes linguísticas 19, 20, 56, 60, 133, 134, 135, 137, 138, 139, 141, 142, 147, 148, 149, 150, 534 atividades intelectuais 14 B Base Nacional Comum Curricular 54, 67, 208, 221, 327, 340, 426, 462, 472 binarismo 29, 39 bissexuais 29, 55, 56, 103, 348, 388, 401 BNCC 54, 157, 208, 209, 316, 327, 328, 329, 330, 339, 409 Brasil colônia 71 A acomodação dialetal 20, 132, 133, 134, 149 AD 343, 349, 359, 360, 362, 374, 379 agenda pedagógica 47 agentes do campo 14 agonística 14 alfabetização 41, 42, 88, 153, 283, 332, 341, 471 alfabetização de pequenos agricultores 153 aluno surdo 166, 167, 169, 170, 171, 172, 173, 174, 175, 176, 187, 188, 190, 191 Análise de Discurso 24, 343, 359, 375, 379, 389, 402, 404, 569 análise linguística 13, 24, 47, 53, 248, 259, 261, 430, 448, 449, 451, 452, 457, 462, 463, 464, 471, 568 antirracista 22, 281, 282, 284, 290, 298 Antologia Nacional 81, 93 antropologia 30, 67, 105, 521 aprendizagem da Língua Portuguesa 10, 20, 166, 167, 173, 174, 175, 176 aquisição/aprendizagem 21, 180, 181, 182, 183, 185, 197, 198 assexuais 29, 348 assujeitamento 19, 96, 345, 346 atitudes linguísticas 19, 20, 56, 60, 133, 134, 135, 137, 138, 139, 141, 142, 147, 148, 149, 150, 534 atividades intelectuais 14 B Base Nacional Comum Curricular 54, 67, 208, 221, 327, 340, 426, 462, 472 binarismo 29, 39 bissexuais 29, 55, 56, 103, 348, 388, 401 BNCC 54, 157, 208, 209, 316, 327, 328, 329, 330, 339, 409 Brasil colônia 71 burocratização 15 C campo do saber 14, 15 campo educacional 113, 209, 215 campo intelectual 14, 16 campos científicos 14 campo social 203 caráter pedagógico 25, 47, 451, 481 cariocas 20, 132, 133, 151 censura 12, 23, 107, 358, 359, 364, 366, 367, 368, 383 Ciência Política 15 Ciências Humanas 209, 356, 513 cis-bissexual 58 Cis/Hétero 61, 63 cis-heteronorma 102, 103 coda silábica 26, 516, 517, 518, 519, 520, 524, 526, 527, 528, 532, 533, 534 colonialidade 16, 153, 285 complexidades 19, 94, 106 comunidade LGBTQIA+ 9, 19, 28, 29, 30, 32, 33, 36, 37, 39, 40, 42, 43, 52, 57, 58, 62, 69, 348, 351, 355 condições sócio-histórico-culturais 95 conscientização 21, 22, 58, 176, 212, 263, 264, 267, 268, 269, 270, 271, 280, 316, 375, 384 contextos de comunicação 459 contextos de formação 47 controle social 51, 99, 131 corpos 39, 57, 59, 102, 103, 105, 108, 114, 121, 355, 388 Covid-19 263, 277, 278 CPI da COVID-19 24, 431, 432, 442 critérios político-sociais 15 S U M Á R I O 572 ensino-aprendizagem 188, 238, 285, 296, 317, 382, 407, 411, 426, 503, 568 ensino de língua 18, 31, 48, 52, 53, 54, 55, 58, 71, 73, 77, 80, 81, 84, 85, 86, 88, 89, 90, 92, 93, 451, 459, 461, 464, 470, 471, 477, 481, 482, 490, 492 ensino de língua portuguesa 71, 80, 81, 85, 88, 89, 90, 92, 93, 477, 492 ensino de línguas 20, 68, 90, 152, 157, 161 ensino e aprendizado 23, 327 ensino fundamental 24, 89, 208, 209, 222, 237, 263, 266, 280, 324, 336, 341, 406, 408, 426, 465, 565 escola pública 21, 88, 202, 221, 223, 231, 263, 323, 565 Escola sem Partido 381, 383, 392, 403 escrita 22, 30, 45, 74, 78, 82, 83, 86, 88, 96, 108, 161, 162, 172, 173, 224, 226, 227, 229, 230, 231, 232, 237, 238, 242, 251, 260, 262, 263, 264, 267, 269, 276, 279, 280, 286, 288, 298, 303, 316, 319, 325, 328, 331, 335, 359, 367, 385, 427, 437, 462, 470, 471 Estrutura da língua portuguesa 533 estruturas gramaticais 25, 195, 451 estudos da linguagem 18, 26, 54, 55, 102, 107, 109, 454 Estudos da Linguagem 68, 92, 95, 96, 200, 512, 564, 566, 569 estudos decoloniais 20, 153 estudos sociolinguísticos 47, 49, 50, 53, 61 exílio 157, 365 F fake news 550 fenômenos sociais 96, 98, 99, 210, 230 filosofia da linguagem 89, 92, 108, 115, 164, 237, 448, 559 financiamentos públicos e privados de pesquisa 15 formação 10, 11, 15, 22, 23, 47, 48, 54, 74, 75, 79, 80, 82, 83, 84, 85, 86, 87, 89, 90, 92, 157, 160, 163, 166, 170, 171, 173, 174, 179, 180, 185, 186, 187, 190, 191, 195, 199, 203, 205, 206, 207, 220, 221, 240, 282, 305, 306, 307, 315, 316, 317, 318, 319, 321, 322, 323, 324, 328, 344, 347, 352, 353, 354, 359, 360, 361, 362, 368, 369, 371, 373, 374, 379, 383, 388, 390, 391, 396, 397, 400, 402, 408, 422, 424, 427, 469, 473, cultura 21, 25, 37, 55, 62, 73, 84, 88, 107, 114, 137, 138, 158, 162, 168, 170, 171, 174, 175, 176, 186, 187, 188, 195, 203, 207, 216, 286, 299, 314, 322, 324, 339, 388, 410, 496, 502, 514, 570 , , , D delírios fundamentalistas 153 descolonialidade 408 desigualdades sociais 90, 394 desumanização 156 dialeto 34, 35, 41, 57, 133, 134, 138, 139, 140, 142, 143, 145, 147, 149, 503, 522 dicionários 82, 91, 500 didática 11, 22, 84, 239, 241, 247, 248, 249, 252, 258, 259, 260, 261, 290, 530 Diretório dos Índios 71 discurso religioso 23, 343, 344, 347, 348, 349, 351, 354, 372, 373 dissertativo-argumentativo 21, 22, 239, 240, 241, 247, 249, 250, 251, 252, 254, 256, 258, 259, 260 ditadura militar 153, 351 diversidade linguística 47, 56, 69, 150 diversidade sexual 69, 130, 343, 393, 394 doutrinação esquerdista 153 E educação bancária 155, 156 educação básica 54, 69, 90, 204, 207, 208, 219, 243, 380, 460, 465, 471, 477, 561 educação bilíngue 169, 171, 175, 176, 177 educação escolar 327, 397, 400 Educação Inclusiva 220, 565 educação libertadora 154, 155, 156, 160 educação linguística 53, 81 Educação Moral 24, 378, 391, 392, 393, 394, 397, 398, 399, 400, 401, 402 educação pública 153 ementas das disciplinas 15 ENEM 45, 56, 69, 222, 223, 229, 231, 233, 235, 236, 237 D S U M Á R I O E 573 histórias em quadrinhos 327, 328, 330, 337, 340, 341 homofobia 57, 103, 127, 131, 348, 352, 369, 393 homossexual 35, 36, 39, 40, 42, 61, 62, 123 HQs 22, 23, 327, 328, 330, 332, 333, 334, 336, 337, 338, 339, 340 I identidade 19, 42, 44, 52, 57, 64, 69, 85, 91, 96, 101, 103, 104, 105, 107, 109, 130, 134, 135, 137, 138, 139, 140, 141, 142, 146, 147, 148, 149, 162, 168, 221, 226, 299, 302, 303, 343, 348, 389, 394, 412, 427, 484, 488, 490, 511, 512, 523 ideologia 22, 73, 75, 79, 81, 113, 153, 162, 228, 282, 344, 345, 346, 347, 350, 351, 352, 353, 354, 355, 360, 361, 362, 368, 374, 380, 396, 397, 399, 400, 401, 402, 410 Ideologia de gênero 393 inclusão 21, 57, 58, 150, 177, 186, 187, 188, 189, 198, 203, 204, 205, 208, 211, 212, 219, 221, 245, 410 Inclusão Social 209 instituição escolar 72, 89 intérprete 10, 20, 23, 165, 166, 167, 169, 170, 171, 172, 173, 174, 175, 176, 205, 213, 566 intersexo 29, 47, 348 J João Pessoa 20, 68, 69, 132, 133, 141, 143, 145, 146, 151, 426, 466, 473, 474, 512, 534 K kit gay 113, 153 L latim 72, 75, 77, 79, 81, 456, 501, 502, 503, 504, 505, 508, 509 legitimação 14, 15, 382 legitimação do saber 14 lei nº 10.436 168, 169 lésbicas 29, 55, 100, 103, 348, 388 letramento literário 407, 422 LGBTfobia 47 LGBTIA+fobia 114, 126, 128 LGBTIA+fóbico 121, 125, 126, 128 histórias em quadrinhos 327, 328, 330, 337, 340, 341 homofobia 57, 103, 127, 131, 348, 352, 369, 393 homossexual 35, 36, 39, 40, 42, 61, 62, 123 HQs 22, 23, 327, 328, 330, 332, 333, 334, 336, 337, 338, 339, 340 I identidade 19, 42, 44, 52, 57, 64, 69, 85, 91, 96, 101, 103, 104, 105, 107, 109, 130, 134, 135, 137, 138, 139, 140, 141, 142, 146, 147, 148, 149, 162, 168, 221, 226, 299, 302, 303, 343, 348, 389, 394, 412, 427, 484, 488, 490, 511, 512, 523 ideologia 22, 73, 75, 79, 81, 113, 153, 162, 228, 282, 344, 345, 346, 347, 350, 351, 352, 353, 354, 355, 360, 361, 362, 368, 374, 380, 396, 397, 399, 400, 401, 402, 410 Ideologia de gênero 393 inclusão 21, 57, 58, 150, 177, 186, 187, 188, 189, 198, 203, 204, 205, 208, 211, 212, 219, 221, 245, 410 Inclusão Social 209 instituição escolar 72, 89 intérprete 10, 20, 23, 165, 166, 167, 169, 170, 171, 172, 173, 174, 175, 176, 205, 213, 566 intersexo 29, 47, 348 J João Pessoa 20, 68, 69, 132, 133, 141, 143, 145, 146, 151, 426, 466, 473, 474, 512, 534 K kit gay 113, 153 L latim 72, 75, 77, 79, 81, 456, 501, 502, 503, 504, 505, 508, 509 legitimação 14, 15, 382 legitimação do saber 14 lei nº 10.436 168, 169 lésbicas 29, 55, 100, 103, 348, 388 letramento literário 407, 422 LGBTfobia 47 LGBTIA+fobia 114, 126, 128 LGBTIA+fóbico 121, 125, 126, 128 histórias em quadrinhos 327, 328, 330, 337, 340, 341 homofobia 57, 103, 127, 131, 348, 352, 369, 393 homossexual 35, 36, 39, 40, 42, 61, 62, 123 HQs 22, 23, 327, 328, 330, 332, 333, 334, 336, 337, 338, 339, 340 freireanas 152 S U M Á R I O G gays 18, 29, 40, 43, 55, 62, 100, 103, 127, 348, 355, 356, 388 língua brasileira 200, 202, 511 linguística 9, 13, 17, 24, 26, 27, 30, 35, 38, 45, 46, 47, 48, 49, 50, 51, 53, 55, 56, 62, 64, 67, 68, 69, 71, 73, 74, 78, 81, 82, 83, 85, 86, 87, 88, 89, 92, 93, 97, 98, 99, 100, 101, 102, 104, 107, 109, 110, 115, 134, 135, 139, 141, 142, 145, 147, 148, 149, 150, 171, 174, 182, 203, 212, 224, 225, 238, 242, 248, 249, 259, 261, 325, 330, 349, 415, 426, 427, 430, 432, 434, 436, 445, 446, 447, 448, 449, 451, 452, 453, 454, 457, 458, 459, 462, 463, 464, 466, 467, 471, 472, 473, 476, 478, 480, 481, 483, 484, 486, 487, 489, 490, 491, 492, 493, 495, 496, 506, 513, 514, 522, 523, 533, 538, 545, 560, 567, 568, 569 Língua Brasileira de Sinais 71, 169, 170, 172, 177, 199, 203, 205, 207, 220, 221, 562, 563 língua de sinais 20, 172, 177, 180, 181, 184, 188, 189, 192, 193, 194, 195, 196, 197, 198, 199, 200 língua de sinais 20, 172, 177, 180, 181, 184, 188, 189, 192, 193, 194, 195, 196, 197, 198, 199, 200 linguagem 16, 17, 18, 19, 20, 21, 22, 23, 24, 26, 30, 32, 35, 36, 44, 45, 47, 48, 49, 50, 51, 53, 54, 55, 56, 57, 58, 59, 65, 66, 68, 89, 91, 92, 96, 97, 100, 101, 102, 103, 107, 108, 109, 110, 113, 114, 115, 116, 119, 128, 129, 131, 158, 161, 162, 163, 164, 172, 183, 184, 200, 221, 224, 225, 226, 229, 232, 237, 238, 248, 249, 251, 252, 253, 254, 255, 256, 258, 259, 280, 282, 283, 284, 285, 286, 295, 300, 303, 307, 308, 309, 310, 311, 316, 318, 322, 323, 324, 325, 330, 331, 333, 337, 339, 341, 352, 360, 386, 389, 404, 408, 409, 411, 413, 415, 426, 428, 430, 433, 434, 435, 436, 437, 446, 447, 448, 451, 453, 454, 456, 457, 458, 459, 460, 462, 463, 471, 473, 479, 484, 492, 497, 503, 521, 559, 569 Linguística Aplicada 18, 28, 30, 44, 45, 50, 68, 91, 92, 304, 307, 325, 411, 426, 474, 476, 560, 564, 567, 568, 569 linguística queer 67, 97, 98, 99, 100, 101, 102, 104, 109, 560 Linguística Queer 19, 50, 67, 94, 95, 96 Linguística Textual 21, 223, 238 literatura 27, 81, 82, 93, 177, 181, 215, 291, 294, 301, 303, 317, 318, 323, 407, 408, 418, 422, 424, 426, 473, 496, 528, 567, 570 livro didático 12, 24, 80, 81, 92, 406, 408, 426, 427, 514 livros didáticos 24, 87, 89, 292, 293, 295, 304, 408, 417, 448 língua(gem) 17, 18, 26, 28, 29, 31, 33, 35, 36, 37, 38, 42, 43, 47, 48, 58, 59, 64, 89, 91, 101, 195, 462 língua geral 71, 72, 73, 74, 76, 77, 81 língua materna 31, 66, 68, 88, 90, 91, 92, 166, 168, 169, 170, 173, 175, 182, 209, 236, 477, 481, 482, 490, 496, 505, 512, 520, 526, 528 gêneros textuais 21, 22, 226, 227, 231, 241, 242, 247, 248, 261, 263, 280, 463, 487, 568 gêneros textuais 21, 22, 226, 227, 231, 241, 242, 247, 248, 261, 263, 280, 463, 487, 568 gramática 24, 25, 68, 77, 78, 79, 81, 82, 83, 84, 85, 86, 87, 88, 91, 93, 236, 336, 338, 340, 415, 433, 434, 435, 442, 447, 448, 450, 451, 454, 455, 456, 457, 458, 459, 460, 461, 464, 466, 467, 468, 469, 470, 471, 472, 473, 474, 476, 477, 478, 479, 481, 484, 485, 489, 490, 491, 492, 493, 495, 496, 497, 499, 500, 502, 503 504 505 508 509 511 512 513 514 574 Língua Portuguesa 20, 53, 54, 71, 77, 85, 88, 165, 166, 167, 169, 170, 171, 172, 173, 174, 175, 176, 223, 224, 226, 229, 231, 238, 240, 322, 330, 407, 448, 450, 474, 513, 560, 562, 563, 564, 565, 568, 569, 570 LGBTQIA+ 18, 19, 28, 29, 30, 32, 33, 35, 36, 37, 39, 40, 42, 43, 47, 52, 56, 57, 58, 59, 60, 61, 62, 63, 64, 69, 343, 346, 347, 348, 351, 355 Língua Portuguesa 20, 53, 54, 71, 77, 85, 88, 165, 166, 167, 169, 170, 171, 172, 173, 174, 175, 176, 223, 224, 226, 229, 231, 238, 240, 322, 330, 407, 448, 450, 474, 513, 560, 562, 563, 564, 565, 568, 569, 570 Libras 10, 21, 71, 165, 166, 169, 170, 171, 172, 174, 175, 177, 179, 180, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 198, 199, 200, 201, 205, 206, 207, 208, 209, 211, 212, 213, 214, 215, 216, 219, 220, 560, 562, 563, 565, 566, 570 língua brasileira 200, 202, 511 R pesquisa 15, 18, 20, 21, 26, 44, 45, 51, 59, 61, 63, 68, 71, 89, 95, 96, 106, 108, 139, 140, 141, 143, 149, 150, 166, 168, 173, 174, 175, 176, 180, 185, 192, 198, 203, 204, 210, 211, 218, 220, 221, 223, 224, 231, 232, 234, 237, 241, 245, 248, 249, 260, 264, 265, 266, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 282, 287, 288, 289, 290, 292, 295, 297, 301, 302, 304, 309, 324, 325, 327, 328, 340, 344, 349, 360, 366, 391, 403, 404, 408, 411, 428, 431, 432, 436, 437, 439, 442, 445, 446, 447, 452, 465, 472, 473, 476, 479, 480, 491, 492, 493, 518, 523, 524, 526, 527, 528, 529, 530, 531, 532, 533, 535, 563, 565, 568, 569, 571 pesquisa 15, 18, 20, 21, 26, 44, 45, 51, 59, 61, 63, 68, 71, 89, 95, 96, 106, 108, 139, 140, 141, 143, 149, 150, 166, 168, 173, 174, 175, 176, 180, 185, 192, 198, 203, 204, 210, 211, 218, 220, 221, 223, 224, 231, 232, 234, 237, 241, 245, 248, 249, 260, 264, 265, 266, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 282, 287, 288, 289, 290, 292, 295, 297, 301, 302, 304, 309, 324, 325, 327, 328, 340, 344, 349, 360, 366, 391, 403, 404, 408, 411, 428, 431, 432, 436, 437, 439, 442, 445, 446, 447, 452, 465, 472, 473, 476, 479, 480, 491, 492, 493, 518, 523, 524, 526, 527, 528, 529, 530, 531, 532, 533, 535, 563, 565, 568, 569, 571 S S sala de aula 12, 19, 20, 21, 23, 24, 36, 50, 51, 53, 56, 57, 58, 59, 65, 66, 68, 150, 159, 162, 166, 167, 169, 170, 171, 172, 173, 174, 175, 181, 186, 188, 190, 203, 204, 207, 209, 210, 218, 219, 230, 238, 299, 307, 317, 319, 321, 327, 328, 330, 336, 337, 338, 339, 340, 341, 381, 382, 383, 395, 397, 402, 408, 409, 421, 422, 459, 463, 487 sexualidade 10, 15, 16, 19, 32, 44, 48, 52, 55, 65, 68, 69, 96, 97, 100, 101, 103, 107, 112, 128, 131, 378, 384, 387, 388, 393, 399, 400, 401, 404, 405, 427 signo linguístico 48, 49, 166 silenciamento 74, 162, 359, 362, 367, 368, 371 sistema capitalista 167 sociedade 11, 16, 23, 26, 30, 37, 43, 44, 45, 51, 52, 53, 56, 58, 59, 72, 101, 105, 149, 157, 163, 168, 169, 173, 177, 180, 198, 203, 205, 209, 212, 218, 219, 220, 226, 227, 237, 250, 251, 263, 264, 267, 270, 275, 276, 277, 278, 279, 280, 281, 283, 285, 295, 300, 306, 315, 349, 351, 355, 363, 364, 369, 372, 374, 375, 379, 381, 382, 384, 385, 388, 389, 397, 400, 403, pessoa com deficiência 177, 204, 221 polissemia 166 política linguística 71, 73, 78, 81, 82, 85, 86, 171 políticas de línguas 19, 71 políticas educacionais 19, 71, 80, 178 pós-modernidade 44, 96, 97 preconceito 53, 55, 57, 66, 69, 103, 145, 149, 208, 212, 300, 356, 364, 401 processo de gramatização 25, 458, 495, 497, 500, 510, 512 produção de texto 239, 250, 260, 408 produtividade discursiva 431 professores 23, 31, 50, 55, 59, 76, 77, 78, 79, 83, 84, 85, 86, 87, 89, 90, 92, 150, 171, 172, 173, 174, 175, 176, 179, 180, 185, 190, 199, 200, 205, 206, 220, 221, 229, língua nacional 74, 77, 78, 81, 82, 85, 88 língua portuguesa 9, 12, 13, 21, 22, 24, 25, 35, 45, 65, 67, 69, 70, 71, 72, 74, 75, 76, 77, 78, 80, 81, 82, 83, 85, 88, 89, 90, 91, 92, 93, 170, 172, 193, 237, 248, 249, 251, 260, 263, 324, 336, 340, 406, 408, 425, 426, 428, 472, 474, 476, 477, 490, 491, 492, 496, 497, 500, 503, 504, 505, 506, 508, 509, 510, 511, 517, 532, 533, 568, 569 575 N neoconservadora 113, 114 neoconservadores 19, 112, 113, 115, 128, 129, 131 O ódio 19, 112, 113, 114, 115, 121, 122, 125, 126, 128, 129, 131, 370 opressores e oprimidas/os 154, 160 P palatalização 133, 148 Paulo Freire 20, 153, 155, 157, 158, 159, 160, 163, 164, 308, 448 pedagogia crítica 154, 304 performativa 62, 96, 103, 115, 123, 125, 128, 130, 131 perspectivas culturais 96 469, 470, 502, 568 professor ouvinte 166 programas de pós-graduação 15 Q queer 32, 47, 62, 67, 97, 98, 99, 100, 101, 102, 103, 104, 106, 107, 108, 109, 110, 111, 385, 387, 388, 560 R raça 15, 16, 65, 97, 98, 100, 103, 109, 212, 296, 300, 356, 397, 410, 412, 413, 416, 418, 419, 424, 427, 428, 570 racismo 22, 103, 114, 127, 156, 163, 282, 283, 284, 285, 300, 301, 303, 304, 348, 357, 412, 419 redação do ENEM 222, 229, 231, 233, 236, 237 representatividade 24, 45, 58, 289, 408, 421, 425, 426 retórica de ruptura 478, 484, 485, 486, 490 rigor científico 451 pessoa com deficiência 177, 204, 221 pessoa com deficiência 177, 204, 221 polissemia 166 política linguística 71, 73, 78, 81, 82, 85, 86, 171 políticas de línguas 19, 71 políticas educacionais 19, 71, 80, 178 pós-modernidade 44, 96, 97 preconceito 53, 55, 57, 66, 69, 103, 145, 149, 208, 212, 300, 356, 364, 401 processo de gramatização 25, 458, 495, 497, 500, 510, 512 produção de texto 239, 250, 260, 408 produtividade discursiva 431 professores 23, 31, 50, 55, 59, 76, 77, 78, 79, 83, 84, 85, 86, 87, 89, 90, 92, 150, 171, 172, 173, 174, 175, 176, 179, 180, 185, 190, 199, 200, 205, 206, 220, 221, 229, 576 407, 408, 409, 413, 445, 463, 470, 482, 483, 496, 498, 509, 511 Sócio-Construtivista 181, 183 sócio-histórico 95, 96, 347, 361, 390, 410 Sociolinguística 9, 18, 19, 38, 44, 46, 47, 48, 49, 50, 51, 52, 64, 65, 66, 67, 68, 69, 150, 451, 476, 477, 478, 479, 481, 482, 483, 484, 487, 520, 521, 523, 524, 533, 534, 560 subversão 19, 44, 96, 103, 130, 386 surdo 21, 165, 166, 167, 169, 170, 171, 172, 173, 174, 175, 176, 186, 187, 188, 190, 191, 196, 197, 198, 200 sutilezas 19, 94, 106 T teoria dialógica da linguagem 24 teóricos do Sul 153 Tradição Sociodiscursiva 477, 482 transexuais 29, 55, 68, 105, 348, 401 transgêneros 29, 103, 388 travestis 29, 41, 42, 55, 68, 103, 105, 348, 355 U Universidade Federal de Uberlândia 95 Universidade Federal do Rio de Janeiro 95 V variação e mudança linguística 478, 486, 487, 490 variáveis linguísticas e extralinguísticas 133, 527 violência 15, 44, 57, 73, 107, 124, 126, 155, 348, 354, 364, 370, 371, 427 407, 408, 409, 413, 445, 463, 470, 482, 483, 496, 498, 509, 511 Sócio-Construtivista 181, 183 sócio-histórico 95, 96, 347, 361, 390, 410 Sociolinguística 9, 18, 19, 38, 44, 46, 47, 48, 49, 50, 51, 52, 64, 65, 66, 67, 68, 69, 150, 451, 476, 477, 478, 479, 481, 482, 483, 484, 487, 520, 521, 523, 524, 533, 534, 560 subversão 19, 44, 96, 103, 130, 386 surdo 21, 165, 166, 167, 169, 170, 171, 172, 173, 174, 175, 176, 186, 187, 188, 190, 191, 196, 197, 198, 200 sutilezas 19, 94, 106 T teoria dialógica da linguagem 24 teóricos do Sul 153 407, 408, 409, 413, 445, 463, 470, 482, 483, 496, 498, 509, 511 Sócio-Construtivista 181, 183 sócio-histórico 95, 96, 347, 361, 390, 410 Sociolinguística 9, 18, 19, 38, 44, 46, 47, 48, 49, 50, 51, 52, 64, 65, 66, 67, 68, 69, 150, 451, 476, 477, 478, 479, 481, 482, 483, 484, 487, 520, 521, 523, 524, 533, 534, 560 subversão 19, 44, 96, 103, 130, 386 surdo 21, 165, 166, 167, 169, 170, 171, 172, 173, 174, 175, 176, 186, 187, 188, 190, 191, 196, 197, 198, 200 sutilezas 19, 94, 106 T teoria dialógica da linguagem 24 teóricos do Sul 153 S U M Á R I O V variação e mudança linguística 478, 486, 487, 490 variáveis linguísticas e extralinguísticas 133, 527 violência 15, 44, 57, 73, 107, 124, 126, 155, 348, 354, 364, 370, 371, 427 T teoria dialógica da linguagem 24 teóricos do Sul 153 577

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Improvements of right ventricular function after intervention with CPAP in patients with obstructive sleep apnoea

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Improvements of right ventricular function after intervention with CPAP in patients with obstructive sleep apnoea Greg Murphy  (  [email protected] ) St. James's Hospital Gerard King  St. James's Hospital Peter Coss  St. James's Hospital Mark Coyle  St. James's Hospital Anne-marie McLaughlin  St. James's Hospital Ross Murphy  St. James's Hospital Greg Murphy  (  [email protected] ) St. James's Hospital Research Article Keywords: Obstructive sleep apnoea, Right heart, Echocardiography, CPAP Posted Date: July 28th, 2023 DOI: https://doi.org/10.21203/rs.3.rs-3158087/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Additional Declarations: No competing interests reported. Improvements of right ventricular function after intervention with CPAP in patients with obstructive sleep 1 apnoea. 2 3 Greg Murphy, Gerard King, Peter Coss, Mark Coyle, Anne-Marie McLaughlin, Ross Murphy 4 5 St James’ Hospital 6 St James street 7 Dublin 8 8 Ireland 9 10 Corresponding author 11 12 Dr Greg Murphy 13 St James’ Hospital 14 St James street 15 Dublin 8 16 Republic of Ireland 17 [email protected] 18 0000-0003-3671-3558 19 20 Abstract 21 22 Background 23 Obstructive sleep apnoea (OSA) is present in 40-80% of patients with cardiovascular morbidity and is 24 associated with adverse effects on cardiovascular health. Continuous positive airway pressure (CPAP) maintains 25 airway patency during sleep and is hypothesised to improve cardiac function. In the present study, we report on 26 the impact of 12 weeks of CPAP and improvements in echocardiographic parameters of the right ventricle (RV). 27 28 Methods 29 Improvements of right ventricular function after intervention with CPAP in patients with obstructive sleep 1 apnoea. 2 3 Greg Murphy, Gerard King, Peter Coss, Mark Coyle, Anne-Marie McLaughlin, Ross Murphy 4 5 St James’ Hospital 6 St James street 7 Dublin 8 8 Ireland 9 10 Corresponding author 11 12 Dr Greg Murphy 13 St James’ Hospital 14 St James street 15 Dublin 8 16 Republic of Ireland 17 [email protected] 18 0000-0003-3671-3558 19 20 Abstract 21 22 Background 23 Obstructive sleep apnoea (OSA) is present in 40-80% of patients with cardiovascular morbidity and is 24 associated with adverse effects on cardiovascular health. Continuous positive airway pressure (CPAP) maintai 25 airway patency during sleep and is hypothesised to improve cardiac function. In the present study, we report o 26 the impact of 12 weeks of CPAP and improvements in echocardiographic parameters of the right ventricle (RV 27 28 Methods 29 Improvements of right ventricu 1 apnoea. Research Article 2 3 Greg Murphy, Gerard King, Pe 4 5 St James’ Hospital 6 St James street 7 Dublin 8 8 Ireland 9 10 Corresponding author 11 12 Dr Greg Murphy 13 St James’ Hospital 14 St James street 15 Dublin 8 16 Republic of Ireland 17 [email protected] 18 0000-0003-3671-3558 19 20 Abstract 21 22 Background 23 Obstructive sleep apnoea (OSA 24 associated with adverse effects 25 airway patency during sleep an 26 the impact of 12 weeks of CPA 27 28 29 1 1 Nineteen newly diagnosed patients with OSA and a respiratory disturbance index (RDI) greater than 10 were 30 enrolled. Echocardiography was performed before treatment and with a follow-up assessment after 12 weeks of 31 CPAP. Echocardiographic and Doppler measurements were made following the American Society for 32 Echocardiography guidelines. The primary outcome was isovolumetric acceleration (IVA). Secondary outcomes 33 include tricuspid annular plane systolic excursion (TAPSE), fractional area change (FAC), RV % strain, TEI 34 index and RV dimension index (RVDI). 35 36 Results 37 There significant improvement in isovolumetric acceleration of 0.5ms2 (P=0.0012 (95% CI -0.72, -0.20)) and 38 significant improvement of 2.05mm in TAPSE (p=0.0379 (95% CI -3.98 - -0.13). There was a trend to 39 improvements in FAC, RV % strain, TEI index or RVDI with twelve weeks of CPAP therapy. 40 41 Conclusion. 42 The present study highlights significant increase in TAPSE and IVA with 12 weeks of CPAP treatment and a 43 trend to improvement in FAC, RVDI and RV % strain. These data indicate favourable characteristics on both 44 load dependent and load independent markers of RV function with CPAP. 45 46 47 48 Keywords. 49 Obstructive sleep apnoea, Right heart, Echocardiography, CPAP 50 51 52 53 54 2 2 60 61 Background 62 63 Obstructive sleep apnoea (OSA) is a common condition characterised by intermittent airway obstruction and 64 apnoeic episodes during sleep. This results in fragmented nonrestorative sleep and significantly impacts 65 cardiovascular health [1]. The frequency among the general population is estimated at 2-7% and is 66 underdiagnosed in the clinical setting. It has been reported to be as high as 40-80% in those with a 67 cardiovascular comorbidity [2, 3]. 68 69 There are multiple physiological mechanisms through which OSA is associated with adverse cardiovascular 70 health outcomes. Firstly, arousal from sleep activates the sympathetic nervous system, increasing blood pressure 71 and heart rate [4]. Research Article Secondly, increased intrathoracic pressure increases afterload [5]. Thirdly, hypoxemia induces 72 tachycardia and may trigger dysrhythmias and demand-supply mismatch [5, 6]. By these mechanisms, OSA has 73 been proven to be strongly associated with cardiovascular comorbidities, including atrial fibrillation, heart 74 failure, pulmonary hypertension and myocardial infarction [7, 8]. Furthermore, Marin et al. demonstrated that 75 untreated severe OSA significantly increased cardiac mortality [9]. 76 77 The right heart acts as a conduit for the lungs and OSA has been associated with altered right heart physiology 78 on echocardiography [10]. However, right ventricular (RV) function remains a challenge to assess given the 79 position in the chest, complex anatomy and the interplay of contractility and load states. Due to these 80 complexities, the best marker for RV function in patients with OSA remains elusive. One meta-analysis of 82 81 60 60 61 Background 62 63 Obstructive sleep apnoea (OSA) is a common condition characterised by intermittent airway obstruction and 64 apnoeic episodes during sleep. This results in fragmented nonrestorative sleep and significantly impacts 65 cardiovascular health [1]. The frequency among the general population is estimated at 2-7% and is 66 underdiagnosed in the clinical setting. It has been reported to be as high as 40-80% in those with a 67 cardiovascular comorbidity [2, 3]. 68 69 There are multiple physiological mechanisms through which OSA is associated with adverse cardiovascular 70 health outcomes. Firstly, arousal from sleep activates the sympathetic nervous system, increasing blood pressure 71 and heart rate [4]. Secondly, increased intrathoracic pressure increases afterload [5]. Thirdly, hypoxemia induces 72 tachycardia and may trigger dysrhythmias and demand-supply mismatch [5, 6]. By these mechanisms, OSA has 73 been proven to be strongly associated with cardiovascular comorbidities, including atrial fibrillation, heart 74 failure, pulmonary hypertension and myocardial infarction [7, 8]. Furthermore, Marin et al. demonstrated that 75 untreated severe OSA significantly increased cardiac mortality [9]. 76 77 The right heart acts as a conduit for the lungs and OSA has been associated with altered right heart physiology 78 on echocardiography [10]. However, right ventricular (RV) function remains a challenge to assess given the 79 position in the chest, complex anatomy and the interplay of contractility and load states. Due to these 80 complexities, the best marker for RV function in patients with OSA remains elusive. Research Article One meta-analysis of 82 81 articles concluded a statistically significant negative impact on myocardial performance index (MPI), tricuspid 82 annular plane excursion (TAPSE), and FAC in patients who have OSA but did not collect data on isovolumetric 83 acceleration time (IVA) or on the impact of intervention with CPAP [11]. 84 85 In the present study, we present the echocardiographic and Doppler features of intervention with continuous 86 positive airway pressure (CPAP) on the mechanics and function of the right heart. The primary outcome is 87 isovolumetric acceleration time (IVA). Secondary outcomes include TAPSE, RV per cent strain, Fractional area 88 change (FAC) and RV dimension index. 89 The right heart acts as a conduit for the lungs and OSA has been associated with altered right heart physiology 78 on echocardiography [10]. However, right ventricular (RV) function remains a challenge to assess given the 79 position in the chest, complex anatomy and the interplay of contractility and load states. Due to these 80 complexities, the best marker for RV function in patients with OSA remains elusive. One meta-analysis of 82 81 articles concluded a statistically significant negative impact on myocardial performance index (MPI), tricuspid 82 annular plane excursion (TAPSE), and FAC in patients who have OSA but did not collect data on isovolumetric 83 acceleration time (IVA) or on the impact of intervention with CPAP [11]. 84 85 In the present study, we present the echocardiographic and Doppler features of intervention with continuous 86 positive airway pressure (CPAP) on the mechanics and function of the right heart. The primary outcome is 87 isovolumetric acceleration time (IVA). Secondary outcomes include TAPSE, RV per cent strain, Fractional area 88 change (FAC) and RV dimension index. 89 3 3 90 91 92 93 94 95 Methods 96 97 Study design 98 99 Nineteen newly diagnosed patients with OSA and a respiratory disturbance index (RDI) greater than 10 were 100 enrolled from St James’ hospital sleep clinic. All patients consented to receive CPAP therapy and to partake in 101 the study. Echocardiography was performed before treatment and with a follow-up assessment after 12 weeks of 102 CPAP. Conventional echocardiographic and Doppler measurements were made following the American Society 103 for Echocardiography guidelines. Patient characteristics can be seen in table 1. 104 At the echocardiographic examination, the subject’s height, weight, heart rate and blood pressure were recorded. Research Article 105 The local hospital ethics committee approved this study, and informed consent was obtained from all participants. 106 All echocardiographic recordings were obtained in digital format and stored for offline analysis. The offline 107 analysis of images was performed using commercially available software “Echo Pac V202 only” 2017- 2018 108 Vingmed Ultrasound on a personal computer workstation. 109 110 Outcomes 111 112 The primary outcome was isovolumetric acceleration (IVA). Myocardial acceleration during the isovolumic 113 phase of contraction (IVA) is a relatively load‐independent marker. 114 IVA was recorded using a 2 mm pulsed‐wave TDI sample volume placed at the annulus and in basal and mid‐ 115 ventricular segments in the apical four‐chamber view and averaged. RV myocardial velocities during peak 116 systole, early and late diastole were recorded and calculated (difference between baseline and peak myocardial 117 systolic velocities divided by the time interval from onset of the myocardial velocity during isovolumic 118 90 91 92 93 94 95 Methods 96 97 Study design 98 99 Nineteen newly diagnosed patients with OSA and a respiratory disturbance index (RDI) greater than 10 were 100 enrolled from St James’ hospital sleep clinic. All patients consented to receive CPAP therapy and to partake in 101 the study. Echocardiography was performed before treatment and with a follow-up assessment after 12 weeks of 102 CPAP. Conventional echocardiographic and Doppler measurements were made following the American Society 103 for Echocardiography guidelines. Patient characteristics can be seen in table 1. 104 At the echocardiographic examination, the subject’s height, weight, heart rate and blood pressure were recorded. 105 The local hospital ethics committee approved this study, and informed consent was obtained from all participants. 106 All echocardiographic recordings were obtained in digital format and stored for offline analysis. The offline 107 analysis of images was performed using commercially available software “Echo Pac V202 only” 2017- 2018 108 4 4 contraction to the time at peak velocity of this wave). The potential effect of respiration was minimised by 119 averaging multiple consecutive beats. 120 121 Secondary outcomes included: 122 1. Tricuspid annular plane systolic excursion (TAPSE), 123 2. Right ventricle dimension size at the tricuspid annulus (RVDI), 124 3. TEI index, 125 4. fractional area change (FAC), 126 5. Research Article right ventricle per cent strain (RV %), 127 128 TAPSE 129 TAPSE was obtained by placing the M-mode line at the lateral tricuspid valve annulus, obtaining an M-mode 130 tracing and measuring the height of the annulus movement during systole. 131 132 TEI index 133 calculated as isovolumic relaxation time plus isovolumic contraction time divided by ejection time. 134 135 Fractional area change 136 Fractional area change was calculated in the apical four-chamber view as the difference in the end-diastolic area 137 and the end-systolic area divided by the end-diastolic area. 138 139 Right ventricle per cent strain 140 RV per cent strain was performed on the apical four-chamber view. We evaluated the average value of the peak 141 systolic strain from all segments of the free wall and septal wall of the RV in the apical four-chamber view, 142 focused on the RV. 143 144 Quality: 145 Studies were completed by a qualified cardiac physiologist and stored offline. The two-dimensional longitudinal 146 strain was measured using 2D deformation imaging (i.e. The per cent systolic deformation relative to the diastolic 147 value) analysis software. Activation of the automatic ROI tracking mode ensured that measurements reflected the 148 5 motion of myocardial tissue segments throughout the cardiac cycle. Images were acquired at an 80s‾1 frame rate 149 and stored digitally on a hard disc for offline analysis by speckle methodology. Not all outcomes could be 150 measured in all studies due to patient factors and image quality. 151 152 153 154 Statistical analysis: 155 Data was blinded after collection and analysed in Microsoft Excel version 16.6. Mean standard deviations were 156 collected. Statistical analysis was performed using the student’s T-test with an alpha of 0.05 using SPSS version 157 27. 158 159 160 161 162 163 164 165 166 167 168 169 170 171 172 173 Results 174 175 Patient Characteristics 176 177 6 6 Patient characteristics can be seen in table 1. Nineteen patients consented to partake in the study. There was a 178 male gender majority (N=17) with a mean BMI in the obese range. Other risk factors are seen in table 1 below. 179 180 Patient characteristics can be seen in table 1. Nineteen patients consented to partake in the study. There was a 178 male gender majority (N=17) with a mean BMI in the obese range. Other risk factors are seen in table 1 below. Research Article 179 180 Patient characteristics can be seen in table 1. Nineteen patients consented to partake in the study. There was a 178 male gender majority (N=17) with a mean BMI in the obese range. Other risk factors are seen in table 1 below. 179 180 N=19 Age 52 yrs. (8) Male gender 70% (N=14) Current smoker 14% Ex-smoker 31% Hypertension 46% Systolic BP 129.3 (20.1) Diastolic BP 88(12.6) ODI (pre) 25(14) ODI (Post) 4.2(2) Epworth score (Pre) 25(14) Epworth score (post) 7(4) Neck Circumference 40cm (2.1) Ejection fraction 65% (8) BMI 32(6) Table 1. Characteristics of participants 181 (SD) = standard deviation 182 ODI = Oxygen desaturation index 183 BMI = Body mass index 184 185 186 187 188 189 190 191 Isovolumetric acceleration. 192 N=19 Age 52 yrs. (8) Male gender 70% (N=14) Current smoker 14% Ex-smoker 31% Hypertension 46% Systolic BP 129.3 (20.1) Diastolic BP 88(12.6) ODI (pre) 25(14) ODI (Post) 4.2(2) Epworth score (Pre) 25(14) Epworth score (post) 7(4) Neck Circumference 40cm (2.1) Ejection fraction 65% (8) BMI 32(6) Table 1. Characteristics of participants 181 (SD) = standard deviation 182 ODI = Oxygen desaturation index 183 BMI = Body mass index 184 185 186 187 188 189 190 191 Isovolumetric acceleration. 192 7 7 193 193 IVA was reported on all included patients in the study (N=19). On diagnosis, the mean was 2.5+/- 1 MS2. After 194 12 weeks of CPAP, the mean was 3 +/- 1.1 MS2 P <0.0012 (95% CI -0.72 - -0.20MS2). There was a significant 195 improvement in IVA with the adaption of CPAP (Figure 1). 196 IVA was reported on all included patients in the study (N=19). On diagnosis, the mean was 2.5+/- 1 MS2. After 194 12 weeks of CPAP, the mean was 3 +/- 1.1 MS2 P <0.0012 (95% CI -0.72 - -0.20MS2). There was a significant 195 improvement in IVA with the adaption of CPAP (Figure 1). 196 197 198 Figure 1. Isovolumetric acceleration (MS2) with pre-CPAP (blue) and post CPAP (Orange) 199 200 198 Figure 1. Isovolumetric acceleration (MS2) with pre-CPAP (blue) and post CPAP (Orange) 199 98 Figure 1. Isovolumetric acceleration (MS2) with pre-CPAP (blue) and post CPAP (Orange) 199 8 8 212 Figure 2. TAPSE in mm with pre-CPAP (blue) and post-CPAP (Orange). 213 214 212 Figure 2. TAPSE in mm with pre-CPAP (blue) and post-CPAP (Orange). 213 212 Figure 2. Research Article TAPSE in mm with pre-CPAP (blue) and post-CPAP (Orange). 213 214 215 216 217 218 RVDI 219 220 RVDI was recorded for all patients in the study. Before, CPAP measurement was 33.16mm SD 4.74, and the 221 post-intervention measurement was 32.37mm. The difference was non-significant (P = 0.3986 (95% CI -1.13 – 222 2.71)) (Figure 3). 223 224 225 Figure 3 – RVDI in mm with pre-CPAP (blue) and post-CPAP (Orange). 226 227 212 Figure 2. TAPSE in mm with pre-CPAP (blue) and post-CPAP (Orange). 213 214 215 216 217 218 RVDI 219 220 RVDI was recorded for all patients in the study. Before, CPAP measurement was 33.16mm SD 4.74, and the 221 post-intervention measurement was 32.37mm. The difference was non-significant (P = 0.3986 (95% CI -1.13 – 222 2.71)) (Figure 3). 223 224 225 Figure 3 – RVDI in mm with pre-CPAP (blue) and post-CPAP (Orange). 226 227 Figure 2. TAPSE in mm with pre-CPAP (blue) and post-CPAP (Orange). 213 224 225 Figure 3 – RVDI in mm with pre-CPAP (blue) and post-CPAP (Orange). 226 227 9 9 228 229 230 231 FAC 232 233 Fractional area change was analysed in 18 patients, with 44.17% (9.17%) and 48.39% (7.45% 234 improvement was non-significant (P=0.1109 (95% CI -9.52 -1.08)) (Figure 4). 235 236 Figure 4 - Fractional area change (%) with pre-CPAP (blue) and post-CPAP (Orange). 237 238 239 240 241 242 243 244 245 TEI index 246 The Tei index was 0.46 (0.13) pre and 0.5 (0.11) post-intervention. Indicating a non-signific 247 TEI (P = 0.4527 95% CI -0.11 - -0.05)(figure 5). 248 249 250 228 229 230 231 FAC 232 233 Fractional area change was analysed in 18 patients, with 44.17% (9.17%) and 48.39% (7.45%) po 234 improvement was non-significant (P=0.1109 (95% CI -9.52 -1.08)) (Figure 4). 235 236 Figure 4 - Fractional area change (%) with pre-CPAP (blue) and post-CPAP (Orange). 237 238 228 229 230 231 FAC 232 233 Fractional area change was analysed in 18 patients, with 44.17% (9.17%) and 48.39% (7.45%) post. Th 234 improvement was non-significant (P=0.1109 (95% CI -9.52 -1.08)) (Figure 4). 235 236 Figure 4 - Fractional area change (%) with pre-CPAP (blue) and post-CPAP (Orange). 237 238 236 Figure 4 - Fractional area change (%) with pre CPAP (blue) and post CPAP (Orange) 237 236 Figure 4 - Fractional area change (%) with pre-CPAP (blue) and post-CPAP (Orange). Research Article 237 238 6 Figure 4 - Fractional area change (%) with pre-CPAP (blue) and post-CPAP (Orange). 237 10 251 Figure 5 – TEI index with pre-CPAP (blue) and post-CPAP (Orange). 252 253 254 RV % strain 255 256 RV per cent strain was analysed in 15 patients. Mean pre intervention strain was 20.56(3.58), post intervention 257 mean 21.11(4.87). The RV per cent strain improvement did not reach significance 0.7370 (95% CI-3.99 – 258 2.88)(Figure 6). 259 260 261 Figure 6 – RV % strain with pre-CPAP (blue) and post-CPAP (Orange). 262 263 264 251 Figure 5 – TEI index with pre-CPAP (blue) and post-CPAP (Orange). 252 251 Figure 5 – TEI index with pre-CPAP (blue) and post-CPAP (Orange). 252 253 254 RV % strain 255 256 RV per cent strain was analysed in 15 patients. Mean pre intervention strain was 20.56(3.58), post interventio 257 mean 21.11(4.87). The RV per cent strain improvement did not reach significance 0.7370 (95% CI-3.99 – 258 2.88)(Figure 6). 259 260 251 Figure 5 – TEI index with pre-CPAP (blue) and post-CPAP (Orange). 252 Figure 5 – TEI index with pre-CPAP (blue) and post-CPAP (Orange). 252 260 260 261 Figure 6 – RV % strain with pre-CPAP (blue) and post-CPAP (Orange). 262 263 260 261 Figure 6 – RV % strain with pre-CPAP (blue) and post-CPAP (Orange). 262 11 11 On Diagnosis After 12 weeks P-Value On Diagnosis After 12 weeks P-Value On Diagnosis After 12 weeks P-Value IVA N=19 2.5± 1 ms2 3.0 +/- 1.1 ms2 <0.0012 (95% CI -0.72 - - 0.20) TAPSE N=19 25.37mm(4.42) 27.42mm (3.91) P= 0.0379 (95% CI -3.98– -0.13) RV % strain N=15 20.56%(3.58) 21.11%(4.87) P= 0.7370 (95% CI-3.99 – 2.88) Fractional area change N=18 44.17%(9.17) 48.39%(7.45) P= 0.1109 (95% CI -9.52 – 1-08) RVDI N=19 33.16mm(4.74) 32.37mm(4.61) P= 0.3986 (95% CI -1.13 – 2.71) Tei index N=18 0.46 (0.13) 0.50 (0.11) P= 0.4001(95% CI -0.11. – 0.05) Table 2 -Interval change in RV function with the use of CPAP in patients with OSA. Research Article 0 *Paired T-test 1 On Diagnosis After 12 weeks P-Value IVA N=19 2.5± 1 ms2 3.0 +/- 1.1 ms2 <0.0012 (95% CI -0.72 - - 0.20) TAPSE N=19 25.37mm(4.42) 27.42mm (3.91) P= 0.0379 (95% CI -3.98– -0.13) RV % strain N=15 20.56%(3.58) 21.11%(4.87) P= 0.7370 (95% CI-3.99 – 2.88) Fractional area change N=18 44.17%(9.17) 48.39%(7.45) P= 0.1109 (95% CI -9.52 – 1-08) RVDI N=19 33.16mm(4.74) 32.37mm(4.61) P= 0.3986 (95% CI -1.13 – 2.71) Tei index N=18 0.46 (0.13) 0.50 (0.11) P= 0.4001(95% CI -0.11. – 0.05) Table 2 -Interval change in RV function with the use of CPAP in patients with OSA. 270 *Paired T-test. 271 272 273 274 275 276 Discussion 277 278 The present study highlights subclinical changes in right ventricular mechanics with the application of CPAP 279 therapy in OSA. There was a significant improvement in IVA and TAPSE with twelve weeks of treatment. 280 There was a favourable trend in RV % strain, FAC, TEI index and RVDI (Table 2). 281 12 282 IVA is a sensitive measure of contractility as a load independent marker of right heart function [12-14]. Arias et 283 al. studied the isovolumetric phase of the left ventricle and demonstrated an improved isovolumetric relaxation 284 time when patients were initiated on CPAP; however they did not report on IVA. They concluded that pressure 285 overload from OSA impacts myocardial relaxation, and by offloading the ventricle with CPAP, myocardial 286 mechanics can improve [15]. The isovolumetric phase has also been studied in other cardiac conditions. Ernande 287 et al. showed that isovolumic contraction peak velocity at the tricuspid annulus >9cm/sec was an independent 288 marker for death in pulmonary hypertension patients [16]. Furthermore, isovolumetric contraction time is an 289 independent subclinical risk factor for heart failure in the general population [17]. To our knowledge, this is the 290 first study of right ventricular IVA in an OSA cohort. 291 Conversely, TAPSE is a highly load dependent marker of RV systolic function and demonstrates a significant 293 improvement with 12 weeks of CPAP (P <0.05). Reduced TAPSE is associated with adverse outcomes in 294 patients with heart failure and is an independent marker of mortality in aortic stenosis [18, 19]. We hypothesise 295 that the significant improvement is likely due to the reduced afterload of CPAP therapy. Research Article 296 Conversely, TAPSE is a highly load dependent marker of RV systolic function and demonstrates a significant 293 improvement with 12 weeks of CPAP (P <0.05). Reduced TAPSE is associated with adverse outcomes in 294 patients with heart failure and is an independent marker of mortality in aortic stenosis [18, 19]. We hypothesise 295 that the significant improvement is likely due to the reduced afterload of CPAP therapy. 296 Conversely, TAPSE is a highly load dependent marker of RV systolic function and demonstrates a significant 293 improvement with 12 weeks of CPAP (P <0.05). Reduced TAPSE is associated with adverse outcomes in 294 patients with heart failure and is an independent marker of mortality in aortic stenosis [18, 19]. We hypothesise 295 that the significant improvement is likely due to the reduced afterload of CPAP therapy. 296 Conversely, TAPSE is a highly load dependent marker of RV systolic function and demonstrates a significant 293 improvement with 12 weeks of CPAP (P <0.05). Reduced TAPSE is associated with adverse outcomes in 294 patients with heart failure and is an independent marker of mortality in aortic stenosis [18, 19]. We hypothesise 295 that the significant improvement is likely due to the reduced afterload of CPAP therapy. 296 Secondary outcomes, including fractional area change, RVDI and RV per cent strain demonstrated a favourable 297 trend to improvement in function. TEI index, a marker of both systolic and diastolic function and an 298 independent marker of mortality, did not show significant improvement [20]. 299 300 The data on RV mechanics with OSA is modest compared to studies of the left ventricle. A meta-analysis by Lu 301 et al. demonstrated OSA to confer a significant reduction in TAPSE, MPI and RV FAC. The Wisconsin sleep 302 study followed patients with OSA for a mean duration of 18 years and did not report a significant change in 303 FAC in patients with OSA [21]. Both of these studies reported on the natural history of sleep apnoea and did not 304 report on the impact of intervention with CPAP. Karamanzias et al. studied echocardiograms after one year of 305 CPAP and found a significant improvement in TAPSE and no significant difference in RV diameter, which is 306 consistent with our findings [22]. 307 308 Speckle tracking and strain imaging allows for subclinical myocardial dysfunction to be identified. Research Article In contrast to 309 our findings, Tadic et al performed a meta-analysis of 337 patients and found a significant improvement in RV 310 Secondary outcomes, including fractional area change, RVDI and RV per cent strain demonstrated a favourable 297 trend to improvement in function. TEI index, a marker of both systolic and diastolic function and an 298 independent marker of mortality, did not show significant improvement [20]. 299 The data on RV mechanics with OSA is modest compared to studies of the left ventricle. A meta-analysis by Lu 301 et al. demonstrated OSA to confer a significant reduction in TAPSE, MPI and RV FAC. The Wisconsin sleep 302 study followed patients with OSA for a mean duration of 18 years and did not report a significant change in 303 FAC in patients with OSA [21]. Both of these studies reported on the natural history of sleep apnoea and did not 304 report on the impact of intervention with CPAP. Karamanzias et al. studied echocardiograms after one year of 305 CPAP and found a significant improvement in TAPSE and no significant difference in RV diameter, which is 306 consistent with our findings [22]. 307 The data on RV mechanics with OSA is modest compared to studies of the left ventricle. A meta-analysis by Lu 301 et al. demonstrated OSA to confer a significant reduction in TAPSE, MPI and RV FAC. The Wisconsin sleep 302 study followed patients with OSA for a mean duration of 18 years and did not report a significant change in 303 FAC in patients with OSA [21]. Both of these studies reported on the natural history of sleep apnoea and did not 304 report on the impact of intervention with CPAP. Karamanzias et al. studied echocardiograms after one year of 305 CPAP and found a significant improvement in TAPSE and no significant difference in RV diameter, which is 306 consistent with our findings [22]. 307 Speckle tracking and strain imaging allows for subclinical myocardial dysfunction to be identified. In contrast to 309 our findings, Tadic et al performed a meta-analysis of 337 patients and found a significant improvement in RV 310 Speckle tracking and strain imaging allows for subclinical myocardial dysfunction to be identified. Research Article In contrast to 309 our findings, Tadic et al performed a meta-analysis of 337 patients and found a significant improvement in RV 310 13 global longitudinal strain (GLS) with CPAP treatment (0.28±0.07, CI 0.15-0.42, p < .0001), highlighting 311 subclinical improvement in function [23]. 312 313 The present study demonstrates significant improvements in TAPSE and IVA, highlighting improved 314 myocardial mechanics across both load dependent and load independent markers of RV function when CPAP is 315 used. These findings support greater screening and use of CPAP in this cohort; however; further data is needed 316 on RV echo parameters in patients on CPAP. 317 318 319 320 Limitations 321 322 A number of limitations should be addressed. Firstly the total number of included patients is modest. Secondly, 323 some RV parameters could not be collected due to patient size and difficulty imaging the right ventricle. Thirdly 324 long-term follow-up data is needed. Fourthly CPAP treatment is contingent on patient adherence, and low 325 uptake has been well described in the literature [24]. 326 327 328 329 Conclusion 330 331 The present study highlights a significant improvement in TAPSE and IVA with CPAP treatment with a positive 332 trend in FAC, TEI index, RVDI and RV % strain. These data indicate favourable characteristics with treatment 333 and adherence to CPAP; however, more data is needed on right heart echocardiographic and Doppler mechanics 334 and the clinical implications of different RV function markers. 335 336 337 1. Arnaud, C., et al., Obstructive sleep apnoea and cardiovascular consequences: 338 Pathophysiological mechanisms. Arch Cardiovasc Dis, 2020. 113(5): p. 350-358. 339 14 2. Punjabi, N.M., The epidemiology of adult obstructive sleep apnea. Proc Am Thorac 340 Soc, 2008. 5(2): p. 136-43. 341 3. Javaheri, S., et al., Sleep apnea: types, mechanisms, and clinical cardiovascular 342 consequences. Journal of the American College of Cardiology, 2017. 69(7): p. 841- 343 858. 344 4. Wszedybyl-Winklewska, M., et al., Central sympathetic nervous system 345 reinforcement in obstructive sleep apnoea. Sleep Med Rev, 2018. 39: p. 143-154. 346 5. Mansukhani, M.P., S. Wang, and V.K. Somers, Chemoreflex physiology and 347 implications for sleep apnoea: insights from studies in humans. Experimental 348 Physiology, 2015. 100(2): p. 130-135. 349 6. Geovanini, G.R. and G. Lorenzi-Filho, Cardiac rhythm disorders in obstructive sleep 350 apnea. J Thorac Dis, 2018. 10(Suppl 34): p. S4221-s4230. 351 7. Peker, Y., J. Carlson, and J. Research Article Hedner, Increased incidence of coronary artery disease in 352 sleep apnoea: a long-term follow-up. Eur Respir J, 2006. 28(3): p. 596-602. 353 8. Peppard, P.E., et al., Prospective study of the association between sleep-disordered 354 breathing and hypertension. N Engl J Med, 2000. 342(19): p. 1378-84. 355 9. Marin, J.M., et al., Long-term cardiovascular outcomes in men with obstructive sleep 356 apnoea-hypopnoea with or without treatment with continuous positive airway 357 pressure: an observational study. Lancet, 2005. 365(9464): p. 1046-53. 358 10. Sanner, B., et al., Right ventricular dysfunction in patients with obstructive sleep 359 apnoea syndrome. European Respiratory Journal, 1997. 10(9): p. 2079-2083. 360 11. Lu, M., et al., Obstructive sleep apnea increases the risk of cardiovascular damage: a 361 systematic review and meta-analysis of imaging studies. Systematic Reviews, 2021. 362 10(1): p. 212. 363 2. Punjabi, N.M., The epidemiology of adult obstructive sleep apnea. Proc Am Thorac 340 Soc, 2008. 5(2): p. 136-43. 341 3. Javaheri, S., et al., Sleep apnea: types, mechanisms, and clinical cardiovascular 342 consequences. Journal of the American College of Cardiology, 2017. 69(7): p. 841- 343 858. 344 4. Wszedybyl-Winklewska, M., et al., Central sympathetic nervous system 345 reinforcement in obstructive sleep apnoea. Sleep Med Rev, 2018. 39: p. 143-154. 346 5. Mansukhani, M.P., S. Wang, and V.K. Somers, Chemoreflex physiology and 347 implications for sleep apnoea: insights from studies in humans. Experimental 348 Physiology, 2015. 100(2): p. 130-135. 349 6. Geovanini, G.R. and G. Lorenzi-Filho, Cardiac rhythm disorders in obstructive sleep 350 6. Geovanini, G.R. and G. Lorenzi-Filho, Cardiac rhythm disorders in obstructive sleep 350 apnea. J Thorac Dis, 2018. 10(Suppl 34): p. S4221-s4230. 351 7. Peker, Y., J. Carlson, and J. Hedner, Increased incidence of coronary artery disease in 352 sleep apnoea: a long-term follow-up. Eur Respir J, 2006. 28(3): p. 596-602. 353 8. Peppard, P.E., et al., Prospective study of the association between sleep-disordered 354 breathing and hypertension. N Engl J Med, 2000. 342(19): p. 1378-84. 355 10. Sanner, B., et al., Right ventricular dysfunction in patients with obstructive sleep 359 apnoea syndrome. European Respiratory Journal, 1997. 10(9): p. 2079-2083. 360 11. Lu, M., et al., Obstructive sleep apnea increases the risk of cardiovascular damage: a 361 systematic review and meta-analysis of imaging studies. Systematic Reviews, 2021. 362 10(1): p. 212. 363 15 12. Research Article Vogel, M., et al., Validation of Myocardial Acceleration During Isovolumic Contraction 364 as a Novel Noninvasive Index of Right Ventricular Contractility. Circulation, 2002. 365 105(14): p. 1693-1699. 366 13. Meluzín, J., et al., Pulsed Doppler tissue imaging of the velocity of tricuspid annular 367 systolic motion; a new, rapid, and non-invasive method of evaluating right 368 ventricular systolic function. Eur Heart J, 2001. 22(4): p. 340-8. 369 14. Lindqvist, P., et al., The use of isovolumic contraction velocity to determine right 370 ventricular state of contractility and filling pressures: A pulsed Doppler tissue imaging 371 study. European Journal of Echocardiography, 2005. 6(4): p. 264-270. 372 15. Arias, M.A., et al., Obstructive Sleep Apnea Syndrome Affects Left Ventricular 373 Diastolic Function. Circulation, 2005. 112(3): p. 375-383. 374 16. Ernande, L., et al., Right Isovolumic Contraction Velocity Predicts Survival in 375 Pulmonary Hypertension. Journal of the American Society of Echocardiography, 376 2013. 26(3): p. 297-306. 377 17. Alhakak, A.S., et al., The cardiac isovolumetric contraction time is an independent 378 predictor of incident heart failure in the general population. International Journal of 379 Cardiology, 2020. 312: p. 81-86. 380 18. Damy, T., et al., Prevalence of, Associations With, and Prognostic Value of Tricuspid 381 Annular Plane Systolic Excursion (TAPSE) Among Out-Patients Referred for the 382 Evaluation of Heart Failure. Journal of Cardiac Failure, 2012. 18(3): p. 216-225. 383 19. Guazzi, M., et al., RV Contractile Function and its Coupling to Pulmonary Circulation 384 in Heart Failure With Preserved Ejection Fraction. JACC: Cardiovascular Imaging, 385 2017. 10(10_Part_B): p. 1211-1221. 386 13. Meluzín, J., et al., Pulsed Doppler tissue imaging of the velocity of tricuspid annular 367 systolic motion; a new, rapid, and non-invasive method of evaluating right 368 ventricular systolic function. Eur Heart J, 2001. 22(4): p. 340-8. 369 14. Lindqvist, P., et al., The use of isovolumic contraction velocity to determine right 370 ventricular state of contractility and filling pressures: A pulsed Doppler tissue imaging 371 study. European Journal of Echocardiography, 2005. 6(4): p. 264-270. 372 15. Arias, M.A., et al., Obstructive Sleep Apnea Syndrome Affects Left Ventricular 373 Diastolic Function. Circulation, 2005. 112(3): p. 375-383. 374 16. Ernande, L., et al., Right Isovolumic Contraction Velocity Predicts Survival in 375 15. Arias, M.A., et al., Obstructive Sleep Apnea Syndrome Affects Left Ventricular 373 Diastolic Function. Circulation, 2005. 112(3): p. 375-383. 374 16. Research Article Ernande, L., et al., Right Isovolumic Contraction Velocity Predicts Survival in 375 Pulmonary Hypertension. Journal of the American Society of Echocardiography, 376 2013. 26(3): p. 297-306. 377 17. Alhakak, A.S., et al., The cardiac isovolumetric contraction time is an independent 378 predictor of incident heart failure in the general population. International Journal of 379 Cardiology, 2020. 312: p. 81-86. 380 16 20. Rodríguez-Arias, J.J., L. Ortega-Paz, and S. Brugaletta, Durable polymer everolimus- 387 eluting stents: history, current status and future prospects. Expert Review of Medical 388 Devices, 2020. 17(7): p. 671-682. 389 21. Korcarz, C.E., et al., Effects of Obstructive Sleep Apnea and Obesity on Cardiac 390 Remodeling: The Wisconsin Sleep Cohort Study. Sleep, 2016. 39(6): p. 1187-95. 391 22. Karamanzanis, G., et al., Impact of continuous positive airway pressure treatment on 392 myocardial performance in patients with obstructive sleep apnea. A conventional and 393 tissue Doppler echocardiographic study. Sleep and Breathing, 2015. 19(1): p. 343- 394 350. 395 23. Tadic, M., et al., The impact of continuous positive airway pressure on cardiac 396 mechanics: Findings from a meta-analysis of echocardiographic studies. The Journal 397 of Clinical Hypertension, 2022. 24(7): p. 795-803. 398 24. Rotenberg, B.W., D. Murariu, and K.P. Pang, Trends in CPAP adherence over twenty 399 years of data collection: a flattened curve. J Otolaryngol Head Neck Surg, 2016. 45(1): 400 p. 43. 401 402 403 404 405 406 Declarations 407 408 The authors declare that no funds, grants, or other support were received during the preparation of this 409 manuscript. The authors have no relevant financial or non-financial interests to disclose. 410 All authors contributed to the study conception and design. Material preparation, data collection and analysis 411 were performed by Gerard King, Peter Coss. The first draft of the manuscript was written by Greg Murphy, 412 20. Rodríguez-Arias, J.J., L. Ortega-Paz, and S. Brugaletta, Durable polymer everolimus- 387 eluting stents: history, current status and future prospects. Expert Review of Medical 388 Devices, 2020. 17(7): p. 671-682. 389 All authors contributed to the study conception and design. Material preparation, data collection and analysis 411 were performed by Gerard King, Peter Coss. The first draft of the manuscript was written by Greg Murphy, 412 17 17 Mark Coyle, Ross Murphy. All authors commented on previous versions of the manuscript. Research Article All authors read and 413 approved the final manuscript 414 This study was performed in line with the principles of the Declaration of Helsinki. Approval was granted by the 415 Ethics Committee of St James Hospital, Dublin 8, Ireland. Informed consent was obtained from all individual 416 participants included in the study and consent to publish 417 418 419 Data Availability 420 421 The data that support the findings of this study are not openly available due to reasons of sensitivity and are 422 available from the corresponding author upon reasonable request. Data are located in controlled access data 423 storage at St James Hospital. 424 18

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1981-2014: os cem números da revista novos estudos

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1981-2014: OS CEM NÚMEROS DA REVISTA NOVOS ESTUDOS Paula Montero Nesta edição de novembro de 2014 estamos cele- brando, com grande alegria, o centésimo número da revista Novos Es- tudos. Foram, até o momento, 33 anos de trabalho ininterrupto e de esforço pródigo de muitos editores e autores. Nesse sentido podemos nos congratular de que a “petição de fé” de Rodrigo Naves em 1991, quando, no número 30, a revista completava seus primeiros dez anos de duração – a de não poupar esforços para manter vivo o projeto edi- torial da Novos Estudos em um país em que tudo é demasiadamente volátil –, tenha produzido resultados. Foram muitos os editores que se empenharam sucessivamente para garantir a qualidade e a regularidade da publicação: Perseu Abra- mo, Juarez Brandão Lopes, Francisco de Oliveira, Rodrigo Naves, Al- varo Comin, Omar Ribeiro Thomaz, Antônio Flávio Pierucci, Flávio Moura e Joaquim Toledo. Cada um deles contribuiu a seu modo para que hoje pudéssemos celebrar nosso centésimo número. Também fizeram sua parte os pesquisadores do Cebrap, que emprestam sua energia intelectual à revista, garantindo sua vitalidade, seriedade e profundidade de análise e reflexão crítica. É preciso reconhecer, ainda, que essa longevidade se deveu também, em grande parte, à generosa contribuição da Fundação Carlos Chagas, que desde 2005 tem apoia- do integralmente o projeto editorial da Novos Estudos, permitindo que a revista enriquecesse, sob a batuta de Flávio Moura, a linguagem visual de seu projeto gráfico. NOVOS ESTUDOS 100 ❙❙ NOVEMBRO 2014 5 Quando considerada em seu conjunto, pode-se ter uma boa pers- pectiva do valor da coleção e de sua transformação ao longo destas últimas décadas. Quanto a seu valor, Novos Estudos fala por si. Defi- nindo-se como um periódico voltado para a cultura e a reflexão crítica, manteve-se sintonizada com as grandes questões do momento nas áreas de política, economia, literatura, ciências humanas e artes e, des- de os primeiros números, serviu de veículo para a divulgação de ideias de grandes pensadores e intelectuais brasileiros e estrangeiros. 6 1981-2014: OS CEM NÚMEROS DA REVISTA NOVOS ESTUDOS ❙❙ Paula Montero 1981-2014: OS CEM NÚMEROS DA REVISTA NOVOS ESTUDOS Sem poder enumerá-los todos nesta curta introdução, poderíamos men- cionar, a título de exemplo, a colaboração internacional de Raúl Pre- bisch nos anos 1980, debatendo candentes questões sobre o modelo econômico da América Latina; o dossiê de Jürgen Habermas de 1987, que marcou época; o dossiê sobre globalização de 1997, que apresen- tou ao público brasileiro autores como Arif Dirlik, Arjun Appadurai e Jean Comaroff; os trabalhos de Fréderic Jameson e Perry Anderson sobre modernidade e pós-modernismo; a contribuição de Adam Przeworski na década de 2000 sobre as relações entre instituições e desenvolvimento; e o dossiê sobre segurança pública de 2008, com uma contribuição inédita de Loïc Wacquant. Embora a revista tenha sempre se transformado ao longo das dé- cadas, adaptando sua linguagem e as linhas editorias segundo o perfil e a preferência dos sucessivos editores, e ao contexto social e político do momento, parece-nos que, ainda assim, é possível assinalar três momentos distintos em sua existência, que podem ser diferenciados pelas mudanças no seu projeto gráfico. O primeiro deles vai de sua fundação, em 1981, até 1989. Nos primeiros dezessete números a re- vista tinha um estilo, quando comparado a seu formato atual, aparen- temente mais artesanal. Com cadernos grampeados de mais ou menos setenta páginas, a publicação já nasce com a convicção da importância da ilustração como linguagem editorial de amplo alcance. Capa de Eli- fas Andreato no primeiro número, ilustrações de Rubens Grilo, Mário Cafiero, fotos, artigos acompanhados de diversas ilustrações e títulos inseridos segundo a imaginação com variedade de tamanhos e diver- sidade de fontes tipográficas. Tomados em seu conjunto, fica bastante nítido que esses números são um espelho do modo como os edito- res e pesquisadores do Cebrap percebiam a conjuntura e as questões mais candentes a ser discutidas. O número de setembro de 1987, que traz o dossiê Habermas, se apresenta, a meu ver, como um marcador importante de mudança na trajetória da revista. Sempre buscando o estreito equilíbrio entre o conhecimento acadêmico, o debate político e a linguagem mais jornalística dos cadernos de cultura dos jornais diários, Novos Estudos se repagina, cresce em número de páginas, ga- nhando um formato mais próximo de um livro, de modo a adaptar-se, segundo seu editor de então, Rodrigo Naves, às necessidades de sua 6 1981-2014: OS CEM NÚMEROS DA REVISTA NOVOS ESTUDOS ❙❙ Paula Montero comercialização. Paula Montero é editora de Novos Estudos. 1981-2014: OS CEM NÚMEROS DA REVISTA NOVOS ESTUDOS A partir desse ano o projeto gráfico se consolida em um padrão que permanece relativamente estável até o presente. Por dezesseis anos o artista Carlos Fajardo ilustrou, com suas cores e seu estilo geométrico, as capas de todas as edições. A partir de 2003, com a chegada de Flávio Moura como novo editor, pode-se perceber a passa- gem para um terceiro momento. O número 70, de dezembro de 2004, pode ser considerado uma boa amostra dessa nova fase. Com apoio do Banco Itaú, a revista enriqueceu seus recursos gráficos introduzindo ensaios visuais de diferentes artistas. Nas palavras de seu editor, a nova disposição gráfica tornou as páginas mais arejadas, valorizou o forma- to das resenhas e refinou o diálogo dos artigos com as artes visuais. Além disso, o número 70 trouxe um índice remissivo completo das mais de cinco centenas de autores brasileiros e estrangeiros que até então haviam colaborado com a revista. Outros desafios se apresentam no momento em que a Novos Estudos chega a seu centésimo número. O campo das revistas científicas mul- tiplicou aos milhares suas publicações, padronizou suas exigências e definiu parâmetros mais estritos de avaliação da qualidade. Manter a personalidade editorial da Novos Estudos, cujo projeto se demarcou pelo desejo de manter o difícil equilíbrio entre a complexidade do co- nhecimento sério e a leveza de uma linguagem agradável e mais aces- sível, exigirá de nós novas habilidades. Parece-nos que, quando visto em perspectiva, o número 100 baliza o período “clássico” da história da Novos Estudos, no qual cada volume é concebido e apresentado como uma unidade material relativamente autônoma e consagrado pela lin- guagem visual que o personaliza. Quisemos marcar esse momento convidando Carlos Fajardo, que tanto contribuiu para a identidade visual da revista, para ilustrar este centésimo número; aos pesquisa- dores e colaboradores do Cebrap, propusemos que contribuíssem ofe- recendo-nos sua percepção analítica das questões mais candentes da atualidade. É preciso ter em conta, no entanto, que, em uma época de hiperco- nectividade, o formato atual da revista não parece mais ser suficiente para manter sua identidade e atrair novos leitores. O desafio a enfren- tar será o de avançar na direção de um projeto editorial que leve em conta a diversidade de plataformas e linguagens nas quais circulam hoje o conhecimento e a informação. NOVOS ESTUDOS 100 ❙❙ NOVEMBRO 2014

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English

Inferring direct regulatory targets from expression and genome location analyses: a comparison of transcription factor deletion and overexpression

BMC genomics

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BioMed Central BioMed Central Open Acc Research article Inferring direct regulatory targets from expression and genome location analyses: a comparison of transcription factor deletion and overexpression p Lin Tang1,3, Xiao Liu1,4 and Neil D Clarke*1,2 Address: 1Biophysics and Biophysical Chemistry, Johns Hopkins School of Medicine, Baltimore, MD, USA, 2Genome Institute of Singapore, Singapore, 3AviaraDX Inc., 2715 Locker West, Carlsbad, CA, USA and 4Developmental Biology, Stanford University School of Medicine, Palo Alto Address: 1Biophysics and Biophysical Chemistry, Johns Hopkins School of Medicine, Baltimore, MD, USA, 2Genome Institute of Singapore, Singapore, 3AviaraDX Inc., 2715 Locker West, Carlsbad, CA, USA and 4Developmental Biology, Stanford University School of Medicine, Palo Alto, CA, USA mail: Lin Tang - [email protected]; Xiao Liu - [email protected]; Neil D Clarke* - [email protected] * Corresponding author Corresponding author Received: 18 May 2006 Accepted: 22 August 2006 Published: 22 August 2006 BMC Genomics 2006, 7:215 doi:10.1186/1471-2164-7-215 Published: 22 August 2006 BMC Genomics 2006, 7:215 doi:10.1186/1471-2164-7-215 This article is available from: http://www.biomedcentral.com/1471-2164/7/215 © 2006 Tang et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. BMC Genomics Open Access Transcription factor binding and gene regulation at low activity We begin our analysis by examining published data on genes whose expression is affected by deletion of LEU3 and genes whose promoters are bound by Leu3 protein [6,7]. We refer to these experiments as "low activity" experiments because both involve Leu3 expressed at endogenous levels. For the transcriptome analysis, expres- sion in a wild-type strain was compared to expression in a leu3Δ strain [6]. For the in vivo binding experiment, chro- matin immunoprecipitation (ChIP) was performed using an epitope-tagged protein expressed from its normal genomic location [7]. Thus, both sets of experiments involve Leu3 expressed from its endogenous promoter. We also refer to these experiments as "low activity" because Leu3, like many transcription factors, requires an activation signal to be fully functional as a transcriptional regulator. In this case, the activation signal that turns the Leu3 DNA binding protein into a transcriptional activator is the binding of a metabolic intermediate, α-isopropyl- malate (α-IPM). It is not clear what fraction of Leu3 is in an activated state under the growth conditions used in the low activity experiments. A comparison of the expression and ChIP datasets has recently been reported by Boer et al. [6]. We present our own evaluation so that a direct com- parison can be made to the new high-activity data reported below. The way a regulatory network is perturbed could have a big effect on the ability to identify direct regulatory targets. The less direct the perturbation, the more likely it is that genes will be regulated in some indirect way. Environ- mental perturbations, for example, could cause signaling events in addition to those that are known and which the experiment was intended to probe. Environmental pertur- bations can also be complicated by time-dependent changes in binding and expression. For these reasons, the most direct perturbation that can be made to a transcrip- tional network is to modify genetically the concentration or activity of a transcription factor. Perturbations of this type are aimed directly at the ultimate effector of gene reg- ulation. In addition, genetic perturbations can be propa- gated for multiple generations before a comparison is made between the baseline condition of the regulatory network (wild-type cell) and its perturbed state (deleted or overexpressed factor). This effectively eliminates kinetic complexities that may otherwise complicate analyses of expression profile differences following an environmental perturbation. http://www.biomedcentral.com/1471-2164/7/215 BMC Genomics 2006, 7:215 Background vation of that binding potential, and the enrichment of functionally related genes. These analyses highlight the utility of both transcription factor deletion and overex- pression in defining direct target genes. The combined analysis of deletion and overexpression experiments also points to a broader physiological role for yeast Leu3 than its historically understood role in branched amino acid metabolism. Transcriptional programs are extremely complicated, and include a great many indirect effects. One of the great challenges in systems biology is to de-convolute complex transcriptional responses to identify the underlying net- work of direct, transcription-factor mediated control. An important step in that direction has been the develop- ment of genome scale chromatin immunoprecipitation assays (ChIP) that map bound transcription factors onto the genome sequence [1,2]. Binding of a transcription fac- tor within a presumptive control region provides evidence that the gene is regulated directly, and the combination of expression analyses and chromatin can be a powerful way of identifying direct targets [3-5]. However, ChIP data may not be sufficient to identify direct targets because genomic binding can be fortuitous and unrelated to gene regulation. There can also be ambiguities in assigning a bound transcription factor to a putative target gene, par- ticularly in higher eukaryotes where regulatory sites can be far away from the affected gene, and can appear 5' to the transcribed sequence, within the sequence, or even 3' to it. Nevertheless, the combination of expression analysis and ChIP localization of bound transcription factors can pro- vide a compelling statistical argument for the enrichment of authentic target genes. The greater the intersection between bound and regulated genes, the greater the confi- dence that some of these genes are truly direct targets. Abstract Background: Effects on gene expression due to environmental or genetic changes can be easily measured using microarrays. However, indirect effects on expression can be substantial. The indirect effects of a perturbation need to be distinguished from the direct effects if we are to understand the structure and behavior of regulatory networks. Results: The most direct way to perturb a transcriptional network is to alter transcription factor activity. Here, for the first time, we compare expression changes and genomic binding in a simple regulon under conditions of both low and high transcription factor activity. Specifically, we assessed the effects on expression and binding due to deletion of the yeast LEU3 transcription factor gene and effects due to elevation of Leu3 activity. Leu3 activity was elevated through overexpression and the introduction of a mutation that renders the protein constitutively active. Genes that are bound and/or regulated by Leu3 under one or both conditions were characterized in terms of their functional annotations and their predicted potential to be bound by Leu3. We also assessed the evolutionary conservation of the predicted binding potential using a novel alignment-independent method. Both perturbations yield genes that are likely to be direct targets of Leu3, including most of the classically defined targets. Additional direct targets are identified by each of the methods. However, experimental and computational criteria suggest that most genes whose expression is affected by the Leu3 genotype are unlikely to be regulated by binding of the protein. Conclusion: Most genes that are differentially expressed by Leu3 are not direct targets despite the exceptional simplicity of the regulon, and the unusually direct nature of the perturbations investigated. These conclusions are reached through computational analyses that support and extend chromatin immunoprecipitation data on the identities of direct targets. These results have implications for the interpretation of expression experiments, especially in cases for which chromatin immunoprecipitation data are unavailable, incomplete, or ambiguous. Page 1 of 13 (page number not for citation purposes) Page 1 of 13 (page number not for citation purposes) http://www.biomedcentral.com/1471-2164/7/215 Transcription factor binding and gene regulation at low activity Gene ontology analyses were performed using the Saccharomyces Genome Database we te [21]. (D–F) These panels are analogous to panels A–C except that the genes were defined based on experiments using onstitutively active protein expressed at higher-than-endogenous levels. /RZ +LJK                         52&$8& *20(5RFFFXSDQF\VFRUH ERXQG RQO\ UHJXODWHG RQO\ ERXQG UHJXODWHG       ERXQG RQO\ UHJXODWHG RQO\ ERXQG UHJXODWHG ERXQG RQO\ UHJXODWHG RQO\ ERXQG UHJXODWHG ERXQG RQO\ UHJXODWHG RQO\ ERXQG UHJXODWHG )UDFWLRQRIJHQHV ERXQG UHJXODWHG ERXQG UHJXODWHG ELQGLQJVLWHHQULFKPHQW ELQGLQJVLWHHQULFKPHQW *2HQULFKPHQW *2HQULFKPHQW 52&$8& *20(5RFFFXSDQF\VFRUH )UDFWLRQRIJHQHV ' ( ) $ % & /RZ +LJK       ERXQG UHJXODWHG ERXQG UHJXODWHG ' $ /RZ    ERXQG UHJXODWHG $ +LJK    ERXQG UHJXODWHG ' '             52&$8& *20(5RFFFXSDQF\VFRUH ERXQG RQO\ UHJXODWHG RQO\ ERXQG UHJXODWHG ERXQG RQO\ UHJXODWHG RQO\ ERXQG UHJXODWHG ELQGLQJVLWHHQULFKPHQW ELQGLQJVLWHHQULFKPHQW 52&$8& *20(5RFFFXSDQF\VFRUH ( %       52&$8& *20(5RFFFXSDQF\VFRUH ERXQG RQO\ UHJXODWHG RQO\ ERXQG UHJXODWHG ELQGLQJVLWHHQULFKPHQW % %       ERXQG RQO\ UHJXODWHG RQO\ ERXQG UHJXODWHG ELQGLQJVLWHHQULFKPHQW 52&$8& *20(5RFFFXSDQF\VFRUH (       ERXQG RQO\ UHJXODWHG RQO\ ERXQG UHJXODWHG )UDFWLRQRIJHQHV *2HQULFKPHQW &       ERXQG RQO\ UHJXODWHG RQO\ ERXQG UHJXODWHG *2HQULFKPHQW )UDFWLRQRIJHQHV ) & ) Comparison of chromatin immunoprecipitation and transcriptome data at low and high Leu3 activity Figure 1 Comparison of chromatin immunoprecipitation and transcriptome data at low and high Leu3 activity. (A) Venn diagram showing the number of genes bound and/or regulated at low concentrations. (B) Association of Leu3 binding sites with the indicated subset of genes defined as bound and/or regulated at low concentrations. All genes were scored for predicted Leu3 binding potential using GOMER [8], and ranked according to this score. Genes in the indicated subset were compared to all other genes in the genome using a receiver operator characteristic curve (ROC), and the value of the area under the curve is shown in this histogram. The histogram starts at a value of 0.5 because that is the value expected by chance. A value of 1.0 would indicate that the predicted binding potential scores perfectly discriminate the experimentally defined subset of genes from all others. Transcription factor binding and gene regulation at low activity Using criteria for Leu3-bound genes [7] and for Leu3-reg- ulated genes [6] as defined by the original authors, only about one third of the genes that are bound by Leu3 are downregulated in a leu3 deletion (Fig 1). The converse is even more striking: only about 3% of the genes whose expression is affected by leu3 deletion are detectably bound by the protein. Boer et al, whose low activity expression data we use here, reached the same conclusion [6]. [6]. Why are so many genes affected by leu3 deletion but not bound by the protein? One possibility is that the ChIP experiment is not sensitive enough. To evaluate this pos- sibility we predicted the potential of the regulated genes to be bound by Leu3. If the predicted potential to be bound is high then the failure to detect binding is likely due to insensitivity of the ChIP assay. If, on the other hand, the Here, for the first time, we compare expression and bind- ing under conditions of both low and high transcription factor activity. The genes that are bound and/or regulated under these conditions are assessed computationally in terms of Leu3 binding potential, the evolutionary conser- Page 2 of 13 (page number not for citation purposes) Page 2 of 13 (page number not for citation purposes) BMC Genomics 2006, 7:215 http://www.biomedcentral.com/1471-2164/7/215 omparison of chromatin immunoprecipitation and transcriptome data at low and high Leu3 activity igure 1 omparison of chromatin immunoprecipitation and transcriptome data at low and high Leu3 activity. (A) Ven agram showing the number of genes bound and/or regulated at low concentrations. (B) Association of Leu3 binding sites wit he indicated subset of genes defined as bound and/or regulated at low concentrations. All genes were scored for predicted eu3 binding potential using GOMER [8], and ranked according to this score. Genes in the indicated subset were compared t l other genes in the genome using a receiver operator characteristic curve (ROC), and the value of the area under the curv shown in this histogram. The histogram starts at a value of 0.5 because that is the value expected by chance. A value of 1.0 ould indicate that the predicted binding potential scores perfectly discriminate the experimentally defined subset of genes om all others. (C) The fraction of genes in each low concentration subset that have Gene Ontology process annotations tha re significantly enriched (P ≤ 1e-6). High activity chromatin-IP analysis identifies additional Leu3 targets missed under low-activity conditions In contrast, for the group of genes whose expression is affected by leu3 deletion but which are not detectably bound by Leu3, we find that the pre- dicted Leu3 binding potential is only slightly greater than what is expected by chance. This effect is attributable to a small number of genes with higher-than-average binding potential (data not shown). Thus, some of these unbound but regulated genes may be direct targets of Leu3 but are undetected in the ChIP experiment for reasons of experi- mental sensitivity. However, most of the unbound genes have binding potentials indistinguishable from unregu- lated genes, and are therefore likely to be indirect targets of Leu3. A search for over-represented motifs among the unbound but regulated genes failed to find any significant motifs. The genes identified by ChIP under high Leu3 activity conditions are almost perfectly a superset of the genes bound at low activity. Of 25 genes whose upstream regions are bound by Leu3 in the low activity experiments (p ≤ 1e-3), we observed binding of 24 at high activity (p ≤ 1e-4). This attests to the quality of the data. Even at a much more stringent confidence level applied to the high activity data (p ≤ 1e-7), 22 of the 25 genes bound at low activity are still found, plus an additional 137 bound genes. In short, nearly all of the genes deemed to be bound at low Leu3 activity are also bound at high activity. More importantly, there are many additional genes that are bound at the same high level of statistical confidence that are not bound in the low activity ChIP experiment. Expression changes are clearly not a reliable indicator of direct regulation because most differentially expressed genes are not detectably bound and do not have promoter sequences that suggest they should be bound. However, expression experiments add considerable value to the chromatin-IP experiments. First, the intersection of the regulated gene set with the bound set, while fairly small, is statistically significant (P < 1e-5). These genes are more likely to be direct targets than genes that are either bound only or regulated only. Second, genes that are both bound and regulated are highly enriched for genes that have related functions. (Fig 1C). High activity chromatin-IP analysis identifies additional Leu3 targets missed under low-activity conditions g y To develop a sense for how transcription factor concentra- tion and activity affects binding and regulation, we per- formed chromatin immunoprecipitation and expression array experiments using a mutant of Leu3 that is constitu- tively active (i.e., not dependent on αIPM) (Methods) [10]. This protein was also expressed from a plasmid at levels about 8–40 fold higher than endogenous Leu3 expression (data not shown). We refer to the data obtained with this strain as "high activity" data in distinc- tion to the low activity data described above. In the case of the ChIP experiments, "high activity" means higher- than-endogenous protein concentrations. In the case of expression experiments, "high activity" refers to both the expression level and the mutation conferring constitutive activation function. The protein was also fused to maltose binding protein (MBP) for affinity purification in ChIP experiments. Details of the ChIP experiments have been submitted elsewhere because they were performed in the context of a separate study on the effects of chromatin on DNA binding site selection (XL, Cheol-Koo Lee, Joshua A. Granek, NDC, and Jason D. Lieb; submitted). In this paper we report the results of a transcriptome analysis using this same construct, compare the genes whose expression is activated with the genes that are bound by Leu3p under the same conditions, and compare these high-activity results with those found previously under low-activity conditions. To calculate the potential to be bound by Leu3, we scored the upstream sequences of all open reading frames (ORFs) in yeast using an equilibrium model for transcription fac- tor binding, implemented in the program GOMER [8], and a position weight matrix (PWM) that we previously defined based on equilibrium dissociation constants for a large number of motif variants [9]. We then ranked all genes by their predicted potential to bind Leu3, and asked whether genes that are bound and/or regulated rank sig- nificantly high in this list. As expected, genes whose pro- moters are bound according to the ChIP experiment are, as a group, enriched in Leu3 binding potential, demon- strating a correlation between predicted binding and observed binding (Fig 1B). This correlation exists both for the genes that are bound and regulated and for the genes that are only bound. Transcription factor binding and gene regulation at low activity (C) The fraction of genes in each low concentration subset that have Gene Ontology process annotations that are significantly enriched (P ≤ 1e-6). Gene ontology analyses were performed using the Saccharomyces Genome Database web site [21]. (D–F) These panels are analogous to panels A–C except that the genes were defined based on experiments using constitutively active protein expressed at higher-than-endogenous levels. Page 3 of 13 (page number not for citation purposes) Page 3 of 13 (page number not for citation purposes) BMC Genomics 2006, 7:215 http://www.biomedcentral.com/1471-2164/7/215 any highly significant enrichment of Gene Ontology (GO) annotations (p ≤ 1e-6; see Methods). predicted potential to be bound is as low as random genes, then there is no expectation that these genes should be bound and it is likely that they are regulated indirectly rather than by Leu3 binding. Page 4 of 13 (page number not for citation purposes) Conservation of binding potential supports the existence of direct target genes among bound and regulated genes Conservation of binding potential supports the existence of direct target genes among bound and regulated genes Enrichment of GO annotations is one way to evaluate whether a gene set is enriched for biologically relevant tar- gets (Fig 1C,1F). Another is to assess the evolutionary con- servation of a gene's predicted binding potential. If genes that are regulated are bound by Leu3 using binding sites that have been selected during evolution, then the pro- moters of those genes will show evidence of conservation for Leu3 binding. To verify this assumption, we first tested the idea on the nine bound and regulated genes identified in the low activity experiments and evaluated the Leu3 binding potential of their promoters compared to all other genes in the genome. The analysis was then repeated for six other Saccharomyces species, using the promoters for genes orthologous to the ones used in the S. cerevisiae analysis. As controls, we derived 20 gene sets whose mem- bers have predicted binding potential in S. cerevisiae that is closely matched to the bound and regulated genes. As expected, there is dramatically greater conservation of binding potential for the Leu3 targets than for the control sequences in the most distantly related species (Fig 2A). Different array platforms were used in our high-activity expression and ChIP experiments, requiring different algorithms for the estimation of statistical significance. To define a set of genes that are both bound and induced, we determined threshold p-values for binding and induction that maximized the fraction of genes that meet both crite- ria, above and beyond the number expected by chance (see Methods). By this criterion, there are 44 genes in common among the top 200 Leu3-bound genes and the top 250 Leu3-induced genes (Fig 1D). These 44 genes are significantly greater than the 9 or 10 that are expected to be in the intersection by chance (p <= 4e-20). We performed binding-potential and GO-enrichment analyses on the bound, regulated and bound and regu- lated gene sets, as described above for the low activity data. The trends are the same (Fig 1E,F). Genes that are bound are associated with higher predicted Leu3 binding potential and genes that are both bound and induced are even higher in Leu3 binding potential. http://www.biomedcentral.com/1471-2164/7/215 http://www.biomedcentral.com/1471-2164/7/215 chance (p < 1e-3 for the null model being no enrichment of leu3Δ-affected genes). This suggests that there are indeed additional direct target genes among the leu3Δ- affected genes that were missed in the low-activity ChIP experiment, perhaps due to insensitivity of the ChIP assay. On the other hand, this is a very modest effect because only a few percent of the leu3Δ-affected genes are bound even under high-activity conditions. This is consistent with the fact that genes affected by leu3 deletion, but which are unbound, tend not to have high predicted Leu3 binding potential. over-expression of constitutively active Leu3 appear to be regulated indirectly rather than by direct binding of the protein because the genes that are induced but not bound have predicted binding potential only slightly greater than that of random genes. Because overexpression may be a non-physiological per- turbation it is possible that the bound and regulated genes identified in this experiment are biologically irrelevant. If that were the case, however, we would not expect these fortuitously expressed genes to share biological functions. It is noteworthy, therefore, that the set of genes bound and induced under these conditions is enriched for certain Gene Ontology annotations (Fig 1F). Indeed, even though the fraction of bound and regulated genes that have signif- icant shared GO process annotations is smaller in these high activity experiments than in the low activity set, the absolute number of genes having GO process annotations in common is higher because there are more genes identi- fied in total (44 vs. 9). Thus, genes identified as possible targets under high activity conditions meet two experi- mental criteria for direct regulation (binding of the factor and differential regulation due to its perturbation) as well as showing a tendency to share biological functions. We conclude that the high-activity data is probably identify- ing at least some new authentic target genes. High activity chromatin-IP analysis identifies additional Leu3 targets missed under low-activity conditions Six of the nine genes that are both bound and regulated have been annotated as being involved in "branched chain family amino acid biosyn- thesis", an enrichment of several hundred fold (p-value ≤ 3e-15). All six of these genes are directly on the committed pathway to leucine or valine biosynthesis. In contrast, nei- ther the bound-only nor the regulated-only gene sets have The bound genes identified in the high-activity ChIP experiment can be used to identify additional direct target genes that were missed in the low-activity analysis. Amongst the several hundred genes identified as being bound only in the high-activity ChIP experiment the number of genes whose expression is affected by leu3 deletion is about twice as great as the number expected by Page 4 of 13 (page number not for citation purposes) Page 4 of 13 (page number not for citation purposes) BMC Genomics 2006, 7:215 http://www.biomedcentral.com/1471-2164/7/215 Combined expression and chromatin-IP analysis under high-activity conditions To determine expression levels under conditions of high- activity Leu3, the same constitutively active, plasmid- expressed MBP-Leu3 fusion strain that was used in the ChIP analysis was analyzed in a microarray-based expres- sion experiment. In contrast to the ChIP analyses, which showed that the genes bound at high Leu3 activity include essentially all the genes bound at low-activity, only about 5–10% of genes whose expression is decreased in the leu3 deletion strain are induced by overexpression of constitu- tively active Leu3 (the exact fraction depends on the crite- ria used to define differential expression). The small number of genes in common is not surprising if most genes that are differentially expressed are the result of indirect effects. Conservation of binding potential supports the existence of direct target genes among bound and regulated genes The enrichment of binding potential in these genes is lower than for the genes identified in the low activity experiment, but this is expected because the genes bound at low Leu3 concentra- tion are more likely to have more and better Leu3 binding sites than the genes bound at high Leu3 concentrations. As in the low activity analysis, most genes induced by We next performed this analysis on the genes identified as bound and regulated in the high-activity experiment. Excluding the nine genes that are identified using only the low-activity data, there are 45 genes identified using some combination of the high or low activity ChIP and expres- Page 5 of 13 (page number not for citation purposes) Page 5 of 13 (page number not for citation purposes) BMC Genomics 2006, 7:215 http://www.biomedcentral.com/1471-2164/7/215 ound and regulated genes are more conserved in predicted binding potential than control genes of comparable binding poten- al Figure 2 Bound and regulated genes are more conserved in predicted binding potential than control genes of compara- ble binding potential. (A) Predicted binding potential for the 600 bp upstream of every protein-coding gene in S. cerevisiae was calculated using GOMER and a Leu3 position weight matrix, and the calculations were repeated for the 600 bp upstream f all orthologous genes from six other species (Methods). The red circle shows the ROC AUC value obtained from compar- ng the 9 S. cerevisiae genes that were identified as bound and regulated in low activity experiments with all other genes in the ame genome; the black circles are the values calculated for orthologous genes in six related species. The red box plot show he distribution of ROC AUC values obtained from 20 sets of 9 control sequences that were selected to closely approximate he predicted binding potential of the bound and regulated genes (Methods). The black box plots are comparable but are calcu- ated using the orthologs of the genes chosen from S. cerevisiae; the open circles are the box plot outliers. For both experi- mental and control genes, if an ortholog was undefined in a genome, that gene was simply omitted from the analysis for that enome. Note that the predicted binding potential of bound and regulated genes is maintained in the distantly related species S. astelli and S. kluyveri to a much greater extent than control sequences. Conservation of binding potential supports the existence of direct target genes among bound and regulated genes For both experi- mental and control genes, if an ortholog was undefined in a genome, that gene was simply omitted from the analysis for that genome. Note that the predicted binding potential of bound and regulated genes is maintained in the distantly related species S. castelli and S. kluyveri to a much greater extent than control sequences. (B) Same as panel A except the genes defined as bound and regulated required high Leu3 activity for their detection in either the ChIP or expression assays, or both. There are 45 such genes, 31 of which required high Leu3 activity for detection in both assays. The remainder were detected at low activ- ity in one of the assays (Fig. 3). Bound and regulated genes are more conserved in predicted binding potential than control genes of comparable binding poten- tial Figure 2 Bound and regulated genes are more conserved in predicted binding potential than control genes of compara- ble binding potential. (A) Predicted binding potential for the 600 bp upstream of every protein-coding gene in S. cerevisiae was calculated using GOMER and a Leu3 position weight matrix, and the calculations were repeated for the 600 bp upstream of all orthologous genes from six other species (Methods). The red circle shows the ROC AUC value obtained from compar- ing the 9 S. cerevisiae genes that were identified as bound and regulated in low activity experiments with all other genes in the same genome; the black circles are the values calculated for orthologous genes in six related species. The red box plot show the distribution of ROC AUC values obtained from 20 sets of 9 control sequences that were selected to closely approximate the predicted binding potential of the bound and regulated genes (Methods). The black box plots are comparable but are calcu- lated using the orthologs of the genes chosen from S. cerevisiae; the open circles are the box plot outliers. For both experi- mental and control genes, if an ortholog was undefined in a genome, that gene was simply omitted from the analysis for that genome. Note that the predicted binding potential of bound and regulated genes is maintained in the distantly related species S. castelli and S. kluyveri to a much greater extent than control sequences. Conservation of binding potential supports the existence of direct target genes among bound and regulated genes (B) Same as panel A except the genes defined as ound and regulated required high Leu3 activity for their detection in either the ChIP or expression assays, or both. There are 5 such genes, 31 of which required high Leu3 activity for detection in both assays. The remainder were detected at low activ- ty in one of the assays (Fig. 3).       6FHU 6SDU 6PLN 6NXG 6ED\ 6FDV 6NOX 52&$8& ERXQGDQGUHJXODWHG DWORZ/HXDFWLYLW\ ERXQGDQGUHJXODWHG EXWUHTXLULQJKLJK/HXDFWLYLW\ WRGHWHFWELQGLQJRUUHJXODWLRQ $ % ERXQGDQG UHJXODWHGJHQHV VHWVRIFRQWUROJHQHV PDWFKHGLQELQGLQJ SRWHQWLDOWRERXQG DQGUHJXODWHGJHQHV RUWKRORJVRI ERXQGDQG UHJXODWHGJHQHV       6FHU 6SDU 6PLN 6NXG 6ED\ 6FDV 6NOX 52&$8& RUWKRORJVRI FRQWUROJHQHV ERXQGDQG UHJXODWHGJHQHV VHWVRIFRQWUROJHQHV PDWFKHGLQELQGLQJ SRWHQWLDOWRERXQG DQGUHJXODWHGJHQHV RUWKRORJVRI ERXQGDQG UHJXODWHGJHQHV RUWKRORJVRI FRQWUROJHQHV       6FHU 6SDU 6PLN 6NXG 6ED\ 6FDV 6NOX 52&$8& ERXQGDQGUHJXODWHG DWORZ/HXDFWLYLW\ $ ERXQGDQG UHJXODWHGJHQHV VHWVRIFRQWUROJHQHV PDWFKHGLQELQGLQJ SRWHQWLDOWRERXQG DQGUHJXODWHGJHQHV RUWKRORJVRI ERXQGDQG UHJXODWHGJHQHV RUWKRORJVRI FRQWUROJHQHV $ ERXQGDQGUHJXODWHG DWORZ/HXDFWLYLW\ ERXQGDQGUHJXODWHG EXWUHTXLULQJKLJK/HXDFWLYLW\ WRGHWHFWELQGLQJRUUHJXODWLRQ %       6FHU 6SDU 6PLN 6NXG 6ED\ 6FDV 6NOX 52&$8& ERXQGDQG UHJXODWHGJHQHV VHWVRIFRQWUROJHQHV PDWFKHGLQELQGLQJ SRWHQWLDOWRERXQG DQGUHJXODWHGJHQHV RUWKRORJVRI ERXQGDQG UHJXODWHGJHQHV RUWKRORJVRI FRQWUROJHQHV % Bound and regulated genes are more conserved in predicted binding potential than control genes of comparable binding poten- tial Figure 2 Bound and regulated genes are more conserved in predicted binding potential than control genes of compara- ble binding potential. (A) Predicted binding potential for the 600 bp upstream of every protein-coding gene in S. cerevisiae was calculated using GOMER and a Leu3 position weight matrix, and the calculations were repeated for the 600 bp upstream of all orthologous genes from six other species (Methods). The red circle shows the ROC AUC value obtained from compar- ing the 9 S. cerevisiae genes that were identified as bound and regulated in low activity experiments with all other genes in the same genome; the black circles are the values calculated for orthologous genes in six related species. The red box plot show the distribution of ROC AUC values obtained from 20 sets of 9 control sequences that were selected to closely approximate the predicted binding potential of the bound and regulated genes (Methods). The black box plots are comparable but are calcu- lated using the orthologs of the genes chosen from S. cerevisiae; the open circles are the box plot outliers. Conservation of binding potential supports the existence of direct target genes among bound and regulated genes (B) Same as panel A except the genes defined as bound and regulated required high Leu3 activity for their detection in either the ChIP or expression assays, or both. There are 45 such genes, 31 of which required high Leu3 activity for detection in both assays. The remainder were detected at low activ- ity in one of the assays (Fig. 3). Page 6 of 13 (page number not for citation purposes) Page 6 of 13 (page number not for citation purposes) http://www.biomedcentral.com/1471-2164/7/215 BMC Genomics 2006, 7:215 consistent with a broader metabolic role for Leu3. Espe- cially relevant is the observation that GCN4 appears to be a direct target of Leu3. GCN4 is the master regulator of general amino acid control and regulates LEU3 expression among many other targets [7,11,12]. Caution is in order as GCN4 could be one of the 9 or 10 genes that are expected to be in the intersection of the bound and expressed genes by chance, and we are unaware of any other evidence for regulation of GCN4 by Leu3. Neverthe- less, our data suggest a positive feedback loop between Leu3 and Gcn4. Such a feedback loop makes physiologi- cal sense because leucine and valine together comprise about 15% of the amino acid residues in proteins, and starvation for branched amino acids could be a general signal for amino acid starvation. Met4 and Met28, which function together to control sulfur and sulfur amino acid metabolism, are also targets of Leu3, and binding to the former has been observed at endogenous concentrations as well [13]. Interestingly, there are a number of Leu3 tar- get genes that have previously been shown to be bound as well by Gcn4 or Met4 [7]. These interactions, summarized in Fig 4B, suggest that Leu3 activates some genes through a feed-forward mechanism in which it both directly con- trols expression of a target gene as well as activating expression of a different transcription factor that targets the same gene. sion data (Fig. 3). Fig 2B shows that the predicted binding potential of Leu3 for these new genes is generally better conserved than genes of similar predicted binding poten- tial that are not bound and regulated. This reinforces the conclusion from GO enrichment analysis that the genes identified in the high activity experiment include novel direct targets that are biologically relevant. Utility of low and high activity perturbations y f g y p The expression levels of nearly all the classically defined targets of Leu3 are affected by both LEU3 deletion and Leu3 over-expression. Indeed, the seven genes that com- prise the pathway for branched amino acid biosynthesis are among the most strongly regulated genes under each condition (Fig 5). This suggests that the primary physio- logical targets can largely be identified from either dele- tion of the transcription factor or its overexpression. On the other hand, GO analysis and the conservation of pre- dicted binding potential both suggest that authentic target genes can be responsive to only one of the perturbations. This is illustrated well by a set of permeases and transport proteins that are bound and regulated by Leu3. As noted above, "organic acid transport" is the second most signif- icant functional annotation among the 54 genes that are bound and regulated (p < 4e-4), with a total for four genes Metabolic functions of target genes imply an expanded physiological role for Leu3 There are 54 genes bound and regulated by Leu3 accord- ing to some combination of ChIP and expression experi- ments at low or high activity (Fig. 3). As this is many more than the number of genes involved in branched amino acid biosynthesis, GO analysis was performed on the full set of 54 genes to help understand the breadth of Leu3 function. Not surprisingly, the GO process enriched with the greatest confidence is "branched chain family amino acid biosynthesis" (7 genes; p = 9e-12). However, there are even larger sets of enriched categories, such as amine biosynthesis (12 genes; p = 2e-11) and carboxylic acid metabolism (15 genes; p = 3e-9). Altogether, there are 17 genes that have GO process annotations with P-values for enrichment of less than 1e-8, and there are 28 genes with GO processes that are enriched with more moderate con- fidence (p ≤ 1e-3). (Fig. 3). The functions of some of the 17 bound and regulated genes that share highly enriched GO process annotations are shown in Fig 4A. This set includes genes for every enzy- matic step on the committed pathway to leucine and valine synthesis, as well as three other genes that lead to the synthesis of other amino acids. GO-enriched genes not represented on this map consist of additional meta- bolic enzymes (SPE2, ALD5), a plasma membrane trans- porter (PDR12), and several transcription factors (MET4, MET28, GCN4, GAT1). These experiments and analyses imply a broader role for Leu3 in cellular physiology than the regulation of branched amino acid biosynthesis that is traditionally ascribed to this transcription factor. Among the transcription factors that appear to be bound and regulated by Leu3 are three that are involved in stress response: HSF1 (heat shock response), MSN2 (binds to stress response elements), and SMP1 (osmotic stress). Since all three of these genes were identified as Leu3 tar- gets only in the high concentration experiments, it is pos- sible that stress is caused by elevated Leu3 activity itself. However, even if metabolic stresses play a role in induc- tion, most of these genes appear to be regulated directly since they are bound by Leu3 as well as being induced. Page 7 of 13 (page number not for citation purposes) A transcriptional regulatory network defined by Leu3 targets Shaded portions of columns identify the genes that are significantly bound or regulated under the indicated experimental condition (low or high activity). The two columns labeled "GO" show the genes whose Gene Ontology process annotations are enriched at the indicated con- fidence levels (1e-3 or 1e-8). Annotations are shown for all genes found with GO annotations enriched with p-value better than 0.05. If more than one annotation was enriched, the most significant annotation is shown. The GO annotation "branched chain family amino acid biosynthesis" has been abbreviated to "branched amino acid biosynthesis". <./: 2$&  GLFDUER[\OLFDFLGWUDQVSRUW <%5& %$3  RUJDQLFDFLGWUDQVSRUW <-5& ,/9   EUDQFKHGDPLQRDFLGELRV\QWKHVLV <(5: $/'   FDUER[\OLFDFLGPHWDEROLVP <1/: 0(7   QLWURJHQFRPSRXQGPHWDEROLVP <25& ,68 FHOOXODUSK\VLRORJLFDOSURFHVV <+5& 6(7 <'5: +20   DPLQHELRV\QWKHVLV <(/& *&1   DPLQHELRV\QWKHVLV <(5& 0(7   DPLQHELRV\QWKHVLV <,5& 0(7   DPLQHELRV\QWKHVLV <1/: /$7   FDUER[\OLFDFLGPHWDEROLVP <2/& 63(   DPLQHELRV\QWKHVLV <3/& 3'5   FDUER[\OLFDFLGPHWDEROLVP <%5& 603  UHJXODWLRQRIWUDQVFULSWLRQ <'5: 53$  WUDQVFULSWLRQ <*/: +6)  UHJXODWLRQRIWUDQVFULSWLRQ </5& ',&  GLFDUER[\OLFDFLGWUDQVSRUW <05& 061  UHJXODWLRQRIWUDQVFULSWLRQ <05: *$7  WUDQVFULSWLRQ <3/& 6.6  UHJXODWLRQRIWUDQVFULSWLRQ <35: 52;  UHJXODWLRQRIWUDQVFULSWLRQ <-5& 56)  UHJXODWLRQRIWUDQVFULSWLRQ <+/& 6+8 FHOOXODUSK\VLRORJLFDOSURFHVV <+5: 0'0 FHOOXODUSK\VLRORJLFDOSURFHVV <.5: 375 FHOOXODUSK\VLRORJLFDOSURFHVV <25& &27 FHOOXODUSK\VLRORJLFDOSURFHVV <%/& 3(7 FHOOXODUSK\VLRORJLFDOSURFHVV <+5& 3$1 SDQWRWKHQDWHELRV\QWKHVLV <./& 37. SRO\DPLQHWUDQVSRUW <//: 732 SRO\DPLQHWUDQVSRUW <'5& *,& UHJXODWLRQRIFHOOXODUSK\VLRORJLFDOSURFHVV <&5: <(5: ,(6 <-/& 35< <.5& <1/& <3/: 50, <'5& $*( FHOOXODUSK\VLRORJLFDOSURFHVV <)/: *$7   QLWURJHQFRPSRXQGPHWDEROLVP <$/& 66$ FHOOXODUSK\VLRORJLFDOSURFHVV <(/: +<3 FHOOXODUSK\VLRORJLFDOSURFHVV <+5& (*' FHOOXODUSK\VLRORJLFDOSURFHVV </5& &&: FHOOXODUSK\VLRORJLFDOSURFHVV <05: 63* <2/: 3+0 <3/& <2/& FHOOXODUSK\VLRORJLFDOSURFHVV A transcriptional regulatory network defined by Leu3 targets g In addition to analyzing Gene Ontology process annota- tions, we also analyzed GO "function" annotations among the 54 bound and regulated genes. Remarkably, the three most significant annotations are related to tran- scriptional regulation, with a total of 10 genes annotated as having transcriptional regulator activity. The next most significant annotation is "organic acid transport" with four genes. The abundance of transcription factor genes among the bound and regulated targets of Leu3 is unex- pected as Leu3 had previously been thought to function as a simple regulator of branched amino acid biosynthesis. However, some of the transcription factor target genes are Page 7 of 13 (page number not for citation purposes) Page 7 of 13 (page number not for citation purposes) BMC Genomics 2006, 7:215 http://www.biomedcentral.com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eu3 targets inferred from at least one combination of ChIP and expression analyses Figure 3 Leu3 targets inferred from at least one combination of ChIP and expression analyses. Genes are grouped accord- ing to the combinations of experiments that support the identification of the gene as a Leu3 target. Leu3 targe Figure 3 3 Leu3 targets inferred from at least one combination of ChIP and expression analyses Figure 3 Leu3 targets inferred from at least one combination of ChIP and expression analyses. Genes are grouped accord- ing to the combinations of experiments that support the identification of the gene as a Leu3 target. Shaded portions of columns identify the genes that are significantly bound or regulated under the indicated experimental condition (low or high activity). The two columns labeled "GO" show the genes whose Gene Ontology process annotations are enriched at the indicated con- fidence levels (1e-3 or 1e-8). Annotations are shown for all genes found with GO annotations enriched with p-value better than 0.05. If more than one annotation was enriched, the most significant annotation is shown. The GO annotation "branched chain family amino acid biosynthesis" has been abbreviated to "branched amino acid biosynthesis". g p y g Leu3 targets inferred from at least one combination of ChIP and expression analyses. Genes are grouped accord- ing to the combinations of experiments that support the identification of the gene as a Leu3 target. Shaded portions of columns identify the genes that are significantly bound or regulated under the indicated experimental condition (low or high activity). The two columns labeled "GO" show the genes whose Gene Ontology process annotations are enriched at the indicated con- fidence levels (1e-3 or 1e-8). Annotations are shown for all genes found with GO annotations enriched with p-value better than 0.05. If more than one annotation was enriched, the most significant annotation is shown. The GO annotation "branched chain family amino acid biosynthesis" has been abbreviated to "branched amino acid biosynthesis". Page 8 of 13 (page number not for citation purposes) BMC Genomics 2006, 7:215 http://www.biomedcentral.com/1471-2164/7/215 tion of selected Leu3 target genes re 4 ction of selected Leu3 target genes. (A) Metabolic pathways in which Leu3 target genes function. Genes that are d and regulated according to at least one combination of low or high activity expression and low or high ChIP analysis subjected to GO process analysis, yielding 17 genes with enriched annotations. Gene names shown here are for those s encoding enzymes directly involved in amino acid biosynthesis. (B) Transcriptional regulatory network involving Leu3. actions of Leu3 with other transcription factor genes are inferred from some combination of expression and ChIP exper- ts as described in the text. Leu3 targe Figure 3 3 Interactions of transcription factors other than Leu3 are inferred from the literature. 7&$ F\FOH DFHW\OFR$ S\UXYDWH /$7 DVSDUWDWH ,/9 /(8 %$7 ,/9 ,/9 /(8 OHXFLQH +20 KRPRVHULQH 0(7 PHWKLRQLQH WKUHRQLQH /(8 /(8 0(7 *&1 0(7 *$7 *$7 $ % 7&$ F\FOH DFHW\OFR$ S\UXYDWH /$7 DVSDUWDWH ,/9 /(8 %$7 ,/9 ,/9 /(8 OHXFLQH +20 KRPRVHULQH 0(7 PHWKLRQLQH WKUHRQLQH /(8 $ 7&$ F\FOH DFHW\OFR$ S\UXYDWH /$7 DVSDUWDWH ,/9 /(8 %$7 ,/9 ,/9 /(8 OHXFLQH +20 KRPRVHULQH 0(7 PHWKLRQLQH WKUHRQLQH /(8 7&$ F\FOH DFHW\OFR$ S\UXYDWH /$7 DVSDUWDWH +20 KRPRVHULQH 0(7 PHWKLRQLQH WKUHRQLQH /(8 0(7 *&1 0(7 *$7 *$7 % Function of selected Leu3 target genes Figure 4 Function of selected Leu3 target genes. (A) Metabolic pathways in which Leu3 target genes function. Genes that are bound and regulated according to at least one combination of low or high activity expression and low or high ChIP analysis were subjected to GO process analysis, yielding 17 genes with enriched annotations. Gene names shown here are for those genes encoding enzymes directly involved in amino acid biosynthesis. (B) Transcriptional regulatory network involving Leu3. Interactions of Leu3 with other transcription factor genes are inferred from some combination of expression and ChIP exper- /(8 0(7 *&1 0(7 *$7 *$7 % % *&1 http://www.biomedcentral.com/1471-2164/7/215 BMC Genomics 2006, 7:215 represented (BAP2, PDR12, OAC1, DIC1). BAP2 (leucine- specific permease) is a well-known target of Leu3 and its expression is strongly affected by Leu3 deletion. However, we found here that BAP2 expression is not induced by ele- vated Leu3 activity. In contrast, DIC1 (mitochondrial dicarboxylate carrier). Is very strongly affected by high Leu3 activity, but is not affected by Leu3 deletion. The other two transport proteins are affected by both perturba- tions. PDR12 is only modestly affected, but OAC1 (mito- chondrial oxaloacetate carrier) is strongly affected. Indeed, its expression changes under both conditions are very similar to the leucine biosynthetic enzyme genes LEU1, LEU2 and BAT1 (Fig 5). of binding is still very low. In between these extremes, however, are the genes that are bound at low concentra- tion but fall below the detection threshold. It is these genes that can be revealed by performing ChIP experi- ments at higher-than-endogenous concentrations. We note that some of the genes that are identified as being novel Leu3 targets arise from the intersection of the high activity ChIP data with the low activity expression data. That is, these genes are detectably bound only at high con- centrations, but the only significant effect on expression is observed when comparing wild-type cells with a leu3 deletion. Our interpretation is that these genes are bound by Leu3 at endogenous levels, but not sufficiently well to be detected in the ChIP experiment. Thus, the failure to detect binding can be attributed to inadequate sensitivity of the ChIP assay for at least some true target genes. For most of the genes we call indirect targets, however, failure to detect binding cannot be attributed to low ChIP sensi- tivity because the predicted binding potential of these genes is no higher than that of average gene in the genome. These examples illustrate the conclusions that we have drawn from the analysis of complete gene sets described above. To summarize, the combination of low activity ChIP and expression experiments identify most of the genes that are the primary physiological targets of Leu3. However, high activity ChIP and expression experiments, either by themselves or in combination with the low activ- ity experiments, identify additional targets. These targets are enriched in authentic biologically relevant targets, as judged by GO annotation analysis and by conservation of predicted binding potential. http://www.biomedcentral.com/1471-2164/7/215 Direct transcription factor perturbation by deletion or overexpression is much simpler than most perturbations. In the case of environmental perturbants, for example, it may not even be known how many transcription factors are involved or how the effect of the perturbation is medi- ated. Nevertheless, it is clear from the analyses presented here that even direct perturbation produces a large number of effects that are not directly related to binding of the transcription factor. Additional experimental meth- ods, such as double mutant analyses, may help to eluci- date networks of direct transcriptional control [14]. Also required are innovative computational methods for com- bining information from expression and ChIP experi- ments [15,16]. Function o Figure 4 Function of selected Leu3 target genes Figure 4 Function of selected Leu3 target genes. (A) Metabolic pathways in which Leu3 target genes function. Genes that are bound and regulated according to at least one combination of low or high activity expression and low or high ChIP analysis were subjected to GO process analysis, yielding 17 genes with enriched annotations. Gene names shown here are for those genes encoding enzymes directly involved in amino acid biosynthesis. (B) Transcriptional regulatory network involving Leu3. Interactions of Leu3 with other transcription factor genes are inferred from some combination of expression and ChIP exper- iments as described in the text. Interactions of transcription factors other than Leu3 are inferred from the literature. Page 9 of 13 (page number not for citation purposes) Page 9 of 13 (page number not for citation purposes) http://www.biomedcentral.com/1471-2164/7/215 Conclusion Expression analyses typically identify a large number of differentially regulated genes, but most such experiments involve systems or perturbations that are inherently more complex than what we have studied here as a model sys- tem. The Leu3 regulon is exceptionally simple by most standards, and the perturbations we have made are argua- bly the most direct perturbations possible. Nevertheless, we find that 10% of all yeast genes have expression levels that are affected by these perturbations, and that almost all of these effects are indirect rather than being due to binding by Leu3. This conclusion is based on the failure to detect binding of Leu3, combined with a computa- tional analysis that shows that most of the unbound genes are not expected to be bound (that is, do not have pre- dicted Leu3 binding potentials that are higher than unreg- ulated genes). Strains and growth conditions                   ORZDFWLYLW\H[SUHVVLRQ IROGFKDQJH=VFRUH KLJKDFWLYLW\H[SUHVVLRQ IROGFKDQJH=VFRUH /(8 /(8 %$7 /(8 ,/9 ,/9 ,/9 2$& ',& %$3 <2/& Relative expression levels of bound genes in low and high activity experiments Figure 5 Relative expression levels of bound genes in low and high activity experiments. Fold-changes in expression leve were normalized to the standard deviation so that a Z-value of 1 corresponds to one standard deviation. Small points show values for the 5000+ genes analyzed in each experiment. Large dots represent the 54 genes that are significantly bound and expressed according to some combination of low and high activity expression and ChIP data. Note that an estimate of stat cal significance was used to identify differentially expressed genes, not the fold-change Z-value shown here. Gene and label shown in red are the seven genes on the committed pathway to leucine and valine biosynthesis. Genes and labels shown in b are annotated as organic acid transporters and are described in the text. The label "YOL155C" indicates a gene that is bou b L 3 d i ti ll t l ff t d t b th l d hi h ti iti b t hi h h k f ti                   ORZDFWLYLW\H[SUHVVLRQ IROGFKDQJH=VFRUH KLJKDFWLYLW\H[SUHVVLRQ IROGFKDQJH=VFRUH /(8 /(8 %$7 /(8 ,/9 ,/9 ,/9 2$& ',& %$3 <2/& Relative ex Figure 5 R l i Relative expression levels of bound genes in low and high activity experiments Figure 5 Relative expression levels of bound genes in low and high activity experiments. Fold-changes in expression levels were normalized to the standard deviation so that a Z-value of 1 corresponds to one standard deviation. Small points show the values for the 5000+ genes analyzed in each experiment. Large dots represent the 54 genes that are significantly bound and expressed according to some combination of low and high activity expression and ChIP data. Note that an estimate of statisti- cal significance was used to identify differentially expressed genes, not the fold-change Z-value shown here. Gene and labels shown in red are the seven genes on the committed pathway to leucine and valine biosynthesis. Genes and labels shown in blue are annotated as organic acid transporters and are described in the text. Strains and growth conditions The LEU3 gene, containing a mutation that confers consti- tutive activation activity on Leu3, was fused to the gene for maltose binding protein on a URA3-containing 2 micron- based plasmid, and transformed into BY4720-leu3Δneo. As a reference strain for expression experiments, we also transformed into BY4720-leu3Δneo a plasmid expressing only the MBP-tagged DNA binding domain of Leu3 (i.e., missing sequences needed for transcription activation). (XL, Cheol-Koo Lee, Joshua A. Granek, NDC, and Jason D. Lieb; submitted). Fusion proteins were expressed from the constitutively active TDH3 promoter. Cells were grown at 30°C in media lacking uracil to maintain selection for the plasmid. Sensitivity of the ChIP assay does seem to be a factor in the failure to identify some true targets. The importance of the high-concentration ChIP analysis is that it gives a sense for what is missing from ChIP analyses performed at endogenous concentrations. At one extreme are genes that are nearly fully occupied at low concentration. Occupancy of these genes is effectively saturated and will not appear to be substantially more bound at higher concentrations. At the other extreme, genes that are bound with vanish- ingly low occupancy at low concentrations will remain undetected at high concentrations because the probability Page 10 of 13 (page number not for citation purposes) http://www.biomedcentral.com/1471-2164/7/215 BMC Genomics 2006, 7:215 Relative expression levels of bound genes in low and high activity experiments Figure 5 Relative expression levels of bound genes in low and high activity experiments. Fold-changes in expression levels were normalized to the standard deviation so that a Z-value of 1 corresponds to one standard deviation. Small points show th values for the 5000+ genes analyzed in each experiment. Large dots represent the 54 genes that are significantly bound and expressed according to some combination of low and high activity expression and ChIP data. Note that an estimate of statist cal significance was used to identify differentially expressed genes, not the fold-change Z-value shown here. Gene and labels shown in red are the seven genes on the committed pathway to leucine and valine biosynthesis. Genes and labels shown in blu are annotated as organic acid transporters and are described in the text. The label "YOL155C" indicates a gene that is bound by Leu3 and is exceptionally strongly affected at both low and high activities, but which has no known function. Expression analysis those cases where there are two or more microarray fea- tures overlapping the 600 bp 5' to an ORF, the microarray feature with the lowest p-value was used to represent pro- moter binding. Enrichment of ORF features was factored into the ranking of genes because preliminary analyses showed that genes that were bound in their promoters and regulated were also enriched in apparent binding to ORFs. While this can be an artifact due to promoter- bound sequences overlapping ORF sequences on the array [1,8], we found that ORFs that were bound and induced were significantly enriched in binding sites relative to unbound ORFs. Thus, detection of binding to ORFs among bound and regulated genes is not entirely an arti- fact of binding to adjacent promoters in this case. p y The same strain used for the chromatin immunoprecipita- tion experiments was used for Affymetrix-based transcrip- tome analysis, and the level of expression of each gene was compared to that of the BY4720-leu3Δneo parent strain carrying a plasmid expressing the Leu3 DNA bind- ing domain only. The cells were cultured in uracil dropout medium (YNB-AA (Sigma) 6.7 g/L, 0.77 g/L, Ura DO Supp. (BD Bioscience, Palo Alto, CA), and 2% glucose supplemented with G418 at 200 mg/L) with shaking at 30°C. The cells were harvested in log-phase growth (A600 ~ 0.7). The total RNA was isolated using RNeasy mini kit (Qiagen, Valancia, CA), following the enzymatic lysis pro- tocol suggested by the manufacture. Briefly, a total of less than 5 × 107 cells were harvested by spinning at 1000 × g for 5 min at 4°C. The cells were resuspended in 2 ml of lysis buffer Y1 (1 M sorbitol, 0.1 M EDTA, pH 7.4, with 0.1% β-mercaptoethanol and 10 U yeast lytic enzyme (MP Biomedicals, Aurora, Ohio)/1 × 107 cells) added just before use. The mixture was gently shaken at 30°C for 10 min, followed by centrifugation at 300 g for 5 min. 350 μl Buffer RLT was added to the pellet and vortexed vigor- ously. 350 μl 70% ethanol was then added, and the total sample was transferred to the RNeasy mini column. Selection of genes bound and regulated at high concentration Direct comparison of ChIP and expression experiments is problematic because both the low and high activity ChIP experiments were performed with spotted PCR product arrays while both the low and high activity expression experiments were performed using Affymetrix oligonucle- otide arrays. For the low activity data we chose to use threshold values provided by the authors of those studies [6,7]. For our own data, we chose to define threshold val- ues for binding and induction that maximize the fraction of genes that are both bound and induced, above and beyond the number that is expected by chance. Specifi- cally, the top B bound genes (50, 100, 150 ...) were com- pared to the top I induced genes (50, 100, 150, ...), and for each combination we determined the value of ((B ∩ I) - (B•I/T))/(B+I), where T is the total number of genes for which there is both expression and binding data. Apply- ing this standard to find the maximal significant overlap, we found 44 genes in common among the top 200 bound genes and the top 250 induced genes. Compared to the number of genes that meet each individual criterion, the 44 genes that are bound and regulated is significantly greater than the 9 or 10 expected by chance (p = 4e-20). A list of the bound genes, regulated genes, and genes that are both bound and regulated by this criterion is provided as Additional file 1. The R statistical packages, rma and affy, were used for back- ground correction, data normalization and estimation of p-values for differential expression [17,18]. Benjamini and Hochberg procedures was also applied for control of the false discovery rate (FDR). There are a total of 9,335 probe sets on the YG_S98 chip, but only 5,592 sets that correspond to protein coding transcripts for which we cal- culated binding probabilities with GOMER [4]. This reduced set of probes was used for subsequent analyses. Expression analysis The column was sequentially washed with 700 μl Buffer RW1, then twice with 500 μl Buffer RPE, followed by centrifuga- tion at greater than 8000 g for 15 seconds Total RNA was collected by eluting at 8000 g for 1 min with 50 μl RNase- free water. Four biological replicates were analyzed for each strain. Total RNA was sent to the Microarray facility of Johns Hopkins University for labeling and hybridiza- tion to the Affymetrix chip YG_S98. http://www.biomedcentral.com/1471-2164/7/215 BMC Genomics 2006, 7:215 Strains and growth conditions The label "YOL155C" indicates a gene that is bound by Leu3 and is exceptionally strongly affected at both low and high activities, but which has no known function. Page 11 of 13 (page number not for citation purposes) http://www.biomedcentral.com/1471-2164/7/215 http://www.biomedcentral.com/1471-2164/7/215 Page 12 of 13 (page number not for citation purposes) Authors' contributions 10. Wang D, Zheng F, Holmberg S, Kohlhaw GB: Yeast transcriptional regulator Leu3p. Self-masking, specificity of masking, and evidence for regulation by the intracellular level of Leu3p. J Biol Chem 1999, 274(27):19017-19024. LT performed and analyzed the expression experiments and was principally responsible for the integration of expression and ChIP data. XL initiated the conservation analysis and helped integrate ChIP and expression analy- ses. NDC helped design the experiments, was involved in all analyses, and wrote the paper. All authors have helped revise the paper and have read and approved the final manuscript. ( ) 11. Natarajan K, Meyer MR, Jackson BM, Slade D, Roberts C, Hinnebusch AG, Marton MJ: Transcriptional profiling shows that Gcn4p is a master regulator of gene expression during amino acid starvation in yeast. Mol Cell Biol 2001, 21(13):4347-4368. ( ) 12. Zhou K, Brisco PR, Hinkkanen AE, Kohlhaw GB: Structure of yeast regulatory gene LEU3 and evidence that LEU3 itself is under general amino acid control. Nucleic Acids Res 1987, 15(13):5261-5273. ( ) 13. Lee TI, Rinaldi NJ, Robert F, Odom DT, Bar-Joseph Z, Gerber GK, Hannett NM, Harbison CT, Thompson CM, Simon I, Zeitlinger J, Jen- nings EG, Murray HL, Gordon DB, Ren B, Wyrick JJ, Tagne JB, Volkert TL, Fraenkel E, Gifford DK, Young RA: Transcriptional regulatory networks in Saccharomyces cerevisiae. Science 2002, 298(5594):799-804. Additional file 1 Bound, regulated and bound+regulated genes at high activity.txt. Tab delimited text file listing the 200 bound genes, 250 regulated gene, and 44 bound and regulated genes determined at high activity and defined as described in the text. Click here for file ( ) 14. Tringe SG, Wagner A, Ruby SW: Enriching for direct regulatory targets in perturbed gene-expression profiles. Genome Biol 2004, 5(4):R29. ( ) 15. Gao F, Foat BC, Bussemaker HJ: Defining transcriptional net- works through integrative modeling of mRNA expression and transcription factor binding data. BMC Bioinformatics 2004, 5:31. [http://www.biomedcentral.com/content/supplementary/1471- 2164-7-215-S1.txt] [http://www.biomedcentral.com/content/supplementary/1471- 2164-7-215-S1.txt] 16. Yang YL, Suen J, Brynildsen MP, Galbraith SJ, Liao JC: Inferring yeast cell cycle regulators and interactions using transcription fac- tor activities. BMC Genomics 2005, 6(1):90. ( ) 17. Gautier L, Cope L, Bolstad BM, Irizarry RA: affy--analysis of Affymetrix GeneChip data at the probe level. Bioinformatics 2004, 20(3):307-315. Acknowledgements 18. Gentleman RC, Carey VJ, Bates DM, Bolstad B, Dettling M, Dudoit S, Ellis B, Gautier L, Ge Y, Gentry J, Hornik K, Hothorn T, Huber W, Iacus S, Irizarry R, Leisch F, Li C, Maechler M, Rossini AJ, Sawitzki G, Smith C, Smyth G, Tierney L, Yang JY, Zhang J: Bioconductor: open software development for computational biology and bioin- formatics. Genome Biol 2004, 5(10):R80. g The experimental work and some of the analysis was performed at Johns Hopkins under NIH grant GM065179 (N.D.C). Subsequent analyses and the writing of the paper by NDC was supported by the Genome Institute of Singapore. 19. Liu X, Noll DM, Lieb JD, Clarke ND: DIP-chip: rapid and accurate determination of DNA-binding specificity. Genome Res 2005, 15(3):421-427. Chromatin immunoprecipitation ( ) Tachibana C, Yoo JY, Tagne JB, Kacherovsky N, Lee TI, Young ET: C bi d l b l l li i l i d i Combined global localization analysis and transcriptome data identify genes that are directly coregulated by Adr1 and Cat8. Mol Cell Biol 2005, 25(6):2138-2146. ( ) 6. Boer VM, Daran JM, Almering MJ, de Winde JH, Pronk JT: Contribu- tion of the Saccharomyces cerevisiae transcriptional regula- tor Leu3p to physiology and gene expression in nitrogen- and carbon-limited chemostat cultures. FEMS Yeast Res 2005, 5(10):885-897. 5(10):885 897. 7. Harbison CT, Gordon DB, Lee TI, Rinaldi NJ, Macisaac KD, Danford TW, Hannett NM, Tagne JB, Reynolds DB, Yoo J, Jennings EG, Zei- tlinger J, Pokholok DK, Kellis M, Rolfe PA, Takusagawa KT, Lander ES, Gifford DK, Fraenkel E, Young RA: Transcriptional regulatory code of a eukaryotic genome. Nature 2004, 431(7004):99-104. 8. Granek JA, Clarke ND: Explicit equilibrium modeling of tran- scription-factor binding and gene regulation. Genome Biol 2005, 6(10):R87. ( ) 7. Harbison CT, Gordon DB, Lee TI, Rinaldi NJ, Macisaac KD, Danford TW, Hannett NM, Tagne JB, Reynolds DB, Yoo J, Jennings EG, Zei- tlinger J, Pokholok DK, Kellis M, Rolfe PA, Takusagawa KT, Lander ES, Gifford DK, Fraenkel E, Young RA: Transcriptional regulatory code of a eukaryotic genome. Nature 2004, 431(7004):99-104. y g ( ) 8. Granek JA, Clarke ND: Explicit equilibrium modeling of tran- scription-factor binding and gene regulation. Genome Biol 2005, 6(10):R87. ( ) 9. Liu X, Clarke ND: Rationalization of gene regulation by a eukaryotic transcription factor: calculation of regulatory region occupancy from predicted binding affinities. J Mol Biol 2002, 323(1):1-8. Chromatin immunoprecipitation The chromatin immunoprecipitation experiments have been submitted elsewhere in the context of an analysis of chromatin effects on binding site selection (XL, Cheol- Koo Lee, Joshua A. Granek, NDC, and Jason D. Lieb; sub- mitted). Enrichment was detected using a microarray that covers nearly all of the yeast genome, with most of the spots corresponding either to ORFs or to intergenic regions [1]. For the analyses in this paper, genes were ranked based on the product of two ChiP enrichment p- values [19], one corresponding to enrichment of the cod- ing sequence and one corresponding to its promoter. In The program GOMER was used to calculate the Leu3 bind- ing potential for all sequences within 600 bp upstream of an ORF, and to compare the potential of genes in an input list (e.g., bound and regulated genes) to other genes in the genome [8]. The binding potential score calculated by GOMER reflects all of the potential binding sites in the regulatory region. The promoters of orthologous genes were scored in an identical manner. Genes that show unu- sually well conserved potential to bind Leu3 typically have binding sites that are themselves orthologous. How- Page 12 of 13 (page number not for citation purposes) Page 12 of 13 (page number not for citation purposes) http://www.biomedcentral.com/1471-2164/7/215 BMC Genomics 2006, 7:215 http://www.biomedcentral.com/1471-2164/7/215 ever, the algorithm does not require that this be the case as no alignment of the promoter regions is involved. Inde- pendently evolved binding sites can contribute to the binding potential score, and to the conservation of this score. The position weight matrix used in these calcula- tions was based on the measurement of Kd values for 50 different Leu3 binding site variants [9]. We also per- formed a motif discovery analysis using the genes that are regulated but not bound under high activity conditions. BioProspector was run on the 600 bp upstream of the reg- ulated genes [20]. Default were used except that widths of 6, 8 10 and 12 were used rather than just the default of 10 bp. Sets of upstream sequences that contained the same number of genes as the experimental set were randomly selected and used as controls to assess significance. scription factor binding sites in the yeast genome. BMC Genomics 2004, 5(1):59. 5. References 1. Lieb JD, Liu X, Botstein D, Brown PO: Promoter-specific binding of Rap1 revealed by genome-wide maps of protein-DNA association. Nat Genet 2001, 28(4):327-334. 1. Lieb JD, Liu X, Botstein D, Brown PO: Promoter-specific binding of Rap1 revealed by genome-wide maps of protein-DNA association. Nat Genet 2001, 28(4):327-334. ( ) 20. Liu X, Brutlag DL, Liu JS: BioProspector: discovering conserved DNA motifs in upstream regulatory regions of co-expressed genes. Pac Symp Biocomput 2001:127-138. ( ) 2. Ren B, Robert F, Wyrick JJ, Aparicio O, Jennings EG, Simon I, Zeitlin- ger J, Schreiber J, Hannett N, Kanin E, Volkert TL, Wilson CJ, Bell SP, Young RA: Genome-wide location and function of DNA bind- ing proteins. Science 2000, 290(5500):2306-2309. 21. Balakrishnan R, Christie KR, Costanzo MC, Dolinski K, Dwight SS, Engel SR, Fisk DG, Hirschman JE, Hong EL, Nash R, Oughtred R, Skrzypek M, Theesfeld CL, Binkley G, Lane C, Schroeder M, Sethura- man A, Dong S, Weng S, Miyasato S, Andrada R, Botstein D, Cherry JM: Saccharomyces Genome Database. [http://www.yeastge nome.org/ ]. g p ( ) 3. Galgoczy DJ, Cassidy-Stone A, Llinas M, O'Rourke SM, Herskowitz I, DeRisi JL, Johnson AD: Genomic dissection of the cell-type- specification circuit in Saccharomyces cerevisiae. Proc Natl Acad Sci U S A 2004, 101(52):18069-18074. 3. Galgoczy DJ, Cassidy-Stone A, Llinas M, O'Rourke SM, Herskowitz I, DeRisi JL, Johnson AD: Genomic dissection of the cell-type- specification circuit in Saccharomyces cerevisiae. Proc Natl Acad Sci U S A 2004, 101(52):18069-18074. ( ) 4. Nagaraj VH, O'Flanagan RA, Bruning AR, Mathias JR, Vershon AK, Sengupta AM: Combined analysis of expression data and tran- ( ) 4. Nagaraj VH, O'Flanagan RA, Bruning AR, Mathias JR, Vershon AK, Sengupta AM: Combined analysis of expression data and tran- Page 13 of 13 (page number not for citation purposes) Page 13 of 13 (page number not for citation purposes)

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Dynamics on the Boston Consulting Group's planning matrices

South African journal of business management

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Dynamics on the Boston Consulting Group's planning matrices C.G. Robinson Graduate School of Business Administration, University of the Witwatersrand, Johannesburg C.G. Robinson Graduate School of Business Administration, University of the Witwatersrand, Johannesburg Graduate School of Business Administration, University of the Witwatersrand, Johannesburg Strategies based on the growth share matrix as a resource allocation tool require that broad categories of businesses are either funded, milked, or divested depending on their strategic positioning on the portfolio chart. Dynamics on the chart are important and this article explores the implications of changing positions of the businesses concerned using the growth gain matrix. The little-used technique of frontier curves, which relates growth rate to cash usage, is elucidated. Because management cannot act in a vacuum and competitive action is inevitable, a checklist for competitive profiling is provided. Competitive dynamics on the growth share matrix are explored least the unwary fall into the trap of conventional strategic thinking. S. Afr. J. Bus. Mgmt. 1985, 16: 109-115 Introduction Two other planning matrices have been developed by the Boston Consulting Group to augment the use of the growth share matrix. While the growth share matrix explores the strategic positioning of a business in an attempt to predict cash flow patterns, the other two matrices explicitly address two key issues, namely • the growth dynamics of a business, whether gaining or losing market share, and • the growth dynamics of a business, whether gaining or losing market share, and • the relationship between growth rate and cash usage of the businesses in the portfolio. • the relationship between growth rate and cash usage of the businesses in the portfolio. Use of the three techniques simultaneously, considerably enhances the signals obtained from the use of the growth share matrix in isolation. A seven-step strategic checklist for using the matrices is advocated. Strategiee wat gebaseer is op die groei-aandeel-matriks as 'n hulpbron-toedelingsinstrument, vereis dat bree kategoriee van besighede 6f befonds, 6f gemelk, 6f gedivest9er word afhangend van hul strategiese posisie op die portefeulje- kaart. Die dinamika van die kaart is dus belangrik en hierdie artikel ondersoek die implikasies wat veranderende posisies op die kaart vir besighede wat die groei-toename-matriks gebruik, mag inhou. Die min gebruikte tegniek van 'frontier curves', of begrensings- kurwes, wat die groeisyfer in verband bring met die gebruik van kontant, word verduidelik. 'n Oorsiglys vir die konstruering van mededingende profiele word verskaf omdat daar geargumenteer word dat bestuur nie in 'n lugleegte kan funksioneer nie en omdat mededinging 'n onlos- maaklike deel van bestuur is. Die mededingingsdinamiek van die groei-aandeel-matriks word verduidelik om te verhoed dat die onbehoedsame persoon in die strik van konvensionele strategiese denke trap. S.-Afr. Tydskr. Bedryfsl. 1985, 16: 109-115 The growth gain matrix Growth gain matrices can be plotted to investigate the relative growth rate of individual businesses compared with the industry average. The business growth rate is plotted on the abscissa while the industry growth rate is plotted on the ordinate as is shown in Figure I . Circles are plotted for 22% 20% 18% 16% CD 14% ! ~ 12% j e c, 10% ; ~ 8% ca ::i 6% 4% 2% 0 0 0 0 0 0;Q 0 ,/0' 0 ,,o , 0,,, , , Maximum sustainable growth rate , t I ,' , , , q/o , ,' I , I ,' I , ,'O I ,' I , , , , ,, , , 0 0 2% 4% 6% 8% 10% 12% 14% 16% 18% 20% 22% Business growth rate 22% 20% 18% 16% CD 14% ! ~ 12% j e c, 10% ; ~ 8% ca ::i 6% 4% 2% 0 0 0 0 0 0;Q 0 ,/0' 0 ,,o , 0,,, , , Maximum sustainable growth rate , t I ,' , , , q/o , ,' I , I ,' I , ,'O I ,' I , , , , ,, , , 0 0 2% 4% 6% 8% 10% 12% 14% 16% 18% 20% 22% Business growth rate Figure I The growth gain matrix: A strong, well-diversified portfolio Maximum sustainable growth rate C.G. Robinson Graduate School of Business Administration, University of the Witwatersrand, P.O. Box 31170, Braamfontein, 2017 Republic of South Africa Figure I The growth gain matrix: A strong, well-diversified portfolio Accepted December 1984 110 S.-Afr. Tydskr. Bedryfsl. 198S, 16(3) S.-Afr. Tydskr. Bedryfsl. 198S, 16(3) Figures I and 2. Obviously the weighted average growth rate of the businesses in the portfolio cannot exceed the maximum sustainable rate of growth without recourse to equity funds. This means the growth rate line must be the centre of gravity of the portfolio and too many of the circles cannot lie too far to the right of the maximum growth rate line. To the extent that they lie to the left an idea is given of the execs., funding capacity of the portfolio. If assets had been used rather than sales on the growth gain matrix the display would give quick optical corroboration as to the feasibility or in- feasibility of projected portfolios. Calculations should be used to corroborate this. Frontier curves A third, though apparently seldom used, technique developed and published by the Boston Consulting Group is that of frontier curve analysis. The technique is essentially an attempt to portray the growth rate of a business in a portfolio graphi- cally as opposed to the cost of funding the growth in terms of the cash used (Moose & Zakon, 1972). / / / / / / Maximum sustainable growth rate + Business growth rate / / / / / / ? / / / / Figure 2 The growth gain matrix: The ideally positioned portfolio (Hammond & Allan, 1975:IO) Maximum sustainable growth rate ( ) The technique uses a two-dimensional grid, Figure 3, on which the annual growth rate in profits is plotted on the ordinate and the cash use as a percentage of earnings after taxes reinvested in the business, effectively the cash use, is plotted on the abscissa. The company's major profit centres are plotted on the grid according to their growth and re- investment characteristics over a chosen period, often five years. Businesses may fall anywhere on the grid. The location, however, is directly related to its value to the company. For example, if a business falls at a 12% growth rate and HX>OJo cash use, the business is using all of its resources to grow at 12%. The business is high growth and self financing and would be considered a star. Similarly, if a company is not increasing its annual growth rate in profits, in real terms discounted for inflation, and is only using 50% of its cash to maintain its competitive position, the remaining 50% of the cash would be available for redeployment and the com- pany could be considered a cash cow. Business growth rate In general cash cows would be expected to have profit growth rates of less than 5% and cash usage rates of 70% Figure 2 The growth gain matrix: The ideally positioned portfolio (Hammond & Allan, 1975:IO) 40 0 30 • Cash generators O Cash users 0 l 20 00 Q) ! The growth gain matrix Note that the calculations must include the effect of corporate overhead. This issue is discussed in the section of frontier curves. individual businesses with the areas being representative of either sales or assets invested. Businesses along the diagonal are those in which the business is growing at the same rate as the market. Businesses gaining share are located below the diagonal and those losing share are located above the diagonal. Cash cows and dogs are clustered on the lower half of the grid while wildcats and stars are in the upper half. An idealized placing of businesses on the growth gain matrix is shown in Figure 2. Cash cows are located on the lower end of the diagonal and are simply maintaining share. Stars are located on the top of the diagonal and slightly below, increasing share slightly. Dogs are situated up against the left- hand ordinate, consistent with the decision to manage for cash at the expense of market share. Two classes of wildcats are found. The wildcats on the left-hand ordinate are being allowed to lose share and are being managed for cash. The wildcats in the upper right comer below the diagonal portray those high-growth, low-share businesses which are being funded aggressively to capture market share. Note that the portfolio shown in Figure I has many of the idealized charac- teristics. The growth gain matrix can also be used to gain competitive insight as to where competitors are placing their emphasis provided a matrix can be drawn for the competitor. Frontier curves 0 i 10 • e Q 'ii 0 :::, C C < -10 0 40 80 120 100 200 Cash use(%) Figure 3 Frontier curves: Growth versus cash use (Moose & Zakon, 1972:66) The company or corporation's maximum sustainable rate of growth is given as D g = E (r-1)p + rp D g = E (r-1)p + rp where g is the sustainable growth rate in assets, D is the debt, E is the equity, r is the return on investment after tax ; is the interest rate after tax, and p is the proportion of eanrlngs retained. P is expressed as a decimal. Debt and equity take their money values and the units for g depend on those used for rand i, percentage or decimal, as discussed before. Note that the formula gives the maximum growth rate sustainable from intemally generated funds and debt leverage without recourse to the equity markets. The maximum sustainable growth rate can be plotted as a vertical line along the business growth axis as shown in Figure 3 Frontier curves: Growth versus cash use (Moose & Zakon, 1972:66) S. Afr. J. Bus. Mgmt. 1985, 16(3) 111 or less. Stars would be expected to be roughly in cash balance with growth rates in profits in excess of lOOJo. Wildcats would be expected to have annual growth rates in profits similar to stars but higher cash usage rates. Dogs, if properly managed, should have low growth rates and be cash generators, albeit small. The theoretical positioning of the menagerie is shown in Figure 4. Businesses that don't conform to the guidelines given are probably misclassified or mismanaged and represent 'cash traps' (Moose & Zakon, 1972:67). Plotting the businesses in the portfolio onto the grid given in Figure 4 highlights growth areas, cash generators and money traps. Thus far cash use has been understated and in reality deductions must be made for corporate cash needs in the form of dividends, interest burden, and overheads. It is assumed tacitly that the individual business, if constituted as a separate company, will have deducted dividend payments before arriving at a figure for cash use. These deductions must be made in order to anive at a realistic appraisal. In general, most companies do not have a system for the allocation of corporate overhead, particularly concerning the thorny issue of dividend payments. Frontier curves The Boston Consulting Group suggests that each business be assessed on the percentage net assets employed and that the given percentage of corporate dividends, interest, and over- head be deducted from profits before per cent reinvested is calculated (Moose & Zakon, 1972:67). Presumably the ap- proach relates to the fact that throughout the Boston Con- sulting Group's analyses, the maximum sustainable growth rate of the asset base of the complete portfolio is a key issue. The approach advocated tends to reduce the number of cash generators and presumably modifies the euphoria associated with portfolio analyses conducted independently of considera- tions associated with corporate overhead, however burden- some and crippling it could be. at each extreme of the cash scale and reduce to pinpoints close to lOOOJo cash use. The hatched circles in Figure 3 represent cash generators and the open circles cash users. It is possible to assess optically whether the corporation has a balance in cash terms or not. In Figure 3 the areas to the right of IOOOJo are greater than those to the left indicating a net cash deficiency and a need for outside financing. Figure 3 gives a guide as to the relative attractiveness of some businesses with respect to others in terms of relative growth in profits and cash flow. The concept can be extended to incorporate the concept of a goal for corporate growth. Given a corporate growth goal and assuming that the corpo- ration invests all of its cash, a frontier line can be drawn on the grid to differentiate attractive from unattractive oppor- tunities for the corporation. The equation derived for the maximum sustainable internal growth rate: D g = E (r-1) p + rp where g is the maximum sustainable growth rate in assets, Dis debt, £ is equity, r is return on net assets after tax, i is interest rate after tax, and p is the proportion of earnings retained, is effectively restated using the suggested method of evaluating the corporate burden involved in overheads and dividends. The Boston Consulting Group restates the equation as where g is the maximum sustainable growth rate in assets, Dis debt, £ is equity, r is return on net assets after tax, i is interest rate after tax, and p is the proportion of earnings retained, is effectively restated using the suggested method of evaluating the corporate burden involved in overheads and dividends. Frontier curves The Boston Consulting Group restates the equation as as D g = E (r-1) + (r-e)p ... (I) D g = E (r-1) + (r-e)p (I) where e is an after-tax charge on assets to cover corporate overhead and dividends (Moose & Zakon, 1972:69). Obviously e must be expressed in the dimension of g, that is as a percentage impact on the after-tax maximum sus- tainable growth rate. The equation as stated above is designed to reflect the total corporate sustainable growth rate and not that of individual businesses or products within the portfolio. Obviously e must be expressed in the dimension of g, that is as a percentage impact on the after-tax maximum sus- tainable growth rate. The equation as stated above is designed to reflect the total corporate sustainable growth rate and not that of individual businesses or products within the portfolio. The equation is a measure for discerning within the portfolio. As such it may not be necessary to calculate e explicitly, as recommended by the Boston Consulting Group. The value for e should be contained in r, the return on assets. Similarly, the extent to which a corporation has a balance between cash users and cash generators cannot be determined just by the numbers of businesses falling to the left or the right of the lOOOJo reinvestment line. The lOOOJo cash line represents an axis about which the cash flows must be in balance, equivalent to an engineering axis with zero inertia. It is necessary to look at the relative size of the cash con- tributions from the cash generators and the voracity of the cash hungry. A rough approximation of the portfolio status can be given visually by marking the positions of the various businesses with circles proportional in area to the amount of cash generated or used. The larger the circle, the larger the cash use or the cash contribution. The circles tend to be larger The equation is a measure for discerning within the portfolio. As such it may not be necessary to calculate e explicitly, as recommended by the Boston Consulting Group. The value for e should be contained in r, the return on assets. For given values of D, E, r, i and e for the corporation and provided these are held constant, equation I reduces to 2 g = Ap Check for internal balance It can be argued that low growth businesses can be leve- raged differently to high growth businesses. There also seems to be evidence that return on investment is not strongly correlated with stage of the product life cycle or the rate of growth in profits (Cvar, 1980). Because of the relative safety of low profit growth rate, mature, cash-generating businesses should support debt equity ratios greater than that of the corporate level. High growth businesses, because of their relative instability in earnings, should be less highly leveraged than the corporation as a whole. The portfolio should be examined for a reasonable distribution of the component businesses in each of the four quadrants of the growth share matrix. Products with the largest amount of sales or capital investment, as depicted by the largest circles, should appear as cash cows or stars. The majority of busi- nesses should appear as cash cows to be used to fund and underwrite the remaining businesses. Only a few businesses should appear as question marks owing to the heavy com- mitment of cash and management time required to transform them into stars. Few businesses should appear as dogs as these are cash traps and require the reinvestment of the little cash they produce. Robinson (1985) illustrated the portfolio of a well-balanced, diversified company. Only a few firms achieve this kind of balance as only one business in any market can be the leader. The average company will have more products in the right-hand half of the matrix than in the left half. Ideas for improving the balance should result from the analysis. Equation I can be used to generate frontier curves for different debt equity ratios as a function of cash usage patterns. The curves are found by taking the required corporate sus- tainable growth rate, g, various return and interest rates, r and i, and varying the ratio of debt, D, to equity, E, across the range of cash usage p. The equation is solved for p. Although not stated explicitly by the Boston Consulting Group this implies being able to express DIE, the debt equity ratio, as a function of p, the cash usage rate. Curves are generated as shown in Figure 6, It should be noted that, in addition to leverage, a small change in cash usage in a mature business tends to have a significant impact on the portfolio as a whole. where A is a constant. When p = l,O or IOOOJo cash retention, g is equal to the sustainable maximum growth rate for the corporation - in this case the desired growth rate. The result is a series of straight lines for various targeted rates as sho\\n in Figure 5. The value of A is easy to ascertain and, given,, i and e, the debt equity ratio can be solved to see if it is consistent with industry norms. When p = l,O or IOOOJo cash retention, g is equal to the sustainable maximum growth rate for the corporation - in this case the desired growth rate. The result is a series of straight lines for various targeted rates as sho\\n in Figure 5. 30 i 20 a, ~ ~ i 10 e * 0 Cl oi ~ C C 0 <( X -10 0 40 80 120 160 200 Cash use(%) 30 i 20 a, ~ ~ i 10 e * 0 Cl oi ~ C C 0 <( X -10 0 40 80 120 160 200 Cash use(%) Figure 4 Idealized positions on the frontier curve The value of A is easy to ascertain and, given,, i and e, the debt equity ratio can be solved to see if it is consistent with industry norms. The frontier curve as shown in Figure 5 allows for dif- ferentiation between businesses. Given a corporate growth goal, say 50Jo, and assuming the company reinvests all of its cash a frontier line can be drawn on the grid. Opportunities alon~ the line are equally attractive, those below the line are less attractive and those above the line are more attractive because of their chance of higher growth with the same cash use. Under reasonable expectations the frontier line becomes a realistic investment decision line. The areas of the circles representing the chosen portfolio above and below the line can be compared to assess the cash flow characteristics of the desired portfolio. The desired corporate growth line must also Cash use(%) Figure 4 Idealized positions on the frontier curve 112 160 200 Cash use(%) Figure S Frontier curves for different corporate growth targets (Moose & Zakon, 1972:67) 30 [ 20 Cl) ~ .c 3 10 e C> ~ ::, C: C: < 0 0 S.-Afr. Tydskr. Bedryfsl. Check for internal balance A low growth business which reduces its cash use from 100% to 50% may sacrifice only one or two percentage points in growth (Moose & Zakon, 1972:68). The same change in cash use for a high growth area may result in a disproportionate loss in growth. A retrenchment of some of the lower growth areas can be translated as a powerful switching of funds to higher growth· areas. where A is a constant. 1985, 16(3) 20% 15% 10% 5% 40 80 120 160 200 Cash usage (%) Figure 6 Frontier curves where the acceptable debt/equity ratio is negatively influenced by high growth rate (Moose & Zakon 1972:68) 112 160 200 Cash use(%) Figure S Frontier curves for different corporate growth targets (Moose & Zakon, 1972:67) S.-Afr. Tydskr. Bedryfsl. 1985, 16(3) 112 112 160 200 Cash use(%) 30 [ 20 Cl) ~ .c 3 10 e C> ~ ::, C: C: < 0 0 20% 15% 10% 5% 40 80 120 160 200 Cash usage (%) Figure 6 Frontier curves where the acceptable debt/equity ratio is negatively influenced by high growth rate (Moose & Zakon, 1972:68) 30 [ 20 Cl) ~ .c 3 10 e C> ~ ::, C: C: < 0 0 20% 15% 10% 5% 40 80 120 160 200 Cash usage (%) [ Cl) ~ .c 3 e C> ~ ::, C: C: < Figure S Frontier curves for different corporate growth targets (Moose & Zakon, 1972:67) Figure 6 Frontier curves where the acceptable debt/equity ratio is negatively influenced by high growth rate (Moose & Zakon, 1972:68) be a rotational axis with a zero moment of inertia for the whole idealized portfolio. frontier curve serves as a means of differentiating favourable investment opportunities from unfavourable areas rather broadly but in a comparable and quantifiable fashion. Given that the I 00% cash use line is an inertial axis as is the desired growth line, the centre of gravity of the portfolio has, both by the laws of statics and by definition, to be at the intersection of the two lines. Competitive analysis Up to now straight lines, rather than curves, have been used for the frontier curve. This assumes that the debt equity ratio is constant across the corporation's businesses and that all businesses, independently of their cash needs, can be leveraged to the same extent. Having represented the company's businesses on the growth share and the growth gain matrices, a seven-step strategic analysis should be carried out (Day, 1977 :29 - 38 and Abell & Hammond, 1979). • divestment candidates. Figure 8 Competitive dynamics on the growth share matrix Company 1 Company 2 CD ! ,:, I ss O> Relative market share Relative market share Competitive logic: Company 1 uses cash cow C to fund A Company 1 discounts in business B to upset company 2 Company 2 engages in price war with cash cow B · and funds for A· dry up prejuducing A· s competitive posi· tion. Figure 9 Competitive dynamics on the growth share matrix .r:. j e Company 1 Company 2 O> f----+--+--.--------- .; I~ Relative market share Relative market share ·conventional logic: Company 1 uses cash cow C to fund A insufficiently and divests itself of B. Com par y 2 uses cash cow B · to fund A· and overtake business A of company 1. Figure 8 Competitive dynamics on the growth share matrix .r:. j e Company 1 O> f----+--+--.--------- .; I~ Relative market share Company 2 Relative market share The next stage is to determine the feasibility of each plan under competitive conditions and financial constraints. Trend analysis The growth share matrix tends to be a static representation of the existing status quo of the portfolio. An equivalent portfolio should be prepared for an earlier period, perhaps three to five years earlier. The two portfolios can then be superimposed upon one another to determine the direction and rate of evolution of each business. Where large changes have occurred during the elapsed time interval it may be necessary to do yearly plots to fix the velocity and direction of movement accurately (Figure 7). In companies which do yearly portfolio planning exercises the trend analysis is simpli- fied as the latest chart only has to be compared with that of the previous year or further back if needed. The curvature of the curve will vary from company to company depending on the earnings trends in mature busi- nesses and the debt equity ratios of similar competitive businesses. The Boston Consulting Group claims that the The trends can be projected for the strategic planning S. Afr. J. Bus. Mgmt. 1985, 16(3) 113 24% .----------,---------~ 22% 20% 18% 16% ., 14% ! .r:. 12% j e "' 8% 6% 4% 2% 10x 6x ,. , , , , , ,. , ,. 4x 2x 1.5x 1 x 0.6x 0,4x Relative market share G-o Forecast position Present position , , , , D , ,,,. , , , 0.2x 0.1x Figure 7 Portfolio dynamics: Forecasting the direction and evolution of the portfolio 24% .----------,---------~ 22% 20% 18% 16% ., 14% ! .r:. 12% j e "' 8% 6% 4% 2% 10x 6x ,. , , , , , ,. , ,. 4x 2x 1.5x 1 x 0.6x 0,4x , , , , D , ,,,. , , , 0.2x 0.1x suffi~ent size to "'.arrant a battle with the market leader, upon question ~ks with sufficient leftward momentum and upon products with leadership positions. Businesses become separated tentatively into four basic strategic sets: • Gr~wth businesses earmarked for market share growth, • maintenance businesses, • harvesting candidates to be milked for maximum cash flow, and · Evaluate competition The growth share matrix and the growth gain matrix should be developed for the firm's major competitors. The displays will not be as reliable as charts for one's own firm but they should at least be a distillation of the best information available on competitors and should, as such, be more useful than the unintegrated data. ·conventional logic: Company 1 uses cash cow C to fund A insufficiently and divests itself of B. Com par y 2 uses cash cow B · to fund A· and overtake business A of company 1. A careful analysis of the competitors' portfolios should reveal what each is doing. An analysis should give insights into their strategies and indicate their cash cows, stars, wild- cats, and dogs; indicate how close they are to their sustainable growth rates; and indicate their strengths and weaknesses and where a major competitor might be so concerned with an area that he would be unlikely to mobilize the resources to hold off an attack in another area. Figure 8 Competitive dynamics on the growth share matrix igure 8 Competitive dynamics on the growth share matrix Figure 8 Competitive dynamics on the growth share matrix Company 1 Company 2 CD ! ,:, I ss O> Relative market share Relative market share Competitive logic: Company 1 uses cash cow C to fund A Company 1 discounts in business B to upset company 2 Company 2 engages in price war with cash cow B · and funds for A· dry up prejuducing A· s competitive posi· tion. Figure 9 Competitive dynamics on the growth share matrix Company 2 Relative market share Taking one business at a time the charts should be com- pared to assess competitive strength where share increases are contemplated. Attempts to gain market share in low growth segments should be done when a business is roughly on a par with the dominant firm in terms of a relative market share, where it has a leadership edge on other strategic criteria, where strong leftward momentum is evident from the trend charts, or where competitors appear not to be investing. In general the decision should be to divest or manage for cash those businesses in the wildcat or dog sectors, particularly where competitors seem to be aggressively expanding strong com- petitive products. • divestment candidates. • divestment candidates. In addition the impact of strategic pricing decisions can also be made particularly with regard to the interplay between one's own portfolio and that of competitors. Two examples are graphically portrayed in Figures 8 and 9. In Figure 8 the firm has three business units, one a largish star, A, a dog, B, and a cash cow, C. The main opponent also has a substantial wildcat, A', and a large cash cow, B'. Conventional strategic planning would advocate that: . • the star, A, be funded, if needed, • the dog, B, be divested or gradually run down, and • the cash cow, C, be used to fund A. In Figure 8 the firm has three business units, one a largish star, A, a dog, B, and a cash cow, C. The main opponent also has a substantial wildcat, A', and a large cash cow, B'. Conventional strategic planning would advocate that: . Relative market share • the star, A, be funded, if needed, This, however, leaves the way right open for the competitor to use his substantial cash flow from his cash cow, B', to fund his wildcat, A', into star status. Potential exists for predatory price cutting by the dog B in competition with B' designed to upset the cash flows from B' and prevent the funding of A' in competition with A. Figure 7 Portfolio dynamics: Forecasting the direction and evolution of the portfolio In Figure 9 a conscious decision has been made to raise prices and margins on the cash cow, C, in order to maximize In Figure 9 a conscious decision has been made to raise prices and margins on the cash cow, C, in order to maximize period, say three to five years, to indicate where each business would be if present policies were maintained for that period. Target charts can then be developed showing the desired future position of the portfolio. Ideas for improving the evolution of the portfolio should result from this, the second stage of the analysis. .r:. j e Company 1 Company 2 O> f----+--+--.--------- .; I~ Relative market share Relative market share ·conventional logic: Company 1 uses cash cow C to fund A insufficiently and divests itself of B. Com par y 2 uses cash cow B · to fund A· and overtake business A of company 1. Check financial balance Detailed pro forma cash flow calculations must be carried out to check the strategic decision chosen. Cash flow needs and generation of each strategy must be checked. Estimates of internally generated and externally available funds must be made and balanced. If a shortage of cash is forecast, then some businesses may have to be reclassified and more dogs and question marks may need to be harvested or abandoned. Some gains in market share may have to be foregone. . . ~ The analysis of industry position should corroborate the trend analysis of step 2 in a graphic manner. Plotting the maximum sustainable growth rate of the portfolio on the vertical axis allows for the selection of business strategies to improve their position relative to the idealized patterns shown on Figure 2. For example a dog due for harvesting which is positioned near the maximum growth rate line can be re- positioned near the ideal zero growth axis by allowing market share to decline in order to maximize cash flow. An analysis of the financial balance of competitors ~ reveal cash flow constraints which will shape their portfolios and their anticipated actions. Consideration of other factors The portfolio display is concerned with a measure of market attractiveness as expressed by market growth rate and com- petitive ability as expressed by relative market share. The location of businesses on the chart indicates their cash charac- teristics. The area of the circle representing a business portrays the relative sales or investment associated with the business. Properly employed, this information is a substantial input to the planning process. Dogs The closer the dog is on the spectrum toward the cash cow business quadrant, the more amenable it is to being treated as a cash cow. The lower the growth rate and the lower the relative market share, the more the dog becomes a candidate for divestment, abandonment, harvesting, or strategic market segmentation. Industry position The growth share matrix uses market growth rate and relative market share as surrogate variables to indicate the cash flow requirements of the portfolio. The analysis can be further refined by plotting the growth gain matrix relating market growth rate to the growth rate of the business under con- sideration (Figure 1). Products above the diagonal represent market growth in excess of product growth, indicating de- clining market share. Ideally only businesses being milked, divested, or phased out and businesses due to receive major efforts to gain market share should lie above the diagonal. If this is not the case then the strategic classifications esta- blished in phase 3 should be revised to account for it. Develop possible target portfolios Target portfolios can now be developed on the strength of the previous four steps, notably checking for balance, trend analysis, evaluation of competition, a consideration of key strategic factors not covered in the portfolio approach, and an analysis of industry position. Tentative strategies for each business can be identified on the basis of their position on the matrix. Cash cows its cash flow in spite of a drift owing to loss of market share. The enhanced cash flow is designated for use in funding a large wildcat, A, into a dominant position over the com- petitor's business. Competitive pricing by the dog B, has had the effect of decreasing the competitor's cash flow from B' owing to predatory price cutting and has eroded his ability to finance A'. its cash flow in spite of a drift owing to loss of market share. The enhanced cash flow is designated for use in funding a large wildcat, A, into a dominant position over the com- petitor's business. Competitive pricing by the dog B, has had the effect of decreasing the competitor's cash flow from B' owing to predatory price cutting and has eroded his ability to finance A'. The usual strategy is to maintain market dominance and use the strong cash flows these businesses generate. Product, technological and price leadership is maintained. Product proliferation and investment in market expansion is guarded against to prevent the sapping of cash flow. 'Exceptions would include expansion to exploit a competitor's weaknesses and new, hard-t0<0py product innovation, or increases in primary demand' (Abell & Hammond, 1979:189). The focus is main- tenance of market and cost dominance while excess cash is channeled into better opportunities. Sound competitive analysis coupled with creativity in asses- sing the strategic implications of moves which woul~ upset the competition is crucial for strategy development. It 1s often the weakest link. It is often seen to be both difficult and highly speculative. Many analysts prefer the easier analysis of internal data. Strategic nirvana, however, is not likely to be reached by introspective contemplation of one's own navel. Problems associated with lack of data must often be overcome by imaginative use of data sources. Stars Market share is either held or grown slightly. Being almost self sufficient, cash infusions are small or infrequent. Building share in a high growth business can easily be obtained by gaining a large proportion of new market business. Holding share is achieved by price reductions, product improvements, production efficiencies and improved market coverage. Evaluate competition Attention should be focused on those products dose to the 1,0 relative market share line and of Competitive logic: Company 1 uses cash cow C to fund A Company 1 discounts in business B to upset company 2 Company 2 engages in price war with cash cow B · and funds for A· dry up prejuducing A· s competitive posi· tion. Competitive logic: Company 1 uses cash cow C to fund A Company 1 discounts in business B to upset company 2 Company 2 engages in price war with cash cow B · and funds for A· dry up prejuducing A· s competitive posi· tion. Competitive logic: Company 1 uses cash cow C to fund A Company 1 discounts in business B to upset company 2 Company 2 engages in price war with cash cow B · and funds for A· dry up prejuducing A· s competitive posi· tion. Figure 9 Competitive dynamics on the growth share matrix 114 S.-Afr. Tydskr. Bedryfsl. 1985, 16(3) Wildcats Most wildcats are in an untenable position because of the poor margins and heavy cash demands. The choices are to expand a few into stars, to focus a few others and to exit the rest. Expansion is geared to gaining a disproportionate share of the growing market or the acquestion of competitors. Domi- nance can be obtained in a market niche by strategic seg- mentation. Exiting can be via divestment as a going concern, harvesting or abandonment. Additional information must be weighed prior to deciding on strategies for each business and on the implementation details of each strategy (Day, 1977 and Abell & Hammond, 1979). Consideration also needs to be given to the acceptance of suboptimal performance to ensure the availability of critical inputs and the need for criteria such as cash flow and profit volatility to be restrained. A detailed discussion follows later. Conclusion The growth gain and frontier curve matrices provide addi· tional insights into the dynamics of analysis and cash alloca- tion around a portfolio. A strategic checklist can be generated to guide the planning process in an attempt to ens.ure ~h~t the unwary do not fall into pitfalls awaiting a too simplistic approach to portfolio planning. Day, G.S. 1977. Diagnosing the product portfolio. J. Mark. vol. 41, 29-38. Hammond, J.S. & Allan, G.B. 1975. A note on the Boston Consulting Group concept of competitive analysis and corporate strategy. Boston, Massachusetts. Harvard Business s. Afr. J. Bus. Mgmt. 1985, 16(3) School, note 9-175-17S. Moose, S.A. & Zakon, A.J. 1972. Frontier curve analysis: As a resource allocation guide. J. Bus. Policy, 63 - 70. Robinson, C.G. 198S. The Boston Consulting Group's strategic menagerie. S. Afr. J. Bus. Mgmt., vol. 16, 76-86. References Abell, D.F. & Hammond, J.S. 1979. Strategic market planning Englewood Cliffs. Prentice Hall. Cvar, M. 1980. Strategic planning: What is it? Cambridge, Massachusetts. Strategic Planning Institute. , g p g g , Massachusetts. Strategic Planning Institute. 115 Day, G.S. 1977. Diagnosing the product portfolio. J. Mark. vol. 41, 29-38. School, note 9-175-17S. Moose, S.A. & Zakon, A.J. 1972. Frontier curve analysis: As a resource allocation guide. J. Bus. Policy, 63 - 70. Robinson, C.G. 198S. The Boston Consulting Group's strategic menagerie. S. Afr. J. Bus. Mgmt., vol. 16, 76-86. Hammond, J.S. & Allan, G.B. 1975. A note on the Boston Consulting Group concept of competitive analysis and corporate strategy. Boston, Massachusetts. Harvard Business

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Portuguese

Associação entre o baixo peso ao nascer e doença periodontal

Revista de saúde pública/Revista de Saúde Pública

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Rev Saúde Pública 2006;40(2):353 Rev Saúde Pública 2006;40(2):353 Carta ao Editor | Letters to the Editor Prezado Editor, Esclarecemos que os autores abaixo discriminados participaram da elaboração da carta ao Editor publicada no vol. 40(1):181-3 da Revista de Saúde Pública, em resposta às questões teóricas, metodoló- gicas e éticas levantadas por Vettori, Sheiham e Peres sobre o artigo “Doença periodontal materna como fator associado com o baixo peso ao nascer”. • Maria Isabel P. Vianna Faculdade de Odontologia. Universidade Federal da Bahia. Salvador, BA, Brasil • Carlos Teles Santos Universidade Estadual de Feira de Santana. Feira de Santana, BA, Brasil • Carlos Teles Santos Universidade Estadual de Feira de Santana. Feira de Santana, BA, Brasil • Simone Seixas da Cruz Departamento de Saúde. Universidade Estadual de Feira de Santana. Feira de Santana, BA, Brasil. Maria da Conceição N. Costa Instituto de Saúde Coletiva, Universidade Federal da Bahia Maria da Conceição N. Costa Instituto de Saúde Coletiva, Universidade Federal da Bahia • Maria da Conceição N. Costa Instituto de Saúde Coletiva. Universidade Fede- • Isaac Suzart Gomes Filho Departamento de Saúde. Universidade Estadual de Feira de Santana. Feira de Santana, BA, Brasil Low birth weight and periodontal disease association Salvador, 20 de fevereiro de 2006. ral da Bahia. Salvador, BA, Brasil Salvador, 20 de fevereiro de 2006. ral da Bahia. Salvador, BA, Brasil • Isaac Suzart Gomes Filho Departamento de Saúde. Universidade Estadual de Feira de Santana. Feira de Santana, BA, Brasil Prezado Editor, Maria da Conceição N. Costa Instituto de Saúde Coletiva, Universidade Federal da Bahia Maria da Conceição N. Costa Instituto de Saúde Coletiva, Universidade Federal da Bahia Dear Editor, We would like to clarify that the authors cited below have participated in the elaboration of the Letter to the Editor published in Revista de Saúde Pública 40(1):184-6, in reply to the ethics, methodological and theoretical questions of Vettori, Sheiham and Peres about the article “Maternal periodontal disease as a factor associated with low birth weight”. • Maria Isabel P. Vianna Faculdade de Odontologia. Universidade Federal da Bahia. Salvador, BA, Brasil • Carlos Teles Santos Universidade Estadual de Feira de Santana. Sal- vador, BA, Brasil • Carlos Teles Santos Universidade Estadual de Feira de Santana. Sal- vador, BA, Brasil • Simone Seixas da Cruz Departamento de Saúde. Universidade Estadual de Feira de Santana. Feira de Santana, BA, Brasil de Feira de Santana. Feira de Santana, BA, Brasil Maria da Conceição N. Costa Instituto de Saúde Coletiva, Universidade Federal da Bahia • Maria da Conceição N. Costa Instituto de Saúde Coletiva. Universidade Fede- ral da Bahia Salvador, BA, Brasil

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Fluxes of lactate into, from, and among gap junction-coupled astrocytes and their interaction with noradrenaline

Frontiers in neuroscience

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METABOLIC, DIAGNOSTIC, AND SIGNALING ROLES OF LACTATE et al., 2006; Gordon et al., 2008), increasing nutrient delivery and by-product removal. Lactate has well-known and intriguing roles in brain function. Its resting concentration (∼0.5–1 mmol/L) doubles during brain activation, and increases ∼10–20-fold during abnormal states (Siesjö, 1978; Mangia et al., 2007). Lactate is generated from pyru- vate when (i) glycolytic flux exceeds the rates of the TCA cycle and the malate-aspartate shuttle (MAS) that transfers reducing equiv- alents from cytoplasmic NADH into mitochondria, or (ii) when oxygen levels are insufficient to sustain oxidative metabolism. Thus, lactate formation is a “safety valve” to quickly regenerate NAD+ from NADH, thereby allowing rapid up-regulation and maintenance of high glycolytic flux. Lactate and pyruvate readily move down their concentration gradients to extracellular fluid, and the lactate/pyruvate concentration ratio in microdialysate is an important diagnostic tool predictive of clinical outcome of patients with traumatic brain injury; the higher the ratio the worse outcome (Nordström et al., 2013). Increased lactate pro- duction to sustain high glycolytic rate is associated with greater lactate release to blood because the brain concentration then exceeds that in blood. High cerebral blood flow maintains this gradient and “pulls” lactate from brain. Lactate in perivascu- lar fluid, presumably mainly released from astrocytic endfeet (Gandhi et al., 2009), stimulates blood flow to activated regions (Laptook et al., 1988; Hein et al., 2006; Lombard, 2006; Yamanishi Conversely, increasing blood lactate concentration by intense physical activity drives lactate down its concentration gradi- ent into all brain cells. Lactate oxidation supplements brain glucose metabolism to an increasing extent with rising blood level (Dalsgaard et al., 2004; Van Hall et al., 2009), and it does not accumulate in brain above resting levels (Dalsgaard et al., 2004). Metabolism of lactate requires its conversion back to pyruvate that, in turn, can have different metabolic fates (conversion to alanine, oxaloacetate, or acetyl CoA), which vary with cell type and metabolic state. Continued net uptake of lactate depends on its oxidation to pyruvate plus NADH and may cause the intracellular redox state to become more reduced, although cytosolic NAD+/NADH ratio is relatively sta- ble in cell lines (Sun et al., 2012). Lactate is co-transported with a proton via equilibrative monocarboxylic acid trans- porters (MCTs) (Poole and Halestrap, 1993), and lactate influx accordingly causes intracellular acidification (Nedergaard and Goldman, 1993). Fluxes of lactate into, from, and among gap junction-coupled astrocytes and their interaction with noradrenaline Leif Hertz 1, Marie E. Gibbs 2 and Gerald A. Dienel 3* 1 Laboratory of Brain Metabolic Diseases, Institute of Metabolic Disease Research and Drug Development, China Medical University, Shenyang, China 2 Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences, Monash University, Clayton, VIC, Australia 3 Department of Neurology, University of Arkansas for Medical Sciences, Little Rock, AR, USA Lactate is a versatile metabolite with important roles in modulation of brain glucose utilization rate (CMRglc), diagnosis of brain-injured patients, redox- and receptor-mediated signaling, memory, and alteration of gene transcription. Neurons and astrocytes release and accumulate lactate using equilibrative monocarboxylate transporters that carry out net transmembrane transport of lactate only until intra- and extracellular levels reach equilibrium. Astrocytes have much faster lactate uptake than neurons and shuttle more lactate among gap junction-coupled astrocytes than to nearby neurons. Lactate diffusion within syncytia can provide precursors for oxidative metabolism and glutamate synthesis and facilitate its release from endfeet to perivascular space to stimulate blood flow. Lactate efflux from brain during activation underlies the large underestimation of CMRglc with labeled glucose and fall in CMRO2/CMRglc ratio. Receptor-mediated effects of lactate on locus coeruleus neurons include noradrenaline release in cerebral cortex and c-AMP-mediated stimulation of astrocytic gap junctional coupling, thereby enhancing its dispersal and release from brain. Lactate transport is essential for its multifunctional roles. MINI REVIEW ARTICLE published: 09 September 2014 doi: 10.3389/fnins.2014.00261 Fluxes of lactate into, from, and among gap junction-coupled astrocytes and their interaction with noradrenaline Edited by: Edited by: Avital Schurr, University of Louisville, USA Reviewed by: Linda H. Bergersen, University of Oslo, Norway Johannes Hirrlinger, University of Leipzig, Germany *Correspondence: Gerald A. Dienel, Department of Neurology, University of Arkansas for Medical Sciences, Slot 500, 4301 W. Markham St. Little Rock, AR 72205, USA e-mail: [email protected] Keywords: astrocyte, acetate, lactate, locus coeruleus, neuron, monocarboxylic acid transporter, memory METABOLIC, DIAGNOSTIC, AND SIGNALING ROLES OF LACTATE Lactate uptake can, therefore, inhibit glycolysis by reducing availability of NAD+ for glycolysis and by acidifi- cation that can inhibit phosphofructokinase, which has a steep pH-activity profile (Dienel, 2012). Widespread lactate signal- ing, especially to neurons, via the receptor GPR81 decreases cAMP (IC50 ∼29 mmol/L), which can decrease glycolysis at high September 2014 | Volume 8 | Article 261 | 1 www.frontiersin.org www.frontiersin.org Multifunctional roles of lactate depend on its transport Hertz et al. extracellular lactate concentrations; a significant effect on cAMP requires ≥10 mmol/L lactate (Lauritzen et al., 2013). Thus, “pushing” lactate into all brain cells from blood provides sup- plementary fuel and evokes regulatory mechanisms that reduce brain glucose utilization when muscular lactate production is high. concentrations. Extracellular pH in brain is normally 7.3, but it is lower in brain slices (∼7.1) incubated at pH 7.3–7.4 (Chesler, 2003). Most results for intracellular pH have been obtained in brain slices or cultured cells and it is generally lower than in extra- cellular fluid although only by 0.2–0.3 pH units, indicating that the H+ concentration is at most two-fold higher intracellularly than extracellularly (e.g., Roos and Boron, 1981). Thus, the H+ gradient only moderately enhances diffusional lactate efflux and reduces its diffusional influx. Lactate can also influence astrocytic and neuronal activities by redox-mediated signaling. Astrocyte calcium signals are regulated by NAD+/NADH redox state (Requardt et al., 2012; Wilhelm and Hirrlinger, 2012), and changes in intracellular NAD+ and NADH levels arising from lactate fluxes may affect their binding to tran- scription factors and influence gene expression (Nakamura et al., 2012). For example, the transcription co-repressor, C-terminal binding protein (CtBP), is a dehydrogenase that undergoes con- formational change with binding of NAD+ and NADH; NADH has a much higher affinity for CtBP, allowing it to serve as a redox sensor that destabilizes interactions with CtBP and tran- scription factors (Kumar et al., 2002; Fjeld et al., 2003). Increased NADH levels are thought to underlie seizure-induced increased expression of brain-derived neurotrophic factor (BDNF) and its receptor TrkB (Garriga-Canut et al., 2006). NAD+ is required for the action of sirtuins, a family of deacetylases that regu- late activities of transcription factors and metabolic cofactors, and important roles for sirtuins in brain development, aging, and neurodegenerative diseases have been identified (Harting and Knoll, 2010; Bonda et al., 2011). METABOLIC, DIAGNOSTIC, AND SIGNALING ROLES OF LACTATE Diffusional uptake is only measurable during very short incu- bation times and contribution of metabolism-driven uptake will distort its kinetics (Hertz and Dienel, 2005). Figure 1A illustrates lactate uptake into cerebellar neurons at 1 mmol/L extracellular lactate. The initial diffusional uptake is very brief (<∼30 s; Figure 1A inset), rapid (∼10 nmol/mg protein or 1 μmol/g wet wt.), and only occurs in cells containing <1 mmol/L lactate. Thereafter, metabolism-driven net uptake takes over and is sustained for ≥1 h at 0.5 nmol lactate/mg protein per min, corresponding to 0.25 nmol glucose equivalent/mg pro- tein per min. Lactate metabolism is lower than measured rates of non-stimulated and stimulated glucose oxidation (1.0 and 2.23 nmol/mg protein per min, respectively) in cerebellar neurons (Peng et al., 1994). The above glucose oxidation rates are minimal values because the assays were based on 14CO2 production, and exchange reactions cause label dilution in amino acid pools, slow- ing 14CO2 release and causing underestimation of oxidation rate. Thus, the potential contribution of any lactate to total CO2 forma- tion in the neurons under activated conditions would be <10% of that from glucose. In cultured astrocytes, diffusional uptake is faster than in neurons (suggesting higher Vmax), but the rate of metabolism-driven uptake is similar (Dienel and Hertz, 2001). To summarize, lactate serves vital functions that include metabolic regulation (sustaining glycolysis by regenerating NAD+ or inhibiting glycolysis by intracellular acidification, NAD+ depletion and signaling), blood flow stimulation, influ- ence on gene transcription via redox state, and signaling via receptor binding. During intense exercise muscle-derived lactate serves as an important metabolite for brain. Movement of lactate to and from cells via MCTs seems to be a central element in its multifunctional roles. Neurons and astrocytes express different MCTs. MCT2 has a Km for lactate of ∼0.7 mmol/L and is predominantly neu- ronal, whereas MCT1 (Km 3–5 mmol/L) and MCT4 (Km 15– 30 mmol/L) are mainly astrocytic (for references see Hertz and Dienel, 2005). These MCT’s do not determine net lactate fluxes, which are mainly metabolism-driven for influx or concentration gradient-driven for efflux (although potentially increased by lac- tate removal by extracellular diffusion or cellular re-uptake), but they may be rate-limiting when concentration gradients develop rapidly. Lactate transport is governed by lactate concentration, Km, and transporter number, and it is enhanced by “transaccel- eration” (Juel, 1991; Juel et al., 1994). METABOLIC, DIAGNOSTIC, AND SIGNALING ROLES OF LACTATE Lactate exit is stimulated by extracellular pyruvate (San Martin et al., 2013), perhaps stimulat- ing a heteroexchange. The lower affinity MCTs in astrocytes may promote astrocytic release and re-uptake even at high concentra- tions. MCTs are inhibited by several drugs, including 4-CIN, and lactate transport is competitively inhibited by D-lactate. These toxins have repeatedly been used to allegedly show the impor- tance of MCT-mediated intercellular transport. However, it has never been demonstrated that these drugs at the same concentra- tions do not also inhibit pyruvate uptake into mitochondria, as shown by McKenna et al. (2001), who demonstrated that incu- bation with 0.25 mmol/L 4-CIN decreased oxidation of glucose to ∼50% of control values in both astrocytes and neurons in pri- mary cultures, although cellular glucose uptake was not inhibited by 4-CIN. Frontiers in Neuroscience | Neuroenergetics, Nutrition and Brain Health MCT TRANSPORTER FUNCTION Lactate is bi-directionally transported across cell membranes by MCT-mediated diffusional, saturable co-transport with H+. In the absence of a transcellular H+ gradient, extracellular lac- tate can increase its intracellular concentration up to, but not beyond the extracellular level and vice versa (Poole and Halestrap, 1993; Juel and Halestrap, 1999). Transporter-mediated diffusional uptake is equilibrative and energy-independent. However, con- tinuing inwardly-directed diffusional net transport (influx) can be achieved by intracellular metabolism that reduces the intra- cellular level of the non-metabolized lactate and maintains a concentration gradient between extra- and intracellular concen- trations of the non-metabolized compound (metabolism-driven uptake). This cannot increase the intracellular concentration of lactate itself. Analogously, continued removal of extracellular lac- tate by diffusion or uptake into other cells can increase net outward transport of lactate (efflux), but not its extracellular con- centration. If extra- and intracellular pH differ, the equilibrium level is determined by the gradients of both lactate anions and H+, and it is reached when the product of intracellular lactate and H+ concentrations equals that of extracellular lactate and H+ September 2014 | Volume 8 | Article 261 | 2 Frontiers in Neuroscience | Neuroenergetics, Nutrition and Brain Health Multifunctional roles of lactate depend on its transport Hertz et al. FIGURE 1 | Influx and gap junction-mediated trafficking of lactate. (A) Diffusional and metabolism-driven lactate uptake. Accumulation of [U-14C]lactate into primary cultures of cerebellar granule cell neurons in lactate/mg protein per min. Slightly modified from Dienel and He ©2001 Wiley-Liss, Inc., with permission of John Wiley and Sons Lactate trafficking among astrocytes Gap junction-coupled astro and gap junction-mediated trafficking of lactate. (A) abolism-driven lactate uptake. Accumulation of primary cultures of cerebellar granule cell neurons in cubated in tissue culture medium of approximately similar lar water phase, shown as a function of time of exposure lactate. The solid line is an extrapolation of the initial, litated diffusion during the first few seconds otein. The inset (right panel) emphasizes the early te uptake. The continued slower uptake of label after the lactate/mg protein per min. Slightly modified from Dienel and ©2001 Wiley-Liss, Inc., with permission of John Wiley and S Lactate trafficking among astrocytes. MCT TRANSPORTER FUNCTION Gap junction-coupled a slices of adult rat brain inferior colliculus were visualized by 5 Lucifer yellow from a micropipette inserted into a single astr yellow labeled the soma (light spots) of as many as 10,000 a up to about 1 mm from the impaled cell (a), and diffusion of astrocytic endfeet surrounding blood vessels caused high pe labeling (b). Scale bars in a and b denote 100 and 25 μm, res FIGURE 1 | Influx and gap junction-mediated trafficking of lactate. (A) lactate/mg protein per min. Slightly modified from Dienel and Hertz (2001), ©2001 Wiley-Liss, Inc., with permission of John Wiley and Sons, Inc. (B) Lactate trafficking among astrocytes. Gap junction-coupled astrocytes in slices of adult rat brain inferior colliculus were visualized by 5 min diffusion of Lucifer yellow from a micropipette inserted into a single astrocyte. Lucifer yellow labeled the soma (light spots) of as many as 10,000 astrocytes located up to about 1 mm from the impaled cell (a), and diffusion of dye into astrocytic endfeet surrounding blood vessels caused high perivasculature labeling (b). Scale bars in a and b denote 100 and 25 μm, respectively. Note that Lucifer yellow is retained within the coupled cells and it reveals the (Continued) lactate/mg protein per min. Slightly modified from Dienel and Hertz (2001), ©2001 Wiley-Liss, Inc., with permission of John Wiley and Sons, Inc. (B) Lactate trafficking among astrocytes. Gap junction-coupled astrocytes in slices of adult rat brain inferior colliculus were visualized by 5 min diffusion of Lucifer yellow from a micropipette inserted into a single astrocyte. Lucifer yellow labeled the soma (light spots) of as many as 10,000 astrocytes located up to about 1 mm from the impaled cell (a), and diffusion of dye into astrocytic endfeet surrounding blood vessels caused high perivasculature labeling (b). Scale bars in a and b denote 100 and 25 μm, respectively. Note that Lucifer yellow is retained within the coupled cells and it reveals the (Continued) lactate/mg protein per min. Slightly modified from Dienel and Hertz (2001), ©2001 Wiley-Liss, Inc., with permission of John Wiley and Sons, Inc. (B) Lactate trafficking among astrocytes. Gap junction-coupled astrocytes in slices of adult rat brain inferior colliculus were visualized by 5 min diffusion of Lucifer yellow from a micropipette inserted into a single astrocyte. FIGURE 1 | Continued size of the syncytium coupled to a single astrocyte. Lactate can enter and leave cells via MCT transporters, and its direct diffusion (i.e., without exit and re-entry) throughout the extent of the entire Lucifer yellow-labeled syncytium is probably less than that of Lucifer yellow. Lactate was directly shown to diffuse through gap junctions to coupled cells located ∼50 μm from the impaled cell (longer distances were not tested; Gandhi et al., 2009). Lactate exit plus re-entry into the same syncytium or to separate nearby syncytia would lead to extensive diffusion of lactate from the point source of the impaled cell. The schematic model for metabolite trafficking (c) illustrates uptake of glucose from blood into interstitial fluid and astrocytic endfeet, followed by diffusion of glucose down its concentration gradient from blood through extracellular fluid and the astrocytic syncytium, ultimately to the cells that are actively metabolizing glucose and creating a local sink toward which unmetabolized glucose diffuses. Detailed studies of (i) rates and capacities for lactate uptake from extracellular fluid into astrocytes and neurons and (ii) shuttling of lactate among gap junction-coupled astrocytes (yellow) compared with shuttling from astrocytes to neurons revealed that astrocytes have faster and greater capacity for lactate uptake and for lactate shuttling within the syncytium compared with neuronal uptake and transfer of lactate to neurons; glucose can also diffuse from an impaled astrocyte to neurons (Gandhi et al., 2009). Thus, astrocytic lactate uptake from interstitial fluid prevails, regardless of the cellular origin of the lactate. Once inside the syncytium (yellow) Acetate is a preferential substrate for astrocytic, but not neu- ronal, MCTs, and it is also metabolized by astrocytes (Muir et al., 1986; Waniewski and Martin, 1998). Acetate may, accord- ingly, serve as an indicator of astrocyte-specific lactate transport. Inhibition of learning in day-old chicks by the non-metabolizable D-lactate can be prevented by administration of acetate at two different time periods, immediately after training and 20 min later (Gibbs and Hertz, 2008). Immediately after training, rescue by acetate requires co-administration of aspartate, which alone has no effect. Twenty min after training acetate by itself res- cues learning; this is a time at which astrocytic metabolism is known to be activated, a further indication that acetate rescues energy metabolism. MCT TRANSPORTER FUNCTION Lucifer yellow labeled the soma (light spots) of as many as 10,000 astrocytes located up to about 1 mm from the impaled cell (a), and diffusion of dye into astrocytic endfeet surrounding blood vessels caused high perivasculature labeling (b). Scale bars in a and b denote 100 and 25 μm, respectively. Note that Lucifer yellow is retained within the coupled cells and it reveals the (Continued) Diffusional and metabolism-driven lactate uptake. Accumulation of [U-14C]lactate into primary cultures of cerebellar granule cell neurons in primary cultures incubated in tissue culture medium of approximately similar pH as the intracellular water phase, shown as a function of time of exposure to 1 mmol/L [U-14C]lactate. The solid line is an extrapolation of the initial, rapid uptake by facilitated diffusion during the first few seconds at ∼10 nmol/mg protein. The inset (right panel) emphasizes the early component of lactate uptake. The continued slower uptake of label after the initial rapid phase represents metabolism-driven uptake, and its rate, indicated by the stippled line, is sustained for at least an hour at 0.5 nmol September 2014 | Volume 8 | Article 261 | 3 www.frontiersin.org Multifunctional roles of lactate depend on its transport Hertz et al. diffusion of lactate down its concentration gradient through gap junctions (purple cylinders) to other coupled astrocytes and their endfeet facilitates lactate discharge to perivascular fluid (blue) where it can be removed from brain by perivascular-lymphatic flow and by discharge into cerebral venous blood. The perivascular fluid space is color coded only to emphasize its location; there is no physical boundary between interstitial fluid and perivascular fluid, although diffusion between these locations is influenced by tortuosity. Isoforms of monocarboxylic acid transporters (MCTs) have different Km values for lactate, and relative rates of lactate transport by these isoforms when lactate concentration rises are illustrated in the table for Km values within the ranges given in the text (i.e., 0.7, 3–5, and 15–30 mmol/L for MCT2, 1, and 4, respectively). The low Km MCT2 in neurons restricts lactate influx and efflux compared with the higher Km isoforms in astrocytes. During brain activation in sedentary subjects, brain lactate level in activated structures is higher than that in blood. Triangles denote outward lactate gradients from intracellular to extracellular fluid, from extracellular fluid to blood, and from intracellular fluid of astrocytes located near cells with high glycolytic activity to endfeet and blood. BRAIN LACTATE FLUXES Because lactate transport is concentration-gradient driven, knowledge of both transport and metabolism is needed to evaluate net fluxes and ultimate fate of transported lactate. Microdialysis and microelectrode studies have shown that extra- cellular lactate levels rise quickly to about twice the resting value of ∼0.5–1 mmol/L during an activating stimulus, then return to normal; up-and-down cycling of extracellular and total lac- tate concentrations occurs with repeated transient stimuli (e.g., Korf and De Boer, 1990; Mangia et al., 2007). Changes in lactate concentration reflect net input and output fluxes to the lactate MCT TRANSPORTER FUNCTION During strenuous physical exercise that greatly increases blood lactate concentration, these gradients would be reversed, driving lactate into all brain cells (not shown). Glc, glucose; Lac, lactate; GLUT, glucose transporter. Modified from Gandhi et al. (2009) ©2009, the authors. Journal compilation ©2009 International Society for Neurochemistry, with permission from John Wiley and Sons, Inc and the authors. FIGURE 1 | Continued These observations identify the affected cells as astrocytes, and the aspartate requirement shows that the res- cue immediately after training is due to formation of glutamate, which is normally formed in astrocytes from lactate/pyruvate by a combination of pyruvate carboxylation to oxaloacetate (which is astrocyte-specific) and pyruvate metabolism via the pyru- vate dehydrogenase. No pyruvate carboxylation is possible with acetate as sole substrate, but co-administration of aspartate abol- ishes this requirement, because aspartate is an alternative oxaloac- etate precursor. Thus, at both times, the rescue by acetate is due to support of astrocytic metabolism impaired by D-lactate, not to MCT-mediated inhibition of neuronal lactate uptake. pool and are not indicators of lactate flux through the lactate pools. Most extracellular lactate produced during brain activation may come from astrocytes (Elekes et al., 1996), but modeling sup- ports a neuronal origin and shuttling to astrocytes (Mangia et al., 2009). Small amounts of lactate, equivalent to ∼5% of the glucose entering brain, are released to blood under resting conditions (Quistorff et al., 2008; Dienel, 2012), whereas during activation considerable quantities of lactate are released from brain to blood, both directly (22% during spreading depression; Cruz et al., 1999) and via the perivascular-lymphatic drainage system (Ball et al., 2010). Lactate efflux causes (i) a large (∼50%) underestimation of the calculated rate of glucose utilization (CMRglc) when assayed with labeled glucose, in contrast to labeled deoxyglucose that is quantitatively trapped after its initial phosphorylation and (ii) a fall in the CMRO2/CMRglc ratio due to greater rise in glu- cose utilization than oxygen consumption (Dienel, 2012). These two events reflect lactate release and occur under various con- ditions, e.g., sensory stimulation (Fox et al., 1988) and mental testing (Madsen et al., 1995) of humans and spreading depression (Adachi et al., 1995; Cruz et al., 1999) and sensory stimula- tion (Madsen et al., 1999; Schmalbruch et al., 2002) of rats. The CMRO2/CMRglc ratio also falls with increased lactate uptake into brain during vigorous exercise (Quistorff et al., 2008). A common factor in all these situations may be an increase in extracellular lactate concentration. BRAIN LACTATE FLUXES FIGURE 2 | Role for trans-astrocytic lactate trafficking in glutamate needed for oxidation and synthesis of glutamate, respectively, according to this model (pathway 4) (lower right corner for OAA and upper left corner for aspartate). Lactate influx (shown in capital letters and with black arrows) could compensate for a lack of trafficking of these two compounds (pathway 4) between spatially separated glutamate-synthesizing and glutamate-oxidizing astrocytes. In addition, provision of lactate-derived pyruvate to astrocytes would provide a faster source than glucose for provision of the precursor carbon skeleton, and if only one of the two glucose molecules is replaced with pyruvate, malate would still be able to enter the mitochondria during glutamate synthesis. Biosynthesis of glutamine is shown in brown, and metabolic degradation of glutamate in blue. Redox shuttling and astrocytic release of glutamine and uptake of glutamate are shown in black, and neuronal hydrolysis of glutamine to glutamate and its release is shown in red. Reactions involving or resulting from transamination between aspartate and oxaloacetate are shown in green. Lactate could provide pyruvate for many of the reactions in these pathways in many astrocytes. AGC1, aspartate-glutamate exchanger, aralar; α-KG, α-ketoglutarate; Glc, glucose; Pyr, pyruvate; OGC, malate/α-KG exchanger. Slightly modified from Hertz (2011), with permission of the author. ©2011 International Society for Cerebral Blood Flow and Metabolism, Inc. FIGURE 2 | Role for trans-astrocytic lactate trafficking in glutamate | y g g turnover. Why would the brain want a lactate transport from one astrocyte to different neighboring astrocytes? One possibility is that lactate-pyruvate interconversions could be of importance for proposed pathways linking glutamate formation, which is astrocyte-specific, with its oxidative degradation, which may also be mainly or exclusively astrocytic (see papers cited in Hertz and Rodrigues, 2014). The proposed pathways linking glutamate synthesis, excitatory neurotransmission, and glutamate oxidation are illustrated in this figure. Pathway 1 (numbered in yellow rectangle) shows the proposed cytosolic-mitochondrial metabolite trafficking associated with astrocytic production of glutamine. Pathway 2 shows glutamine transfer from astrocytes to glutamatergic neurons and extracellular release of transmitter glutamate. Pathway 3 illustrates subsequent re-uptake of glutamate and its oxidative metabolism in astrocytes. Pathway 4 provides the necessary aspartate- and oxaloacetate-dependent connections between pathways 1 and 3, with all pathways located in the same cell. A major problem with this model is that glutamate formation and oxidation may not occur in the same astrocyte, but, instead, in spatially-separated astrocytes. CELLULAR LACTATE UPTAKE SHUTTLING To compare astrocytic and neuronal rates and capacities for uptake of lactate from extracellular fluid and for its transcellu- lar shuttling, Gandhi et al. (2009) devised a real-time, selective, sensitive assay to measure lactate concentration in single cells in adult rat brain slices. At 2 mmol/L extracellular lactate, the approximate concentration during brain activation, initial rates of lactate uptake into astrocytes were twice those of neurons, and over the range 2–40 mmol/L the initial rate of diffusional September 2014 | Volume 8 | Article 261 | 4 Frontiers in Neuroscience | Neuroenergetics, Nutrition and Brain Health Multifunctional roles of lactate depend on its transport Hertz et al. lactate uptake into astrocytes was four-fold greater than that into neurons. The capacity for lactate uptake into astrocytes was also double that of neurons over this range. Because as many as ten thousand astrocytes are coupled via gap junctions (Ball et al., 2007) (Figures 1Ba,b), lactate can diffuse down its concentra- tion gradient to other astrocytes within the large syncytium, as shown directly for coupled cells located ∼50 μm apart (Gandhi et al., 2009). The initial rate of transfer among coupled astrocytes increased with lactate concentration from 0 to 5 mmol/L, whereas there was no concentration dependence of lactate transfer to neu- rons; net lactate transfer to another astrocyte was about five-fold greater than transfer to an equidistant neuron. capillaries and are also connected together via gap junctions (Figure 1Bb). Some of the lactate diffuses via its concentration gradient within the syncytium to endfeet where it can be released to perivascular fluid and ultimately to cerebral venous blood (Figure 1Bc) (Gandhi et al., 2009; Dienel, 2012), where it can stimulate blood flow that also washes out lactate from perivascu- lar space fluid. Because more glucose is delivered to brain than is phosphorylated, release of a portion of excess fuel as lactate is not an energetic waste when viewed from a whole-body perspective. Other organs oxidize the released lactate. The reduced CMRO2/CMRglc ratio during activation is prevented by propranolol, an inhibitor of β-adrenergic signaling. In control rats, the CMRO2/CMRglc ratio fell from 6.1 to 4.0 after stimu- lation of brain activity by release from their shelter boxes, and it rose back to 5.8 after the animals re-entered the box. After Together, these findings demonstrate that astrocytes avidly take up extracellular lactate, and quickly distribute the lac- tate to other astrocytes within the syncytium. CELLULAR LACTATE UPTAKE SHUTTLING There is a small, slower uptake of extracellular lactate by neurons and low transfer rate from astrocytes to neurons. Astrocytic endfeet surround needed for oxidation and synthesis of glutamate, respectively, according to this model (pathway 4) (lower right corner for OAA and upper left corner for aspartate). Lactate influx (shown in capital letters and with black arrows) could compensate for a lack of trafficking of these two compounds (pathway 4) between spatially separated glutamate-synthesizing and glutamate-oxidizing astrocytes. In addition, provision of lactate-derived pyruvate to astrocytes would provide a faster source than glucose for provision of the precursor carbon skeleton, and if only one of the two glucose molecules is replaced with pyruvate, malate would still be able to enter the mitochondria during glutamate synthesis. Biosynthesis of glutamine is shown in brown, and metabolic degradation of glutamate in blue. Redox shuttling and astrocytic release of glutamine and uptake of glutamate are shown in black, and neuronal hydrolysis of glutamine to glutamate and its release is shown in red. Reactions involving or resulting from transamination between aspartate and oxaloacetate are shown in green. Lactate could provide pyruvate for many of the reactions in these pathways in many astrocytes. AGC1, aspartate-glutamate exchanger, aralar; α-KG, α-ketoglutarate; Glc, glucose; Pyr, pyruvate; OGC, malate/α-KG exchanger. Slightly modified from Hertz (2011), with permission of the author. ©2011 International Society for Cerebral Blood Flow and Metabolism, Inc. FIGURE 2 | Role for trans-astrocytic lactate trafficking in glutamate Trans-astrocytic lactate transport and its subsequent conversion to pyruvate and carboxylation would allow rapid synthesis of oxaloacetate (OAA) and aspartate that are September 2014 | Volume 8 | Article 261 | 5 www.frontiersin.org Multifunctional roles of lactate depend on its transport Hertz et al. (Yu et al., 1982; McKenna et al., 1996). However, the Figure 2 schematic shows cycling of aspartate and oxaloacetate within one astrocyte, and a problem with this model is that glutamate synthesis and its subsequent oxidation may occur in different astrocytes. Lactate transport between synthesizing and degrading astrocytes could rectify this problem by providing a substrate for rapid synthesis of both oxaloacetate and aspartate in the cells receiving lactate (Figure 2, black arrows), which could also partly replace glucose in α-ketoglutarate/glutamate synthesis. The huge flux in this cycle (Sibson et al., 1998; Rothman et al., 2011) and high rates of glutamate neosynthesis, accounting for 15–30% of the flux are consistent with the major trans-astrocytic lactate fluxes indicated by the large difference between glucose oxidation and total glucose utilization rates determined with glucose and deoxyglucose, which was described above. propranolol administration, the CMRO2/CMRglc ratio remained unaltered during rest, stimulation, and recovery (6.2, 6.3, 6.4) (Schmalbruch et al., 2002). Thus, (i) stimulation activates gly- colysis in stimulated region(s) with much less effect on oxidative metabolism, (ii) this effect is dependent on β-adrenergic stim- ulation, and (iii) there must be efflux of a glucose metabolite, e.g., lactate, from the stimulated area. Part of the reduction in CMRO2/CMRglc ratio during brain activation may also reflect retention of some glucose in tissue by (i) an increase in lac- tate, (ii) use of glucose for glycogen synthesis, and (iii) increased pyruvate carboxylation (Öz et al., 2004) leading to enhanced glu- tamate formation (Gibbs et al., 2007; Mangia et al., 2012). The reduced CMRO2/CMRglc ratio during exercise is also inhibited by propranolol (Quistorff et al., 2008; Gam et al., 2009). p p Inhibition by propranolol of an activation-induced fall in CMRO2/CMRglc ratio is consistent with a recent demonstra- tion that specifically locus coeruleus (LC) neurons (the principal source of noradrenaline to brain cortex (Moore and Bloom, 1979), including astrocytes (Bekar et al., 2008), are stimulated by L-lactate, independent of its caloric value (Tang et al., 2014). REFERENCES Adachi, K., Cruz, N. F., Sokoloff, L., and Dienel, G. A. (1995). Labeling of metabolic pools by [6-14C]glucose during K(+)-induced stimulation of glucose utiliza- tion in rat brain. J. Cereb. Blood Flow Metab. 15, 97–110. doi: 10.1038/jcbfm. 1995.11 Balazs, R. (1965). Control of glutamate oxidation in brain and liver mitochondrial systems. Biochem. J. 95, 497–508. Ball, K. K., Cruz, N. F., Mrak, R. E., and Dienel, G. A. (2010). Trafficking of glu- cose, lactate, and amyloid-beta from the inferior colliculus through perivascular routes. J. Cereb. Blood Flow Metab. 30, 162–176. doi: 10.1038/jcbfm.2009.206 There might be additional beneficial effects of an adrenergically-stimulated, gap junction-mediated astrocyte- to-astrocyte lactate trafficking. Subsequent conversion of lactate to pyruvate would boost synthesis of oxaloacetate since pyru- vate carboxylation in liver (and probably also in astrocytes) is stimulated by α-adrenergic activity (Garrison and Borland, 1979). Oxaloacetate is rapidly converted to aspartate which causes a 50% increase of astrocytic glutamate production (Pardo et al., 2011), consistent with increased mitochondrial gluta- mate formation by aspartate addition (Von Korff et al., 1971). Based on this aspartate dependence of glutamate formation and consistent with rapid astrocytic oxidative degradation of glutamate (McKenna, 2013; Whitelaw and Robinson, 2013), an interaction between glutamate synthesis and degradation has been suggested (Hertz, 2011). This interaction, illustrated in Figure 2 and described in its legend, would make the aspartate formed during glutamate oxidation available during glutamate synthesis. Moreover, use of aspartate transaminase, rather than of glutamate dehydrogenase in the inter-conversion between α-ketoglutarate and glutamate is consistent with predominant transamination-dependent glutamate degradation in brain mitochondria (Balazs, 1965; Dennis et al., 1977) vs. extensive use of glutamate dehydrogenase by cultured, isolated astrocytes Ball, K. K., Gandhi, G. K., Thrash, J., Cruz, N. F., and Dienel, G. A. (2007). Astrocytic connexin distributions and rapid, extensive dye transfer via gap junctions in the inferior colliculus: implications for [(14)C]glucose metabolite trafficking. J. Neurosci. Res. 85, 3267–3283. doi: 10.1002/jnr.21376 Bekar, L. K., He, W., and Nedergaard, M. (2008). Locus coeruleus alpha-adrenergic- mediated activation of cortical astrocytes in vivo. Cereb. Cortex 18, 2789–2795. doi: 10.1093/cercor/bhn040 Bonda, D. J., Lee, H. G., Camins, A., Pallas, M., Casadesus, G., Smith, M. A., et al. (2011). The sirtuin pathway in ageing and Alzheimer disease: mechanistic and therapeutic considerations. Lancet Neurol. 10, 275–279. doi: 10.1016/s1474- 4422(11)70013-8 Chesler, M. (2003). Regulation and modulation of pH in the brain. Physiol. Rev. 83, 1183–1221. doi: 10.1152/physrev.00010.2003 Cruz, N. CONCLUDING REMARKS Lactate transport between brain cells is mainly among astrocytes and occurs both via gap junctions and release to extracellular space. The latter mechanism is important for LC-adrenergic sig- naling, and it also leads to a significant exit of lactate from the brain via peri-capillary flux and the lymphatic system. Adrenergic signaling plays a role in regulating lactate fluxes, and inter- astrocytic lactate flux may assist glutamate production and degra- dation in the glutamate-glutamine cycle. FIGURE 2 | Role for trans-astrocytic lactate trafficking in glutamate Release of L-lactate from cultured astrocytes excites LC neurons and triggers release of noradrenaline, and physiologically-relevant concentrations of exogenous L-lactate (EC50 ∼0.5 mmol/L) mimics these effects (Tang et al., 2014). The effects of L-lactate were stereo-selective, independent of its uptake into neurons, and involved a cAMP-mediated step. In vivo injections of L-lactate in the LC evoked arousal similar to the excitatory transmit- ter, L-glutamate. Because (i) lactate release is associated with activation-induced decreases in CMRO2/CMRglc ratio (inhibited by propranolol) and (ii) astrocytic gap junction conductivity is up-regulated by cAMP, an intermediate in β-adrenergic signal- ing (Enkvist and McCarthy, 1994) blockade by propranolol may reduce gap junction-mediated lactate transport and release from brain. 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September 2014 | Volume 8 | Article 261 | 8 REFERENCES doi: 10.3389/fnins.2014.00261 This article was submitted to Neuroenergetics, Nutrition and Brain Health, a section of the journal Frontiers in Neuroscience. Copyright © 2014 Hertz, Gibbs and Dienel. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, dis- tribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Received: 09 July 2014; accepted: 04 August 2014; published online: 09 September 2014. Citation: Hertz L, Gibbs ME and Dienel GA (2014) Fluxes of lactate into, from, and among gap junction-coupled astrocytes and their interaction with noradrenaline. Front. 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Frontiers in Neuroscience | Neuroenergetics, Nutrition and Brain Health REFERENCES Preferential utilization of acetate by astrocytes is attributable to transport. J. Neurosci. 18, 5225–5233. Whitelaw, B. S., and Robinson, M. B. (2013). Inhibitors of glutamate dehydroge- nase block sodium-dependent glutamate uptake in rat brain membranes. Front. Endocrinol. (Lausanne) 4:123. doi: 10.3389/fendo.2013.00123 Roos, A., and Boron, W. F. (1981). Intracellular pH. Physiol. Rev. 61, 296–434. Wilhelm, F., and Hirrlinger, J. (2012). Multifunctional Roles of NAD(+) and NADH in Astrocytes. Neurochem. Res. 37, 2317–2325. doi: 10.1007/s11064-012- 0760-y Rothman, D. L., De Feyter, H. M., De Graaf, R. A., Mason, G. F., and Behar, K. L. (2011). 13C MRS studies of neuroenergetics and neuro- transmitter cycling in humans. NMR Biomed. 24, 943–957. doi: 10.1002/ nbm.1772 Yamanishi, S., Katsumura, K., Kobayashi, T., and Puro, D. G. (2006). 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Stroke 33, 251–255. doi: 10.1161/hs0102.101233 Conflict of Interest Statement: The authors declare that the research was con- ducted 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 con- ducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Sibson, N. R., Dhankhar, A., Mason, G. F., Rothman, D. L., Behar, K. L., and Shulman, R. G. (1998). Stoichiometric coupling of brain glucose metabolism and glutamatergic neuronal activity. Proc. Natl. Acad. Sci. U.S.A. 95, 316–321. doi: 10.1073/pnas.95.1.316 Received: 09 July 2014; accepted: 04 August 2014; published online: 09 September 2014. Citation: Hertz L, Gibbs ME and Dienel GA (2014) Fluxes of lactate into, from, and among gap junction-coupled astrocytes and their interaction with noradrenaline. Front. Neurosci. 8:261. REFERENCES No use, distribution or reproduction is permitted which does not comply with these terms. Van Hall, G., Stromstad, M., Rasmussen, P., Jans, O., Zaar, M., Gam, C., et al. (2009). Blood lactate is an important energy source for the human brain. J. Cereb. Blood Flow Metab. 29, 1121–1129. doi: 10.1038/jcbfm.2009.35 September 2014 | Volume 8 | Article 261 | 8 Frontiers in Neuroscience | Neuroenergetics, Nutrition and Brain Health

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DOMESTICATION, DEGENERATION, AND THE ESTABLISHMENT OF THE ADDO ELEPHANT NATIONAL PARK IN SOUTH AFRICA, 1910s–1930s

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The Historical Journal, , (), pp. – © The Author(s), . Published by Cambridge University Press. This is an Open Access article, distributed under the terms of the Creative Commons Attribution licence (http://creativecommons.org/licenses/by/./), which permits unrestricted re-use, distribution, and reproduction in any medium, provided the original work is properly cited. The Historical Journal, , (), pp. – © The Author(s), . Published by Cambridge University Press. This is an Open Access article, distributed under the terms of the Creative Commons Attribution licence (http://creativecommons.org/licenses/by/./), which permits unrestricted re-use, distribution, and reproduction in any medium, provided the original work is properly cited. doi:./SX * I am grateful to Jim Secord, Richard Staley, Harriet Ritvo, Saul Dubow, Pippa Skotnes, Laura Brassington, and two anonymous reviewers for providing invaluable feedback on this article in its various forms. I thank Jane Carruthers, Nigel Penn, Anandaroop Sen, and Bodhi Kar for their helpful suggestions. Lastly, my thanks to Sujit Sivasundaram and the editor- ial team of the Historical Journal. Department of History and Philosophy of Science, University of Cambridge, Cambridge, CBRH jasb@ cam.ac.uk Department of History and Philosophy of Science, University of Cambridge, Cambridge, CBRH jasb@ cam.ac.uk DOMESTICATION, DEGENERATION, AND THE ESTABLISHMENT OF THE ADDO ELEPHANT NATIONAL PARK IN SOUTH AFRICA, s–s* J U L E S S K O T N E S - B R O W N Department of History and Philosophy of Science, University of Cambridge A B S T R A C T. This article examines conflict between farmers and elephants in the Addo region in s–s South Africa to explore the porosity of the concepts ‘wild’, ‘tame’, and ‘domestic’, and their relationship to race, degeneration, nature conservation, and colonialism. In the s, settler farmers indicted the ‘Addo Elephants’, as ‘vicious’ thieves who raided crops and ‘hunted’ farmers. This view conflicted with a widespread perception of elephants as docile, sagacious, and worthy of protection. Seeking to reconcile these views, bureaucrats were divided between exterminating the animals, creating a game reserve, and drawing upon the expertise of Indian mahouts to domes- ticate them. Ultimately, all three options were attempted: the population was decimated by hunter Phillip Jacobus Pretorius, an elephant reserve was created, the animals were tamed to ‘lose their fear of man’ and fed oranges. Despite the presence of tame elephants and artificial feeding, the reserve was publicized as a natural habitat, and a window onto the prehistoric. This was not para- doxical but provokes a need to rethink the relationship between wildness, tameness, and domesticity. These concepts were not implicitly opposed but existed on a spectrum paralleling imperialist hierarch- ies of civilization, race, and evolution, upon which tame elephants could still be considered wild. In , the Addo Elephant National Park, a reserve near the city of Port Elizabeth, was founded with a unique zoological purpose: the protection of a single ‘race’ of elephants. The park has since become an important attraction, and is home to several hundred elephants, antelope, carnivora, and thousands In , the Addo Elephant National Park, a reserve near the city of Port Elizabeth, was founded with a unique zoological purpose: the protection of a single ‘race’ of elephants. The park has since become an important attraction, and is home to several hundred elephants, antelope, carnivora, and thousands  https://doi.org/10.1017/S0018246X19000761 Published online by Cambridge University Press  J U L E S S K O T N E S - B R O W N of birds. Despite its renown, little has been written on its history. John Pringle, The conservationists and the killers (Johannesburg, ); C. S. Stokes, Sanctuary (Cape Town, ), pp. –; Jane Meiring, The Sundays River Valley (Cape Town, ), pp. –; Die Addo Olifante (Pretoria, ); Anthony Hall-Martin, ‘Elephant survivors’, Oryx, (), pp. –. J pp Raf De Bont, ‘A world laboratory: framing the Albert National Park’, Environmental History, (), p. . For examples of ‘scientific’ parks, see Melissa Harper and Richard White, ‘How national were the first national parks?’, in Bernhard Gissibl, Sabine Höhler, and Patrick Kupper, eds., Civilizing nature (New York, NY, ), p. ; Patrick Kupper, ‘Science and national parks: a transatlantic perspective on the interwar years’, Environmental History, (), pp. –; Carruthers, National park science. J p g  pp  For discussions of elephant sagacity, see Harriet Ritvo, The animal estate (Cambridge, MA, ), p. ; Susan Nance, Entertaining elephants (Baltimore, MD, ), pp. –. For a provocative analysis into whether working elephants were colonizers or colonized in Burma, see Jonathan Saha, ‘Colonizing elephants: animal agency, undead capital and imperial science in British Burma’, BJHS Themes, (), pp. –. https://doi.org/10.1017/S0018246X19000761 Published online by Cambridge University Press y (  ) pp  Jane Carruthers, National park science (Cambridge, ), pp. –. For a provocative analysis into whether working elephants were colonizers or colonized in Burma, see Jonathan Saha, ‘Colonizing elephants: animal agency, undead capital and imperial science in British Burma’, BJHS Themes, (), pp. –. For discussions of elephant sagacity, see Harriet Ritvo, The animal estate (Cambridge, MA ), p. ; Susan Nance, Entertaining elephants (Baltimore, MD, ), pp. –. Jane Carruthers, National park science (Cambridge, ), pp. –. For discussions of elephant sagacity, see Harriet Ritvo, The animal estate (Cambridge, MA, ), p. ; Susan Nance, Entertaining elephants (Baltimore, MD, ), pp. –. For a provocative analysis into whether working elephants were colonizers or colonized in Burma, see Jonathan Saha, ‘Colonizing elephants: animal agency, undead capital and imperial science in British Burma’, BJHS Themes, (), pp. –. Raf De Bont, ‘A world laboratory: framing the Albert National Park’, Environmental History, (), p. . For examples of ‘scientific’ parks, see Melissa Harper and Richard White, ‘How national were the first national parks?’, in Bernhard Gissibl, Sabine Höhler, and Patrick Kupper, eds., Civilizing nature (New York, NY, ), p. ; Patrick Kupper, ‘Science and national parks: a transatlantic perspective on the interwar years’, Environmental History, (), pp. –; Carruthers, National park science. DOMESTICATION, DEGENERATION, AND THE ESTABLISHMENT OF THE ADDO ELEPHANT NATIONAL PARK IN SOUTH AFRICA, s–s* Most accounts depict its genesis as a process of enlightenment: ‘killers’ turned their guns from wildlife and became ‘conservationists’, realizing that nature-protection was ethical.Jane Carruthers has published a short, nuanced account of the park, which contextualizes it as the first example of a trend in s southern African wildlife-protection of creating sanctuaries protecting particular species. Behind this success story lies a bloody history of conflict amongst farmers, zoologists, bureaucrats, and elephants. In the s, the ‘Addo Elephants’ roamed the Addo region, ignoring human-inscribed boundaries between farm and bushveld, trampling crops, fences, and occasionally humans in the process. Settler farmers denounced them as ‘vicious’, and demanded they be eliminated, which brought them into conflict with various scientists and metro- politan publics, who were persuaded by a widespread Anglo perception of the elephant as sagacious and peaceful.Bureaucrats, big-game hunters, and zool- ogists saw African elephants as potential imperial agents: highly intelligent animals who could be domesticated for labour as in South Asia.For farmers, they were ‘vermin’ who competed with their livelihood. These conflicts between farmers on the ground, bureaucrats in offices, and zoologists in museums, spawned a twenty-year controversy into the status of these animals as wild or domestic, ‘noble’ or ‘rogue’, oppressed victims, or violent by nature. Ultimately, the founding of the park was shaped by debates about ele- phant domestication, and fears of elephant degeneration. Structured into three sections representing different strategies of elephant management – proposals to domesticate the elephants in the s, the deci- sion to eliminate them in , and, finally, to protect them in a game reserve in – this article seeks to make two contributions to the histories of African conservation, animals, and natural history. First, it adds to growing interest in connecting histories of science and national parks. For analyses of evolution, labour, and apartheid in South Africa, see Dubow, Scientific racism in modern South Africa; Saul Dubow, ‘Human origins, race typology and the other Raymond Dart’, African Studies, (), pp. –. For the relationship between colonized Africans and animals, see Achille Mbembe, On the postcolony (Berkeley, CA, ), pp. –; Ritvo, The animal estate, p. ; Clapperton Mavhunga, ‘Vermin beings: on pestiferous animals and human game’, Social Text, (), pp. –; Lance van Sittert, ‘Routinising genocide: the politics and practice of vermin extermination in the Cape Province c. –’, Journal of Contemporary African Studies, (), pp. –. Jane Carruthers, The Kruger National Park: a social and political history (Pietermaritzburg, ), pp. –.  Dubow, Scientific racism in modern South Africa, p. . These policies would culminate in the Natives Land Act, in which only per cent of the land was reserved for Africans. William Beinart and Peter Delius, ‘The historical context and legacy of the Natives Land Act of ’, Journal of Southern African Studies, (), pp. –. Jan Smuts, Africa and some world problems (Oxford, ), p. . Peder Anker, ‘The politics of ecology in South Africa on the radical left’, Journal of the History of Biology, (), pp. –; Peder Anker, Imperial ecology: environmental order in the British empire, –(Cambridge, MA, ). Smuts, Africa and some world problems, . See also Saul Dubow, A commonwealth of knowledge: science, sensibility, and white South Africa, –(Oxford, ), pp. –, –; Anker, Imperial ecology, pp. –.  Carruthers, National park science, p. xxiii.  p   g Saul Dubow, Scientific racism in modern South Africa (Cambridge, ), p. . Ibid., pp. –. J. H. Pim, , qu. in ibid., p. . Carruthers, National park science, p. xxiii.  Jane Carruthers, The Kruger National Park: a social and political history (Pietermaritzburg, ), pp. –. Carruthers, National park science, p. xxiii. Peder Anker, ‘The politics of ecology in South Africa on the radical left’, Journal of the History of Biology, (), pp. –; Peder Anker, Imperial ecology: environmental order in the British empire, –(Cambridge, MA, ). Saul Dubow, Scientific racism in modern South Africa (Cambridge, ), p. . Ibid., pp. –. J. H. Pim, , qu. in ibid., p. . Dubow, Scientific racism in modern South Africa, p. . These policies would culminate in the Natives Land Act, in which only per cent of the land was reserved for Africans. William Beinart and Peter Delius, ‘The historical context and legacy of the Natives Land Act of ’, Journal of Southern African Studies, (), pp. –. Jan Smuts, Africa and some world problems (Oxford, ), p. . Jan Smuts, ‘Science in South Africa’, Nature, (), p. . Smuts, Africa and some world problems, . See also Saul Dubow, A commonwealth of knowledge: science, sensibility, and white South Africa, –(Oxford, ), pp. –, –; Anker, Imperial ecology, pp. –. For analyses of evolution, labour, and apartheid in South Africa, see Dubow, Scientific racism in modern South Africa; Saul Dubow, ‘Human origins, race typology and the other Raymond Dart’, African Studies, (), pp. –. For the relationship between colonized Africans and animals, see Achille Mbembe, On the postcolony (Berkeley, CA, ), pp. –; Ritvo, The animal estate, p. ; Clapperton Mavhunga, ‘Vermin beings: on pestiferous animals and human game’, Social Text, (), pp. –; Lance van Sittert, ‘Routinising genocide: the politics and practice of vermin extermination in the Cape Province c. –’, Journal of Contemporary African Studies, (), pp. –. Jan Smuts, ‘Science in South Africa’, Nature, (), p. .  DOMESTICATION, DEGENERATION, AND THE ESTABLISHMENT OF THE ADDO ELEPHANT NATIONAL PARK IN SOUTH AFRICA, s–s* Although, as Raf de Bont has argued, it is now a ‘cliché’ that conservation ‘should be “science based”’, parks were not always considered scientific spaces. https://doi.org/10.1017/S0018246X19000761 Published online by Cambridge University Press T H E A D D O E L E P H A N T S I N S O U T H A F R I C A  Scientists were at times actively opposed to African parks on the grounds that they were vast reservoirs of livestock diseases.As Carruthers has noted, the ‘his- toriography of nature conservation as science in South Africa…has hardly been touched upon by professional historians’.This article addresses this lacuna by arguing that the Addo Elephant National Park was founded with explicit scien- tific racist intentions. Peder Anker has observed close links between politics and ecology in South Africa in relation to evolutionary theories and the perceived place of human ‘races’ in society.Saul Dubow, similarly, has investigated how fears of African ‘racial deterioration’in cities shaped apartheid policies of creating rural ‘native reserves’.In these spaces, the pastoral African ‘veneer of civilization’ could be ‘protected’ from ‘the urban environment’.In the s–s, Prime Minister Jan Smuts argued that Africa was a ‘human laboratory’ in which racial evolution was an ‘experiment’.In South Africa, Smuts thought that multiple stages of human ‘evolution’ were preserved and ready for study: from ‘living fossil’hunter-gatherers, to the developing ‘Transvaal Boer… type’.Although such racist evolutionary and ecological thinking was typically associated with humans, elephants were also subjected to it.Racial taxonomy, neo-Lamarckian interpretations of evolution, and fears of degeneration shaped every elephant-management policy taken by the Cape Province. Concepts of racial degeneration justified brutal violence against the animals, and Smuts, Africa and some world problems, . See also Saul Dubow, A commonwealth of knowledge: science, sensibility, and white South Africa, –(Oxford, ), pp. –, –; Anker, Imperial ecology, pp. –.  https://doi.org/10.1017/S0018246X19000761 Published online by Cambridge University Press  J U L E S S K O T N E S - B R O W N preservationist thinking was mobilized to argue for the creation of the park in ways paralleling the development of segregation legislature and ‘native reserves’. My second aim is to bring the history of the wild/domestic border into greater conversation with the history of national parks, and the intellectual history of race.Environmental historians have devoted considerable attention to prob- lematizing the perceived ‘wildness’ of African parks. For a related example from Southeast Asia, see Jonathan Saha, ‘Do elephants have souls? Animal subjectivities and colonial encounters’, in Deana Heath and Stephen Legg, eds., South Asian governmentalities: Michel Foucault and the question of postcolonial orderings (Cambridge, ), pp. –. Here, Saha shows how regimes of colonial governmentality were extended to elephants in Burma. p For work that has attempted to historicize and refute the wild/domestic binary, see Harriet Ritvo, ‘The domestic stain, or maintaining standards’, in The Multispecies Editing Collective, ed., Troubling species: care and belonging in a relational world (Munich, ), pp. –; Harriet Ritvo, ‘Calling the wild: selection, domestication, and species’, in Angelique Richardson, ed., After Darwin: animals, emotions, and the mind (New York, NY, ), pp. –; Harriet Ritvo, Noble cows and hybrid zebras: essays on animals and history (Charlottesville, VA, ), pp. –; Jamie Lorimer, ‘Elephants as companion species: the lively biogeographies of Asian elephant conservation in Sri Lanka’, Transactions of the Institute of British Geographers, (), pp. –; Jamie Lorimer and Sarah Whatmore, ‘After the “king of beasts”: Samuel Baker and the embodied historical geographies of elephant hunting in mid-nineteenth-century Ceylon’, Journal of Historical Geography, (), pp. –. Much has been written on this. Select examples include Roderick Neumann, Imposing wil- derness (Berkeley, CA, ); Terence Ranger, ‘Whose heritage? The case of the Matobo National Park’, Journal of Southern African Studies, (), pp. –; Shirley Brooks, ‘Images of “wild Africa”: nature tourism and the (re)creation of Hluhluwe Game Reserve, –’, Journal of Historical Geography, (), pp. –; Carruthers, The Kruger National Park; Gissibl, Höhler, and Kupper, ‘Introduction’; John MacKenzie, The empire of nature: hunting, conservation and British imperialism (Manchester, ). For a critique of MacKenzie’s work, see Lance Van Sittert, ‘Bringing in the wild: the commodification of wild animals in the Cape Colony/Province c. –’, Journal of African History, (), pp. –. pp Gissibl, Höhler, and Kupper, ‘Introduction’, p. . Bernhard Gissibl, Sabine Höhler, and Patrick Kupper, ‘Introduction’, in Gissibl, Höhler, and Kupper, eds., Civilizing nature, p. . For a related example from Southeast Asia, see Jonathan Saha, ‘Do elephants have souls? Animal subjectivities and colonial encounters’, in Deana Heath and Stephen Legg, eds., South Asian governmentalities: Michel Foucault and the question of postcolonial orderings (Cambridge, ), pp. –. Here, Saha shows how regimes of colonial governmentality were extended to elephants in Burma. For work that has attempted to historicize and refute the wild/domestic binary, see Harriet Ritvo, ‘The domestic stain, or maintaining standards’, in The Multispecies Editing Collective, ed., Troubling species: care and belonging in a relational world (Munich, ), pp. –; Harriet Ritvo, ‘Calling the wild: selection, domestication, and species’, in Angelique Richardson, ed., After Darwin: animals, emotions, and the mind (New York, NY, ), pp. –; Harriet Ritvo, Noble cows and hybrid zebras: essays on animals and history (Charlottesville, VA, ), pp. –; Jamie Lorimer, ‘Elephants as companion species: the lively biogeographies of Asian elephant conservation in Sri Lanka’, Transactions of the Institute of British Geographers, (), pp. –; Jamie Lorimer and Sarah Whatmore, ‘After the “king of beasts”: Samuel Baker and the embodied historical geographies of elephant hunting in mid-nineteenth-century Ceylon’, Journal of Historical Geography, (), pp. –. Bernhard Gissibl, Sabine Höhler, and Patrick Kupper, ‘Introduction’, in Gissibl, Höhler, and Kupper, eds., Civilizing nature, p. . Much has been written on this. Select examples include Roderick Neumann, Imposing wil- derness (Berkeley, CA, ); Terence Ranger, ‘Whose heritage? The case of the Matobo National Park’, Journal of Southern African Studies, (), pp. –; Shirley Brooks, ‘Images of “wild Africa”: nature tourism and the (re)creation of Hluhluwe Game Reserve, –’, Journal of Historical Geography, (), pp. –; Carruthers, The Kruger National Park; Gissibl, Höhler, and Kupper, ‘Introduction’; John MacKenzie, The empire of nature: hunting, conservation and British imperialism (Manchester, ). For a critique of MacKenzie’s work, see Lance Van Sittert, ‘Bringing in the wild: the commodification of wild animals in the Cape Colony/Province c. –’, Journal of African History, (), pp. –. Gissibl, Höhler, and Kupper, ‘Introduction’, p. . DOMESTICATION, DEGENERATION, AND THE ESTABLISHMENT OF THE ADDO ELEPHANT NATIONAL PARK IN SOUTH AFRICA, s–s* These spaces were not ‘untouched’ windows onto the prehistoric, but constructed landscapes, deeply entangled with colonial attempts to ‘civilize’ nature, biopolitics, and racism.Created initially to preserve animals for hunters, and later to entertain tourists, national parks were often established at the expense of indigenous Africans who were forcibly removed from their lands.Nevertheless, such work has largely operated according to the idea that conservationists predicated the park ideal as a ‘fundamental separation of nature, usually understood as wil- derness, from society and culture’.Animals living within parks have been treated as necessarily ‘wild’, rather than as ‘domestic’, or ‘tame’. Yet the histori- cization of these actors’ categories reveals that not all advocates of national parks were thinking within these binaries. Parks were not always constructed p For work that has attempted to historicize and refute the wild/domestic binary, see Harriet Ritvo, ‘The domestic stain, or maintaining standards’, in The Multispecies Editing Collective, ed., Troubling species: care and belonging in a relational world (Munich, ), pp. –; Harriet Ritvo, ‘Calling the wild: selection, domestication, and species’, in Angelique Richardson, ed., After Darwin: animals, emotions, and the mind (New York, NY, ), pp. –; Harriet Ritvo, Noble cows and hybrid zebras: essays on animals and history (Charlottesville, VA, ), pp. –; Jamie Lorimer, ‘Elephants as companion species: the lively biogeographies of Asian elephant conservation in Sri Lanka’, Transactions of the Institute of British Geographers, (), pp. –; Jamie Lorimer and Sarah Whatmore, ‘After the “king of beasts”: Samuel Baker and the embodied historical geographies of elephant hunting in mid-nineteenth-century Ceylon’, Journal of Historical Geography, (), pp. –. Much has been written on this. Select examples include Roderick Neumann, Imposing wil- derness (Berkeley, CA, ); Terence Ranger, ‘Whose heritage? The case of the Matobo National Park’, Journal of Southern African Studies, (), pp. –; Shirley Brooks, ‘Images of “wild Africa”: nature tourism and the (re)creation of Hluhluwe Game Reserve, –’, Journal of Historical Geography, (), pp. –; Carruthers, The Kruger National Park; Gissibl, Höhler, and Kupper, ‘Introduction’; John MacKenzie, The empire of nature: hunting, conservation and British imperialism (Manchester, ). For a critique of MacKenzie’s work, see Lance Van Sittert, ‘Bringing in the wild: the commodification of wild animals in the Cape Colony/Province c. –’, Journal of African History, (), pp. –. For a problematization of this, see William Adams, Against extinction (London, ), pp. –.   For a provocative discussion of agency and animal predation on humans, see Brett Walker, ‘Animals and the intimacy of history’, History and Theory, (), pp. –. y y y y   pp   The idea of allowing animals to ‘speak’ is derived from Timothy Mitchell, Rule of experts: Egypt, techno-politics, modernity (Berkeley, CA, ), pp. –. For a critique of the use of modern science to ventriloquize animals, see Abigail Woods et al., Animals and the shaping of modern medicine: one health and its histories (Cham, ), p. . For a historiographical overview of animal agency, see Hilda Kean, ‘Challenges for histor- ians writing animal–human history: what is really enough?’, Anthrozoös, (), pp. –. For a historiographical overview of animal agency, see Hilda Kean, ‘Challenges for histor- ians writing animal–human history: what is really enough?’, Anthrozoös, (), pp. –. See Cape Town Archives Repository (KAB), PAS /; KAB, /UIT /, /, // Chris Philo and Chris Wilbert, eds., ‘Animal spaces, beastly places: an introduction’, in Animal spaces, beastly places (London, ), pp. –. https://doi.org/10.1017/S0018246X19000761 Published online by Cambridge University Press For a critique of the use of modern science to ventriloquize animals, see Abigail Woods et al., Animals and the shaping of modern medicine: one health and its histories (Cham, ), p. . Clapperton Chakanetsa Mavhunga, The mobile workshop: the tsetse fly and African knowledge production (Cambridge, MA, ). g y y g  pp  See Cape Town Archives Repository (KAB), PAS /; KAB, /UIT /, /, // . p g f p  Clapperton Chakanetsa Mavhunga, The mobile workshop: the tsetse fly and African knowledge production (Cambridge, MA, ). For a problematization of this, see William Adams, Against extinction (London, ), pp. –. For a historiographical overview of animal agency, see Hilda Kean, ‘Challenges for histor- ians writing animal–human history: what is really enough?’, Anthrozoös, (), pp. –. See Cape Town Archives Repository (KAB), PAS /; KAB, /UIT /, /, // . For a provocative discussion of agency and animal predation on humans, see Brett Walker, ‘Animals and the intimacy of history’, History and Theory, (), pp. –. The idea of allowing animals to ‘speak’ is derived from Timothy Mitchell, Rule of experts: Egypt, techno-politics, modernity (Berkeley, CA, ), pp. –. For a critique of the use of modern science to ventriloquize animals, see Abigail Woods et al., Animals and the shaping of modern medicine: one health and its histories (Cham, ), p. . Clapperton Chakanetsa Mavhunga, The mobile workshop: the tsetse fly and African knowledge production (Cambridge, MA, ). Chris Philo and Chris Wilbert, eds., ‘Animal spaces, beastly places: an introduction’, in Animal spaces, beastly places (London, ), pp. –. DOMESTICATION, DEGENERATION, AND THE ESTABLISHMENT OF THE ADDO ELEPHANT NATIONAL PARK IN SOUTH AFRICA, s–s* https://doi.org/10.1017/S0018246X19000761 Published online by Cambridge University Press  T H E A D D O E L E P H A N T S I N S O U T H A F R I C A as wild places in which the domestic was excluded, nor were their animal inha- bitants necessarily considered wild. The Addo Elephant National Park was publicized a ‘wild’ space, but also one where ‘tame’ animals could be viewed in their ‘natural state’, bathing in bore- hole water and feasting upon cultivated oranges. This does not mean that con- servationists placed a veil of wilderness over a constructed landscape,but that tame, domestic, and wild were not neatly bifurcated actors’ categories, and could co-exist within early twentieth-century parks. Paying closer attention to these categories, along with their inextricably linked concepts of degeneration, race, and evolution, can potentially transform how we think about animal- protection in this period. But where does the agency of the elephants fit into a story of colonial dis- course and environmental alteration?Farmers, bureaucrats, and zoologists were aware of the capacity of elephants to shape Addo, the danger they posed to humans, and were hesitant to enrage them for fear of retaliation. For settlers and bureaucrats in the region who struggled to contain their move- ments, elephant agency was self-evident and limiting it was their primary aim. The difficulty here is not allowing elephants to ‘speak’, but considering how they trumpeted louder than colonial representations of their behaviour. Rather than utilizing neo-materialist theory or modern science to ascribe agency to the elephants, I treat agency as an exercise of writing.Here, I am inspired by Clapperton Chakanetsa Mavhunga’s use of the term ‘mobility’ in interpreting the actions of tsetse flies and the knowledge produced in response to their movements.A key motif in this article is boundary crossing, and I dem- onstrate how the physical mobility of elephants (roaming, trampling, wallow- ing) upset colonial understandings of elephant behaviour and transformed ‘Addo Elephant’ into a conceptually mobile category. Not only did it render the Addo region a ‘beastly place’,but the mobilities of the elephants shaped processes of knowledge production. A longer history of this dispossession is beyond the scope of this article. For a history and sociology of land and identity in the region, see Teresa K. Connor, ‘The frontier revisited: dis- placement, land and identity among farm labourers in the Sundays River Valley’, Journal of Contemporary African Studies, (), pp. –. p y f  pp  Mr Ross, qu. in ‘Union of South Africa: minutes of the provincial council of the province of the Cape of Good Hope. Third session () – second council’, Apr. , KAB, PAS g Ibid., p. .  P. J. Pretorius, Jungle man: the autobiography of Major P. J. Pretorius (London, ), p. . My thanks to Nigel Penn for this observation. For a brief account of battles within the bush, see Johan de Villiers, ‘Perspective on John Graham and the Fourth Cape Eastern Frontier War’, New Contree, (), pp. –. M. T. Hoffman, ‘Major P. J. Pretorius and the decimation of the Addo elephant herd in –: important reassessments’, Koedoe, (), p. . Meiring, The Sundays River Valley, pp. –. DOMESTICATION, DEGENERATION, AND THE ESTABLISHMENT OF THE ADDO ELEPHANT NATIONAL PARK IN SOUTH AFRICA, s–s* The animals repeatedly under- mined official representations of their behaviour, creating moments of https://doi.org/10.1017/S0018246X19000761 Published online by Cambridge University Press  J U L E S S K O T N E S - B R O W N controversy and forcing officials to rethink their categorization as noble beasts, domestic labourers, wild animals, or ferocious rogues. controversy and forcing officials to rethink their categorization as noble beasts, domestic labourers, wild animals, or ferocious rogues. P. J. Pretorius, Jungle man: the autobiography of Major P. J. Pretorius (London, ), p. . My thanks to Nigel Penn for this observation. For a brief account of battles within the bush, see Johan de Villiers, ‘Perspective on John Graham and the Fourth Cape Eastern Frontier War’, New Contree, (), pp. –. M. T. Hoffman, ‘Major P. J. Pretorius and the decimation of the Addo elephant herd in –: important reassessments’, Koedoe, (), p. . Meiring, The Sundays River Valley, pp. –. Ibid., p. . A longer history of this dispossession is beyond the scope of this article. For a history and sociology of land and identity in the region, see Teresa K. Connor, ‘The frontier revisited: dis- placement, land and identity among farm labourers in the Sundays River Valley’, Journal of Contemporary African Studies, (), pp. –. Mr Ross, qu. in ‘Union of South Africa: minutes of the provincial council of the province of the Cape of Good Hope. Third session () – second council’, Apr. , KAB, PAS I In the s, the area known as the ‘Addo Bush’ was a region of approximately forty-two square kilometres, characterized by dense bush and low rainfall. In the early twentieth century, the bush was thought to be uninhabited by humans and impenetrable: it could only be traversed by cutting it down to clear a path (Figure ). In the s, the area known as the ‘Addo Bush’ was a region of approximately forty-two square kilometres, characterized by dense bush and low rainfall. In the early twentieth century, the bush was thought to be uninhabited by humans and impenetrable: it could only be traversed by cutting it down to clear a path (Figure ). Despite this, the bush and its surrounds had a long history of amaXhosa, amaFengu and Khoekhoe occupation and dispossession. The bush itself was a site in which the first major conflicts of the fourth Xhosa frontier war took place.Xhosa leader Ndlambe had a stronghold within the bush, and between and , a series of skirmishes between his forces and those of Colonel John Graham took place.By , the colonial government had firm control over the area, and divided the bush into two forest reserves in an attempt to ‘protect’ it from human use.At the end of the century, although the bush was thought to be unoccupied, between and ,indigenous Africans were still farming in the surrounding valley.In the s, their inde- pendence was steadily eroded: a succession of companies purchased large tracts of land for development, and in , the Strathsomers Estate Company passed a motion forbidding all but white settlers from purchasing their estates. This, according to Jane Meiring, reduced the ‘status of the native’ to ‘that of a labourer entirely dependent upon the European farmer for his living’.By the s, indigenous Africans had largely been dispossessed of their lands, and were living primarily as tenants on white farms, or as labourers. This longer history of dispossession in the Addo region wrote African land-ownership out of the history of the bush.Bureaucrats, zoologists, and other white civi- lians in the s assumed that the bush was a primordial landscape and the home of the elephants ‘from time immemorial’. https://doi.org/10.1017/S0018246X19000761 Published online by Cambridge University Press T H E A D D O E L E P H A N T S I N S O U T H A F R I C A  Fig. . /; G. J. Franks, ‘The romance of the Addo Bush’, Rand Daily Mail, Jan. , p. ; Janisch to Chabaud, Apr. , KAB, /UIT /. R. G. D. to provincial secretary, Sept. , KAB, PAS /. Berry to district commandant, Grahamstown, Apr. , KAB, PAS /; MacQueen to Chabaud, Aug. , KAB, PAS /. Meiring, The Sundays River Valley, p. . I Teams of labourers were required to cut through the bush. Cape Sundays River Settlements (Cape Town, ), p. . Fig. . Teams of labourers were required to cut through the bush. Cape Sundays River Settlements (Cape Town, ), p. . Because of its density and the presence of wild elephants, the bush was thought to be extremely dangerous. Even Frederick Selous, the most famous African big-game hunter of the nineteenth century, feared for his safety in the region, and declined the opportunity to shoot an Addo Elephant.Some African labourers outright refused to enter the bush, while others would only do so if armed.Surrounding the bush was the Sundays River Valley, a farming district situated between two major cities, Port Elizabeth and Grahamstown (now Makhanda). Unlike the protected bush, the valley had a relatively long history of colonial agriculture. White settlers had been farming in the region from at least ,and since the late nineteenth century, the Cape government and several private developers had attempted to convert the region into a citrus, lucerne, and grain-farmer’s paradise. These schemes brought more settlers into the area, and as agriculturalists encroached upon https://doi.org/10.1017/S0018246X19000761 Published online by Cambridge University Press  J U L E S S K O T N E S - B R O W N the borders of the bush, farmers encountered herds of aggressive, highly mobile elephants. the borders of the bush, farmers encountered herds of aggressive, highly mobile elephants. In , these farmers complained about the elephants to the Alexandria and Uitenhage magistrates, as well as the Cape provincial administration. These great beasts were crossing boundaries between bushveld and farm, drink- ing borehole water, trampling fences that impeded their path, and occasionally attacking humans. The four government bureaucrats adjudicating farmers’ complaints, Lewis Mansergh and Don Janisch of the Cape provincial administra- tor’s office, C. W. Chabaud, the magistrate of Uitenhage, and the magistrate of Alexandria, were sceptical of such claims. The idea that elephants were terror- izing the countryside contradicted a widespread perception of elephants amongst metropolitan elites. These great pachyderms were considered docile, intelligent, and co-operative with humans. From the mid-nineteenth to the early twentieth centuries, elephants were not thought to be typical pests in the Anglo-world. Elephants fascinated artists, writers, and scientists alike. Wise, compassionate, noble, and humane were some of the adjectives used to describe these great beasts. Ibid., pp. –. Janisch to Chabaud, Apr. , KAB, /UIT /; Chabaud to Janish, June , KAB, /UIT /, /, //; Mansergh, ‘Farms Strathmore and Mentone, Alexandria’, July , KAB, PAS /. Sujit Sivasundaram, ‘Trading knowledge: the East India Company’s elephants in India and Britain’, Historical Journal, (), p. . Ritvo, The animal estate, pp. –, . Nathaniel Shaler, Domesticated animals: their relation to man and to his advancement in civiliza- tion (New York, NY, ), p. . See also Georg Hartwig, The tropical world (London, ), p. . Sh l D i d i l These writers were unaware of the brutal training process that transformed Asian ele- phants into colonial labourers. See Saha, ‘Colonizing elephants’. Shaler, Domesticated animals, p. . Nathaniel Shaler, Domesticated animals: their relation to man and to his advancement in civiliza- tion (New York, NY, ), p. . See also Georg Hartwig, The tropical world (London, ), p. . I In Britain and India, as Sujit Sivasundaram has shown, elephants were considered to possess near-human intelligence, human-hand-like trunks, and a penchant for working with humans.According to Harriet Ritvo, the elephant was often con- sidered one of the most intelligent of all animal species.This animal, wrote Harvard geology professor Nathaniel Shaler, is ‘innately domesticable, and best fitted by nature for companionship with man, of all our great quadru- peds’.Being ‘innately domesticable’ was unique: in all other domestic animals except the dog, obedience had been ‘slowly developed by thousands of years of selection’.Elephants, on the other hand, could supposedly be caught in the wild and easily domesticated. Despite their putative partiality to humans, elephants were also thought capable of treachery. The noble elephant could transform into a ferocious and pathological ‘rogue’. According to the Oxford English dictionary, this term first entered British naturalists’ vocabularies in traveller James Holman’s A voyage around the world (). Holman defined the ‘rogue’ as ‘a large male https://doi.org/10.1017/S0018246X19000761 Published online by Cambridge University Press T H E A D D O E L E P H A N T S I N S O U T H A F R I C A  who has been driven from the herd, after losing a contest for mastery of the whole; or a female wandering from it in quest of her calf’.Unlike other ele- phants in Ceylon, rogues were ‘cunning and daring’, and ‘a plague and a terror to the neighbourhood in which they prowl’.By the s, this concept had been directly linked to agricultural depredations. In the  edition of Encyclopaedia Britannica, rogues were defined as ‘solitary bulls…of a spiteful disposition’ that were ‘permanently separated from their kind’, due to ‘their partiality for cultivated crops’. As Chris Roche has argued, settlers living alongside elephants in Knysna, South Africa, had a similarly dynamic opinion of the beasts. From the s to the s, the settlers’ conceptions of the elephants shifted between cultur- ally valued animals and pests as economic systems changed. By the s, gov- ernment bureaucrats thought that the Knysna herd, along with the Addo Elephants, were the last remnants of a population of elephants that once abounded the Cape. Settlers in Knysna who encountered them rejected the idea of elephant sagacity. James Holman, A voyage around the world, III (London, ), p. . Ibid. ‘Elephant’, in Encyclopaedia Britannica (New York, NY, ), p. . Chris Roche, ‘“The elephants at Knysna” and “the Knysna elephants”: from exploitation to conservation: man and elephants at Knysna, –’ (BA Hons thesis, Cape Town, ), qu. on p. . Discussions of this legislature are beyond the scope of this article. For an analysis of these legal categories, see MacKenzie, The empire of nature, pp. –. Roche, ‘“The elephants at Knysna” and “the Knysna elephants”’, pp. , –. Ibid., pp. –. Chabaud to Janisch, June , KAB, /UIT /, /, //. Ibid. James Holman, A voyage around the world, III (London, ), p. . Chris Roche, ‘“The elephants at Knysna” and “the Knysna elephants”: from exploitation to conservation: man and elephants at Knysna, –’ (BA Hons thesis, Cape Town, ), qu. on p. . ‘Elephant’, in Encyclopaedia Britannica (New York, NY, ), p. . James Holman, A voyage around the world, III (London, ), p. . Ibid. Discussions of this legislature are beyond the scope of this article. For an analysis of these legal categories, see MacKenzie, The empire of nature, pp. –. Chabaud to Janisch, June , KAB, /UIT /, /, //. Ibid. g g p f pp  Roche, ‘“The elephants at Knysna” and “the Knysna elephants”’, pp. , –. Ibid., pp. –. I The Knysna Elephants destroyed their crops and har- assed people, and were viewed as vermin that ‘infested’ the forest in the mid- to late nineteenth century.Despite their protests, Roche argues, as the wildlife- protection movement grew, well-funded game-protection lobbies were founded, and in elephants were legally classified as ‘Royal Game’. Under this classification, they could only be hunted with a Royal Game licence and permission from the governor. Like the Knysna Elephants, as the Addo Elephants crossed the physical borders between farm and veld, they crossed the conceptual margins between Royal Game and vermin. White farmers bitterly resented their classification as Royal Game: the elephants could only legally be shot if they destroyed crops or injured livestock. Yet in this case, the onus of proof lay upon the farmers, and action against an elephant could only be taken after the damage had been done.Elephant depredations were so severe that farmers insisted that the animals be exterminated.Yet government officials were unwilling to sanc- tion the death of Royal Game based on this testimony alone. Information was needed from an ‘experienced man’ as to why the Addo Elephants were behav- ing in ways that undermined existing knowledge of elephant behaviour. https://doi.org/10.1017/S0018246X19000761 Published online by Cambridge University Press  J U L E S S K O T N E S - B R O W N In , Chabaud, the magistrate of Uitenhage, suggested that domestication might prove a means of reconciling the human and elephant conflict. Although African elephants were not usually domesticated, he insisted that this was pos- sible, and worth attempting.Seeking to confirm his views, he contacted Captain James MacQueen, ‘a gentleman who has done a lot of exploration work in Central Africa and who is well acquainted with the habits of ele- phants’.In his initial conversation with Chabaud, MacQueen connected the concept of a rogue elephant with ideas about degeneration. Ibid. Chabaud to Janish, July , KAB, /UIT /, /, //. Ibid. Edwin Ray Lankester, Degeneration: a chapter in Darwinism (London, ), p. . Ibid. Daniel Pick, Faces of degeneration (Cambridge, ), pp. –. Lankester, Degeneration, p. . Chabaud to Janish, July , KAB, /UIT /, /, //; MacQueen to Chabaud, Aug. , KAB, /UIT /, /, //. Chabaud to Janish, July , KAB, /UIT /, /, //. Ibid. Ibid. Lankester, Degeneration, p. . T H E A D D O E L E P H A N T S I N S O U T H A F R I C A  T H E A D D O E L E P H A N T S I N S O U T H A F R I C A In August , MacQueen surveyed the bush with two white farmers, and a ‘native guide called Bartman’, and his observations confirmed his specula- tions.The Addo Elephants’ mischief was the result of ‘rogue’ elephants who had become ‘vicious’ by exposure to ‘civilization’, and farmers who shot indiscriminately, inevitably wounding them.Bullet-wounds, he speculated, were key in turning an elephant from sagacious to rogue, because wounded ele- phants ‘cannot keep pace with the herd, and will end up taking the vagrant occupation of a rogue’.Human violence had caused the elephant-degeneracy problem. Addo Elephants were not malicious and destructive agents: their mobility was a response to human brutality. They were forced to cross the line between peaceful and pest, as farmers unjustly extended the boundaries between their farms and the bush. This said, MacQueen acknowledged that elephant mobility needed to be cur- tailed and thought that benevolent elephant management provided the solu- tion. Like Chabaud, he suggested that the province create an elephant ‘paddock’, and ‘reclaim’ the animals from the wild – domesticate them as had been the norm in India for centuries. MacQueen’s ‘reclaiming project’, which may seem antithetical to nature conservation, has a long history in Anglo-imperial thought.It was posed in an intellectual milieu which bifur- cated the sagacious elephant from the rogue and correlated the degeneration of the African elephant with the putative decadence of the African continent from its ‘heights’ of Roman and Carthaginian antiquity. Although Asian elephants had long been utilized by humans in war and for- estry, African elephants had not. In the nineteenth century, naturalists from Georges Cuvier to William Jardine, to Francis Galton had speculated whether the African elephant could be ‘domesticated’, and why no domestication had taken place in Africa for centuries.As early as , travellers to South Africa such as missionary Henry Methuen had commented on why African ele- phants had never been ‘tame’. MacQueen to Chabaud, Aug. , KAB, /UIT /, /, //. Ibid. Ibid. Ibid. Ibid. William Jardine, The natural history of the pachydermes (London, ), p. ; Georges Cuvier, ‘Memoir upon living and fossil elephants’, The Philosophical, (), pp. –; Francis Galton, ‘The first steps towards the domestication of animals’, Transactions of the Enthological Society of London, (), pp. –. Henry Methuen, Life in the wilderness: or, wanderings in South Africa (London, ), p. . Ibid, p. . https://doi.org/10.1017/S0018246X19000761 Published online by Cambridge University Press Henry Methuen, Life in the wilderness: or, wanderings in South Africa (London, ), p. . Ibid, p. . I MacQueen was ‘surprised to hear that they are said to be dangerous, as the elephant is inoffen- sive by nature’ and thought that there ‘must be some “rogue” elephants – degenerates – among them’, who were corrupting the herds.According to British zoologist Edwin Lankester, degeneration constituted a ‘gradual change of the structure in which the organism becomes adapted to less varied and less complex conditions of life’.This left the ‘whole animal in a lower con- dition…than was the ancestral form with which we are comparing it’.By raiding farms, the Addo Elephants were placed in a ‘lower condition’ by com- parison with the sagacious elephants that MacQueen had encountered in Central Africa. According to Daniel Pick, in Britain, France, and Italy, degener- ation in humans was a symptom of advancing civilization and social ‘progress’ in the nineteenth and twentieth centuries. Through alienation from nature, pro- cesses of elaboration were being reversed, producing ‘degenerate’ people. Animals could also fall prey to degeneration. In his monograph, Degeneration, Lankester argued that any ‘new set of conditions occurring to an animal which render its food and safety easily attained, seem to lead as a rule to Degeneration…as Rome degenerated when possessed of the riches of the ancient world’. Evidently convinced by such ideas, MacQueen speculated that the cause of roguery was the combination of advancing civilization upon the bush, and a very poor diet (rather than a lavish Roman one).In his opinion, one means of resolving the conflict would be to plough up the bush, and cultivate ‘sweet pota- toes and bananas, of which elephants are very fond’.If this was done, they would ‘become docile and almost domesticated in time’.Impressed by his knowledge, Chabaud hired MacQueen to visit Addo and assess the situation. https://doi.org/10.1017/S0018246X19000761 Published online by Cambridge University Press J. Emerson Tennent, The wild elephant and the method of capturing and taming it in Ceylon (London, ), p. .  p John Crawfurd, ‘On the physical and mental characteristics of the Negro’, Transactions of the Ethnological Society of London, (), p. . By this point, debates about the natural history of race and evolution were increasingly brought into the public eye in Britain. Sadiah Qureshi, ‘Peopleing natural history’, in Helen Curry et al., eds., Worlds of natural history (Cambridge, ), pp. –. y f y g pp  Clements Markham, The travels of Pedro de Cieza de Léon, A.D. –(London, ), p. xxiv.  For a history of the war elephant in Africa, see Thomas Trautmann, Elephants and kings: an environmental history (Chicago, IL, ), ch. ; Methuen, Life in the wilderness, p. . Charles Andersson, The lion and the elephant, ed. L. Lloyd (London, ), pp. –; John Steel, A manual of the diseases of the elephant and of his management and uses (Madras, ), p. xii.  y g  Methuen, Life in the wilderness, p. .  Shaler, Domesticated animals, pp. –. Samuel Baker, Wild beasts and their ways (London, ), p. .  For a history of the war elephant in Africa, see Thomas Trautmann, Elephants and kings: an environmental history (Chicago, IL, ), ch. ; Methuen, Life in the wilderness, p. . Methuen, Life in the wilderness, p. . By this point, debates about the natural history of race and evolution were increasingly brought into the public eye in Britain. Sadiah Qureshi, ‘Peopleing natural history’, in Helen Curry et al., eds., Worlds of natural history (Cambridge, ), pp. –. Clements Markham, The travels of Pedro de Cieza de Léon, A.D. –(London, ), p. xxiv. John Crawfurd, ‘On the physical and mental characteristics of the Negro’, Transactions of the Ethnological Society of London, (), p. . J. Emerson Tennent, The wild elephant and the method of capturing and taming it in Ceylon (London, ), p. . Charles Andersson, The lion and the elephant, ed. L. Lloyd (London, ), pp. –; John Steel, A manual of the diseases of the elephant and of his management and uses (Madras, ), p. xii. Andrew Wilson, ‘Elephants’, Belgravia, , p. . Samuel Baker, Wild beasts and their ways (London, ), p. . Shaler, Domesticated animals, pp. –. f f p f g Andrew Wilson, ‘Elephants’, Belgravia, , p. . T H E A D D O E L E P H A N T S I N S O U T H A F R I C A For Methuen, the reasons were cultural: the Africans he encountered (amaXhosa) hunted elephants rather than training them.Methuen thought elephant hunting was cruel and insisted that the animal ‘be enlisted in the service of man’.Not only did Indian elephant trai- ners (mahouts) provide a model of expertise here, but in antiquity, this feat had been accomplished by Hannibal of Carthage, who trained African elephants for https://doi.org/10.1017/S0018246X19000761 Published online by Cambridge University Press  J U L E S S K O T N E S - B R O W N warfare.Like the Carthaginians, settlers could utilize elephants in conquering the environment and its inhabitants, by employing elephants to eat and trample the ‘Fish river bush’, a stronghold of the amaXhosa. In the mid- to late nineteenth century, such discussions became less about cul- tural difference, and more about hierarchies of race.In , president of the Royal Geographical Society, Clements Markham, argued that ‘The inferiority of the African, as compared with the Hindu, is demonstrated by the latter having domesticated the elephant…while the former…has merely destroyed the elephant for the sake of his ivory tusks.’This view seems to have been widespread, and ex- colonial administrator of Singapore John Crawfurd (),as well as James Tennent ()offered near identical arguments. Others, such as big-game hunter Charles Andersson (), and veterinarian John Steel (), thought the lack of African elephant domestication was connected with the ‘decline’ of the continent from its ‘heights’ of Roman and Carthaginian civilization. During and after the Berlin Conference of –, the African elephant became captive to British developmentalist rhetoric: a symbol of, and a tool for, ‘civilizing’ the continent. Numerous British scholars saw the domestication of the elephant as a means of reclaiming the putative glory of North African antiquity. The new ‘superior’ race in British Africa – the Anglo-Saxons – could ‘civilize’ the beast. According to such thinkers, if domesticated, elephants, like Africans, would not degenerate as a result of advancing civilization but stood to benefit: domestication would accelerate their mental evolution. Wilson, ‘Elephants’, p. . T H E A D D O E L E P H A N T S I N S O U T H A F R I C A In , Scottish zoologist Andrew Wilson suggested elephants were ‘susceptible of higher development, through domestication’.Domesticated elephants would benefit colonial survey-teams by serving as steeds that could traverse impenetrable jungles without fear of predators.These animals could even assist in securing Rhodes’s vision of Cape-to-Cairo: British imperialists could train elephants to construct railway lines across the continent (Figure ). Samuel Baker, Wild beasts and their ways (London, ), p. .  https://doi.org/10.1017/S0018246X19000761 Published online by Cambridge University Press  T H E A D D O E L E P H A N T S I N S O U T H A F R I C A Fig. . Frontispiece to Shaler’s monograph on domestication and civilization. Here an explicit link between the African elephant, civilization, and domestication is drawn. Nathaniel Shaler, Domesticated animals: their relation to man and to his advancement in civilization (New York, NY, ). Image from the Biodiversity Heritage Library. Contributed by the Webster Family Library of Veterinary Medicine, <https://doi.org/./bhl.title.>. Warrington-Smyth to Mansergh, May , KAB, PAS /. In South Asia, elephants were typically captured through the creation of a keddah enclos- ure. For more on this, see Saha, ‘Colonizing elephants’; Trautmann, Elephants and kings. Mansergh, ‘Farms Strathmore and Mentone, Alexandria’. J. H. Stone, ‘M. K. Gandhi: some experiments with truth’, Journal of Southern African Studies, (), pp. –; Maureen Swan, ‘The Natal Indian Strike’, Journal of Southern African Studies, (), pp. –; Ramachandra Guha, Gandhi before India (London, ). Mansergh, ‘Farms Strathmore and Mentone, Alexandria’. Ibid. Warrington-Smyth to Mansergh, May , KAB, PAS /.  g y g y    In South Asia, elephants were typically captured through the creation of a keddah enclos- ure. For more on this, see Saha, ‘Colonizing elephants’; Trautmann, Elephants and kings. Mansergh, ‘Farms Strathmore and Mentone, Alexandria’. Ibid. Fig. . Frontispiece to Shaler’s monograph on domestication and civilization. Here an explicit link between the African elephant, civilization, and domestication is drawn. Nathaniel Shaler, Domesticated animals: their relation to man and to his advancement in civilization (New York, NY, ). Image from the Biodiversity Heritage Library. Contributed by the Webster Family Library of Veterinary Medicine, <https://doi.org/./bhl.title.>. For these British imperialists, as well as Chabaud and MacQueen in the Cape Province, the African elephant was thus not a naturally ‘wild’ species that should be left undisturbed. Elephants were powerful imperial agents, who wanted the benefits of ‘civilized life and employment’.Domestication represented the apex of elephant evolution, and their ‘decline’ in Africa was a result of contin- ental degeneration. While Hindus had successfully domesticated the animals, indigenous Africans had not. This ‘inability’ to master a docile animal that was naturally predisposed to co-operating with humans was taken as evidence of biological African inability to manage nature. To address this, a multispecies civilizing mission was needed: both African humans and elephants could be ‘civilized’ through employment. MacQueen’s domestication proposal needs to be viewed within this intellectual landscape – like much of Africa, the Addo Bush was constructed as a backward, wild, and violent space from which the noble and ‘innately domesticable’ elephant could be uplifted. https://doi.org/10.1017/S0018246X19000761 Published online by Cambridge University Press J U L E S S K O T N E S - B R O W N  I I Mansergh, Farms Strathmore and Mentone, Alexandria . J. H. Stone, ‘M. K. Gandhi: some experiments with truth’, Journal of Southern African Studies, (), pp. –; Maureen Swan, ‘The Natal Indian Strike’, Journal of Southern African Studies, (), pp. –; Ramachandra Guha, Gandhi before India (London, ). I I Unlike MacQueen and Chabaud, other Cape provincial bureaucrats were less convinced by nineteenth-century literature on elephant domestication. In May , MacQueen’s domestication proposal was debated. Secretary of the Cape provincial administration J. Warrington-Smyth made enquiries into whether the animals could ‘be kraaled and tamed’ by mahouts.In the course of his enquiries, he realized that this was not economically feasible. Capturing the animals would be difficult because of the dense bush, and the ele- phants were too expensive as labourers, given their voracious appetite.Lewis Mansergh, a bureaucrat in the Cape provincial administrator’s office drew upon racial science to dismiss the proposal. Economic issues aside, although he recog- nized that the ‘taming and employment of the elephants for services such as they render to man in India…might open a new era’, there was insufficient Indian labour to perform ‘such an experiment’.Perhaps concerned about growing anti-Indian sentiment amongst whites, Gandhi’s political activism in Natal, and the recent Natal Indian Strike of November ,he also felt it was not a ‘suitable time to import men from India’, but was willing to employ a ‘trustworthy European’.However, the issue was complicated by a greater labour problem: in the absence of Indian labourers, indigenous South Africans would be required to direct the elephants. Such labourers, in Mansergh’s view, were unsuitable for this task: Handling the animals is not natural to the Native of South Africa, and in his present stage of development it is unlikely that he would readily adapt himself to a calling which, to be successfully proceeded with, one would think would involve some her- editary qualities or, at least traditional if not some natural or national characteristics. Mansergh’s ideas were presented as common-sense but require further analysis. Drawing upon Anglo-imperialist rhetoric which had correlated elephant domes- tication with hierarchies of civilization, Mansergh suggested that over centuries of co-evolution elephants had learned to respect Indians, and such characteris- tics were passed down generations in elephant populations. Indigenous South Africans, contrastingly, had never domesticated elephants, and thus were not naturally predisposed to this task. Even if they learned from mahouts, the https://doi.org/10.1017/S0018246X19000761 Published online by Cambridge University Press T H E A D D O E L E P H A N T S I N S O U T H A F R I C A  elephants would not obey them because African elephants had no hereditary characteristics rendering them amenable to African control. There are parallels in British Burma in this period. Burmese elephant trainers were con- sidered racially suited to the task on account of their skin which could supposedly withstand exposure to rough elephant hair without causing rashes. Jonathan Saha, ‘Among the beasts of Burma: animals and the politics of colonial sensibilities, c. –’, Journal of Social History, (), pp. –. Walton to secretary of the divisional council, Alexandria, May , KAB, PAS /. ‘Addo farmers petition to Sir Frederic de Waal’, Sept. , KAB PAS /. P. L. Sclater, ‘The domestication of the African elephant’, Journal of the Society for the Preservation of the Wild Fauna of the Empire, (), pp. –; James Stevenson-Hamilton, Animal life in Africa: book II, the vegetarians (London, ), pp. –. f f g ( ) pp Frank Melland, Elephants in Africa (London, ), p. .  ‘Killed by elephants’, Queenstown Weekly Review, Feb. . ‘Union of South Africa: minutes of the provincial council of the province of the Cape of Good Hope. Third session () – second council’, Apr. , KAB, PAS /. Ibid. History, (), pp. –. P. L. Sclater, ‘The domestication of the African elephant’, Journal of the Society for the Preservation of the Wild Fauna of the Empire, (), pp. –; James Stevenson-Hamilton, Animal life in Africa: book II, the vegetarians (London, ), pp. –. Frank Melland, Elephants in Africa (London, ), p. . ‘Union of South Africa: minutes of the provincial council of the province of the Cape of Good Hope. Third session () – second council’, Apr. , KAB, PAS /. Ibid. ‘Killed by elephants’, Queenstown Weekly Review, Feb. . Walton to secretary of the divisional council, Alexandria, May , KAB, PAS /. ‘Addo farmers petition to Sir Frederic de Waal’, Sept. , KAB PAS /. There are parallels in British Burma in this period. Burmese elephant trainers were con- sidered racially suited to the task on account of their skin which could supposedly withstand exposure to rough elephant hair without causing rashes. Jonathan Saha, ‘Among the beasts of Burma: animals and the politics of colonial sensibilities, c. –’, Journal of Social History, (), pp. –. P. L. Sclater, ‘The domestication of the African elephant’, Journal of the Society for the Preservation of the Wild Fauna of the Empire, (), pp. –; James Stevenson-Hamilton, Animal life in Africa: book II, the vegetarians (London, ), pp. –. Frank Melland, Elephants in Africa (London, ), p. . ‘Union of South Africa: minutes of the provincial council of the province of the Cape of Good Hope. Third session () – second council’, Apr. , KAB, PAS /. Ibid. ‘Killed by elephants’, Queenstown Weekly Review, Feb. . Walton to secretary of the divisional council, Alexandria, May , KAB, PAS /. ‘Addo farmers petition to Sir Frederic de Waal’, Sept. , KAB PAS /. f f , g ( , ), pp  Frank Melland, Elephants in Africa (London, ), p. . ‘Union of South Africa: minutes of the provincial council of the province of the Cape of Good Hope. Third session () – second council’, Apr. , KAB, PAS /. Ibid. ‘Killed by elephants’, Queenstown Weekly Review, Feb. . Walton to secretary of the divisional council, Alexandria, May , KAB, PAS /. ‘Add f i i Si F d i d W l’ S KAB PAS /  I I In other words, in thousands of years of selection, the relationship between indigenous South Africans and elephants had never been one of master and servant: to Mansergh, indigenous South Africans were biologically incapable of command- ing elephants. In , evidence to the contrary of Mansergh’s view already existed. In the s, the Belgian Congo government, with the assistance of Indian mahouts, had established a keddah (elephant training establishment) and trained ele- phants for timber hauling. Zande peoples were employed as mahouts.Yet this could also be explained in terms of racial difference: the Azande had ‘Hamatic and Berberine ancestry’, which rendered them ‘natural born mahouts of Africa, the lineal descendants of Hannibal’s Nubians’, who had domesticated African elephants in antiquity.Without any South African ‘natives’ sharing such ancestry, the domestication proposal was discarded by the Cape provincial government. On April , the provincial council of the Cape met to formulate an alternative. Politician Mr Ross was anxious to pre- serve the elephants, because they were ‘the last surviving remnant in the Southern portion of the Continent of the great African fauna’.In an attempt to stabilize the status of the Addo Elephants as a protected species, and not a pest, the council resolved to create an elephant reserve. This would allow them to curtail elephant mobility, regulate livestock, and prevent cattle from wandering into the area and ‘possibly irritate the elephants’.By mid- , this proposal had irritated farmers, who complained that the elephants continued to lay waste to their properties. They claimed to be defenceless against the ‘wily brutes’ who had already killed and ‘shockingly mutilated’ several farmers in the area.If the Cape provincial administration insisted on protecting the Addo Elephants, argued farmer Louis Walton, they should compensate the farmers financially.In September, thirteen farmers signed a petition demanding the extermination of the elephants. https://doi.org/10.1017/S0018246X19000761 Published online by Cambridge University Press  J U L E S S K O T N E S - B R O W N As the elephants continued to trample crops and fences while moving between bush and farm, this physical mobility forced state officials to reconsider MacQueen’s categorization of the species as naturally peaceful. The ‘degener- ate’ rogue elephants were too dangerous to be allowed to live, but killing any elephant, they feared, would enrage the other elephants, and drive the entire population to roguery. R. G. D. to provincial secretary, Cape Town, Sept. , KAB, PAS /; ‘Addo farmers petition to Sir Frederic de Waal’, Sept. , KAB PAS /. R. G. D. to provincial secretary, Cape Town, Sept. , KAB, PAS /; ‘Addo farmers petition to Sir Frederic de Waal’, Sept. , KAB PAS /. Meiring, The Sundays River Valley, pp. –. Mansergh to secretary for lands, Pretoria,  May , KAB, PAS /; ‘Union of South Africa: minutes of the provincial council of the province of the Cape of Good Hope. Second session () – third council’, Apr. , KAB, /UIT /. Anonymous report, likely Lewis Mansergh, ‘The elephants in the Addo Bush’, Apr. , KAB, PAS /. Brauer, ‘Provincial council, Cape Town’, Apr. , KAB, PAS /. Reeler, ‘Addo Elephants: report of //’, KAB, PAS /. Ibid. Ibid. Pretorius, Jungle man, p. ; Reeler, ‘Addo Elephants: report of //’, KAB, PAS / . p p    Meiring, The Sundays River Valley, pp. –. Mansergh to secretary for lands, Pretoria,  May , KAB, PAS /; ‘Union of South Africa: minutes of the provincial council of the province of the Cape of Good Hope. Second session () – third council’, Apr. , KAB, /UIT /. Anonymous report, likely Lewis Mansergh, ‘The elephants in the Addo Bush’, Apr. , KAB, PAS /. Pretorius, Jungle man, p. ; Reeler, ‘Addo Elephants: report of //’, KAB, PAS / . ‘Fate of the Addo Elephants: Science Association’s views’, Eastern Province Herald, Aug. , p. . Paul Matschie, ‘Ueber Geographische Abarten Des Afrikanischen Elefanten’, Sitzungsberichte der Gesellschaft Naturforschender Freunde zu Berlin, , pp. –. Ibid., pp. –. Richard Lydekker, ‘. The ears as a race-character in the African elephant’, Proceedings of the Zoological Society of London (Aug. ), p. . For preservationism, eugenics, and human evolution, see Dubow, ‘Human origins, race typology and the other Raymond Dart’; Dubow, Scientific racism in modern South Africa, pp. –. For more on SA, see Dubow, A commonwealth of knowledge, pp. –. Emphasis added, ‘Fate of the Addo Elephants: Science Association’s views’, p. . Ibid. ‘Depredations of Addo Elephants’, Eastern Province Herald, Aug. , p. . ‘Depredations of Addo Elephants’, Eastern Province Herald, Aug. , p. . f g pp Emphasis added, ‘Fate of the Addo Elephants: Science Association’s views’, p. . Ibid.  I I Wholesale extermination was the only safe course of action.More importantly, in , the Sundays River Settlement Scheme, an irrigation and town-building project, began attracting farmers from across the world.The Sundays River settlements could not afford to be destroyed by elephant mobility, and the enclosure of the bush was too expensive to enter- tain.Given these new economic constrains, elephant ‘civilization’ was no longer a possibility. Unable to prevent the elephants from crossing physical boundaries between bush and farm, the provincial government could not prevent them from crossing conceptual margins between ‘Royal Game’ and ‘vermin’. On April , the provincial government under Frederic de Waal resolved to exterminate the animals. One month later, the task was given to famed elephant hunter Phillip Jacobus Pretorius.Pretorius came at a price but managed to convince the administra- tion that the operation would be profitable. The sale of elephant skeletons and skins for museums, ivory and meat for the market, and the capture of calves for zoos would fetch a total of £,.At the end of May, the hunt began. This was the first step towards physically (but not conceptually) transforming the bush from a wasteland into a semi-domestic space and involved carving roads in a four-by-four grid, and erecting shooting platforms.Pretorius’s hunt was mired with difficulties. He battled to recruit African labour – with an abun- dance of agricultural jobs in the region, few labourers were willing to risk their lives hunting elephants – and only a group of desperate ex-convicts agreed to work for him.Although it had been intended as a quick and profitable hunt, the operation dragged on for more than a year, and by mid-, zoolo- gists in South Africa, Britain, and the USA began campaigning for the cessation of the slaughter. https://doi.org/10.1017/S0018246X19000761 Published online by Cambridge University Press T H E A D D O E L E P H A N T S I N S O U T H A F R I C A T H E A D D O E L E P H A N T S I N S O U T H A F R I C A  While some of these scientists made economic arguments for elephant domestication, most mobilized racial science to articulate their value to zoology. This ultimately became the primary argument for elephant-protection and is directly correlated with the foundation of the park. The South African Association for the Advancement of Science (SA) – the largest science society of the country – began pushing a taxonomic argument in favour of their preservation.In , Paul Matschie, a German zoologist, had com- pared eighteen elephant specimens, and suggested that African elephants exhibited considerable skull and ear variation relative to their environments. In his view, they needed to be divided into four regional races.In , British zoologist Richard Lydekker suggested that further sub-races should be created on the basis of ear-comparison. One of these was what he referred to as the ‘Addo Bush, or East Cape Elephant’.The Addo Bush elephant was categorized as a unique type (Figure ). In August , SAissued a statement, drawing upon this taxonomic argu- ment and infusing it with eugenic tropes of ‘dying races’ that were already in circulation across South Africa, but typically applied to humans.At a meeting in July, J. R. L. Kingon proposed that SAadopt a resolution that ‘this Association views with great regret the decision…to exterminate the herd of Cape elephants, relics of a dying race, now preserved in the Addo Bush’.Subsequently, SAcirculated a memorandum, suggesting that the remaining elephants be preserved in the interests of science, and the ‘young animals…be trained for labour’, while the ‘rogues, should, of course be shot’.SAsuggested that the elephants were not all ‘degenerates’, but a unique race of importance to zoology that could be preserved in peace. One month later, the Uitenhage and Port Elizabeth farmers associations met to discuss this, and other protests against the slaughter of the elephants. In this meeting, the status of the Addo Elephants as a unique racial type did little to change the farmers’ perspectives. They insisted that the elephants were natur- ally violent, and that it was a choice between ‘protecting human life or protect- ing animal life’.They were also declared to be prone to criminality: farmer Jack Harvey insisted that the elephant was ‘always a thief, and a thief of the https://doi.org/10.1017/S0018246X19000761 Published online by Cambridge University Press ) and West Cape Elephant (p. ). Richard Lydekker, ‘. The ears as a race-chara n (Aug. ). Image from the Biodiversity Heritage Library. Ibid. Ibid. Ibid. ‘The elephants in the Addo Bush’, Apr. , KAB, PAS /. Ibid. ‘Scientific events: destruction of elephants in Cape Colony’, Science, (), p. ; John Hamlyn, ‘The Addo Elephants’, Hamlyn’s Menagerie Magazine, Nov. . Hoffman, ‘Major P. J. Pretorius and the decimation of the Addo elephant herd in – ’, p. ; ‘Depredations of Addo Elephants’. Alwin Karl Haagner, South African mammals: a short manual for the use of field naturalists, sportsmen and travellers (Cape Town, ), pp. –, qu. on p. . Van Hoogstraten to Mentz, Oct. , KAB, PAS /. Anonymous, ‘The elephants in the Addo Bush’, KAB, PAS /; ‘Resume of interview between the administrator and Major Pretorius’, Nov. , KAB, PAS /; ‘Reply by Major Pretorius to wire no. proposing alternative arrangement’, Feb. , KAB, PAS /. Hoffman, ‘Major P. J. Pretorius and the decimation of the Addo elephant herd in – ’, p. ; ‘Depredations of Addo Elephants’. T H E A D D O E L E P H A N T S I N S O U T H A F R I C A Contributed by the Natu on, <www.biodiversitylibrary.org/bibliography/>. ) and West Cape Elephant (p. ). Richard Lydekker, ‘. The ears as a race n (Aug. ). Image from the Biodiversity Heritage Library. Contributed by t on, <www.biodiversitylibrary.org/bibliography/>. est Cap ). Im iodiver ) and W n (Aug.  on, <www.b T H E A D D O E L E P H A N T S I N S O U T H A F R I C A  worst nature’.Another farmer, C. H. Mackay, rose in support of Harvey, claiming that the elephants ‘played football’ with his pumpkins.In his opinion, the ‘scientific value’ of the herd could be preserved by the supply of ‘representative specimens free of cost’ to the Port Elizabeth Museum. Despite such arguments, the operation was becoming costly. In order to sell the specimens to zoologists, Pretorius agreed to skin the animals, collect their bones and ivory, and make measurements of their morphology. This was time consuming, and nearing the end of October, Pretorius had killed only thirteen elephants. Although the government had assumed the elephant remains would be valu- able, insects and carnivores feasted upon them in the field, and few museums were willing to pay for those that survived. Ivory sales were negligible: almost all the elephants had minute or absent tusks. A mere £of £,expend- iture was recovered (Figure ). By this point, SA’s resolution that the elephants were a ‘dying race’ had found traction in North America and Britain, and the government was under pressure to prevent what Science called a ‘zoological calamity’.Directors of three major South African Natural History Museums, Louis Peringuey (South African Museum), Frederick FitzSimons (Port Elizabeth Museum), and Ernest Warren (Natal Museum), were all vocally opposed to the hunt.Alwin Karl Haagner, of the National Zoological Gardens, also condemned it as a ‘calamity’ and propagated a plea for the government to reconsider domestication (Figure ). With the elephants worthless as dead specimens, their living value to science provided a new rationale for their preservation. Under pressure from zoologists, the expense of the hunt did not seem justified. Seeking to ‘pocket’ their losses,the administration renegotiated with Pretorius. ‘Scientific events: destruction of elephants in Cape Colony’, Science, (), p. ; John Hamlyn, ‘The Addo Elephants’, Hamlyn’s Menagerie Magazine, Nov. . https://doi.org/10.1017/S0018246X19000761 Published online by Cambridge University Press ‘Proclamation: game reserve’, Dec. , KAB, PAS /. Magistrate of Addo to secretary for lands, Cape Town, July , KAB, /UIT /, /, //. J. J. Millard to superintendent of the Sundays River Settlement, reports for the months  Aug. – Nov. , KAB, PAS /; J. J. Millard to superintendent of the Sundays River Settlement, reports for the months Nov. to Aug. , KAB, PAS /. This is discussed frequently. See Cooper to provincial secretary Cape Town, Feb. , PAS /; Chisnall to secretary of lands, Pretoria, Nov. , KAB, PAS /; questions in T H E A D D O E L E P H A N T S I N S O U T H A F R I C A The ultimate outcome of the negotiations was that Pretorius would stop collecting specimens, and kill all but sixteen elephants, at a rate of £per elephant.In December , an elephant reserve for the remaining sixteen was created in an attempt p p p Alwin Karl Haagner, South African mammals: a short manual for the use of field naturalists, sportsmen and travellers (Cape Town, ), pp. –, qu. on p. . p p  pp q p Van Hoogstraten to Mentz, Oct. , KAB, PAS /.  Anonymous, ‘The elephants in the Addo Bush’, KAB, PAS /; ‘Resume of interview between the administrator and Major Pretorius’, Nov. , KAB, PAS /; ‘Reply by Major Pretorius to wire no. proposing alternative arrangement’, Feb. , KAB, PAS /. https://doi.org/10.1017/S0018246X19000761 Published online by Cambridge University Press J U L E S S K O T N E S - B R O W N  Fig. . Pretorius supervises his team of labourers as they skin an Addo Elephant in the field. Exact date unknown, –. P. J. Pretorius, Jungle man: the autobiography of Major P. J. Pretorius (London, ), p. . Fig. . Pretorius supervises his team of labourers as they skin an Addo Elephant in the field. Exact date unknown, –. P. J. Pretorius, Jungle man: the autobiography of Major P. J. Pretorius (London, ), p. . to stabilize their categorization as a scientifically valuable species. Under the ward of the province, no humans nor livestock could enter the reserve. Human and elephant spaces were thoroughly segregated – any human entering the reserve was to be prosecuted, and any elephants leaving their ‘sanctuary’ could be shot. From to , although economic concerns were of importance, every decision was justified by recourse to interlinked ideas about race, evolution, civ- ilization, and degeneration. Because indigenous South Africans were consid- ered incapable of domesticating the elephants, the provincial government attempted to segregate the animals in a ‘wild’ space. Facing opposition from farmers, officials subsequently resolved to eliminate the population to protect humans from ‘degenerate’ rogues. Now, protecting the elephants against the wishes of the farmers had been justified according to eugenic concerns about the elephants’ distinct character as a ‘dying race’ (Figure ). T H E A D D O E L E P H A N T S I N S O U T H A F R I C A https://doi.org/10.1017/S0018246X19000761 Published online by Cambridge University Press T H E A D D O E L E P H A N T S I N S O U T H A F R I C A  Fig. . Haagner’s field guide, South African mammals: a short manual for the use of field naturalists, sportsmen and travellers (Cape Town, ), p. , included photographs of an elephant pulling a plough and a cart under the direction of an African labourer in Mozambique. Such photographs served as evidence of the possibility and profitability of domestication. Image from the Biodiversity Heritage Library. Contributed by the American Museum of Natural History Library, <https://doi.org/./bhl.title.>. Fig. . Haagner’s field guide, South African mammals: a short manual for the use of field naturalists, sportsmen and travellers (Cape Town, ), p. , included photographs of an elephant pulling a plough and a cart under the direction of an African labourer in Mozambique. Such photographs served as evidence of the possibility and profitability of domestication. Image from the Biodiversity Heritage Library. Contributed by the American Museum of Natural History Library, <https://doi.org/./bhl.title.>. p   g    This is discussed frequently. See Cooper to provincial secretary Cape Town, Feb. , PAS /; Chisnall to secretary of lands, Pretoria, Nov. , KAB, PAS /; questions in https://doi.org/10.1017/S0018246X19000761 Published online by Cambridge University Press I I I By the time the reserve was created, the Addo Bush had been physically pacified. The elephant population was a fraction of its former size, a section of the bush had been cut into a grid, and its boundaries were delineated. Yet the monthly reports of elephant caretaker J. J. Millard suggest that the Addo Elephants wanted nothing to do with the reserve.This, most commentators thought, was the result of a lack of water in the region.Others offered https://doi.org/10.1017/S0018246X19000761 Published online by Cambridge University Press  J U L E S S K O T N E S - B R O W N Fig. . Pretorius used morphological charts to take measurements for taxonomy and taxidermy. A total of six annotated copies have survived, five with measurements, and this one, with a humorous poem. The author requested that their poem be deposited in the Addo Elephant files, and it appears without any further context in KAB, PAS /. Image reproduced with permission from the Cape Archives Repository, Cape Town. For a brief account of the other five, see M. T. Hoffman, ‘Major P. J. Pretorius and the decimation of the Addo elephant herd in : important reassessments’ Koedoe () p  Fig. . Pretorius used morphological charts to take measurements for taxonomy and taxidermy. A total of six annotated copies have survived, five with measurements, and this one, with a humorous poem. The author requested that their poem be deposited in the Addo Elephant files, and it appears without any further context in KAB, PAS /. Image reproduced with permission from the Cape Archives Repository, Cape Town. For a brief account of the other five, see M. T. Hoffman, ‘Major P. J. Pretorius and the decimation of the Addo elephant herd in –: important reassessments’, Koedoe, (), p. . https://doi.org/10.1017/S0018246X19000761 Published online by Cambridge University Press T H E A D D O E L E P H A N T S I N S O U T H A F R I C A  psychological interpretations. Zoologist Frederick FitzSimons thought the animals were traumatized by Pretorius’s devastation of their friends, and no longer wished to reside in a space violated by memories of terror. Bureaucrats began to recognize that the area designated as wild (the Addo Bush) would never be appealing to animals accustomed to drinking farmers’ water supplies and eating their crops. provincial council re elephants in Addo Bush, Aug. , KAB, PAS /; J. D. de V. to secretary for lands, Pretoria, July , KAB, PAS /.     Chabaud to Janish, July , KAB, /UIT /, /, //. y J y    J. D. de V. to secretary for lands, Pretoria, July , KAB, PAS /. Jamie Lorimer has made a similar point in the case of present-day Asian elephants. Lorimer, ‘Elephants as companion species’, p. . provincial council re elephants in Addo Bush, Aug. , KAB, PAS /; J. D. de V. to secretary for lands, Pretoria, July , KAB, PAS /. J. D. de V. to secretary for lands, Pretoria, July , KAB, PAS /. Jamie Lorimer has made a similar point in the case of present-day Asian elephants. Lorimer, ‘Elephants as companion species’, p. . The process of constructing and maintaining these boreholes is meticulously documen- ted in Millard’s reports in KAB, PAS /and PAS /. Trollope’s original report is reproduced in this publication. Die Addo Olifante, pp. –. Carruthers, National Park Science, pp. –. ‘Port Elizabeth Publicity Association: minutes of informal meeting held at Hotel Elizabeth’, Aug. , KAB, /UIT, Part , //. Trollope to secretary of Port Elizabeth Publicity Association, Aug. , KAB, /UIT, Part , //. Trollope, ‘Report on the Addo National Park covering period th September to date’, Feb. , KAB, /UIT, Part , //. Chabaud to Janish, July , KAB, /UIT /, /, //. Trollope, ‘Report on the Addo National Park covering period th September to date’, Feb. , KAB, /UIT, Part , //. The process of constructing and maintaining these boreholes is meticulously documen- ted in Millard’s reports in KAB, PAS /and PAS /. Trollope’s original report is reproduced in this publication. Die Addo Olifante, pp. –. Carruthers, National Park Science, pp. –. ‘Port Elizabeth Publicity Association: minutes of informal meeting held at Hotel Elizabeth’, Aug. , KAB, /UIT, Part , //. Trollope to secretary of Port Elizabeth Publicity Association, Aug. , KAB, /UIT, Part , //. Trollope, ‘Report on the Addo National Park covering period th September to date’, Feb. , KAB, /UIT, Part , //. Ch b d t J i h J l KAB /UIT /  / / / p p p  The process of constructing and maintaining these boreholes is meticulously documen- ted in Millard’s reports in KAB, PAS /and PAS /. Trollope’s original report is reproduced in this publication. Die Addo Olifante, pp. –. Carruthers, National Park Science, pp. –. ‘Port Elizabeth Publicity Association: minutes of informal meeting held at Hotel Elizabeth’, Aug. , KAB, /UIT, Part , //. Trollope to secretary of Port Elizabeth Publicity Association Aug KAB /UIT Trollope to secretary of Port Elizabeth Publicity Association, Aug. , KAB, /UIT, Part , //.  p Part , //. I I I Throughout the s, the administra- tion continued transforming the bush into a semi-domestic space by erecting boreholes and reservoirs, in the hope that elephants would utilize these water supplies, rather than those belonging to farmers.Once sufficient water was provided, in , Harold Trollope, a game ranger from the Kruger National Park, and a team of African labourers drove the elephants into the reserve. Shortly after the drive was complete, the area was declared a national park, with Trollope as its warden.The elephants, who had once freely roamed the Sundays River Valley, were now to be confined in a small reserve. At this point, seeking to capitalize upon the zoological curiosity of the ele- phants, and their close proximity to Port Elizabeth, the Port Elizabeth Publicity Board attempted to transform the park into a tourist spectacle. The near-impenetrable bush rendered this difficult: nobody could see the ele- phants, and the animals dared not approach humans. In order to solve this problem, attempts were made to tame the elephants. Between and , Trollope commenced a ‘feeding experiment’,in which he tried to train the animals to venture into the open by feeding them oranges, in the hope that they might ‘lose their fear of man’. Between and , elephant management in the Addo Bush followed MacQueen’s original suggestion for rendering the elephants ‘docile and almost domesticated in time’:in order to regain their trust, they were given a paddock and offered sustenance. Yet this was never referred to as a domestica- tion project. Instead, the word ‘tame’, once interchangeable with ‘domesti- cated’, was used exclusively and began to take on a new meaning: the https://doi.org/10.1017/S0018246X19000761 Published online by Cambridge University Press  J U L E S S K O T N E S - B R O W N training of elephants to become docile and trusting of humans. By , the elephants were beginning to appear at viewing platforms, eagerly awaiting their food.In February , the Rand Daily Mail reported that the elephants were ‘getting tamer…and have shown a fancy for the forage’.A year later, the same newspaper reported that the ‘TAME ELEPHANTS’ were a ‘TREAT FOR TOURISTS’, and that the reserve would be opening to the public shortly, in which the ‘com- paratively tame’ elephants ‘will amuse visitors by eating oranges at their feeding places’.In , Jane Meiring dubbed them ‘the pampered pets of the Parks Board’. The construction of African parks as windows onto the prehistoric is relatively common in this period. See Bernhard Gissibl, ‘A Bavarian Serengeti’, in Gissibl, Höhler, and Kupper, eds., Civilizing nature, pp. –. The idea that South Africa, as a country, could offer glimpses into the deep past, likewise, was widely publicized by statesman Jan Smuts. See Dubow, A commonwealth of knowledge, pp. –. p p Emphasis added, ‘Addo Bush Elephants’, Rand Daily Mail, Feb. , p. . ‘Tame elephants treat for tourists’, Rand Daily Mail, July , p.  Lamarckism remained an influential theory of evolution in the s–s. In the case of the Addo Elephants, it seems to have maintained its popularity well into the s, some- times in synthesis with ideas of natural selection. This is likely a result of the popularity of Prime Minister Jan Smuts’s philosophical treatise, Holism and evolution (London, ), which used Lamarckism to criticize mechanistic interpretations of evolution (p. ). See Peter J. Bowler, The eclipse of Darwinism: anti-Darwinian evolution theories in the decades around (Baltimore, MD, and London, ), pp. –, , , , . ‘South African elephants’, Eastern Province Herald, Apr. , p. . See for example ‘Looking after the elephant’, Rand Daily Mail, Mar. , p. . Meiring, The Sundays River Valley, p. . Trollope, ‘Report on the Addo National Park’. Ibid., p. , qu. on p. . Pretorius, Jungle man, p. .  Trollope, ‘Report on the Addo National Park’. Trollope, ‘Report on the Addo National Park’. Emphasis added, ‘Addo Bush Elephants’, Rand Daily Mail, Feb. , p. . ‘Tame elephants treat for tourists’, Rand Daily Mail, July , p. . Meiring, The Sundays River Valley, p. . The construction of African parks as windows onto the prehistoric is relatively common in this period. See Bernhard Gissibl, ‘A Bavarian Serengeti’, in Gissibl, Höhler, and Kupper, eds., Civilizing nature, pp. –. The idea that South Africa, as a country, could offer glimpses into the deep past, likewise, was widely publicized by statesman Jan Smuts. See Dubow, A commonwealth of knowledge, pp. –. Lamarckism remained an influential theory of evolution in the s–s. In the case of the Addo Elephants, it seems to have maintained its popularity well into the s, some- times in synthesis with ideas of natural selection. This is likely a result of the popularity of Prime Minister Jan Smuts’s philosophical treatise, Holism and evolution (London, ), which used Lamarckism to criticize mechanistic interpretations of evolution (p. ). See Peter J. Bowler, The eclipse of Darwinism: anti-Darwinian evolution theories in the decades around (Baltimore, MD, and London, ), pp. –, , , , . Pretorius, Jungle man, p. . Ibid., p. , qu. on p. . ‘South African elephants’, Eastern Province Herald, Apr. , p. . See for example ‘Looking after the elephant’, Rand Daily Mail, Mar. , p. . ‘South African elephants’, Eas Deneys Reitz, ‘Are the Addo Elephants to become extinct?’, Cape Argus, May , p. . Ibid. Ibid. Ibid. Such views were also articulated in C. S. Stokes’s bestselling account of South African nature conservation, Sanctuary (), p. . I I I (Figure ) Yet despite this physical domestication of the Addo Elephant National Park, and the presence of ‘tame elephants’, the bush retained its conceptual status as a window onto the deep past.In the s, various South African naturalists, influenced by neo-Lamarckian ideas, argued that the area was of considerable interest to evolutionary biology.It provided an example of thousands of years of adaptation to a new environment producing a new species (for some), or a degenerate race (for others), through Lamarckian use-inheritance. The greatly diminished or missing tusks, characteristic of the Addo Elephants, was key to their arguments. Perhaps seeking to justify his hunt, Pretorius argued that they were a degenerate ‘family of elephants’.Based on field-comparisons with a Knysna Elephant he shot, he argued that the elephants had lost their tusks due to inbreeding, and lack of use in the dense and ‘horrible’ Addo Bush. Zoologist Louis Peringuey of the South African Museum offered a near-identical argument. These ideas caught on quickly, and from to the s, proponents of the reserve increasingly stressed its scientific significance.These publicists argued that the elephants had not degenerated but become perfectly Lamarckism remained an influential theory of evolution in the s–s. In the case of the Addo Elephants, it seems to have maintained its popularity well into the s, some- times in synthesis with ideas of natural selection. This is likely a result of the popularity of Prime Minister Jan Smuts’s philosophical treatise, Holism and evolution (London, ), which used Lamarckism to criticize mechanistic interpretations of evolution (p. ). See Peter J. Bowler, The eclipse of Darwinism: anti-Darwinian evolution theories in the decades around (Baltimore, MD, and London, ), pp. –, , , , .  https://doi.org/10.1017/S0018246X19000761 Published online by Cambridge University Press T H E A D D O E L E P H A N T S I N S O U T H A F R I C A  Fig. . As late as the s, the elephants were still being viewed feeding on oranges on the outskirts of the park. Visitors were aware of the artificial provision of water. National Parks Board of Trustees, Die Addo Olifante (Pretoria, ), insert at p. . Reproduced with permission from University of Cape Town Libraries. See also C. S. Stokes, Sanctuary (Cape Town, ), p. . Fig. . Ibid. Such views were also articulated in C. S. Stokes’s bestselling account of South African nature conservation, Sanctuary (), p. . p p p Emphasis added, ‘State to be game reserves’, Rand Daily Mail, Sept. , p. . Emphasis added, Trollope, ‘Report on the Addo National Park’. I I I As late as the s, the elephants were still being viewed feeding on oranges on the outskirts of the park. Visitors were aware of the artificial provision of water. National Parks Board of Trustees, Die Addo Olifante (Pretoria, ), insert at p. . Reproduced with permission from University of Cape Town Libraries. See also C. S. Stokes, Sanctuary (Cape Town, ), p. . adapted to their environment. In a newspaper article, former minister of lands Deneys Reitz argued that the elephants exhibited no signs of degeneration, as they ‘remained enormous animals’, and did not lag ‘intellectually behind the rest of the elephant family’.Instead, they had developed unique characteris- tics on account of hundreds of thousands of years of use-inheritance for the bush, and become a ‘sub-species’.Elephant tusks, he argued, were needed in forests to strip bark and dig up roots, but not in the Addo Bush. On the con- trary, they posed a handicap in ‘tangled and thick’ vegetation, and thus ‘nature’ was ‘eliminating’ their tusks.Yet, ‘nature’ had compensated by providing the elephants with ‘greater bulk’ to push through the ‘impenetrable thicket which would defeat any other pachyderm’. Evidently, a tourist spectacle of deep time was incompatible with earlier visions of domesticated elephants on farms. Yet the taming of the elephants did not undermine the idea of ‘natural conditions’. Trollope still referred to the area as one in which elephants lived under ‘natural conditions which were one https://doi.org/10.1017/S0018246X19000761 Published online by Cambridge University Press  J U L E S S K O T N E S - B R O W N of South Africa’s greatest attractions in the past’.Likewise, in a seemingly contradictory sentence, T. C. White of the Port Elizabeth Publicity Association argued that the Addo ‘elephants were becoming quite tame’ and that soon tour- ists would ‘be able to see the elephants in their natural state’.This seems to pose a paradox: commentators framed the park as a ‘natural habitat’, yet it had been cut into a grid, irrigated, and the elephants were referred to as ‘tame’. Human interference in the area had initially caused the roguery problem, but now promised its solution. These apparent contradictions were never explained, nor problematized by contemporary commentators. I V In , Northern https://doi.org/10.1017/S0018246X19000761 Published online by Cambridge University Press T H E A D D O E L E P H A N T S I N S O U T H A F R I C A  Rhodesian bureaucrat Frank Melland argued that the ‘elephant problem’ and the ‘native problem’, meant ‘much the same thing…the attempt to bring an old, indigenous and very different form of life into harmony with our own life and aims’.In South Africa specifically, ‘native reserves’ were justified as spaces in which Africans were ‘protected’ from the supposedly degenerative effects of urbanization.To Jan Smuts, South Africa was also a ‘laboratory’ in which racial evolution at different stages could be studied – from hunter gath- erers to Boers.Similar thinking shaped the development of the Addo Elephant National Park: the elephants were confined in a sanctuary which ‘pro- tected’ their ‘race’ from ‘civilization’ and offered a site for the study of elephant racial evolution. Rhodesian bureaucrat Frank Melland argued that the ‘elephant problem’ and the ‘native problem’, meant ‘much the same thing…the attempt to bring an old, indigenous and very different form of life into harmony with our own life and aims’.In South Africa specifically, ‘native reserves’ were justified as spaces in which Africans were ‘protected’ from the supposedly degenerative effects of urbanization.To Jan Smuts, South Africa was also a ‘laboratory’ in which racial evolution at different stages could be studied – from hunter gath- erers to Boers.Similar thinking shaped the development of the Addo Elephant National Park: the elephants were confined in a sanctuary which ‘pro- tected’ their ‘race’ from ‘civilization’ and offered a site for the study of elephant racial evolution. When viewed through racialized lenses of domestication and degeneration, the paradox at play in the Addo Elephant National Park ceases to be a contra- diction. Historicizing actors’ categories of ‘domestic’, ‘tame’, and ‘wild’, as well as their connotations of natural and unnatural, reveals that national parks were not necessarily constructed as spaces in which wild animals roamed free of human interference. Taming the elephants was not antithetical to seeing them under ‘natural conditions’: it was a strategy to transform the area into a spectacle of the deep past, and simultaneously to put them on a path towards increasing mental complexity, while protecting them from advancing ‘civiliza- tion’. I V It might be easy to dismiss the construction of the Addo Bush as a site of wilder- ness as merely artifice: preserving the animals under ‘natural conditions’ was impossible, and this was a compromise. The reality is more complex: the cre- ation of the park was shaped by ideas of domestication and degeneration. In this period, elephants were perceived as a fundamentally domesticable species in Anglo-imperialist thought. In zoological and imperial discourse, ele- phants sat on the fence between wild and domestic, peaceful and pestilent, and could cross to either side when confronted with ‘civilized’ peoples, just like dogs or ‘wild’ humans. Yet as they crossed physical borders between bush and farm in search of nutrition and fulfilment, the Addo Elephants appeared to resist any stable classification. In response to their mobilities, colonial bureaucrats were repeatedly forced to rethink their categorization of wild or tame, peaceful or pestilent. This boundary-straddling, amidst racist discussions of a wild-continent waiting to be tamed, created the liminal status of the ‘race’, suggesting that the elephants were naturally domesticable and peaceful animals, living in a wild and violent space. Although the elephants were tamed, they were never ‘civilized’. Due to a series of scientific racist ideas, preservationist and segregationist arguments proved more powerful. With domestication proposals deemed unprofitable or impossible on the grounds of race, scientists gathered momentum for the cre- ation of the park by depicting the elephants as a ‘dying race’ that had adapted to the bush over millennia. Such discourse naturalized the bush as their ancestral home and working elephants could hardly be publicized as a spectacle of the prehistoric. The bush itself had supposedly produced their racial peculiarities and training them for labour would remove them from the very environment to which they were suited. While elephant domestication was a facet of the nineteenth-century British ‘civilizing mission’, the creation of this park had parallels with twentieth- century segregationist thought in southern Africa. Dubow, Scientific racism in modern South Africa, p. . Melland, Elephants in Africa, p. .  Smuts, ‘Science in South Africa’, p. ; Smuts, Africa and some world problems, p. . https://doi.org/10.1017/S0018246X19000761 Published online by Cambridge University Press Smuts, ‘Science in South Africa’, p. ; Smuts, Africa and some world problems, p. . I V In this space, concepts of tame, domestic, and wild were not bifurcated, but entangled within a network of scientific racist ideas. Wild was considered to be a stage in the evolution of the elephant, as it was in the development of ‘the African’. Melland, Elephants in Africa, p. .  https://doi.org/10.1017/S0018246X19000761 Published online by Cambridge University Press

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English

Focal Seizures with Corresponding Neuroimaging and Electroencephalographic Findings in a Patient with Scolex Remnants within a Calcified Cysticercus

˜The œAmerican journal of tropical medicine and hygiene

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Images in Clinical Tropical Medicine Focal Seizures with Corresponding Neuroimaging and Electroencephalographic Findings in a Patient with Scolex Remnants within a Calcified Cysticercus Oscar H. Del Brutto1*† and Naoum P. Issa2† 1School of Medicine, Universidad Esp´ıritu Santo—Ecuador, Guayaquil, Ecuador; 2Department of Neurology, University of Chicago, Chicago, Illinois A 31-year old woman was admitted to the hospital with a cluster of seizures in the context of a 4-year history of epilepsy. Seizures presented in clusters over 1 or 2 weeks followed by remittances lastingseveralmonths.Duringthe clusters,seizures occurred several times a day and always started withinvoluntary twitchingoftherightsideofthe face,sometimesassociated with secondary generalization. She had been diagnosed with neu- rocysticercosis (image not available) at another hospital and started on antiepileptic drugs without improvement. On present admission, neurological examination was unremarkable. Neu- roimaging studies showed a calcified cysticercus in the left frontal lobe with heterogeneous content due to presence of scolex remnants (Figure 1). An electroencephalogram (EEG) revealed focal epileptiform interictal discharges corresponding with the location of the calcification (Figure 2). Administration of intravenouscorticosteroidsrapidlyabortedtheclusterofseizures. The association between neurocysticercosis and epilepsy has been demonstrated in large clinical series and population- based studies.1,2 However, the lack of correlation be- tween location of parasites, EEG findings, and seizure semiology—noticed in some cases—made some authors to question this relationship, arguing that both conditions may just occur by chance. This report provides proof-of-concept that epilepsy is causally related to neurocysticercosis and reinforces previous studies suggesting that calcified cysti- cerci may cause recurrent unprovoked seizures when trap- ped antigenic parasitic remnants get exposed to the host immune system.3–5 Received May 10, 2018. Accepted for publication June 6, 2018. Financial support: Study supported by Universidad Esp´ıritu Santo— Ecuador, Guayaquil, Ecuador. Authors’ addresses: Oscar H. Del Brutto, School of Medicine, Uni- versidad Esp´ıritu Santo—Ecuador, Guayaquil, Ecuador, E-mail: [email protected]. Naoum P. Issa, University of Chicago Medical Center, Chicago, IL, E-mail: [email protected]. Authors’ addresses: Oscar H. Del Brutto, School of Medicine, Uni- versidad Esp´ıritu Santo—Ecuador, Guayaquil, Ecuador, E-mail: [email protected]. Naoum P. Issa, University of Chicago Medical Center, Chicago, IL, E-mail: [email protected]. 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. FIGURE 1. Left panel: Computed tomography showing a superficial single calcified cysticercus in the lower left frontal lobe (thick arrow). Left- central panel: fluid attentuated inversion recovery magnetic resonance imaging sequence showed the calcification surrounded by mild perilesional edema (small arrows). Am. J. Trop. Med. Hyg., 99(4), 2018, pp. 815–816 doi:10.4269/ajtmh.18-0400 Am. J. Trop. Med. Hyg., 99(4), 2018, pp. 815–816 doi:10.4269/ajtmh.18-0400 Copyright © 2018 by The American Society of Tropical Medicine and Hygiene * Address correspondence to Oscar H. Del Brutto, Air Center 3542, P.O. Box 522970, Miami, FL 33152-2970. E-mail: oscardelbrutto@ hotmail.com † Th th t ib t d ll t thi k hotmail.com † These authors contributed equally to this work. Received May 10, 2018. Accepted for publication June 6, 2018. Financial support: Study supported by Universidad Esp´ıritu Santo— Ecuador, Guayaquil, Ecuador. Authors’ addresses: Oscar H. Del Brutto, School of Medicine, Uni- versidad Esp´ıritu Santo—Ecuador, Guayaquil, Ecuador, E-mail: [email protected]. Naoum P. Issa, University of Chicago Medical Center, Chicago, IL, E-mail: [email protected]. Perilesional oedema and seizure activity in patients with calcified neurocysticercosis:aprospectivecohortandnestedcase-control study. Lancet Neurol 7: 1099–1105. Images in Clinical Tropical Medicine Focal Seizures with Corresponding Neuroimaging and Electroencephalographic Findings in a Patient with Scolex Remnants within a Calcified Cysticercus Right-central panel: T2-weighted sequence showing the calcification to be located in a cortical sulcus; the interior of the calcification is heterogeneous because of the presence of scolex remnants (thick arrow). Right panel: Echo-gradient sequence confirming the presence of scolex remnants within the calcification (thick arrow) and showing another solid calcification in the upper ipsilateral frontal lobe. This figure appears in color at www.ajtmh.org. FIGURE 1. Left panel: Computed tomography showing a superficial single calcified cysticercus in the lower left frontal lobe (thick arrow). Left- central panel: fluid attentuated inversion recovery magnetic resonance imaging sequence showed the calcification surrounded by mild perilesional edema (small arrows). Right-central panel: T2-weighted sequence showing the calcification to be located in a cortical sulcus; the interior of the calcification is heterogeneous because of the presence of scolex remnants (thick arrow). Right panel: Echo-gradient sequence confirming the presence of scolex remnants within the calcification (thick arrow) and showing another solid calcification in the upper ipsilateral frontal lobe. This figure appears in color at www.ajtmh.org. 815 816 DEL BRUTTO AND ISSA FIGURE 2. A 21-channel digital electroencephalogram showed focal epileptiform interictal discharges in the left frontotemporal region (circle), corresponding to the location of the calcification with scolex remnants. This figure appears in color at www.ajtmh.org. FIGURE 2. A 21-channel digital electroencephalogram showed focal epileptiform interictal discharges in the left frontotemporal region (circle), corresponding to the location of the calcification with scolex remnants. This figure appears in color at www.ajtmh.org. FIGURE 2. A 21-channel digital electroencephalogram showed focal epileptiform interictal discharges in the corresponding to the location of the calcification with scolex remnants. This figure appears in color at www.ajtm FIGURE 2. A 21-channel digital electroencephalogram showed focal epileptiform interictal discharges in the left frontotemporal region (circle), corresponding to the location of the calcification with scolex remnants. This figure appears in color at www.ajtmh.org. y 4. Ooi WW, Wijemanne S, Thomas CB, Quezado CB, Quezado M, Brown CR, Nash TE, 2011. A calcified Taenia solium granuloma associated with recurrent perilesional edema causing re- fractory seizures: histopathological features. Am J Trop Med Hyg 85: 460–463. yg 5. Nash TE, Bartelt LA, Korpe PS, Lopes B, Houpt ER, 2014. Calcified neurocysticercus, perilesional edema, and histologic inflammation. Am J Trop Med Hyg 90: 318–321. REFERENCES 1. Del Brutto OH, Santib ´añez R, Noboa CA, Aguirre R, D´ıaz E, Alarc ´on TA, 1992. Epilepsy due to neurocysticercosis: analysis of 203 patients. Neurology 42: 389–392. 4. Ooi WW, Wijemanne S, Thomas CB, Quezado CB, Quezado M, Brown CR, Nash TE, 2011. A calcified Taenia solium granuloma associated with recurrent perilesional edema causing re- fractory seizures: histopathological features. Am J Trop Med Hyg 85: 460–463. p gy 2. Del BruttoOH,ArroyoG,DelBruttoVJ,ZambranoM,GarciaHH,2017. On the relationship between calcified neurocysticercosis and epi- lepsy in an endemic village: a large-scale, computed tomography- based population study in rural Ecuador. Epilepsia 58: 1955–1961. p gy 2. Del BruttoOH,ArroyoG,DelBruttoVJ,ZambranoM,GarciaHH,2017. On the relationship between calcified neurocysticercosis and epi- lepsy in an endemic village: a large-scale, computed tomography- based population study in rural Ecuador. Epilepsia 58: 1955–1961. yg 5. Nash TE, Bartelt LA, Korpe PS, Lopes B, Houpt ER, 2014. Calcified neurocysticercus, perilesional edema, and histologic inflammation. Am J Trop Med Hyg 90: 318–321. y 3. Nash TE, Pretell EJ, Lescano AG, Bustos JA, Gilman RH, Gonzalez AE, Garcia HH; Cysticercosis Working Group in Per´u, 2008.

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https://link.springer.com/content/pdf/10.1007%2F978-3-030-71281-5_2.pdf

English

Rotator Cuff

IDKD Springer series

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Key Points • Knowledge of anatomy and biomechanics of rotator cuff tears is essential for understanding tear patterns. Fig. 2.1  Anatomy of the shoulder, sagittal PD FSE fat sat WI, 1 sub- scapularis; 2 long head of the biceps tendon; 3 supraspinatus; 4 infra- spinatus; 5 teres minus; 6 coracoid process; 7 acromioclavicular joint • Rotator cuff should be divided into anterior and posterior tendon injuries. • Reports should include location, size, retraction, pattern, and fatty atrophy. • Reports should include location, size, retraction, pattern, and fatty atrophy. Rotator Cuff Eva Llopis, Alexeys Perez, and Luis Cerezal Eva Llopis, Alexeys Perez, and Luis Cerezal Eva Llopis, Alexeys Perez, and Luis Cerezal Its anatomy allows abduction, adduction, flexion, exten- sion, and medial and lateral rotation but sacrifices stability. Rotator cuff tendons and muscles are the dynamic stabilizers of the shoulder helping the bone discrepancy between the glenoid and the humeral head to avoid shoulder dislocation. The origin of the rotator cuff is the scapula, and they insert into the humeral head forming parallel structures (Fig. 2.1). Although the rotator cuff is separate at the origin, the rotator cuff is organized in a five-layer structure where they approach each other near the insertion. This layered structure explains the appearance of delaminating tears. In the lower level, there are some perpendicular lineal fibers that represent an exten- sion of the coracohumeral ligament and extend from the rota- tor interval through the supraspinatus and the infraspinatus. It is called the rotator cable and has biomechanical implications Learning Objectives • Review new anatomical concepts of the rotator cuff. • Review new anatomical concepts of the rotator cuff. • Review characteristics of the anterior rotator cuff, subscapularis and posterior rotator cuff, and supra- spinatus and infraspinatus for partial- and full-­ thickness and massive tears. • Describe the more important differential diagnosis when facing a MR request for shoulder pain. • Learn how to do standardized reports and what information should be included in a report for decision-making. Key Points Rotator Cuff 2.1 2.1 The shoulder is the most flexible and movable synovial joint allowing a huge ROM. The original version of this chapter was revised. The cross-referenced chapter incorrect Surname and Given name in reference 18 has been corrected. The correction to this chapter is available at https://doi.org/10.1007/978-3-030-71281-5_21 © The Author(s) 2021, corrected publication 2021 J. Hodler et al. (eds.), Musculoskeletal Diseases 2021–2024, IDKD Springer Series, https://doi.org/10.1007/978-3-030-71281-5_2 E. Llopis (*) Hospital de la Ribera, Hospital IMSKE, Valencia, Spain A. Perez CST, Consorsi Sanitari de Terrassa, Terrassa, Spain L. Cerezal DMC, Diagnostico mÉdico Cantabria, Santander, Spain Fig. 2.1  A scapularis; spinatus; 5 © The Author(s) 2021, corrected publication 2021 J. Hodler et al. (eds.), Musculoskeletal Diseases 2021–2024, IDKD Springer Series, https://doi.org/10.1007/978-3-030-71281-5_2 E. Llopis (*) Hospital de la Ribera, Hospital IMSKE, Valencia, Spain A. Perez CST, Consorsi Sanitari de Terrassa, Terrassa, Spain L. Cerezal DMC, Diagnostico mÉdico Cantabria, Santander, Spain Fig. 2.1  A scapularis; spinatus; 5 E. Llopis (*) Hospital de la Ribera, Hospital IMSKE, Valencia, Spain A. Perez CST, Consorsi Sanitari de Terrassa, Terrassa, Spain Fig. 2.1  Anatomy of the shoulder, sagittal PD FSE fat sat WI, 1 sub- scapularis; 2 long head of the biceps tendon; 3 supraspinatus; 4 infra- spinatus; 5 teres minus; 6 coracoid process; 7 acromioclavicular joint L. Cerezal DMC, Diagnostico mÉdico Cantabria, Santander, Spain 11 © The Author(s) 2021, corrected publication 2021 J. Hodler et al. (eds.), Musculoskeletal Diseases 2021–2024, IDKD Springer Series, https://doi.org/10.1007/978-3-030-71281-5_2 E. Llopis et al. 12 Fig. 2.3  Coronal PD fat sat WI, demonstrates high signal intensity within the insertion of the supraspinatus without disruption Fig. 2.2  Anatomy of the shoulder, sagittal PD FSE fat sat WI, rotator cable is shown as an extension of the coracohumeral ligament crossing the underlying fibers of the supraspinatus and infraspinatus Fig. 2.3  Coronal PD fat sat WI, demonstrates high signal intensity within the insertion of the supraspinatus without disruption Fig. 2.2  Anatomy of the shoulder, sagittal PD FSE fat sat WI, rotator cable is shown as an extension of the coracohumeral ligament crossing the underlying fibers of the supraspinatus and infraspinatus humeral head and between the joint capsule and the gle- noid rim, the tendons experience structural changes. These changes represent histologically a continuum model of different progressive changes that start in reactive tendinopathy and continue into tendon disre- pair and degenerative tendinopathy and end in tendon rupture. 2.1 Initially those changes are reversible but shift to degeneration and rupture when the capacity of the tissue to repair is not enough. Our role would be to be sensitive enough to depict changes that are reversible. Initially the reaction of the tendon to load, friction, and activity results in small changes with disorganized extracellular matrix and subtle inflammatory reaction around the tendon that can be seen on imaging as peri- tendinitis and focal thickness of the tendon on high- resolution MR or US. In an attempt to heal matrix breakdown with collagen, separation and proliferation of abnormal tenocytes and increase of ­vascularization occur, which on imaging can be seen as low echo- genicity areas on US and focal areas of high signal intensity on fluid-sensitive MRI. Progressively histo- logical changes evolve to cellular and matrix changes with mucoid degeneration, chondral metaplasia, and amyloid deposition together with reparative changes and inflammation such as an increase of fibroblastic cells and neovascularization. These changes represent degenerative tendinopathy, and they are precursors of tendon tears. On imaging an increase in tendon thick- ness and changes of intrasubstance echogenicity on US and signal intensity on MR can be depicted [11, 12] (Fig. 2.3). in stress shielding, acting like a suspension bridge. It can be depicted on US and MR, especially in ABER position where the fibers of the supraspinatus relax [1–7] (Fig. 2.2). The posterior rotator cuff tendons include the supraspina- tus and infraspinatus that have a bursal and an articular site. The teres minus is rarely injured, and its function on the bio- mechanics of the shoulder is still to be determined. The supraspinatus inserts onto the anterior part of the greater tuberosity. It is important to realize that it has some anterior extensions to the lesser tuberosity reinforcing the rotator interval and merging with the subscapularis. The infraspina- tus origins in the infraspinatus fossa at the inferior part of the scapula and inserts into the greater trochanter with a trape- zoidal shape appearance. It has a wide insertion covering part of the supraspinatus insertion [8, 9]. The subscapularis has the largest tendon, with many fas- cicles. Its superior fascicles have a tendinous insertion, many of which insert into the lesser tuberosity, while some cross toward the greater tuberosity, whereas its inferior fascicles have a muscular insertion onto the humerus. 2.1 Its function is anterior stabilization of the shoulder and of the long head of the biceps tendon [10]. 2.2 Posterior Rotator Cuff, Supraspinatus and Infraspinatus 1. Tendinopathy. Secondary to repetitive contact of the tendons with movements between the acromioclavicular arch and the 2  Rotator Cuff 13 2. Partial Tears. higher probability to progression to a full-thickness tear than those that are smaller than 50% of the thickness. Tears that are smaller than 50% are usually treated con- servatively; surgical treatment is only recommended when conservative measures fail [13, 14]. The incapacity to heal and restore the native structure of the tendon leads to partial tear with scar formation with disorganized tissue that decreases mechanical properties. On imaging this is seen as focal areas of fluid echo- genicity or high signal intensity on fluid-sensitive sequences, particularly PD fat saturation or T2 fat satura- tion sequences. They can be divided depending on the location, in articular, intrasubstance, or bursal partial tears (Fig. 2.4). Tears that are bigger than 50% have Bursal-side tears are associated with subacromial and coracohumeral arch degenerative changes. The presence of fluid in the subacromial bursa facilitates the radiologi- cal diagnosis. These tears are occult to arthroscopy. Because of their adequate blood supply, they have a ten- dency to heal [13, 14]. a b Fig. 2.4  Two different cases on coronal PD fat sat FSE WI. (a) Articular side partial (arrows) rupture of more than 50% of tendon thickness; (b) bursal side partial rupture (arrows) affecting less than 50% of the tendon thickness Partial articular surface tears are the more frequent; they have been named as PASTA (partial articular supraspinatus tendon avulsion) lesions or rim rent tears. They don’t heal properly and have a tendency to progress to full-thickness tears. Insertional tears occur in younger population and are related to traumatic events, whereas more proximal tears in the critical zone are associated with degenerative changes. Partial tears have a vertical component that should be mea- sured in mm and is easy to be seen in conventional projec- tions; its horizontal component is more difficult, and ABER position helps to depict its full extension. Partial articular surface tears occurring within the 2nd and 3rd layer may extend horizontally and might involve the supraspinatus and the infraspinatus in a delaminating type of injury with- out retraction [15–18] (Fig. 2.5). a a a a Fig. 2.5  Coronal PD fat sat WI, showing delaminating partial rupture with fluid between the different layers without disruption of its articular side insertion b Fig. 2.4  Two different cases on coronal PD fat sat FSE WI. 2. Partial Tears. (a) Articular side partial (arrows) rupture of more than 50% of tendon thickness; (b) bursal side partial rupture (arrows) affecting less than 50% of the tendon thickness b b Fig. 2.4  Two different cases on coronal PD fat sat FSE WI. (a) Articular side partial (arrows) rupture of more than 50% of tendon thickness; (b) bursal side partial rupture (arrows) affecting less than 50% of the tendon thickness Fig. 2.5  Coronal PD fat sat WI, showing delaminating partial rupture with fluid between the different layers without disruption of its articular side insertion 14 E. Llopis et al. Intrasubstance tears occur within the tendon and usu- ally are associated with intense pain. and 4 cm, or greater than 5 cm. The retraction is defined as no retraction, small retraction if it doesn’t reach the level of the acromioclavicular joint, and large when it passes the AC joint. Arthroscopically the pattern of the tear is described as crescent, U-, or L-shaped [15–18] (Figs. 2.6 and 2.7). Intrasubstance tears occur within the tendon and usu- ally are associated with intense pain. 3. Full-Thickness Tears. Full-thickness tears are those tears that extend from the articular side to the bursal side; they don’t have to involve the entire tendon in other planes. It is important to describe the size of the tear, the number of tendons affected, the retraction, and the shape of the tear. The size of the tear is categorized as smaller than 2 cm, between 3 3. Full-Thickness Tears. Full-thickness tears are those tears that extend from the articular side to the bursal side; they don’t have to involve the entire tendon in other planes. It is important to describe the size of the tear, the number of tendons affected, the retraction, and the shape of the tear. The size of the tear is categorized as smaller than 2 cm, between 3 Fat atrophy has implication on the therapeutic approach and patient prognosis. If the atrophy is greater than 50% of the muscle, there is a high rate of recurrence after a a a b Fig. 2.6  Full-thickness tear of the supraspinatus, coronal (a) and sagit- tal (b) PD fat sat WI a b Fig. 2.7  MRI arthrography of the shoulder coronal (a) and sagittal (b) T1 FSE fat sat WI demonstrating full-thickness complete rupture of the supraspinatus a a a b Fig. 2.6  Full-thickness tear of the supraspinatus, coronal (a) and sagit- tal (b) PD fat sat WI b Fig. 2.6  Full-thickness tear of the supraspinatus, coronal (a) and sagit- tal (b) PD fat sat WI b b b Fig. 2.7  MRI arthrography of the shoulder coronal (a) and sagittal (b) T1 FSE fat sat WI demonstrating full-thickness complete rupture of the supraspinatus Fig. 2.6  Full-thickness tear of the supraspinatus, coronal (a) and sagit- tal (b) PD fat sat WI 2  Rotator Cuff 15 Fig. Intrasubstance tears occur within the tendon and usu- ally are associated with intense pain. 2.9  Coronal PD FSE WI of a massive rupture of the rotator cuff showing superior migration of the humeral head (black arrow), remod- elation of the acromioclavicular arch (black arrow head), glehohumeral degenerative changes, and fatty atrophy of the supraspinatus (*) Fig. 2.8  Sagittal FSE T1WI showing moderate atrophy of the supra- spinatus [1] and severe atrophy of the infraspinatus Fig. 2.9  Coronal PD FSE WI of a massive rupture of the rotator cuff showing superior migration of the humeral head (black arrow), remod- elation of the acromioclavicular arch (black arrow head), glehohumeral degenerative changes, and fatty atrophy of the supraspinatus (*) Glenohumeral arthrosis can be seen as loss of the joint space and presence of inferior humeral osteophytes. In the last stages and on imaging, we can depict subchondral cyst formation, secondary areas of avascular necrosis and bone marrow edema, and ultimately collapse of the humeral head [13, 21–23]. Glenohumeral arthrosis can be seen as loss of the joint space and presence of inferior humeral osteophytes. In the last stages and on imaging, we can depict subchondral cyst formation, secondary areas of avascular necrosis and bone marrow edema, and ultimately collapse of the humeral head [13, 21–23]. For treatment purposes it is important to describe the morphology and bone stock of the glenoid, the deltoid, and the alignment [21–23] (Fig. 2.9). 2.3 Fig. 2.8  Sagittal FSE T1WI showing moderate atrophy of the supra- spinatus [1] and severe atrophy of the infraspinatus Subscapularis tendon tears are more frequent than previ- ously thought. They have been found in cadavers in more than 30%. They are underestimated in all imaging tech- niques. The sagittal plane is the best for its MR evaluation. On MR arthrography we strongly recommend external forced rotation for better depiction of partial articular side tears. Most missed are partial- and full-thickness tears of the posterior fibers of the subscapularis. They are closely related to the rotator interval and the anterior crossing fibers of the supraspinatus. Lafosse classification is the most accepted one; it is divided into five grades (Table 2.2). Subscapularis tears are associated with anterosuperior impingement. Anterosuperior impingement was described by Gerber arthroscopically revealing impingement of the undersurface of the subscapularis tendon against the anterosuperior gle- noid rim in the position of flexion and internal rotation. It is less common than posterosuperior impingement. There are repair. There have been different classifications for fat atrophy. The Goutallier system is based on the percentage of fat within the muscle on CT axial planes; Zanetti and Thomazeau analyzed the supraspinatus fossa on T1-weighted sagittal MR images, and recently ISAKOS defined four different grades [13, 19–21] (Table  2.1) (Fig. 2.8). repair. There have been different classifications for fat atrophy. The Goutallier system is based on the percentage of fat within the muscle on CT axial planes; Zanetti and Thomazeau analyzed the supraspinatus fossa on T1-weighted sagittal MR images, and recently ISAKOS defined four different grades [13, 19–21] (Table  2.1) (Fig. 2.8). repair. There have been different classifications for fat atrophy. The Goutallier system is based on the percentage of fat within the muscle on CT axial planes; Zanetti and Thomazeau analyzed the supraspinatus fossa on T1-weighted sagittal MR images, and recently ISAKOS defined four different grades [13, 19–21] (Table  2.1) (Fig. 2.8). repair. There have been different classifications for fat atrophy. The Goutallier system is based on the percentage of fat within the muscle on CT axial planes; Zanetti and Thomazeau analyzed the supraspinatus fossa on T1-weighted sagittal MR images, and recently ISAKOS defined four different grades [13, 19–21] (Table  2.1) (Fig. 2.8). 4. Massive Rotator Cuff Tears, Rotator Cuff Arthropathy. A massive rotator cuff tear is characterized by the involvement of two or more tendons or a retraction greater than 5  cm. 2.3 As the rotator cuff centralizes the humeral head into the glenoid and serves as the fulcrum when the deltoid abducts and elevates the arm, its deficiency changes the biomechanics. There is progressive migration of the humeral head superiorly causing adaptive changes on the coracoacromial arch with acetabulization. 4. Massive Rotator Cuff Tears, Rotator Cuff Arthropathy. A massive rotator cuff tear is characterized by the involvement of two or more tendons or a retraction greater than 5  cm. As the rotator cuff centralizes the humeral head into the glenoid and serves as the fulcrum when the deltoid abducts and elevates the arm, its deficiency changes the biomechanics. There is progressive migration of the humeral head superiorly causing adaptive changes on the coracoacromial arch with acetabulization. E. Llopis et al. 16 Table 2.2  Fatty atrophy Grade 0 Normal muscle Grade 1 Some fatty streaks Grade 2 Less than 50% of fatty infiltration Grade 3 50% equal fat to muscle Grade 4 More than 50% fatty infiltration ISAKOS adopted Goutallier and Fuchs classification of the fatty atro- phy valid for CT and MRI associated rotator interval lesions, long head of the biceps tendon lesions and dislocation, and SLAP lesions [24–26] (Fig. 2.10). associated rotator interval lesions, long head of the biceps tendon lesions and dislocation, and SLAP lesions [24–26] (Fig. 2.10). 2.4 Differential Diagnosis ISAKOS adopted Goutallier and Fuchs classification of the fatty atro- phy valid for CT and MRI ISAKOS adopted Goutallier and Fuchs classification of the fatty atro- phy valid for CT and MRI It is important to know some entities that clinically can mimic rotator cuff lesions. Fig. 2.10  Axial arthro-MRI FSE T1 fat sat WI demonstrates partial articular side tear of the subscapularis 1. Calcified Tendinopathy. Crystal deposition, especially CPPD deposits on the tendon, causes inflammation and is an important cause of shoulder pain in young adults. Its cause is still unknown although microtrauma, ischemia, and metaplasia have been proposed as causes. It is usually a self-limited ­disease with spontaneous resolution in a high percentage of cases. Different clinical stages have been described: a precalcification phase which is clinically silent; the calci- fication phase in which crystal deposition occurs, subse- quently the start of a resorptive phase with inflammatory reaction that causes severe pain; and the end a post-­ calcification phase (Fig. 2.11). The deposits of CPPD might migrate to the subacromial bursa causing bursitis or less frequent into the bone at the lesser or greater tuberos- ity. It is important to know this potential involvement of the numeral head to avoid confusion with a tumor. MRI shows a sclerotic or lytic lesion surrounded by peripheral bone marrow edema. The key for the diagnosis is the association with calcifications within the proximal tendon [27, 28] (Fig. 2.12). Fig. 2.10  Axial arthro-MRI FSE T1 fat sat WI demonstrates partial articular side tear of the subscapularis a b Fig. 2.11  Rotator cuff hydroxyapatite deposition disease. (a) AP radiograph shows periarticular calcifications; (b) axial GET2; (c) coronal PD FSE fat sat WI shows coarse ovoid calcification in the subacromial subdeltoid bursa surrounded by fluid indicating inflammation a b a b a Fig. 2.11  Rotator cuff hydroxyapatite deposition disease. (a) AP radiograph shows periarticular calcifications; (b) axial GET2; (c) coronal PD FSE fat sat WI shows coarse ovoid calcification in the subacromial subdeltoid bursa surrounded by fluid indicating inflammation 2  Rotator Cuff 17 a b Fig. 2.12  Intrabone migration of hydroxyapatite deposition, (a) AP radiographs demonstrate calcifications in the greater tuberosity sur- rounded by a radiolucency area (*). Calcifications within the rotator cuff space help for the diagnosis of hydroxyapatite deposition disease. (b) Coronal FSE PD fat sat WI shows the same features of calcifications within the greater tuberosity (*) surrounded by a hyperintense halo (arrow) c c a c a c a b Fig. 2.11  (continued) 2. Adhesive Capsulitis. 2. Adhesive Capsulitis. Adhesive capsulitis is a clinical diagnosis character- ized by pain and marked decrease of the range of motion, especially to external rotation. MRI findings are usually not specific but can correlate with the clinical stages. In the acute inflammatory phase, MRI can show axillary capsular thickening and capsular edema and rotator inter- val synovitis. Progressively hypervascularization and fibrosis occur, which may be reflected on MRI images by thickening of the coracohumeral ligament, subcoracoid fibrosis, and capsular thickening. On MR arthrography classic findings are low volume injection, often less than 8 ml, and thickening of the structures [29, 30] (Fig. 2.13). p Adhesive capsulitis is a clinical diagnosis character- ized by pain and marked decrease of the range of motion, especially to external rotation. MRI findings are usually not specific but can correlate with the clinical stages. In the acute inflammatory phase, MRI can show axillary capsular thickening and capsular edema and rotator inter- val synovitis. Progressively hypervascularization and fibrosis occur, which may be reflected on MRI images by thickening of the coracohumeral ligament, subcoracoid fibrosis, and capsular thickening. On MR arthrography classic findings are low volume injection, often less than 8 ml, and thickening of the structures [29, 30] (Fig. 2.13). 3 N D i S d 3. Nerve Denervation Syndromes. . Nerve Denervation Syndromes. There are two main nerve denervation syndromes around the shoulder secondary to the suprascapular nerve and to the axillary nerve. Suprascapular neuropathy can be related to compression of an associated paralabral cyst in a superior labrum injury. When there is no compression, there are two main origins: a viral inflammation and or overuse in athletes with overhead activities. On imaging in the acute phase, supraspinatus and infraspinatus muscle edema is seen, whereas in chronic phases fatty atrophy and volume loss of the muscle are shown [31] (Fig. 2.14). The causes of axillary nerve denervation can be sec- ondary to injury of the axillary nerve in anterior inferior shoulder dislocation especially in patients older than 40 years of age; can be secondary to compression due to a lesion in the quadrilateral space; or can be idiopathic. The role of MRI is to rule out compression causes and There are two main nerve denervation syndromes around the shoulder secondary to the suprascapular nerve and to the axillary nerve. Suprascapular neuropathy can be related to compression of an associated paralabral cyst in a superior labrum injury. When there is no compression, there are two main origins: a viral inflammation and or overuse in athletes with overhead activities. On imaging in the acute phase, supraspinatus and infraspinatus muscle edema is seen, whereas in chronic phases fatty atrophy and volume loss of the muscle are shown [31] (Fig. 2.14). Fig. 2.12  Intrabone migration of hydroxyapatite deposition, (a) AP radiographs demonstrate calcifications in the greater tuberosity sur- rounded by a radiolucency area (*). Calcifications within the rotator cuff space help for the diagnosis of hydroxyapatite deposition disease. (b) Coronal FSE PD fat sat WI shows the same features of calcifications within the greater tuberosity (*) surrounded by a hyperintense halo (arrow) The causes of axillary nerve denervation can be sec- ondary to injury of the axillary nerve in anterior inferior shoulder dislocation especially in patients older than 40 years of age; can be secondary to compression due to a lesion in the quadrilateral space; or can be idiopathic. The role of MRI is to rule out compression causes and associated injuries in shoulder dislocation. MRI will demonstrate secondary to the lesion of the axillary nerve edema of the teres minus or fatty atrophy on chronic stages [31] (Fig. 2.15). E. Llopis et al. 18 Fig. 3. Nerve Denervation Syndromes. 2.13  Patient with decreased external rotation, sagittal FSE PD fat sat WI shows thickening of the coracohumeral ligament with soft tissue edema lated Greater Tuberosity Fractures. Isolated fractures of the greater tuberosity can be sec- dary to shoulder direct or indirect trauma or in older pulation to minor trauma in osteoporotic patients. hen there is little or no displacement (<5  mm), they ght be difficult to see on plain films especially if only e projection is seen. They can be seen on US as a dis- ntinuity of cortical bone line and are easy to be diag- sed on MRI [32] (Fig. 2.16). Conclusions diological report in rotator cuff injuries plays a critical n the decision-making process for the surgeon and the t outcome. Spend time recognizing the pattern. The should be simple and standardized. The radiologist be aware of the treatment implications of the different of tear and understand the parameters used to evaluate r cuff tears on MRI. agnosis of concomitant lesions and knowing the dif- ial diagnosis are very important. 3  Patient with decreased external rotation, sagittal FSE PD fat shows thickening of the coracohumeral ligament with soft tissue a b Fig. 2.14  (a) axial GE T2 WI and (b) sagittal FSE T2 WI of a paral- abral cyst (*) in the supraglenoid notch causing entrapment of the suprascapularis nerve and secondary edema in the infraspinatus (arrow) a a a a b Fig. 2.14  (a) axial GE T2 WI and (b) sagittal FSE T2 WI of a paral- abral cyst (*) in the supraglenoid notch causing entrapment of the suprascapularis nerve and secondary edema in the infraspinatus (arrow) b Fig. 2.13  Patient with decreased external rotation, sagittal FSE PD fat sat WI shows thickening of the coracohumeral ligament with soft tissue edema 4. Isolated Greater Tuberosity Fractures. Isolated fractures of the greater tuberosity can be sec- ondary to shoulder direct or indirect trauma or in older population to minor trauma in osteoporotic patients. When there is little or no displacement (<5  mm), they might be difficult to see on plain films especially if only one projection is seen. They can be seen on US as a dis- continuity of cortical bone line and are easy to be diag- nosed on MRI [32] (Fig. 2.16). 2.5 Conclusions 2.5 Our radiological report in rotator cuff injuries plays a critical role in the decision-making process for the surgeon and the patient outcome. Spend time recognizing the pattern. The report should be simple and standardized. The radiologist must be aware of the treatment implications of the different types of tear and understand the parameters used to evaluate rotator cuff tears on MRI. Fig. 2.14  (a) axial GE T2 WI and (b) sagittal FSE T2 WI of a paral- abral cyst (*) in the supraglenoid notch causing entrapment of the suprascapularis nerve and secondary edema in the infraspinatus (arrow) Diagnosis of concomitant lesions and knowing the dif- ferential diagnosis are very important. 2  Rotator Cuff 19 a b c Fig. 2.15  A 48-year-old patient with anterior and inferior dislocation of the shoulder and associated complications like partial tear of the supraspinatus, greater tuberosity fracture, and axillary nerve injury with soft tissue edema of the teres minor. (a) Sagittal FSE PD fat sat WI shows anteroinferior glenoid-labral complex lesion (white arrow) soft tissue edema and effusion in the axillary recess [1] and edema in the teres minus [2]; (b) coronal FSE PD fat sat WI demonstrates small par- tial tear of the supraspinatus (*), non-displaced fracture of the greater tuberosity (white arrows), and soft tissue edema of the axillary recess (black arrow); (c) posterior plane on coronal FSE PD fat sat WI demon- strates edema of the teres minor secondary to axillary nerve lesionf b b a b a b a a c c teres minus [2]; (b) coronal FSE PD fat sat WI demonstrates small par- tial tear of the supraspinatus (*), non-displaced fracture of the greater tuberosity (white arrows), and soft tissue edema of the axillary recess (black arrow); (c) posterior plane on coronal FSE PD fat sat WI demon- strates edema of the teres minor secondary to axillary nerve lesion Fig. 2.15  A 48-year-old patient with anterior and inferior dislocation of the shoulder and associated complications like partial tear of the supraspinatus, greater tuberosity fracture, and axillary nerve injury with soft tissue edema of the teres minor. (a) Sagittal FSE PD fat sat WI shows anteroinferior glenoid-labral complex lesion (white arrow) soft tissue edema and effusion in the axillary recess [1] and edema in the Fig. Take Home Messages • Reports of rotator cuff tears should follow a struc- tured form including size of the lesion, retraction, shape, and atrophy. • It is important to remember that despite the rotator cuff muscles origin are separate when they insert, they weave together being these areas the more dif- ficult to depict on MRI. • Differential diagnosis should include other causes of shoulder pain that might be clinically mimickers. 2.5 Conclusions 2.15  A 48-year-old patient with anterior and inferior dislocation of the shoulder and associated complications like partial tear of the supraspinatus, greater tuberosity fracture, and axillary nerve injury with soft tissue edema of the teres minor. (a) Sagittal FSE PD fat sat WI shows anteroinferior glenoid-labral complex lesion (white arrow) soft tissue edema and effusion in the axillary recess [1] and edema in the E. Llopis et al. 20 a References b b Fig. 2.16  A 45-year-old male with a request of US to rule out rotator cuff lesion after indirect trauma; (a) US of the shoulder shows discontinuity of the cortical bone of the greater tuberosity (arrow); (b) AP radiograph performed later demonstrates the small non-displaced fracture (arrows) 1. Clark JM, Harryman DT 2nd. Tendons, ligaments, and capsule of the rotator cuff. Gross and microscopic anatomy. J Bone Joint Surg Am. 1992;74(5):713–25. b 2. De Franco MJ, Cole BJ. Current perspective on rotator cuff anat- omy. J Arthroscop Rel Surg. 2009;25(3):305–20. 3. Llopis E, Montesinos P, Guedez MT, et  al. Normal Shoulder MRI and MR arthrography: anatomy and technique. Semin Musculoskelet Radiol. 2015;19(3):212–30. 4. Lee SY, Lee JK. Horizontal component of partial-thickness tears of rotator cuff: imaging characteristics and comparison of ABER view with oblique coronal view at MR arthrography initial results. Radiology. 2002;224(2):470–6. gy ( ) 5. Burkhart SS, Esch JC, Jolson RS. The rotator crescent and rota- tor cable: an anatomic description of the shoulder’s “suspension bridge”. Arthroscopy. 1993;9(6):611–6. g py 6. Morag Y, Jamadar DA, Boon TA. Ultrasound of the rotator cable: prevalence and morphology in asymptomatic shoulders. Am J Roentgenol. 2012;198:W27–30. 7. Gyftopoulos S, Bencardino J, Nevsky G, et al. Rotator cable: MRI study of its appearance in the intact rotator cuff with anatomic and histologic orrelation. Am J Roentgenol. 2013;200(5):1101–005. g g 8. Moser TP, Cardinal E, Bureau NJ, et al. The aponeurotic expansion of the supraspinatus tendon: anatomy and prevalence in a series of 150 shoulder MRIs. Skelet Radiol. 2015;44:223–31. 9. Curtis AS, Burbank KM, Tierney JJ, et  al. The insertional footprint of the rotator cuff: an anatomic study. Arthroscopy. 2006;22(6):609–11. Fig. 2.16  A 45-year-old male with a request of US to rule out rotator cuff lesion after indirect trauma; (a) US of the shoulder shows discontinuity of the cortical bone of the greater tuberosity (arrow); (b) AP radiograph performed later demonstrates the small non-displaced fracture (arrows) 10. Morag Y, Jamadar DA, Miller B, et al. The subscapularis: anatomy, injury and imaging. Skelet Radiol. 2011;40(3):255–69. 11. Buck F, Grehn H, Hilbe M, et al. Magnetic resonance histologic correlation in rotator cuff tendons. J MRI. 2010;32:165–72. 12. Cook JL, Rio E, Purdam CR, et al. Revisiting the continuum model of tendon pathology: what is its merit in clinical practice and research. Br J Sports Med. 2016;0:1–7. Key Points • Anterior rotator cuff, subscapularis and posterior rotator cuff, and supraspinatus and infraspinatus are slightly different. p 13. Imhoff AB, Savoie FH. Rotator cuff across the life span ISAKOS consensus book. Berlin, Heidelberg, New York: Springer; 2019. 14. Morag Y, Jacobson JA, Miller B, et  al. MR imaging of rota- tor cuff injury: What the clinician need to know. Radiographics. 2006;26(4):1045–65. • Superior fibers of the subscapularis and anterior fibers of the supraspinatus weaved together are closely related to biceps tendon and rotator interval tears. 15. Tuite MJ, Turnbull JR, Orwin JF. Anterior versus posterior, and rim-rent rotator cuff tears: prevalence and MR sensitivity. Skelet Radiol. 1998;27:237–43. 16. Fukuda H, Hamada K, Nakajima T, et al. Partial-thickness tears of the rotator cuff. A clinicopathological review based on 66 surgically verified cases. Int Orthop. 1996;20(4):257–65. • New patterns of injuries such as delamination tears can be depicted with high-resolution MRI. • Reports should include pattern, size, retraction, shape, and fatty atrophy. 17. Cha SW, Lee CK, Sugaya H, et al. Retraction pattern of delami- nated rotator cuff tears: dual-layer rotator cuff repair. J Orthop Surg Res. 2016;11:75–85. 2  Rotator Cuff 21 18. Choo HJ, SJ Lee, Kim JH, et al. Delaminated tears of the rota- tor cuff: prevalence, characteristics, and diagnostic accuracy using indirect MR arthrography. Am J Roentgenol. 2015;204:360–6.i 25. Lafosse L, Jost B, Reiland Y, et al. Structural integrity and clinical outcomes after arthroscopic repair of isolated subscapularis tears. J Bone Jt Surg A. 2007;89(6):1184–93. 19. Davidson J, Burkhart SS. The geometric classification of rotator cuff tears: a system linking tear pattern to treatment and prognosis. Arthroscopy. 2010;26(3):417–24. 26. Habermeyer P, Krieter C, Tang KL, et al. A new arthroscopic clas- sification of articular-sided supraspinatus footprint lesions: a pro- spective comparison with Snyder’s and Ellman’s classification. J Shoulder Elb Surg. 2008;17(6):909–13. 20. Goutallier D, Postel JM, Gleyze P, et  al. Influence of cuff mus- cle fatty degeneration on anatomic and functional outcomes after simple suture of full thickness tears. J Shoulder Elb Surg. 2003;12(6):550–4. 27. Siegal D, Wu JS, Newman JS, et al. Calcific tendinitis: a pictorial review. Can Assoc Radiol J. 2009;60(5):263–72.i 28. Speed CA, Hazleman BL. Calcific tendinitis of the shoulder. N Engl J Med. 1999;340(20):1582–4. 21. Zanetti M, Gerber C, Hodler J. Quantitative assessment of the muscles of the rotator cuff with MRI. Investig Radiol. 1998;33(3):163–70. 29. Park S, Lee DH, Yoon SH, et al. Open Access  This chapter is licensed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons. org/licenses/by/4.0/), which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropri- ate credit to the original author(s) and the source, provide a link to the Creative Commons license and indicate if changes were made. The images or other third party material in this chapter are included in the chapter's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the chapter's Creative Commons license and your intended use is not permitted by statu- tory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. Key Points Evaluation of adhesive capsulitis of the shoulder with fat-suppressed T2-weighted MRI: associa- tion between clinical features and MRI finding. Am J Roentgenol. 2016;207:135–41. 22. Di Benedetto P, Beltrame A, Cicuto C, et al. Rotator cuff tears rep- arability index based on preoperative MRI: our experience. Acta Biomed. 2019;90:36–46. 30. Lee SY, Park J, Song SW. Correlation of MR arthrographic findings and range of shoulder motion in patients with frozen shoulder. Am J Roentgenol. 2012;198:173–9. 23. Samim M, Walsh P, Gyftopoulos S, et  al. 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Dlx5-augmentation in neural crest cells reveals early development and differentiation potential of mouse apical head mesenchyme

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Dlx5‑augmentation in neural crest cells reveals early development and differentiation potential of mouse apical head mesenchyme Tri H. Vu1,3, Masaki Takechi1,3, Miki Shimizu2, Taro Kitazawa2, Hiroki Higashiyama2, Akiyasu Iwase2, Hiroki Kurihara2 & Sachiko Iseki1* Tri H. Vu1,3, Masaki Takechi1,3, Miki Shimizu2, Taro Kitazawa2, Hiroki Higashiyama2, Akiyasu Iwase2, Hiroki Kurihara2 & Sachiko Iseki1* Neural crest cells (NCCs) give rise to various tissues including neurons, pigment cells, bone and cartilage in the head. Distal-less homeobox 5 (Dlx5) is involved in both jaw patterning and differentiation of NCC-derivatives. In this study, we investigated the differentiation potential of head mesenchyme by forcing Dlx5 to be expressed in mouse NCC (NCCDlx5). In NCCDlx5 mice, differentiation of dermis and pigment cells were enhanced with ectopic cartilage (ec) and heterotopic bone (hb) in different layers at the cranial vertex. The ec and hb were derived from the early migrating mesenchyme (EMM), the non-skeletogenic cell population located above skeletogenic supraorbital mesenchyme (SOM). The ec developed within Foxc1+-dura mater with increased PDGFRα signalling, and the hb formed with upregulation of BMP and WNT/β-catenin signallings in Dermo1+-dermal layer from E11.5. Since dermal cells express Runx2 and Msx2 in the control, osteogenic potential in dermal cells seemed to be inhibited by an anti-osteogenic function of Msx2 in normal context. We propose that, after the non-skeletogenic commitment, the EMM is divided into dermis and meninges by E11.5 in normal development. Two distinct responses of the EMM, chondrogenesis and osteogenesis, to Dlx5-augmentation in the NCCDlx5 strongly support this idea. Neural crest cells (NCCs) are mesenchymal cells that originate from the dorsal part of neural tube by epithelial- to-mesenchymal transition. NCCs then migrate to different regions of the embryo, where they give rise to various cell types such as bone and cartilage of the skull, sensory neurons, pericytes, melanocytes and smooth ­muscles1. NCCs and paraxial mesodermal cells (MES) cooperatively form the craniofacial structure. NCCs contribute to the rostral craniofacial skeleton including the pharyngeal skeleton while MES give rise to caudal ­cranium2–4. The boundary between NCC and MES in the calvarium corresponds to the coronal suture between the frontal and parietal ­bones3–5. p The NCC is accurately regulated by a complex gene network from the appearance to migration and ­differentiation1. Distal-less homeobox 5 (Dlx5) is expressed early at the neural plate border for establishing the area of NCC delamination, but it does not control NCC specification or ­migration1,6. Dlx5 is required for patterning and differentiation of the ­NCC7. Expression of Dlx5 and its co-functional member of the Dlx gene family, Dlx6, are involved in jaw ­patterning8,9. www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports Scientific Reports | (2021) 11:2092 Results Dl 5 Dlx5 expression and NCC distribution in the NCCDlx5. NCC-specific forced expression of Dlx5 was confirmed in Wnt1-Cre;R26RCAG-flox-Dlx5/+ (hereafter NCCDlx5) mice at E9.5 with ectopic Dlx5 expression in the pharyngeal ­arch11. We further examined the expression of Dlx5 in later stages by comparison with X-gal stain- ing of Wnt-Cre;R26RlacZ/+ (NCCLacZ) (n = 3) to demonstrate the NCC distribution. NCCs of wild-type located at the maxillary process and supraorbital ridge at E10.5, but hardly detected in the surrounding of the brain at the vertex (Fig. 1a). At E11.5, NCCs made up the mandibular and maxillary processes, also the head mesenchyme surrounding the brain (Fig. 1b). Endogenous Dlx5 expression in head mesenchyme at E10.5 was found only in the mandibular process, whilst in the NCCDlx5, Dlx5 was additionally expressed in the maxillary process and the SOM (n = 3) (Fig. 1c,d). At E11.5, endogenous Dlx5 expression was seen in the frontal bone primordium of the SOM (Fig. 1e). In the NCCDlx5, Dlx5 was expressed in the EMM besides the SOM (n = 3) at E11.5 (Fig. 1f, arrow- head). Therefore, Dlx5 expression was successfully induced in NCCs, including EMM, in the NCCDlx5. Dl h p y g We examined the NCC distribution in the NCCDlx5 by whole-mount in situ hybridization (WISH) for Snail family transcriptional repressor 1 (Snai1), a NCC ­specifier1 , at E9.0 (n = 4). Snai1 expression was shown in a comparable pattern between the control and the NCCDlx5 (Fig. 1g,h). We next examined the Snai1 expression by section ISH (n = 3). We confirmed that post-migratory NCC-derived mesenchyme at the surrounding of the forebrain and the mandibular process similarly expressed Snai1 in both of the control and the NCCDlx5 (Fig. 1i,j). In the dorsal region of the rhombencephalon, Snai1 expression was detected in migrating NCCs in the control and the NCCDlx5 (Fig. 1k,l), the data revealed no difference between the two in this area. These results indicated that Dlx5-overexpression do not affect the migration and distribution of cranial NCCs. Predisposition of NCC differentiation in the NCCDlx5 mouse. Dlx5 is normally expressed in the trigeminal ­ganglion14, and the size is reduced in Dlx5 knock-out ­mice7. Acetylated tubulin staining at E11.5 demonstrated that neuron localization did not show obvious difference between the control and the NCCDlx5 (n = 3) (Fig. 2a–d). Results Dl 5 Reconstructed trigeminal ganglion from the serial histological sections at E17.5 illustrated the similar shape and size of the control and the NCCDlx5 (Fig. 2e,f) and no significant difference in volume (n = 3) (Fig. 2g). We also examined the pigment cell, another NCC-derivative1, by expression of dopachrome tautomerase (Dct)34 on frontal sections at E15.5 (n = 3) (Fig. 2h,i). The number of Dct-positive cells in the head dermis was significantly higher in the NCCDlx5 than that of the control (p < 0.05) (Fig. 2j), suggesting that the NCC potential for pigment cell differentiation was enhanced by Dlx5-augmentation. p p gf y g We next examined bone and cartilage formation in the calvarium. In the control, chondrocranium cartilages were observed at the skull base and lateral walls, while no cartilage and bone was observed at the apical part of the head at E13.5 and E14.5 (Fig. 2k,m). Interestingly, cartilage was formed at the vertex of the NCCDlx5 (Fig. 2l,n). This calvarial cartilage was newly introduced to the region that usually has no cartilage, hence it is an ectopic ­cartilage35 (hereafter ec). The ec did not connect with cartilages at the skull base or the lateral wall and appeared like a bridge connecting bilateral hemispheres at E13.5 (n = 4) and E14.5 (n = 4) (Fig. 2l,n, arrowhead). As the frontal and parietal bones further developed at E14.5, the coronal suture was identified between the frontal bone and parietal bone in the control (Fig. 2m). The ec was seen anterior to the prospective coronal suture and seemed to outline the posterior border of NCC-derived frontal bone (Fig. 2n, arrowhead). The ec remained unossified (n = 5) (Fig. 2p, arrowhead), whereas no cartilage was detected at the same region in the control even at postnatal day 0 (P0) (Fig. 2o).h p y g At P0, calvarial bone formation was distinct between the control and the NCCDlx5. The frontal bone developed toward the midline, forming the interfrontal suture in the control (Fig. 2o). In the NCCDlx5, not only ec but bony islands were also found in the interfrontal suture (Fig. 2p, asterisk) and the posterior part of frontal bone form- ing area (Fig. 2p, double asterisk). In some NCCDlx5 mice, “patchy” bones with holes (Supplementary Fig. S1a,b) were formed randomly in the frontal bone and interfrontal area. www.nature.com/scientificreports/ chondrogenic lineage and chondrocyte maturation in the ­chick20,21. In mice, the calvarium and chondrocranium malformations have been shown to associate with Dlx5-downregulation7,22,23. Meanwhile, cranial base cartilages derived from NCC are enlarged by Dlx5-overexpression, but calvarial bones have not been ­examined11. In calvarial development, formation of the frontal and parietal bones start with the aggregation of mesen- chymal cells in the area of the supraorbital ridge at embryonic day (E) 10.53, referred to as the supraorbital ridge ­mesenchyme5,24–26 or supraorbital mesenchyme (SOM)27. The SOM proliferates and differentiates into osteoblasts from E11.5, then intrinsically expands to the apex of the head to form the bone from E13.54,5. Importantly, due to the intimate association and mutual support of cranial bones and the dura mater, the defects in the dura mater affect calvarial bone formation and ­maintenance28–31. Before the SOM begins apical growth, a population of head mesenchyme, termed as early migrating ­NCC32 or early migrating mesenchyme (EMM)27, is established above the SOM to contribute to the sutures or soft tissue layers such as the dermis and the ­meninges4,25,32. Transcrip- tome analysis revealed that the SOM and the EMM exhibit different gene expression profiles by E12.533, and the development of the skull vault is achieved by interactions between the apical (EMM) and basal (SOM) cell ­populations28. Although the EMM is normally non-osteogenic, previous reports demonstrated that the EMM can generate bone in genetic ­disorders27,32.iff g g NCC-specific Dlx5-augmentation results in a switch of the jaw ­identity11, but the effect on NCC differentia- tion potential has not been examined. In this study, we further investigated the effect of Dlx5-overexpression in NCCs with special reference to early development and differentiation potential of the EMM. Dlx5‑augmentation in neural crest cells reveals early development and differentiation potential of mouse apical head mesenchyme Tri H. Vu1,3, Masaki Takechi1,3, Miki Shimizu2, Taro Kitazawa2, Hiroki Higashiyama2, Akiyasu Iwase2, Hiroki Kurihara2 & Sachiko Iseki1* In jaw development, Dlx5/6 works downstream of Endothelin1 (Edn1), localized in the mandibular process while Dlx5/6 are absent in the maxillary ­process8–12. Double knock-out of Dlx5/6 in mice causes mandible transformation into maxilla-like ­structure8. Reversely, forced Dlx5 expression in NCCs in mice (NCCDlx5) induces ectopic Dlx5 expression in the maxillary process leading to upregulation of mandibular- specific genes and appearance of several phenotypic hallmarks of the mandible in the maxilla ­region11.f pi g pp p yp g Dlx5 is also expressed in differentiation stages of NCC-derivatives: ganglion of cranial nerves, cartilage and ­bone13,14. In osteoblast differentiation, Dlx5 is induced by BMP signalling, then Dlx5 enhances Runt-related tran- scription factor 2 (Runx2), a master transcriptional regulator for ­osteogenesis15–17. DLX5 directly binds to SP7, a downstream of Runx2, to promote osteoblast ­differentiation18. Calvarial osteoblasts isolated from Dlx5 deleted mice show reduced proliferation and ­differentiation19. Dlx5 is also expected to induce recruitment of fibroblasts to 1Section of Molecular Craniofacial Embryology, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University (TMDU), 1‑5‑45 Yushima, Bunkyo‑ku, Tokyo 113‑8549, Japan. 2Department of Physiological Chemistry and Metabolism, Graduate School of Medicine, The University of Tokyo, 7‑3‑1 Hongo, Bunkyo‑ku, Tokyo 113‑0033, Japan. 3These authors contributed equally: Tri H. Vu and Masaki Takechi *email: s.iseki.emb@ tmd.ac.jp | https://doi.org/10.1038/s41598-021-81434-x Scientific Reports | (2021) 11:2092 www.nature.com/scientificreports/ Results Dl 5 fr, frontal bone; md, mandibular process; mx, maxillary process; so, supraorbital ridge; EMM, early migrating mesenchyme; SOM, supraorbital mesenchyme; op, optic vesicle; ot, otic vesicle, rho, rhombencephalon. Scale bars; 500 μm (a, b, c, e), 200 μm (g), and 100 μm (i, k). but the relative position of bilateral parietal bones in the cranium was abnormal at P0, and the anterior edge of parietal bone, which comprises the coronal suture was more inclined to the posterior (Fig. 2p). We reason that the inclination was caused by the appearance of ec and hb that interfered the normal parietal bone development. Given the significant effects of Dlx5 on NCC, we attempted to analyze MES-specific Dlx5-augmented mice by crossing Mesp1-Cre ­mice36 and R26RCAG-flox-Dlx5/+. Four litters at E11.5–13.5 were examined, but all Mesp1- Cre;R26RCAG-flox-Dlx5/+ fetuses were lethal, making following analyses impossible. but the relative position of bilateral parietal bones in the cranium was abnormal at P0, and the anterior edge of parietal bone, which comprises the coronal suture was more inclined to the posterior (Fig. 2p). We reason that the inclination was caused by the appearance of ec and hb that interfered the normal parietal bone development. Given the significant effects of Dlx5 on NCC, we attempted to analyze MES-specific Dlx5-augmented mice b i M 1 C i 36 d R26RCAG flox Dlx5/+ F li E11 13 i d b ll M 1 NCC potential for chondrogenesis and osteogenesis was increased in the calvaria. Skeletal staining analysis of the NCCDlx5 revealed that both chondrogenesis and osteogenesis were promoted simultane- ously at the same region of the calvarium, which has not been reported in any other calvarial bone mutants. We thus further analysed the phenotype. Because the ec and hb were present at the NCC-MES junction, we first confirmed the cell origin of the misregulated structures. The NCC domain was visualized by enhanced yel- low fluorescent protein (EYFP) in the Wnt1-Cre;R26RCAG-flox-Dlx5/EYFP (NCCDlx5/EYFP) and in the littermate control Wnt1-Cre;R26REYFP/+ (NCCEYFP) at E17.5.i ( ) In bright-field images, the baso-lateral part of the coronal suture was comparable between the NCCEYFP and the NCCDlx5/EYFP (n = 5) (Fig. 3a,b, brackets). However, the coronal suture at the vertex seemed to be shifted more posterior in the NCCDlx5/EYFP compared to the NCC EYFP (Fig. 3a,b, dashed line). Results Dl 5 These irregular bones seemed to fuse to the frontal bone, therefore we called them heterotopic ­bones35 (hereafter hb). In summary, Dlx5 ectopic expression in head mesenchyme induced ec and hb formations. The development of the endogenous frontal bone was com- parable between the control and the NCCDlx5 at P0 (Fig. 2o,p). In the NCCDlx5, Dlx5 was not augmented in MES https://doi.org/10.1038/s41598-021-81434-x Scientific Reports | (2021) 11:2092 | www.nature.com/scientificreports/ Figure 1. Ubiquitous Dlx5 expression in NCCs and the effect on the distribution of NCCs in the NCCDlx5. (a, b) X-gal staining of NCCLacZ counterstained by nuclear fast red to visualize NCCs at E10.5 and E11.5. (c–f) Dlx5 expression on the frontal section of the control and the NCCDlx5 at E10.5 (c, d) and E11.5 (e, f). In the NCCDlx5, Dlx5 is ectopically expressed in maxilla and supraorbital mesenchyme at E10.5 (d), and in apical head mesenchyme at E11.5 (f, arrowhead). (g, h) Snai1 expression, a marker for NCC, by WISH of the control and the NCCDlx5 at E9.0. (i–l) Snai1 expression by ISH on frontal sections of the control and the NCCDlx5 at E9.0, at planes corresponding to hatched lines in (g, h), showing the forebrain, the mandibular process (i, j), and the dorsal area (k, l). fr, frontal bone; md, mandibular process; mx, maxillary process; so, supraorbital ridge; EMM, early migrating mesenchyme; SOM, supraorbital mesenchyme; op, optic vesicle; ot, otic vesicle, rho, rhombencephalon. Scale bars; 500 μm (a, b, c, e), 200 μm (g), and 100 μm (i, k). Figure 1. Ubiquitous Dlx5 expression in NCCs and the effect on the distribution of NCCs in the NCCDlx5. (a, b) X-gal staining of NCCLacZ counterstained by nuclear fast red to visualize NCCs at E10.5 and E11.5. (c–f) Dlx5 expression on the frontal section of the control and the NCCDlx5 at E10.5 (c, d) and E11.5 (e, f). In the NCCDlx5, Dlx5 is ectopically expressed in maxilla and supraorbital mesenchyme at E10.5 (d), and in apical head mesenchyme at E11.5 (f, arrowhead). (g, h) Snai1 expression, a marker for NCC, by WISH of the control and the NCCDlx5 at E9.0. (i–l) Snai1 expression by ISH on frontal sections of the control and the NCCDlx5 at E9.0, at planes corresponding to hatched lines in (g, h), showing the forebrain, the mandibular process (i, j), and the dorsal area (k, l). Results Dl 5 Under the fluorescent microscope, we found that the frontal bone of the NCCEYFP was highlighted while the parietal bone was not, and the "NCC tongue"3 protruded to the sagittal suture (n = 5) (Fig. 3c, arrowheads). The ec and hb were formed within the fluorescent NCC-derived domain in the NCCDlx5/EYFP (n = 5) (Fig. 3d). In sagittal sections, the ec and hb were clearly detectable by fluorescence (n = 3) (Fig. 3f). The hb was formed in line with the frontal bone and parietal bone, and the ec was always seen underneath the bone-forming layer (Fig. 3f). In the NCCEYFP, osteogenic fronts https://doi.org/10.1038/s41598-021-81434-x Scientific Reports | (2021) 11:2092 | www.nature.com/scientificreports/ Figure 2. Dlx5-augmentation in NCCs modifies NCC-derivatives. (a–d) Immunohistochemical staining for acetylated tubulin (c, d), nuclear counterstained by Hoechst (a, b) in the trigeminal ganglion. (e, f) 3-D reconstruction of trigeminal ganglion of the control (e) and the NCCDlx5 (f). (g) Statistical analysis of the trigeminal ganglion volume measured after reconstruction. (h, i) Dct localization, a pigment cell specifier, by ISH on frontal section of E15.5 heads. Insets in (h, i) are high magnified images of the boxed areas. (j) Statistical analysis of the number of Dct-positive cells. (k–p) Skeletal staining for cartilage (alcian blue) and bone (alizarin red). Lateral views at E13.5 (k, l), dorsal views (skull base removed) of calvaria at E14.5 (m, n) and P0 (o, p) of skeletal staining in the control and the NCCDlx5. Ectopic cartilage is induced in the NCCDlx5 (arrowheads in l, n, p) from E13.5. The developing heterotopic bone overlaps with the ectopic cartilage (double asterisks in p) at P0. Dashed lines contour cranial bones. Two-tailed t-test; *p < 0.05; ns, not significant. cs, coronal suture; fr, frontal bone; pa, parietal bone; pc, pigment cell; tg, trigeminal ganglion. Scale bar; 200 μm (a–d, h, i), 500 μm (e, f) and Figure 2. Dlx5-augmentation in NCCs modifies NCC-derivatives. (a–d) Immunohistochemical staining for acetylated tubulin (c, d), nuclear counterstained by Hoechst (a, b) in the trigeminal ganglion. (e, f) 3-D reconstruction of trigeminal ganglion of the control (e) and the NCCDlx5 (f). (g) Statistical analysis of the trigeminal ganglion volume measured after reconstruction. (h, i) Dct localization, a pigment cell specifier, by ISH on frontal section of E15.5 heads. Insets in (h, i) are high magnified images of the boxed areas. (j) Statistical analysis of the number of Dct-positive cells. Results Dl 5 (k–p) Skeletal staining for cartilage (alcian blue) and bone (alizarin red). Lateral views at E13.5 (k, l), dorsal views (skull base removed) of calvaria at E14.5 (m, n) and P0 (o, p) of skeletal staining in the control and the NCCDlx5. Ectopic cartilage is induced in the NCCDlx5 (arrowheads in l, n, p) from E13.5. The developing heterotopic bone overlaps with the ectopic cartilage (double asterisks in p) at P0. Dashed lines contour cranial bones. Two-tailed t-test; *p < 0.05; ns, not significant. cs, coronal suture; fr, frontal bone; pa, parietal bone; pc, pigment cell; tg, trigeminal ganglion. Scale bar; 200 μm (a–d, h, i), 500 μm (e, f) and 1 mm (k–p). Figure 2. Dlx5-augmentation in NCCs modifies NCC-derivatives. (a–d) Immunohistochemical staining for acetylated tubulin (c, d), nuclear counterstained by Hoechst (a, b) in the trigeminal ganglion. (e, f) 3-D reconstruction of trigeminal ganglion of the control (e) and the NCCDlx5 (f). (g) Statistical analysis of the trigeminal ganglion volume measured after reconstruction. (h, i) Dct localization, a pigment cell specifier, by ISH on frontal section of E15.5 heads. Insets in (h, i) are high magnified images of the boxed areas. (j) Statistical analysis of the number of Dct-positive cells. (k–p) Skeletal staining for cartilage (alcian blue) and bone (alizarin red). Lateral views at E13.5 (k, l), dorsal views (skull base removed) of calvaria at E14.5 (m, n) and P0 (o, p) of skeletal staining in the control and the NCCDlx5. Ectopic cartilage is induced in the NCCDlx5 (arrowheads in l, n, p) from E13.5. The developing heterotopic bone overlaps with the ectopic cartilage (double asterisks in p) at P0. Dashed lines contour cranial bones. Two-tailed t-test; *p < 0.05; ns, not significant. cs, coronal suture; fr, frontal bone; pa, parietal bone; pc, pigment cell; tg, trigeminal ganglion. Scale bar; 200 μm (a–d, h, i), 500 μm (e, f) and 1 mm (k–p). https://doi.org/10.1038/s41598-021-81434-x Scientific Reports | (2021) 11:2092 | www.nature.com/scientificreports/ Figure 3. Ectopic cartilage and heterotopic bone in the NCCDlx5 formed in NCC-derived head mesenchyme. Skull vault of E17.5 NCCEYFP (a, c, e) and NCCDlx5/EYFP (b, d, f) with brightfield (a, b) and fluorescent (c–f) images. (c) and (d) are the fluorescent images of (a) and (b), respectively. Green fluorescence implies NCC originating cells. Results Dl 5 Scale bars; 1 mm (a–d, g–j) and 100 μm (e, f). of the frontal and parietal bones normally overlap at the coronal suture (Fig. 3e), however, in the NCCDlx5/EYFP, the suture was established in a widely opened end-to-end type (Fig. 3f, arrowheads). Therefore, the extended NCC-derived area hosted the NCC-derived ec and hb, and the position of the coronal suture was shifted back- wards. Computed X-ray microtomography (μCT) data of P0 (n = 3) revealed that the NCC-derived frontal bone length at the midline of calvaria in the NCCDlx5 increased significantly by 18.5% compared to control (p < 0.001) (Fig. 3g–k). Furthermore, the NCC-derived calvarial bone volume rose by 10.2% (p < 0.05) (Fig. 3l). The vol- ume of MES-derived parietal bone, meanwhile, did not significantly decrease (n = 3) (Supplementary Fig. S1c). Besides, the ec was found at the cranial vertex, the chondrogenic potential of the NCCDlx5 obviously increased in this area. Thus, the NCC-derived apical head mesenchyme increased chondrogenic and osteogenic potentials in response to Dlx5-overexpression. of the frontal and parietal bones normally overlap at the coronal suture (Fig. 3e), however, in the NCCDlx5/EYFP, the suture was established in a widely opened end-to-end type (Fig. 3f, arrowheads). Therefore, the extended NCC-derived area hosted the NCC-derived ec and hb, and the position of the coronal suture was shifted back- wards. Computed X-ray microtomography (μCT) data of P0 (n = 3) revealed that the NCC-derived frontal bone length at the midline of calvaria in the NCCDlx5 increased significantly by 18.5% compared to control (p < 0.001) (Fig. 3g–k). Furthermore, the NCC-derived calvarial bone volume rose by 10.2% (p < 0.05) (Fig. 3l). The vol- ume of MES-derived parietal bone, meanwhile, did not significantly decrease (n = 3) (Supplementary Fig. S1c). Besides, the ec was found at the cranial vertex, the chondrogenic potential of the NCCDlx5 obviously increased in this area. Thus, the NCC-derived apical head mesenchyme increased chondrogenic and osteogenic potentials in response to Dlx5-overexpression. The ec developed inside of the dura mater. Histological analysis at E15.5 showed that cranial bones had not reached to the midline, and cartilage was absent in the vertex in the control (Fig. 4a–c). In the NCCDlx5, the thickness of the ec was comparable to that of cranial base cartilages (n = 3) (Fig. 4d–f). The hb was ossified on top of the ec in the calvaria (Fig. 4d–f). Results Dl 5 Brackets and dashed lines in dorsal views (a, b) indicate the coronal suture at the lateral side and the prospective coronal sutures at the apex, respectively. Arrowheads in (c) point to the “NCC tongue” between the parietal bones. (e, f) Parasagittal sections of the control (NCCEYFP) and mutant (NCCDlx5/EYFP) at planes indicated in (a) (a’–a’’) for (e) and (b) (b’–b’’) for (f). Nuclei are counterstained by Hoechst. Brackets and arrowheads in (e, f) indicate the coronal sutures of the control and the opened end-to-end junction at the coronal suture of the mutant, respectively. (g–j) μCT images of calvarial bone of the control (g, i) and the NCCDlx5 (h, j) at P0. Double-head arrows in (g–j) demonstrate the NCC-derived bone length to be measured. (k, l) Statistical analyses of NCC-derived bone length (k) and volume (l) (n = 3). Two-tailed t-test, *p < 0.05; ***p < 0.001. cs, coronal suture; ec, ectopic cartilage; fr, frontal bone; hb, heterotopic bone; ip, interparietal bone; pa, parietal bone. Scale bars; 1 mm (a–d, g–j) and 100 μm (e, f). Figure 3. Ectopic cartilage and heterotopic bone in the NCCDlx5 formed in NCC-derived head mesenchyme. Skull vault of E17.5 NCCEYFP (a, c, e) and NCCDlx5/EYFP (b, d, f) with brightfield (a, b) and fluorescent (c–f) images. (c) and (d) are the fluorescent images of (a) and (b), respectively. Green fluorescence implies NCC originating cells. Brackets and dashed lines in dorsal views (a, b) indicate the coronal suture at the lateral side and the prospective coronal sutures at the apex, respectively. Arrowheads in (c) point to the “NCC tongue” between the parietal bones. (e, f) Parasagittal sections of the control (NCCEYFP) and mutant (NCCDlx5/EYFP) at planes indicated in (a) (a’–a’’) for (e) and (b) (b’–b’’) for (f). Nuclei are counterstained by Hoechst. Brackets and arrowheads in (e, f) indicate the coronal sutures of the control and the opened end-to-end junction at the coronal suture of the mutant, respectively. (g–j) μCT images of calvarial bone of the control (g, i) and the NCCDlx5 (h, j) at P0. Double-head arrows in (g–j) demonstrate the NCC-derived bone length to be measured. (k, l) Statistical analyses of NCC-derived bone length (k) and volume (l) (n = 3). Two-tailed t-test, *p < 0.05; ***p < 0.001. cs, coronal suture; ec, ectopic cartilage; fr, frontal bone; hb, heterotopic bone; ip, interparietal bone pa, parietal bone. Results Dl 5 As expected from the skeletal staining data (Fig. 2), the endogenous frontal bone and parietal bone of the NCCDlx5 observed on HE sections illustrated similar bone quality in terms https://doi.org/10.1038/s41598-021-81434-x Scientific Reports | (2021) 11:2092 | www.nature.com/scientificreports/ Figure 4. Histological analysis of the ectopic cartilage and heterotopic bone in the NCCDlx5 at E15.5. (a–f) Frontal sections of E15.5 control (a–c) and NCCDlx5 (d–f) stained by HE and Alcian blue. (b, c, e, f) are high magnification images of boxed areas in (a, d). Heterotopic bone is induced at the vertex of the NCCDlx5, ectopic cartilage locates under the bone forming layer (e, f). (g, h) Semi-thin sections stained by toluidine blue on frontal sections of the control (g) and the NCCDlx5 (h). (i, j) TEM analysis of the control (i) and the NCCDlx5 (j) at the white boxed areas in (g, h). Arrows and arrowheads in (i, j) point to longitudinal-arranged fibroblasts and collagen fibrils, respectively. Ectopic cartilage appears in the meninges and is flanked by dura mater (j). ar, arachnoid mater; br, brain; du, dura mater; ec, ectopic cartilage; fr, frontal bone; hb, heterotopic bone; pa, parietal bone; pi, pia mater. Scale bars; 500 μm (a, d), 100 μm (b, c, e, f), 2 μm (i, j). Figure 4. Histological analysis of the ectopic cartilage and heterotopic bone in the NCCDlx5 at E15.5. (a–f) Frontal sections of E15.5 control (a–c) and NCCDlx5 (d–f) stained by HE and Alcian blue. (b, c, e, f) are high magnification images of boxed areas in (a, d). Heterotopic bone is induced at the vertex of the NCCDlx5, ectopic cartilage locates under the bone forming layer (e, f). (g, h) Semi-thin sections stained by toluidine blue on frontal sections of the control (g) and the NCCDlx5 (h). (i, j) TEM analysis of the control (i) and the NCCDlx5 (j) at the white boxed areas in (g, h). Arrows and arrowheads in (i, j) point to longitudinal-arranged fibroblasts and collagen fibrils, respectively. Ectopic cartilage appears in the meninges and is flanked by dura mater (j). ar, arachnoid mater; br, brain; du, dura mater; ec, ectopic cartilage; fr, frontal bone; hb, heterotopic bone; pa, parietal bone; pi, pia mater. Scale bars; 500 μm (a, d), 100 μm (b, c, e, f), 2 μm (i, j). of thickness or degree of mineralization compared to the counterpart of the control (Supplementary Fig. S2). www.nature.com/scientificreports/ In the control, expression of Forkhead Box C1 (Foxc1), transcribed in all three meningeal ­layers40, was broadly detected in the mesenchyme, but the signal was not seen or at much lower levels just underneath the epidermis at E11.5 (Fig. 6a, arrowheads). Importantly, expres- sion domains of Foxc1 and Dermo1, molecular markers for the meninges and the ­dermis41,42, respectively, were mutually exclusive (Fig. 6a,b). Control mice showed no expression of Sox9 in the EMM (Fig. 6c), but Runx2 was expressed in the Dermo1 expressing mesenchyme as a thin layer (Fig. 6b,d). Msx1 expression was found in the whole head mesenchyme (Fig. 6e). Msx2 expression domain was localized to the outer layer of the EMM, includ- ing a part of the meninges and the dermis (Fig. 6f, compared to 6a,b). l g p g g p In the NCCDlx5, the Foxc1 expression domain appeared to contain the ec primordium marked by Sox9 expres- sion (Fig. 6g,i), which is consistent with the phenotype in which the ec is surrounded by the dura mater (Fig. 4j). Remarkably, Dermo1 expression was highly upregulated in the NCCDlx5 compared to the control (Fig. 6h), indi- cating that Dlx5-augmentation enhanced the dermis formation. Runx2 expression of the NCCDlx5 was more evident compared to the control (Fig. 6d,j). Sox9 expression domain was included in the Runx2 domain (Fig. 6i,j). Because the hb developed outside of the ec (Fig. 4f), Runx2 expressing cells outside of Sox9-positive layer were thought to differentiate into osteoblasts. Importantly, these osteoblasts also expressed Dermo1 (Fig. 6h,j), sug- gesting that the hb was derived from the dermal layer. In contrast, the ec shown by Sox9 expressing domain seemed not to show Dermo1 expression (Fig. 6h,i). Moreover, Msx1 expression was present in the arachnoid and the pia mater, and was not expressed in other parts of the EMM (Fig. 6k). Msx2 was downregulated at some areas of head mesenchyme, however, expressed in the ec and hb (Fig. 6l). Since Lmx1b loss-of-function induced hb ­formation27, we also examined Lmx1b expression. Lmx1b was expressed in both the ec and hb (Supplementary Fig. S4), suggesting that since Dlx5 is a downstream of Lmx1b27, Dlx5-overexpression does not affect Lmx1b expression. PDGFRα, WNT/β‑catenin and Bmp2 signals are upregulated in the NCCDlx5. Platelet-derived growth factor receptor Alpha (Pdgfra) augmented in NCCs generated ec at the coronal suture, which was simi- lar to the ec of the NCCDlx543. www.nature.com/scientificreports/ We explored the gene expression change that led to ec and hb formations. At E10.5, few mesenchymal cells were detected at the apical head, and histological difference between the control and the NCCDlx5 was not noticed (Fig. 1a,b). We found that there were differences in gene expression as well as histology from E11.5. SRY-Box tran- scription factor 9 (Sox9) and Runt-related transcription factor 2 (Runx2) were used for evaluating mesenchymal condensation of cartilage and bone, respectively. At E11.5, Sox9 and Runx2 were substantially upregulated in the EMM region of the NCCDlx5 compared to the control (Fig. 5a–d, arrowheads). Mesenchymal condensation for the ec and hb was found at E11.5, which was around the same time with the beginning of original cranial base and calvarial development. The ectopic Sox9 expression domain was not connected to any part of the future skull base domain (n = 5) (Fig. 5b). This result confirmed that the ectopic Sox9 expression was not due to the extension of skull base primordium. In contrast, Runx2 expression in the EMM seemed to be continuous with the SOM by a thin expression line in the NCCDlx5 (n = 4) (Fig. 5c,d). To test whether the developing hb was independent of the SOM, we examined expression of Sp7, an early osteoblast marker and downstream of Runx2, at E14.5 by WISH (n = 5). The development of frontal and parietal bones was visualized by Sp7 expressing domain at this stage (Fig. 5e,f, dotted line). In the EMM area of the NCCDlx5, several Sp7 expression islands were independent of the SOM (Fig. 5f, arrowheads). These results strongly suggested that the hb in the NCCDlx5 is formed in the EMM and independent of the endogenous frontal bone.h p g The EMM layer was thickened in the NCCDlx5 at E11.5, which contained expanded Sox9 and Runx2 expres- sion domains (Fig. 5g,h). Our BrdU incorporation assay showed a significantly increased ­BrdU+ cells in the EMM of E11.5 NCCDlx5 (n = 3, p < 0.001) (Fig. 5i). Immunohistochemical (IHC) staining for cell death showed no signals in the EMM of both the control and the NCCDlx5 at E11.5 (n = 3) (Supplementary Fig. S3). Therefore, the thickened EMM in the NCCDlx5 was caused by increased cell proliferation. Early development of the EMM in the control and the NCCDlx5. We examined gene expression in early development of the ec and hb (n = 4) at E11.5. Results Dl 5 We conducted a more detailed investigation by using transmission electron microscope (TEM) at E15.5 (n = 3). Using toluidine blue stained semi-thin sections, we chose the relevant area of the control and the NCCDlx5 for TEM analysis (Fig. 4g–j). In the control, the dura mater located just underneath the bone layer, characterized by longitudinally arranged fibroblast-like cells (Fig. 4i, arrow), and collagen bundles filling intercellular spaces (Fig. 4i, arrowhead)37. The arachnoid mater was clearly seen next to the dura mater, which contains more loosely attached cells, and numerous free ­ribosomes28,37,38 (Fig. 4i). In the NCCDlx5, the ec occupied a large space between the bone and the brain (Fig. 4j). On its outer and inner surfaces, similar structures that had the characteristics of the dura mater were found (Fig. 4j). Besides, the arachnoid mater was recognized under the dura mater structure (Fig. 4j). Therefore, our histological analyses demonstrated that the ec developed within the dura mater. The ec and hb were derived from the EMM. It was reported that apical mesenchyme has both osteo- genic and chondrogenic potential in vitro39. Double conditional knock-out of Msh homeobox 1/2 (Msx1/2) in the mouse NCC (Msx1/2cko/cko) generated heterotopic bones from the EMM at abnormal positions including the suture ­area32. More recently, in vivo loss and gain of function experiments of LIM homeobox transcription factor 1 beta (Lmx1b), which is expressed in the EMM but not in the SOM, demonstrated the inhibitory function of Lmx1b on osteogenic specification in the ­EMM27. Lmx1b loss-of-function in head mesenchyme (Lmx1b ­LOFHM) induced osteogenic marker expression in the vertex mesenchyme, future interfrontal suture and expanded bone- forming area resulting in ­synostosis27. These previous studies suggest that the EMM has osteogenic potential, which is inhibited in normal context. https://doi.org/10.1038/s41598-021-81434-x Scientific Reports | (2021) 11:2092 | www.nature.com/scientificreports/ www.nature.com/scientificreports/ We performed double imunnofluorescent staining for PDGFRα and SOX9 at E11.5 (n = 3), PDGFRα was present in the outer portion of the EMM in the control, while SOX9 signal was not detected (Fig. 7a–d). In the NCCDlx5, PDGFRα expression levels were more intensive in the dermal and SOX9- positive layers (Fig. 7e–h). Semi-quantitative analysis on immunofluorescent staining showed that PDGFRα sig- nal was intensified by Dlx5-augmentation (Fig. 7u). WNT/β-catenin signalling induces osteoblast differentiation in intramembranous ­ossification44,45. The conditional β-catenin loss-of-function in the dermis using Dermo1- Cre or Engrailed1-Cre driver resulted in the loss of dermis and cranial bones. Instead, cartilages were induced between the epidermis and the thinner ­meninges42. Reversely, the NCCDlx5 had thickened dermis (Fig. 6h) and hb (Fig. 4f). We conducted double immunofluorescent  staining for β-catenin and RUNX2 signals at E11.5 (n = 3). In the control, β-catenin and RUNX2 were sparsely expressed below the epidermis (Fig. 7i–l). In the NCCDlx5, RUNX2 signal illustrated the hb (Fig. 7m,n), β-catenin signal in the dermis and hb forming area were clearly upregulated (Fig. 7m–p). Semi-quantitative analysis for β-catenin showed that the signal was significantly upregulated by Dlx5-augmentation (Fig. 7v). Bone morphogenetic protein (Bmp) is involved in hb formation in the interfrontal ­suture32,46 and it regulates Runx2 expression through DLX547. Our data showed that Bmp2 was not expressed in the control head mes- enchyme at E11.5 (Fig. 7q,r). However, it was ectopically induced in hb forming area in the NCCDlx5 (n = 3) (Fig. 7s,t). https://doi.org/10.1038/s41598-021-81434-x Scientific Reports | (2021) 11:2092 | www.nature.com/scientificreports/ com/scientificreports/ Figure 5. EMM developed the ectopic cartilage and heterotopic bone. (a–d) Expression of chondrocyte marker- Sox9 (a, b) and osteoblast marker-Runx2 (c, d) on frontal sections of E11.5 control (a, c) and NCCDlx5 (b, d) by ISH. Arrowheads indicate ectopic expressions of Sox9 (b) and Runx2 (d) in the EMM of the NCCDlx5. (e, f) Lateral views of Sp7 expression, an early osteoblast marker and downstream of Runx2, by WISH on heads of E14.5 control (e) and NCCDlx5 (f). Dotted lines in (e, f) outline developing frontal and parietal bones. Arrowheads in f point bony islands independent of the frontal bone primordium in the NCCDlx5. (g, h) BrdU incorporation assay in the EMM at E11.5 of the control (g) and the NCCDlx5 (h). BrdU positive cells were counted in the areas between the dashed lines of (g, h). Discussion Dl 5 i Dlx5 expression in the NCC is involved in jaw patterning. Aside from that, Dlx5 is expressed in the several NCC-derived head components and is related to their ­differentiation1, thus, we further examined predisposi- tion of the NCC affected by Dlx5-augmentation. Investigations of Snai1 expression indicated that the migration and distribution of NCCs were unaffected by Dlx5-overexpression (Fig. 1). There was little effect on trigeminal ganglion development, but the number of pigment cells was increased in the NCCDlx5 (Fig. 2), which possibly corresponds to enhanced dermal cell proliferation in the NCCDlx5 (Figs. 5h, 6h). Despite the extra skeletogenesis in the NCCDlx5, non-skeletal NCC-derivatives such as trigeminal ganglion, dermis, and pigment cells were not attenuated, suggesting that NCCs did not fluctuate between non-skeletogenic and skeletogenic fates.i l Previous reports showed that modifications in molecular cascades in mouse head mesenchyme resulted in either ec or hb formation in the skull vault. In particular, Msx1/2cko/cko and Lmx1b ­LOFHM caused hb formation at the posterior of the frontal bone similar to the NCCDlx5, but ec formation was not reported in those ­mutants27,32. By contrast, Pdgfra upregulation in NCCs generated ec in the coronal suture, meanwhile, the frontal bone appeared ­unchanged43. Some mutants demonstrated that cartilages replaced calvarial bones in mice, such as β-catenin knock-out42,45 and fibroblast growth factor 8 (Fgf8) gain-of-function48. Therefore, chondrogenesis could be upregulated at the expense of osteogenesis. In this study, we showed that chondrogenesis and osteogenesis were promoted simultaneously in the NCCDlx5 calvaria (Fig. 2). Ec and hb formation in the NCCDlx5 occurred in the meningeal and dermal layers of the EMM, respectively (Figs. 4, 6). We also found that, in normal development, the EMM seems to be committed to dermal and meningeal layers by E11.5 (Fig. 6). The distinct cell differentia- tion of apical head mesenchyme in response to Dlx5-augmentation in NCCs strongly support the idea that there are different cell populations in the EMM by this stage.h f p p y g The development of the ec in dura mater layer highly suggests that head mesenchyme which was origi- nally destined to be meningeal precursor cells could be turned into chondrocytes. The ec formation within the dura mater was previously reported; residual cartilages are occasionally formed above the trigeminal ganglia in ­mammals49, and the pila antotica near the ala temporalis in therians develops inside the dura mater as atavistic ­relics50. www.nature.com/scientificreports/ www.nature.com/scientificreports/ Figure 6. Gene expression patterns in the EMM at E11.5. Expression pattern of Foxc1-meningeal marker (a, g), Dermo1-dermal marker (b, h), Sox9-chondrocyte marker (c, i), Runx2-osteoblast marker (d, j), and osteogenic regulators: Msx1 (e, k), Msx2 (f, l) on frontal sections of E11.5 control (a–f) and NCCDlx5 (g–l) by ISH. Arrowheads in (a) point cells under the epidermis that weakly express Foxc1. Foxc1 and Dermo1 demonstrate complementary expression in head mesenchyme in the control (a, b), Dermo1 and Runx2 are co-expressed in a thin cell layer under the epidermis (b, d). Msx1 is expressed in the whole head mesenchyme (e), while Msx2 is mainly expressed at the outer layer of head mesenchyme (f). In the NCCDlx5, Sox9 expression in EMM illustrates the forming ectopic cartilage (i). Foxc1 is expressed in the meninges and faintly in the ectopic cartilage (g). Dermo1 and Runx2 are upregulated; Runx2 expression is enclosed by Dermo1 expression (h, j). Msx1 is not expressed in ectopic cartilage and heterotopic bone, whereas Msx2 is expressed in both misregulated structures. Dashed lines in (g–l) outline the ectopic cartilage forming region estimated by Sox9 expression (i). br, brain; ec, ectopic cartilage; hb, heterotopic bone. Scale bars; 50 μm. Figure 6. Gene expression patterns in the EMM at E11.5. Expression pattern of Foxc1-meningeal marker (a, g), Dermo1-dermal marker (b, h), Sox9-chondrocyte marker (c, i), Runx2-osteoblast marker (d, j), and osteogenic regulators: Msx1 (e, k), Msx2 (f, l) on frontal sections of E11.5 control (a–f) and NCCDlx5 (g–l) by ISH. Arrowheads in (a) point cells under the epidermis that weakly express Foxc1. Foxc1 and Dermo1 demonstrate complementary expression in head mesenchyme in the control (a, b), Dermo1 and Runx2 are co-expressed in a thin cell layer under the epidermis (b, d). Msx1 is expressed in the whole head mesenchyme (e), while Msx2 is mainly expressed at the outer layer of head mesenchyme (f). In the NCCDlx5, Sox9 expression in EMM illustrates the forming ectopic cartilage (i). Foxc1 is expressed in the meninges and faintly in the ectopic cartilage (g). Dermo1 and Runx2 are upregulated; Runx2 expression is enclosed by Dermo1 expression (h, j). Msx1 is not expressed in ectopic cartilage and heterotopic bone, whereas Msx2 is expressed in both misregulated structures. Dashed lines in (g–l) outline the ectopic cartilage forming region estimated by Sox9 expression (i). br, brain; ec, ectopic cartilage; hb, heterotopic bone. Scale bars; 50 μm. www.nature.com/scientificreports/ (i) Statistical analysis of the percentage of BrdU positive cells over the total number of EMM cells (n = 3). Two- t il d t t t ***p<0 001 EMM l i ti h f f t l b i t l b SOM bit l Figure 5. EMM developed the ectopic cartilage and heterotopic bone. (a–d) Expression of chondrocyte marker- Sox9 (a, b) and osteoblast marker-Runx2 (c, d) on frontal sections of E11.5 control (a, c) and NCCDlx5 (b, d) by ISH. Arrowheads indicate ectopic expressions of Sox9 (b) and Runx2 (d) in the EMM of the NCCDlx5. (e, f) Lateral views of Sp7 expression, an early osteoblast marker and downstream of Runx2, by WISH on heads of E14.5 control (e) and NCCDlx5 (f). Dotted lines in (e, f) outline developing frontal and parietal bones. Arrowheads in f point bony islands independent of the frontal bone primordium in the NCCDlx5. (g, h) BrdU incorporation assay in the EMM at E11.5 of the control (g) and the NCCDlx5 (h). BrdU positive cells were counted in the areas between the dashed lines of (g, h). (i) Statistical analysis of the percentage of BrdU positive cells over the total number of EMM cells (n = 3). Two- tailed t-test,***p < 0.001. EMM, early migrating mesenchyme; fr, frontal bone; pa, parietal bone; SOM, supraorbital mesenchyme. Scale bars; 200 μm (a–d), 500 μm (e, f), 50 μm (g, h). https://doi.org/10.1038/s41598-021-81434-x Scientific Reports | (2021) 11:2092 | Discussion Dl 5 i In the clinical aspect, some meningeal chondrosarcomas, which are tumours containing cartilaginous islands, in the dura mater are ­reported51. Additionally, when the dura mater is transplanted to the trunk in contact with mesodermal elements, the transplant sometimes develops ­cartilage52. In addition, explants of mouse mes- enchymal cells from the vertex area of the head (corresponding to the EMM) at E12.5–14.0 show the potential for bone and cartilage ­formations39. These reports support our idea of dura-to-cartilage transformation. l gh p pp g In the NCCDlx5, the ec develops in the frontal bone area in proximity to the coronal suture, which is the NCC- MES boundary, and some small cartilages in the interfrontal suture. Loss of β-catenin is one of the causes of ec induction during calvarial ­development42. However, β-catenin was detected in the ec forming area in the NCCDlx5 (Fig. 7f,o), suggesting that formation of ec was not attributed to change in WNT/β-catenin signalling. Pdgfra upregulation in the NCC in mice exhibits ec formation in the coronal and interfrontal suture, which is similar to the ec in the NCCDlx543. We found that PDGFRα signal was increased in the NCCDlx5. Although the layer of ec formation was not studied in the Pdgfra ­mutant43, it is suggested that the interfrontal area, the NCC-MES boundary show potential for cartilage differentiation when stimulated by PDGFRα (Fig. 8b). f Our gene expression analyses suggest that, in the control, Runx2-expressing layer just below the epider in the EMM does not associate with the meninges at E11.5. The Runx2-expressing layer is present within Scientific Reports | (2021) 11:2092 | https://doi.org/10.1038/s41598-021-81434-x www.nature.com/scientificreports/ Figure 7. PDGFRα, β-catenin and Bmp2 signals in the EMM at E11.5. (a–h) Double immunofluoresce SOX9 and PDGFRα on frontal sections at E11.5, nuclear counterstained by Hoechst of the control (a–d (e–h). Arrows in f, h point to the ectopic cartilage. PDGFRα signal is shown in the SOX9-positive area immunofluorescent staining for RUNX2 and β-catenin on frontal sections at E11.5, nuclear counterstai control (i–l) and the NCCDlx5 (m–p). Arrows in (n, p) point to the heterotopic bone. β-catenin signal is positive area (p). (q–t) Bmp2 expression by ISH on frontal sections at E11.5 of the control (q, r) and the are high magnified images of the boxed area in (q, s). (u, v) Semi-quantification analysis on immunoflu PDGFRα and β-catenin. Two-tailed t-test;**p < 0.01;***p < 0.001. Discussion Dl 5 i Foxc1 is expressed underneath the dermal layer to induce meningeal cell differentiation. In the NCCDlx5 (b), Dlx5 upregulates Pdgfra in the dura mater to generate the ectopic cartilage at the interfrontal and NCC-MES boundary area. In the dermis layer, Dlx5 inhibits the anti-osteogenic function of Msx2 [Msx2 (in)] but support the pro-osteogenic function of Msx2 [Msx2 (ac)] by upregulating Bmp2 and β-catenin, resulting in Runx2 upregulation. Consequently, osteogenesis is activated in the apical head mesenchyme to form the heterotopic bone. ac, activator; in, inhibitor. expression domain of Dermo1, a dermal cell marker, and this expression domain does not express Foxc1, a meningeal marker. Based on these results, we propose that the cells expressing both Runx2 and Dermo1 in the EMM are dermal progenitors (Fig. 8a). Furthermore, the Runx2 expression in mesenchymal cells suggests the intrinsic osteogenic potential of the dermis. Anti-osteogenic functions of Msx2 and Lmx1b are likely to suppress the osteogenic potential in the ­EMM27,32. g Msx2 can act as either osteogenic inhibitor or activator (Fig. 8, Msx2 (in), Msx2 (ac))53. MSX2 inhibits Runx2 transcriptional ­activity54,55, and competes with RUNX2 in binding to a regulatory sequence of Osteocalcin (Ocn), an osteogenic induction ­gene56 (Msx2 (in)). The anti-osteogenic activity of Msx1 and Msx2 in the EMM explained the hb in the Msx1/2cko/cko at the early stage of calvarial development (~ E12.5)32. In the Lmx1b ­LOFHM, Msx2 expression was downregulated in the EMM, and the phenotype could be explained similarly to the Msx1/2cko/cko27. We thus propose that Msx2 inhibits Runx2 osteogenic induction in the dermis in normal situation (Fig. 8a), and possibly Msx1 is also related to this function to some extent. p y Contrary to the anti-osteogenic function above mentioned, Msx2 has been shown to promote both prolif- eration (undifferentiated condition) and differentiation of osteoblast lineage ­cells57,58 (Msx2 (ac)). Despite the similar hb formation to the Msx1/2cko/cko, Msx2 was expressed in the hb forming region in the NCCDlx5 (Fig. 6). Dlx5 is an important factor to antagonize Msx2 anti-osteogenic ­function55,59,60, it therefore seems that Dlx5- overexpression suppressed the anti-osteogenic function of Msx2 in the NCCDlx5 (Fig. 8b). By contrast, Dlx5 appears to promote the pro-osteogenic function of Msx2 in concert with other osteoblast activators such as Bmp2 and β-catenin (Fig. 8b, Msx2(ac)). Dlx5 is a downstream target of Bmp247, and we observed increased Bmp2 expression in head mesenchyme of the NCCDlx5. Discussion Dl 5 i ec, ectopic cartilage; hb, heterotopic bo (a, e, i, m, r, t); 200 μm (q, s). Figure 7. PDGFRα, β-catenin and Bmp2 signals in the EMM at E11.5. (a–h) Double immunofluorescent staining for SOX9 and PDGFRα on frontal sections at E11.5, nuclear counterstained by Hoechst of the control (a–d) and the NCCDlx5 (e–h). Arrows in f, h point to the ectopic cartilage. PDGFRα signal is shown in the SOX9-positive area (h). (i–p) Double immunofluorescent staining for RUNX2 and β-catenin on frontal sections at E11.5, nuclear counterstained by Hoechst of the control (i–l) and the NCCDlx5 (m–p). Arrows in (n, p) point to the heterotopic bone. β-catenin signal is shown in the RUNX2- positive area (p). (q–t) Bmp2 expression by ISH on frontal sections at E11.5 of the control (q, r) and the NCCDlx5 (s, t). (r, t) are high magnified images of the boxed area in (q, s). (u, v) Semi-quantification analysis on immunofluorescent staining for PDGFRα and β-catenin. Two-tailed t-test;**p < 0.01;***p < 0.001. ec, ectopic cartilage; hb, heterotopic bone. Scale bars; 50 μm (a, e, i, m, r, t); 200 μm (q, s). https://doi.org/10.1038/s41598-021-81434-x Scientific Reports | (2021) 11:2092 | www.nature.com/scientificreports/ Figure 8. Proposed regulatory molecular cascades in EMM development at E11.5. In the control (a), the EMM develops into dermal and meningeal layers. The dermis is marked by a dermal marker-Dermo1. Runx2 and Msx2 are expressed in the dermis and Msx2 [Msx2 (in)] inhibits osteogenic induction of Runx2, consequently bone is not formed in the dermis. Foxc1 is expressed underneath the dermal layer to induce meningeal cell differentiation. In the NCCDlx5 (b), Dlx5 upregulates Pdgfra in the dura mater to generate the ectopic cartilage at the interfrontal and NCC-MES boundary area. In the dermis layer, Dlx5 inhibits the anti-osteogenic function of Msx2 [Msx2 (in)] but support the pro-osteogenic function of Msx2 [Msx2 (ac)] by upregulating Bmp2 and β-catenin, resulting in Runx2 upregulation. Consequently, osteogenesis is activated in the apical head mesenchyme to form the heterotopic bone. ac, activator; in, inhibitor. Figure 8. Proposed regulatory molecular cascades in EMM development at E11.5. In the control (a), the EMM develops into dermal and meningeal layers. The dermis is marked by a dermal marker-Dermo1. Runx2 and Msx2 are expressed in the dermis and Msx2 [Msx2 (in)] inhibits osteogenic induction of Runx2, consequently bone is not formed in the dermis. Materials and methods i All experiments were carried out in accordance with the relevant guidelines and regulations of the Tokyo Medical and Dental University and the University of Tokyo. Animal studies were conducted following the ARRIVE guidelines. In situ hybridization (ISH). Embryos of E9.0–12.5 were collected in ice-cold phosphate-buffered saline (PBS). Samples were fixed with 4% paraformaldehyde/PBS overnight at 4 °C. For frozen sections, samples were incubated in 25% sucrose in PBS, finally embedding in O.C.T. compound (Sakura Finetek, Japan) and stored at − 80 °C. Heads of E15.5 were frozen freshly in O.C.T. compound. Frozen sections were cut at 12 μm thick- ness (Leica CM1850, Germany). For whole-mount in situ hybridization (WISH) purposes, the fixed samples were dehydrated in a graded series of methanol and stored in 100% methanol at − 20 °C. DNA fragments of mouse Bmp2, Dct, Dlx5, Dermo1, Foxc1, Lmx1b, Msx1, Msx2, Runx2, Snai1, Sox9, and Sp7 shown in Supple- mentary Table S1 were subcloned into the pGEM-easy vector (Promega, USA), or pCRII vector (Invitrogen, USA). Digoxigenin (DIG)-labelled RNA probes complementary to the cDNA were synthesized by Sp6 or T7 RNA polymerase (Roche Diagnostics, Germany) and used for section or whole-mount in situ hybridization. Gene expression was visualized by nitro blue tetrazolium chloride and 5-Bromo-4-chloro-3-indolyl phosphate (Roche Diagnostics, Germany). In situ hybridization experiments were performed at least three times on differ- ent samples. Immunohistochemical (IHC) and immunofluorescent staining. Bromodeoxyuridine (BrdU) (Roche Diagnostics) was used for BrdU cell proliferating assay. BrdU in PBS was injected intraperitoneally to the pregnant mice at a dose of 100 mg BrdU/kg body weight one hour before dissection. E11.5 fetal heads were col- lected, fixed with 4% PFA, and prepared to be embedded in the O.C.T. compound. Other immunohistochemical analyses were performed on untreated fixed samples. Samples were cut at 12 μm. Sections were incubated with mouse anti-BrdU (1:200, 11170376001, Roche Diagnostics), rabbit anti-cleaved caspase3 (1:200, #9661, Cell Signaling Technology), or anti-Acetylated Tubulin (1:200, T7451, Sigma-Aldrich), double immunohistochemi- cal staining with mixtures of rat anti-PDGFRα (1:500, 14-1401, eBioscience) and rabbit anti-SOX9 (1:1500, cat.#AB5535, Millipore), mouse anti- β-catenin (1:500, 610153, BD BioSciences) and rabbit anti-RUNX2 (1:150, #12556, Cell Signaling Technology). SOX9 and RUNX2 fluorescence signals were detected by anti-Rabbit Alexa Fluor 555 (1:300, A31572, Molecular Probes). Discussion Dl 5 i These results suggest that Dlx5 activates Bmp2 through positive feedback. Since Msx2 is also a main downstream of Bmp261, maintenance of Msx2 expression in the NCCDlx5 could be caused by Bmp2 induction. WNT/β-catenin promotes intramembranous bone formation and dermal layer ­differentiation42. We demonstrated that β-catenin levels were increased in the forming hb and probably associated with the enhanced dermis differentiation (Fig. 7m–p). Our data are consistent with previous reports that Bmp2 and β-catenin synergistically induce Msx262,63. Bmp2 upregulation in the NCCDlx5 is consistent with Msx1/Msx2cko/cko and Lmx1b ­LOFHM ­mutants27,32. Altogether, the hb formation in the NCCDlx5 was caused by enhanced osteogenic induction of Bmp2 and β-catenin signalling pathways that involve Msx2 (Fig. 8b). It will be intriguing to clarify in more detail about the molecular mechanism for the phenotype, involved in the dual function of Msx2 in the future.i It was reported that Lmx1b prevents ossification of EMM from E9.527. In this study, we found that expression patterns of Dermo1 and Foxc1 are mutually complementary at E11.5 in the control (Fig. 6a,b). Taken together, we propose that after non-skeletogenic commitment, the EMM is divided into two populations, dermal and menin- geal layers by E11.5 in normal development (Fig. 8a). However, this commitment is not irreversible because the cell fate can be altered to cartilage and bone by responding to pro-skeletogenic signals such as augmentation of Dlx5. Given that the EMM has differentiation potentials to both cartilage and bone, it should be carefully evalu- ated which mesenchyme (EMM or SOM) contributes to ectopic and heterotopic skeletogenesis in the calvarium when mutant mice are examined. Apical-basal patterning in cranial development by the interaction between the EMM and SOM has yet to be fully elucidated. Although the molecular basis of EMM differentiation in its early development is still unknown, our findings provide a more detailed picture of the EMM sublayers together with their potentials, shedding light on developmental mechanisms of cranial development. Scientific Reports | (2021) 11:2092 | https://doi.org/10.1038/s41598-021-81434-x www.nature.com/scientificreports/ Materials and methods i Mice. Mice with Dlx5 conditional expression reporter allele by utilizing Cre-LoxP recombination system, R26RCAG-flox-Dlx5/+, were described ­previously11. R26RCAG-flox-Dlx5/+ mice were maintained on ICR genetic back- ground. Wnt1-Cre (#022501) mice were obtained from the Jackson Laboratory (#022501, Maine, USA) and maintained on ICR genetic background. Wnt1-Cre driver targets the ­NCC64. R26RCAG-flox-Dlx5/+ mice were crossed with Wnt1-Cre mice to constitutively activate Dlx5 expression in NCC. Wnt1-Cre;R26RCAG-flox-Dlx5/+ mice were used as the mutant, NCCDlx5, and mice without Wnt1-Cre or R26RCAG-flox-Dlx5/+ allele were studied as the control. R26RlacZ mice were described ­previously65. β-galactosidase staining of Wnt-Cre;R26RlacZ/+ (NCCLacZ) visualizes the NCC. Wnt1-Cre;R26REYFP/+ mice expressing enhanced yellow fluorescent protein (EYFP) were crossed with R26RCAG-flox-Dlx5/+ mice to generate Wnt1-Cre;R26RCAG-flox-Dlx5/EYFP (NCCDlx5/EYFP), in which NCC expresses EYFP with NCCDlx5 phenotype. The Wnt1-Cre;R26REYFP/+ (NCCEYFP) control littermates express EYFP in the NCC. The morning on which a vaginal plug was found was designated as embryonic day 0 (E0). Animal procedures were approved by the Institutional Animal Care and Use Committee of Tokyo Medical and Dental University (0170238A, A2018-48C, A2019-060A) and the University of Tokyo (P19-043). All experiments were carried out in accordance with the relevant guidelines and regulations of the Tokyo Medical and Dental University and the University of Tokyo. Animal studies were conducted following the ARRIVE guidelines. Mice. Mice with Dlx5 conditional expression reporter allele by utilizing Cre-LoxP recombination system, R26RCAG-flox-Dlx5/+, were described ­previously11. R26RCAG-flox-Dlx5/+ mice were maintained on ICR genetic back- ground. Wnt1-Cre (#022501) mice were obtained from the Jackson Laboratory (#022501, Maine, USA) and maintained on ICR genetic background. Wnt1-Cre driver targets the ­NCC64. R26RCAG-flox-Dlx5/+ mice were crossed with Wnt1-Cre mice to constitutively activate Dlx5 expression in NCC. Wnt1-Cre;R26RCAG-flox-Dlx5/+ mice were used as the mutant, NCCDlx5, and mice without Wnt1-Cre or R26RCAG-flox-Dlx5/+ allele were studied as the control. R26RlacZ mice were described ­previously65. β-galactosidase staining of Wnt-Cre;R26RlacZ/+ (NCCLacZ) visualizes the NCC. Wnt1-Cre;R26REYFP/+ mice expressing enhanced yellow fluorescent protein (EYFP) were crossed with R26RCAG-flox-Dlx5/+ mice to generate Wnt1-Cre;R26RCAG-flox-Dlx5/EYFP (NCCDlx5/EYFP), in which NCC expresses EYFP with NCCDlx5 phenotype. The Wnt1-Cre;R26REYFP/+ (NCCEYFP) control littermates express EYFP in the NCC. The morning on which a vaginal plug was found was designated as embryonic day 0 (E0). Animal procedures were approved by the Institutional Animal Care and Use Committee of Tokyo Medical and Dental University (0170238A, A2018-48C, A2019-060A) and the University of Tokyo (P19-043). www.nature.com/scientificreports/ at room temperature. Samples were then washed by distilled water and optically cleared by glycerol in 0.5% KOH until the bone and cartilage were visible. at room temperature. Samples were then washed by distilled water and optically cleared by glycerol in 0.5% KOH until the bone and cartilage were visible. Computed X‑ray microtomography (μCT). Heads of P0 mice were fixed in 70% ethanol overnight. μCT was taken by inspeXio SMX100CT (Shimadzu, Japan). The data were analyzed by Avizo 6.3. μCT scans were uploaded to Avizo 6.3 as DICOM files and visualized using Isosurface in Avizo 6.3. The NCC-derived bone length was measured in three-dimension at the midline, tracing the top of the calvaria (Fig. 3g–j, n = 3). The bone volume was calculated by Avizo 6.3, including all the bone components within the frontal bone forming area. Histological analysis. Heads of fetuses at E15.5 were fixed in Bouin’s fixative solution for 48 h. The samples were washed by 70% ethanol, then dehydrated in a gradient of ethanol until 100%, followed by xylene treatment and embedded in paraffin. Sections were cut at 5 μm thickness (Leica RM2235, Germany), then stained by 1% alcian blue (Sigma, 05500-10G) in 3% acetic acid, followed by Mayer’s Hematoxylin and 1% Eosin Y solution. Comparisons were made among at least three independent littermates. Transmission electron microscopy (TEM). Heads of E15.5 fetuses were trimmed to collect the targeted tissues, then fixed in 2.5% glutaraldehyde/0.1 M phosphate buffer (PB) for 72 h. After washing in PB overnight at 4 °C, samples were postfixed with Osmium tetroxide (OsO4) for 2 h. Samples were then dehydrated in ethanol, followed by infiltration of epon resin and propylene oxide catalyst, then embedded in epon resin. Semi-thin sec- tions at 1 μm and toluidine blue staining were utilized to examine the samples. Ultrathin sections at 80 nm were collected and double-stained with uranyl acetate and lead citrate on carbon-coated copper grids. Sections were observed by transmission electron microscopy (Hitachi H-7100, Japan) (n = 3). Received: 15 May 2020; Accepted: 5 January 2021 Received: 15 May 2020; Accepted: 5 January 2021 References 1. Green, S. A., Simoes-Costa, M. & Bronner, M. E. Evolution of vertebrates as viewed from the crest. 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This work was supported by grants-in-aid from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (18K06821 to M.T., 17H04357 and 18K19605 to S.I.). Author contributions T.H.V, M.T designed, conducted experiments, analyzed data and wrote manuscript. M.S, T.K provided prelimi- nary data. H.H, A.I and H.K advised on the experiments, managed mice. S.I designed, conducted experiments, analyzed data, revised and approved manuscript. www.nature.com/scientificreports/ Regulation of bone and cartilage development by network between BMP signalling and transcription factors. J. Biochem. https​://doi.org/10.1093/jb/mvs00​4 (2012).f g g g j 2. Hussein, S. M., Duff, E. K. & Sirard, C. Smad4 and β-catenin co-activators functionally interact with lymphoid-enhancing factor to regulate graded expression of Msx2. J. Biol. Chem. https​://doi.org/10.1074/jbc.M3054​72200​ (2003). b l l l β d bl d ff d b f ll h g g p p g j 63. Mbalaviele, G. et al. β-catenin and BMP-2 synergize to promote osteoblast differentiation and new bone formation. J. Cell. Biochem. https​://doi.org/10.1002/jcb.20253​ (2005). https://doi.org/10.1038/s41598-021-81434-x Scientific Reports | (2021) 11:2092 | Competing interests h p g The authors declare no competing interests. Additional informationh Additional information Supplementary Information The online version contains supplementary material available at https​://doi. org/10.1038/s4159​8-021-81434​-x. Correspondence and requests for materials should be addressed to S.I. Reprints and permissions information is available at www.nature.com/reprints. Publisher’s note  Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Open Access  This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creat​iveco​mmons​.org/licen​ses/by/4.0/. © The Author(s) 2021 https://doi.org/10.1038/s41598-021-81434-x Scientific Reports | (2021) 11:2092 |

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Wish Mango Tree: hybrid experimentation and creation

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Wish Mango Tree: Hybrid experimentation and creation Keywords Keywords Art, Design and Technology, Artistic Process, Augmented Reality, Hybrid Creation Process, Wish Mango Tree. Art, Design and Technology, Artistic Process, Augmented Reality, Hybrid Creation Process, Wish Mango Tre This essay presents the process of creating the artistic project at augmented reality “Wish Mango Tree” is configured as a specific site / public intervention for installing in the georeferenced mango trees of the city of Belém a technological device for electronic labeling and the creation of a hollow steel plate at mango tree. This process involves experimentation and creation related to Visual Arts, Design and programming, experienced through a mobile application, being a physical hybrid intervention. “Wish Mango Tree” is inspired by the work “Wish Tree”, a series of art installations in process, started in 1981, by the Japanese artist, and member of the Fluxus group, Yoko Ono. She chooses a tree native to a place, or plants one under her guidance. The public is invited to tie a wish in writing and hang it on the tree. Yoko has already installed this work in some cities in the world. “Wish Mango Tree” proposes an action similar to the public and passers-by of the mango trees in Belém. The project promotes interaction between individuals: humans and mango trees. Digital content can be viewed in the augmented reality app at the site specific where mango leaf wishes can be accessed by anyone. “Mangueira Desejo” seeks to fill a gap or lack identified: the invisibility of mango trees, seeking to use technology to give visibility to a social and ecological problem. The political dimension of the project is revealed in giving visibility to the mango trees, activating the collective memory and provoking questions and commitments from the individuals involved: trees, people, and institutions. This artistic project aimed at experimentation and creation related to Visual Arts and Design, experienced through a mobile application. And evokes the affective memory of its participants, seeking to enhance, strengthen and maintain the identity and cultural memory of Pará through digital media. The project fits into the artistic and cultural area: Visual Arts, with the creation of proposals in the Visual Arts area, through the areas: installation, intervention mechanisms, specific site, urban art, digital art, new media, photography, being a hybrid proposal between art and digital design. English English 1 9 2 A B S T R A C T | DOI Number 10.24135/link 2021.v2i1.109 Wish Mango Tree: Hybrid experimentation and creation In addition to addressing the experience of the visual arts in their technical, formal and conceptual reflections, of creation, diffusion, training and memory. In this sense, the project promotes creation, experimentation and design associated with a historical, social, cultural, sustainable and / or technological context, which can be translated into propositional actions that address graphic design, interactive media, web design/applications, design of games. The mango trees themselves are objects of public interest, the app invites everyone to “hang” their desires on the “Wish Mango Trees ”, promoting the transformation of the mango trees into a receptacle for the aspirations of the people who cross it. The app will promote remotely a network experience that triggers a physical experience in the main mango trees of the square, when approaching a mango tree to start the action, which will give visibility to the cloud of annotations at mango leaves through mobile app.

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Visual Interpretation of Synthetic Aperture Radar Sea Ice Imagery by Expert and Novice Analysts: An Eye Tracking Study. Alexandru Gegiuc1, Juha Karvonen1, Jouni Vainio2, Eero Rinne1,3, Roman Bednarik4, and Marko Mäkynen1 1Finnish Meteorological Institute(FMI), Marine Research, Erik Palménin aukio 1, 00560 Helsinki, Finland 2Finnish Meteorological Institute(FMI), Ice Service, Erik Palménin aukio 1, 00560 Helsinki, Finland 3University Centre in Svalbard (UNIS), Arctic Geophysics, Longyearbyen, Norway 4University of Eastern Finland (UEF), Joensuu, Finland Correspondence: Alexandru Gegiuc ([email protected]) https://doi.org/10.5194/tc-2022-8 Preprint. Discussion started: 25 February 2022 c⃝Author(s) 2022. CC BY 4.0 License. https://doi.org/10.5194/tc-2022-8 Preprint. Discussion started: 25 February 2022 c⃝Author(s) 2022. CC BY 4.0 License. Abstract. Mapping of sea ice types and parameters in the SAR imagery is therefore subject to an analyst’s performance which may lead to inconsistencies between the ice charts. By measuring the fixation duration over different sea ice types we can identify the features in a SAR image that require more cognitive effort in classification, and thus are more prone to miss-classification. 10 Ambiguities in classification were found especially for regions less restrictive for navigation, consisting of mixed sea ice properties and uneven thicknesses. We also show that the experts are able to correctly map large sea ice covered areas only by looking at the SAR images. Based on the eye movement data, ice categories with most of the surface covered by ice, i.e. in ice charts fast ice and very close ice, were easier to classify than areas with mixed ice thicknesses such as open ice or very open ice. 15 ice. 15 Visual Interpretation of Synthetic Aperture Radar Sea Ice Imagery by Expert and Novice Analysts: An Eye Tracking Study. Alexandru Gegiuc1, Juha Karvonen1, Jouni Vainio2, Eero Rinne1,3, Roman Bednarik4, and Marko Mäkynen1 1Finnish Meteorological Institute(FMI), Marine Research, Erik Palménin aukio 1, 00560 Helsinki, Finland 2Finnish Meteorological Institute(FMI), Ice Service, Erik Palménin aukio 1, 00560 Helsinki, Finland 3University Centre in Svalbard (UNIS), Arctic Geophysics, Longyearbyen, Norway 4University of Eastern Finland (UEF), Joensuu, Finland Correspondence: Alexandru Gegiuc ([email protected]) 1 1 https://doi.org/10.5194/tc-2022-8 Preprint. Discussion started: 25 February 2022 c⃝Author(s) 2022. CC BY 4.0 License. https://doi.org/10.5194/tc-2022-8 Preprint. Discussion started: 25 February 2022 c⃝Author(s) 2022. CC BY 4.0 License. 1 Introduction Here we pay attention to at the time the analysts use for analyzing the images and the sea ice types or features identified, interaction with the SAR images such as zooming or panning the image content and other differences or similarities between experts and novices. conditions are required by the analysts to be able to translate the SAR information into relevant geophysical ice characteristics. 25 Furthermore a temporal gap in the SAR or additional sea ice data available to analysts results in increased uncertainty (Gegiuc et al., 2018). We used an eye tracker to record and compute the gaze points on the screen from each analyst, while looking at, identifying and classifying sea ice features in five SAR images acquired over the Baltic Sea. Our study is divided in two parts. First we investigate the interaction between both novice and expert analysts and SAR imagery during visual identification and 30 classification of sea ice features. Here we pay attention to at the time the analysts use for analyzing the images and the sea ice types or features identified, interaction with the SAR images such as zooming or panning the image content and other differences or similarities between experts and novices. conditions are required by the analysts to be able to translate the SAR information into relevant geophysical ice characteristics. 25 Furthermore a temporal gap in the SAR or additional sea ice data available to analysts results in increased uncertainty (Gegiuc et al., 2018). We used an eye tracker to record and compute the gaze points on the screen from each analyst, while looking at, identifying and classifying sea ice features in five SAR images acquired over the Baltic Sea. Our study is divided in two parts. First we investigate the interaction between both novice and expert analysts and SAR imagery during visual identification and 30 classification of sea ice features. Here we pay attention to at the time the analysts use for analyzing the images and the sea ice types or features identified, interaction with the SAR images such as zooming or panning the image content and other differences or similarities between experts and novices. We used an eye tracker to record and compute the gaze points on the screen from each analyst, while looking at, identifying and classifying sea ice features in five SAR images acquired over the Baltic Sea. Our study is divided in two parts. Abstract. We demonstrate the use of eye tracking methodology as a non-invasive way to identify elements behind uncertainties typi- cally introduced during the process of sea ice charting using satellite synthetic aperture radar (SAR) imagery. To our knowledge, this is the first time eye tracking is used to study the interpretation of satellite SAR images over sea ice. We describe differences and similarities between expert and novice analysts while visually interpreting a set of SAR sea ice images. 5 In ice charting, SAR imagery serves as the base layer for mapping the sea ice conditions. Linking the backscatter signatures in the SAR imagery and the actual sea ice parameters is a complex task which requires highly trained experts. Mapping of sea ice types and parameters in the SAR imagery is therefore subject to an analyst’s performance which may lead to inconsistencies between the ice charts. By measuring the fixation duration over different sea ice types we can identify the We demonstrate the use of eye tracking methodology as a non-invasive way to identify elements behind uncertainties typi- cally introduced during the process of sea ice charting using satellite synthetic aperture radar (SAR) imagery. To our knowledge, this is the first time eye tracking is used to study the interpretation of satellite SAR images over sea ice. We describe differences and similarities between expert and novice analysts while visually interpreting a set of SAR sea ice images. 5 We demonstrate the use of eye tracking methodology as a non-invasive way to identify elements behind uncertainties typi- cally introduced during the process of sea ice charting using satellite synthetic aperture radar (SAR) imagery. To our knowledge, this is the first time eye tracking is used to study the interpretation of satellite SAR images over sea ice. We describe differences and similarities between expert and novice analysts while visually interpreting a set of SAR sea ice images. 5 In ice charting, SAR imagery serves as the base layer for mapping the sea ice conditions. Linking the backscatter signatures in the SAR imagery and the actual sea ice parameters is a complex task which requires highly trained experts. Mapping 5 In ice charting, SAR imagery serves as the base layer for mapping the sea ice conditions. Linking the backscatter signatures in the SAR imagery and the actual sea ice parameters is a complex task which requires highly trained experts. 1 Introduction Maritime shipping in cold regions requires up-to-date information about sea ice conditions, typically over large areas. This information is usually provided by experts trained for analyzing sea ice. They rely on satellite imagery and in-situ observations to describe the ice conditions by means of ice types and parameters. In the Baltic Sea, daily ice charts produced during winter by the Finnish Ice Service (FIS) contain manually drawn polygons of distinct ice types (WMO-JCOMM, 2014) and related 20 parameters, such as sea ice concentration, thickness or degree of ridging, indicated by symbols or values on the ice chart. These charts are mainly based on the visual interpretation of synthetic aperture radar (SAR) imagery and messages from ice- breakers. Visual discrimination of sea ice features in SAR imagery is a complex task because the SAR radar signature is mainly dependent on sea ice surface roughness. Thus, additional knowledge on the regional and local weather, topography and sea ice by the Finnish Ice Service (FIS) contain manually drawn polygons of distinct ice types (WMO-JCOMM, 2014) and related 20 parameters, such as sea ice concentration, thickness or degree of ridging, indicated by symbols or values on the ice chart. These charts are mainly based on the visual interpretation of synthetic aperture radar (SAR) imagery and messages from ice- breakers. Visual discrimination of sea ice features in SAR imagery is a complex task because the SAR radar signature is mainly dependent on sea ice surface roughness. Thus, additional knowledge on the regional and local weather, topography and sea ice conditions are required by the analysts to be able to translate the SAR information into relevant geophysical ice characteristics. 25 Furthermore a temporal gap in the SAR or additional sea ice data available to analysts results in increased uncertainty (Gegiuc et al., 2018). We used an eye tracker to record and compute the gaze points on the screen from each analyst, while looking at, identifying and classifying sea ice features in five SAR images acquired over the Baltic Sea. Our study is divided in two parts. First we investigate the interaction between both novice and expert analysts and SAR imagery during visual identification and 30 classification of sea ice features. 1 Introduction First we investigate the interaction between both novice and expert analysts and SAR imagery during visual identification and 30 classification of sea ice features. Here we pay attention to at the time the analysts use for analyzing the images and the sea ice types or features identified, interaction with the SAR images such as zooming or panning the image content and other differences or similarities between experts and novices. 2 2 https://doi.org/10.5194/tc-2022-8 Preprint. Discussion started: 25 February 2022 c⃝Author(s) 2022. CC BY 4.0 License. In the second part we explore the relationship between gaze data of experts analyzing SAR features and the actual sea ice classification results. We were interested in identifying individual differences between experts in their classification results and how these are linked with their eye movements. We also compare the eye tracking fixation data to the automated analysis of the SAR image local complexity. 1.1 Sea ice charting in the Baltic Sea 5 1.1 Sea ice charting in the Baltic Sea 5 Figure 1. An example of SAR mosaic with 500 m pixel size over Baltic Sea (Bay of Bothnia), processed from RADARSAT-2 SAR HH polarization imagery (left), and the corresponding Finnish Ice Chart (right) (© Finnish Meteorological Institute) from 15th of March 2013 Figure 1. An example of SAR mosaic with 500 m pixel size over Baltic Sea (Bay of Bothnia), processed from RADARSAT-2 SAR HH- polarization imagery (left), and the corresponding Finnish Ice Chart (right) (© Finnish Meteorological Institute) from 15th of March 2013. The basis for the Baltic Sea ice charting at FIS is daily SAR mosaic which is constructed automatically by merging together independent SAR images. The mosaic is updated once per day, typically in the morning, to include most recent available SAR images. Figure 1 shows an example of the daily SAR mosaics and the corresponding sea ice chart manually drawn by the FIS analysts. 10 When constructing an ice chart, historical information is always used as the base layer for the analysis. Using the most recent data, experts classify SAR mosaic regions into several sea ice types as defined in (WMO-JCOMM, 2014). The new chart consists of polygon boundaries and the ice type within each ice polygon based on the available information, most of which comes from a fresh SAR frame. Due to time constraints, FIS analysts typically draw large polygons i.e. tens of kilometers while suppressing small scale features such as leads or cracks. Parameters such as the degree of ridging in a qualitative scale is 3 https://doi.org/10.5194/tc-2022-8 Preprint. Discussion started: 25 February 2022 c⃝Author(s) 2022. CC BY 4.0 License. then added on top of each polygon as symbols. Similarly, sea ice thickness is added to the chart as average level ice thickness for each polygon. The visual interpretation of a sea ice SAR frame is known to be a complex task, as it requires extensive knowledge on the typical sea ice dynamics, current and historical weather and ice conditions as well as good understanding of the relation between the radar backscatter signature and various sea ice types in different weather conditions. 1.1 Sea ice charting in the Baltic Sea 5 Subsequently, analysts have 5 The visual interpretation of a sea ice SAR frame is known to be a complex task, as it requires extensive knowledge on the typical sea ice dynamics, current and historical weather and ice conditions as well as good understanding of the relation The visual interpretation of a sea ice SAR frame is known to be a complex task, as it requires extensive knowledge on the typical sea ice dynamics, current and historical weather and ice conditions as well as good understanding of the relation between the radar backscatter signature and various sea ice types in different weather conditions. Subsequently, analysts have 5 to combine their information from SAR imagery with other available information and apply their own expertise to be able to correctly classify a sea ice region. A detailed description of the ice charting practices can be found in the ice charting manual (MANICE, 2005). 5 between the radar backscatter signature and various sea ice types in different weather conditions. Subsequently, analysts have 5 to combine their information from SAR imagery with other available information and apply their own expertise to be able to correctly classify a sea ice region. A detailed description of the ice charting practices can be found in the ice charting manual (MANICE, 2005). For the Baltic Sea, ice concentration, mean level-ice thickness and ridge density are the three For the Baltic Sea, ice concentration, mean level-ice thickness and ridge density are the three main parameters reported in the FIS ice charts. The colors in the ice charts can be regarded as semaphore lights for shipping, distinguishing between different 10 ice types (see Table 1). Without ice-breakers’ assistance the red color represents "stop" or "no go" and consists of consolidated or very close ice. With ice-breakers’ assistance navigation in these areas is possible. Close ice marked with orange color may be restrictive (difficult) for weaker ice class ships and it often contains mixed ice types difficult to predict. Yellow color marks regions of open ice where weak ice class ships should "proceed with care". Green and other colors are all considered navigable areas with no restrictions. Consolidated land fast ice is marked with gray color. 15 FIS ice charts. The colors in the ice charts can be regarded as semaphore lights for shipping, distinguishing between different 10 ice types (see Table 1). 1.2 Visual interpretation of imagery and digital images 20 These values are however computed in simple tasks, for example recognition of easy objects. For example, in (Castoldi and Du¸t˘a, 2012) it was found that in crisis response and damage assessment the average fixation duration is about 1 second (998 ms) while trying to asses the building damage level in satellite and aerial images with high density features. In the case of SAR imagery it is not yet known the typical fixation duration employed by experts charting sea ice. A typical fixation duration of a human observer is around 0.3 s (Yarbus, 1967) and in general varies between 0.2 and 0.8 s. 20 These values are however computed in simple tasks, for example recognition of easy objects. For example, in (Castoldi and Du¸t˘a, 2012) it was found that in crisis response and damage assessment the average fixation duration is about 1 second (998 ms) while trying to asses the building damage level in satellite and aerial images with high density features. In the case of SAR imagery it is not yet known the typical fixation duration employed by experts charting sea ice. 25 In our study we focus on the visual interpretation of SAR images with sea ice content, aiming to demonstrate the possibility 25 of using eye tracking in this field, where monitoring and measuring the quality of the sea ice information by visual means of classification has also a great impact in safety of winter navigation. In our study we focus on the visual interpretation of SAR images with sea ice content, aiming to demonstrate the possibility 25 of using eye tracking in this field, where monitoring and measuring the quality of the sea ice information by visual means of classification has also a great impact in safety of winter navigation. In our study we focus on the visual interpretation of SAR images with sea ice content, aiming to demonstrate the possibility 25 of using eye tracking in this field, where monitoring and measuring the quality of the sea ice information by visual means of classification has also a great impact in safety of winter navigation. 1.1 Sea ice charting in the Baltic Sea 5 Even though both manual and automated charts aim to have high accuracy, consistency and reliability, significant differences between the two products have been reported in literature (Agnew and Howell (2011),CIS (2006),Gegiuc et al. (2018),Kar- vonen et al. (2015)). Karvonen et al. (2015) presents a comparison between the ice concentrations estimated manually and automatically. The automatic analysis is aimed to support the ice charting work and perhaps replace the manual estimates in the future. In their study, differences in ice concentration estimates between expert groups were found, together with differ- 5 ences between manual and automated estimates. This underlines that uncertainties and inconsistencies in the ice charting can be introduced by experts doing the ice analysis. However, there are currently no means of measuring this kind of uncertainty introduced by the analysts in the ice charts. Even though both manual and automated charts aim to have high accuracy, consistency and reliability, significant differences between the two products have been reported in literature (Agnew and Howell (2011),CIS (2006),Gegiuc et al. (2018),Kar- vonen et al. (2015)). Karvonen et al. (2015) presents a comparison between the ice concentrations estimated manually and automatically. The automatic analysis is aimed to support the ice charting work and perhaps replace the manual estimates in the future. In their study, differences in ice concentration estimates between expert groups were found, together with differ- 5 ences between manual and automated estimates This underlines that uncertainties and inconsistencies in the ice charting can the future. In their study, differences in ice concentration estimates between expert groups were found, together with differ- 5 ences between manual and automated estimates. This underlines that uncertainties and inconsistencies in the ice charting can be introduced by experts doing the ice analysis. However, there are currently no means of measuring this kind of uncertainty introduced by the analysts in the ice charts. 5 1.1 Sea ice charting in the Baltic Sea 5 Without ice-breakers’ assistance the red color represents "stop" or "no go" and consists of consolidated or very close ice. With ice-breakers’ assistance navigation in these areas is possible. Close ice marked with orange color may be restrictive (difficult) for weaker ice class ships and it often contains mixed ice types difficult to predict. Yellow color marks regions of open ice where weak ice class ships should "proceed with care". Green and other colors are all considered navigable areas with no restrictions. Consolidated land fast ice is marked with gray color. 15 Table 1. Ice Chart - traffic color lights with the corresponding recommendation per ice class. Table 1. Ice Chart - traffic color lights with the corresponding recommendation per ice class. Table 1. Ice Chart - traffic color lights with the corresponding recommendation per ice class. Ice class IC (%) Recommendation Very close ice 9-10/10 No Go, Stop. IB OK Close ice 7-8/10 Proceed with IB guidance, IB OK Open ice 4-6/10 Proceed with care, IB OK Very open ice 1-3/10 Go, OK Open water < 1 Go, OK New ice 71% Go, OK Level ice 91% Go, OK Fast ice 100% No Go, Stop. IB OK IC = ice concentration; IB = ice-breaker. Automated classification methods of sea ice parameters from SAR imagery exist as well, see an overview in Zakhvatkina et al. (2019).They typically employ a similar approach as the manual ice charting. First pixels with similar texture features, intensities, patterns are grouped into segments which are statistically analyzed and classified based on their correlation degree with known ice classes or parameters. The classification decision can be based on training data, or on reference data (e.g. Karvonen et al. (2015)). One example of the automated Baltic Sea ice products based on the SAR imagery is recently developed 20 degree of ice ridging product in Gegiuc et al. (2018). There a direct comparison with the manual ice chart is also shown and discussed in more detail. 20 Karvonen et al. (2015)). One example of the automated Baltic Sea ice products based on the SAR imagery is recently developed 20 degree of ice ridging product in Gegiuc et al. (2018). There a direct comparison with the manual ice chart is also shown and discussed in more detail. 4 https://doi.org/10.5194/tc-2022-8 Preprint. Discussion started: 25 February 2022 c⃝Author(s) 2022. CC BY 4.0 License. 1.2 Visual interpretation of imagery and digital images Human visual interpretation of scenes has been studied for long in many domains, including aviation (Wickens et al., 2005), 10 medicine (Ahmidi MS et al., 2012; Eivazi et al., 2012; Erridge, 2018) or human behaviour studies (Gordon et al., 2007). In aviation, for example, errors in plane manoeuvres that lead to fatalities are mostly linked with errors in situation assessment, and more specifically errors related to attention allocation of the pilots (Wickens et al., 2005). Visual interpretation of complex scenery plays an important role in medicine, where for example neurosurgeons perform Human visual interpretation of scenes has been studied for long in many domains, including aviation (Wickens et al., 2005), 10 medicine (Ahmidi MS et al., 2012; Eivazi et al., 2012; Erridge, 2018) or human behaviour studies (Gordon et al., 2007). In aviation, for example, errors in plane manoeuvres that lead to fatalities are mostly linked with errors in situation assessment, and more specifically errors related to attention allocation of the pilots (Wickens et al., 2005). Visual interpretation of complex scenery plays an important role in medicine, where for example neurosurgeons perform Visual interpretation of complex scenery plays an important role in medicine, where for example neurosurgeons perform critical decision-making tasks during surgery procedures. In those cases, even the smallest error can be fatal to patients (Eivazi 15 et al., 2012; Erridge, 2018). In image-based diagnosis or any other medical diagnosis which is based on the visual interpretation of a medical scan, the quality and speed of visual information processing are crucial with a direct impact on people’s health. Consequently, by measuring the level of expertise of analysts, their focus or cognitive effort, can help increase safety or minimize errors in the performed visual recognition tasks. critical decision-making tasks during surgery procedures. In those cases, even the smallest error can be fatal to patients (Eivazi 15 et al., 2012; Erridge, 2018). In image-based diagnosis or any other medical diagnosis which is based on the visual interpretation of a medical scan, the quality and speed of visual information processing are crucial with a direct impact on people’s health. Consequently, by measuring the level of expertise of analysts, their focus or cognitive effort, can help increase safety or minimize errors in the performed visual recognition tasks. A typical fixation duration of a human observer is around 0.3 s (Yarbus, 1967) and in general varies between 0.2 and 0.8 s. 2 Study Area, Datasets and Experiment Subjects Our study area is the Baltic Sea of Fig. 2, located approximately between the 54◦and 66◦of Our study area is the Baltic Sea of Fig. 2, located approximately between the 54◦and 66◦of northern latitude and between 10◦and 31◦of eastern longitude. Most parts of the Baltic Sea have seasonal ice cover and ice information is necessary for the 30 winter ship navigation. This information is provided in the form of daily ice charts. The FIS ice analysts have experience of 10◦and 31◦of eastern longitude. Most parts of the Baltic Sea have seasonal ice cover and ice information is necessary for the 30 winter ship navigation. This information is provided in the form of daily ice charts. The FIS ice analysts have experience of 5 https://doi.org/10.5194/tc-2022-8 Preprint. Discussion started: 25 February 2022 c⃝Author(s) 2022. CC BY 4.0 License. sea ice charting from the SAR imagery since the availability of the ERS-1 SAR imagery in early 1990’s. The analysts are also familiar with typical Baltic Sea ice conditions and are able to provide valuable insight regarding the ice charting process. Figure 2. Baltic Sea, the study area. Figure 2. Baltic Sea, the study area. 2.2 Experiment Subjects In total four persons took part in the study. Of these four, two were experts (denoted as E1 and E2) in the SAR sea ice analysis; E2 with an experience of ten years, and E1 with over 25 years in operational ice charting and other projects related to the sea ice analysis using SAR and other imagery, see Table 3. Experts are hard to recruit, as on a national scale there may be only a few available. 5 The experts have acquired their experience and developed their skills in ice charting and SAR sea ice analysis through various ways. E1 focused the analysis mainly on the Baltic Sea area, having worked at the Finnish Meteorological Institute (FMI) already when the first SAR data became available for ice charting in early 1990’s, while E2 focused mainly on the Caspian Sea and Greenland waters, when working at Danish Meteorological Institute (DMI). 10 Two novices (N1 and N2) with little or no familiarity with classification of SAR sea ice imagery participated to the study. N1 10 had been familiar with Geographical Information System (GIS) software and was a contributor to the development of FMI’s own sea ice charting software. At the time of recording N1 was working under FIS and had very little practice on the ice charting, but was somewhat familiar with the ice charting practices and SAR analysis. N2 had no experience with the SAR sea ice analysis, but was somewhat familiar with remote sensing of sea ice in general. 2.1 Stimulus images We used five RADARSAT-2 (RS-2) ScanSAR Wide images covering different regions of Baltic sea across three different winter seasons. The RS-2 SAR images with their acquisition time and central coordinates are listed in Table 3. The images 5 were selected so that different ice conditions were present from easy to recognize open-water and sea ice areas, to complex texture patterns. They are also shown in 3. 5 In FIS, the original SAR images are typically reduced in size for easier manipulation and saving disk space. This reduces the amount of detail available for analysis. The 100 m resolution of the SAR imagery used by the FIS analysts is lower than In FIS, the original SAR images are typically reduced in size for easier manipulation and saving disk space. This reduces the amount of detail available for analysis. The 100 m resolution of the SAR imagery used by the FIS analysts is lower than the original resolution. Here we used the same down-scaled resolution for the RS-2 SAR images. 10 the original resolution. Here we used the same down-scaled resolution for the RS-2 SAR images. 10 Due to the complexity of the visual SAR texture features of sea ice images, we included for reference five non-SAR images with easy-to-recognize content: a human face, a flower, a fish, a cat and a bird. These are referred to as natural images. They were selected from a publicly available eye tracking database (Judd et.al, 2009). Natural images were used as stimuli to establish a reference gaze behaviour for the analysts when viewing images that they are familiar with, with easy to recognize objects, regardless of their expertise level. 15 Due to the complexity of the visual SAR texture features of sea ice images, we included for reference five non-SAR images with easy-to-recognize content: a human face, a flower, a fish, a cat and a bird. These are referred to as natural images. They were selected from a publicly available eye tracking database (Judd et.al, 2009). Natural images were used as stimuli to establish a reference gaze behaviour for the analysts when viewing images that they are familiar with, with easy to recognize objects, regardless of their expertise level. 15 6 https://doi.org/10.5194/tc-2022-8 Preprint. Discussion started: 25 February 2022 c⃝Author(s) 2022. CC BY 4.0 License. 3.1 Hardware and software for Eye Tracking Experiment To record the eye movements, we used a Tobii X2-30 Eye Tracker (30 Hz) (Tobii Technology AB, 2014), in connection with an ordinary PC and an external monitor with a 22" diagonal size, similar to the ones used in the operational ice charting at the time of recording. Verbal protocols were recorded for the entire session, and used later for data filtering and analysis. All users gave their consent for recording their voice and eye movements. 20 gave their consent for recording their voice and eye movements. 20 We instructed the participants to look at the selected images and interpret the content verbally. For the non-SAR images, the participants were instructed to recognize the objects from the background by naming the category which belongs to and give a short description of the object. When looking at the SAR images, the participants had the task to describe their content freely by identifying sea ice types and features and classify them as they would in a typical ice charting routine. We instructed the participants to look at the selected images and interpret the content verbally. For the non-SAR images, the participants were instructed to recognize the objects from the background by naming the category which belongs to and give a short description of the object. When looking at the SAR images, the participants had the task to describe their content freely by identifying sea ice types and features and classify them as they would in a typical ice charting routine. To display the stimuli and record the eye movements data we used the Tobii Studio software (Tobii AB, 2016). Due to the 25 differences in complexity and image size and resolution, natural and SAR sea ice images were viewed using different tools. The natural images were shown through the Tobii Studio recording software as image stimulus, while the SAR images were opened and viewed with an image viewing program (Irfan View) which allowed users to freely change the scale or pan the viewed images. To display the stimuli and record the eye movements data we used the Tobii Studio software (Tobii AB, 2016). Due to the 25 differences in complexity and image size and resolution, natural and SAR sea ice images were viewed using different tools. 3.1 Hardware and software for Eye Tracking Experiment The natural images were shown through the Tobii Studio recording software as image stimulus, while the SAR images were opened and viewed with an image viewing program (Irfan View) which allowed users to freely change the scale or pan the viewed images. 7 7 https://doi.org/10.5194/tc-2022-8 Preprint. Discussion started: 25 February 2022 c⃝Author(s) 2022. CC BY 4.0 License. 3.2 Eye Tracking Experiment Procedure At the beginning of each recording session (one session per user), the users were required to perform a 9-point calibration rou- tine, by focusing their gaze onto the displayed calibration points in predefined position on the screen. After a good calibration was obtained, the recording started with a page showing lines of text with instructions for the user. Then, the images were displayed one by one in an alternate manner, so that a natural image was displayed before each SAR image. There was no time 5 limit for displaying an image and each user had the power to take their time visually inspecting each scene. The users were asked to manually change the displayed stimulus when finished analysing it, by simply pressing a keyboard key or using the mouse by clicking the Close button. 3.3 Data analysis methodology Eye movement data, verbal explanations and the interaction with the shown imagery were used in our analysis. 10 Here we study how the two experts perform in identifying and classifying sea ice regions in the SAR imagery without any additional information. More specifically we want to see if there are clear differences between their results and how could these be related to their experience and training. Eye movement data, verbal explanations and the interaction with the shown imagery were used in our analysis. 10 Here we study how the two experts perform in identifying and classifying sea ice regions in the SAR imagery without any additional information. More specifically we want to see if there are clear differences between their results and how could these be related to their experience and training. W i ti t f ll i h ti We investigate following research questions: 1. Are experts able to identify/classify ice types in the SAR images without additional ice information? If yes, which ice 15 types/features they can identify based on SAR data alone? Are they in agreement with each other and with the official FIS ice chart? 2. What visual strategies, if any, they follow during the visual interpretation of the sea ice features in the SAR imagery (e.g., order preference, time to get familiar with each SAR image before the actual analysis)? 3. Can eye movements distinguish between experts and novices? 4. Are the eye movements of experts during SAR analysis any different than when they look at the natural images, or can their gaze distinguish between easy or difficult to interpret areas? 20 We divided the gaze data into segments that correspond to the scanning phase and the analysis phase. The scanning phase refers to the beginning of each image visualization, when users first look at the SAR images just before starting their analysis. The analysis phase refers to the time when the users have started their analysis by providing verbal explanations and focusing their attention on one region at a time. We computed the average dwell time, fixation duration mean, standard deviation, and fixation density (number of fixations per ice area). 25 the natural images, or can their gaze distinguish between easy or difficult to interpret areas? 20 We divided the gaze data into segments that correspond to the scanning phase and the analysis phase. 3.3 Data analysis methodology The scanning phase refers to the beginning of each image visualization, when users first look at the SAR images just before starting their analysis. The analysis phase refers to the time when the users have started their analysis by providing verbal explanations and focusing their attention on one region at a time. We computed the average dwell time, fixation duration mean, standard deviation, and fixation density (number of fixations per ice area). 25 Table 2. Participants in the eye tracking experiment. E1 E2 N1 N2 age 56 39 28 24 experience 25 10 1 0 visual defects no no no no E = sea ice analysis expert; N = novice; experience = nr. of years 8 8 Figure 3. RADARSAT-2 SAR images listed in the Table 3 Table 3. RADARSAT-2 ScanSAR images over the Baltic Sea used in the eye tracking experiment. https://doi.org/10.5194/tc-2022-8 Preprint. Discussion started: 25 February 2022 c⃝Author(s) 2022. CC BY 4.0 License. https://doi.org/10.5194/tc-2022-8 Preprint. Discussion started: 25 February 2022 c⃝Author(s) 2022. CC BY 4.0 License. Fi 3 RADARSAT 2 SAR i li t d i th T bl 3 Figure 3. RADARSAT-2 SAR images listed in the Table 3 4 Results First, we discuss the qualitative observations and interaction with SAR imagery by the four analysts. This includes the SAR image analysis strategies employed by the two experts. Moreover, the gaze behaviours and performance measures are reported, more specifically we discuss the actual classification results by the two experts, including similarities and differences in the sea ice recognition / classification results. Further, we look into how the two experts performed when compared to the official ice 5 chart. Lastly, we look into the fixation statistics in relation to the SAR image complexity. 5 4.1 Qualitative observations and interaction with SAR imagery In general, the two novice users focused their gaze especially onto the most 20 obvious (salient) features in the images, such as islands, ship tracks or clear ice boundaries. much, their answers being confusing and limited. In general, the two novice users focused their gaze especially onto the most 20 obvious (salient) features in the images, such as islands, ship tracks or clear ice boundaries. Because the geographical span and the ice conditions varied with each SAR image shown, the experts also changed the viewed scale of the stimuli by zooming-in and out. For the SAR images 1 and 2, E1 did not change the scale at all, the ice covered areas being visible and distinguishable. For the SAR images 3,4 and 5, E1 did change the scale from 16 % to 28 %, Because the geographical span and the ice conditions varied with each SAR image shown, the experts also changed the viewed scale of the stimuli by zooming-in and out. For the SAR images 1 and 2, E1 did not change the scale at all, the ice covered areas being visible and distinguishable. For the SAR images 3,4 and 5, E1 did change the scale from 16 % to 28 %, 21 % and 21 % respectively. In these cases, the ice covered areas were much smaller compared with the SAR images 1 and 25 2. For all five images, E1 increased the scale 0.9 times the initial scale, in average per image. On the other hand, E2 required much more increased scale for all of the images, although he was able to provide some general description of the ice situation at the default scale. He did increase the scale in each image several times, reaching an average per image of five times the initial scale. With the increase of the scale, only part of the image remained visible, thus forcing the users to change the viewed area by 30 panning the image (almost constantly at higher scales). In general, a higher scale (i.e., the smaller area viewed) corresponded with a higher number of change of view (COV) times. To see how different participants reacted in average per image, we With the increase of the scale, only part of the image remained visible, thus forcing the users to change the viewed area by 30 panning the image (almost constantly at higher scales). Table 3. RADARSAT-2 ScanSAR images over the Baltic Sea used in the eye tracking experiment. able 3. RADARSAT-2 ScanSAR images over the Baltic Sea used in the eye tracking experiment. Image Date Time (UTC) Latitude Longitude 1 2010-02-12 16:16 61.252003 19.829067 2 2011-02-27 05:04 59.394806 21.461995 3 2012-02-06 15:59 60.009427 24.561313 4 2012-02-13 15:54 59.986025 25.619694 5 2012-02-20 15:50 60.219841 26.569200 central image coordinates are given in WGS84. Image Date Time (UTC) Latitude Longitude 1 2010-02-12 16:16 61.252003 19.829067 2 2011-02-27 05:04 59.394806 21.461995 3 2012-02-06 15:59 60.009427 24.561313 4 2012-02-13 15:54 59.986025 25.619694 5 2012-02-20 15:50 60.219841 26.569200 central image coordinates are given in WGS84. 9 https://doi.org/10.5194/tc-2022-8 Preprint. Discussion started: 25 February 2022 c⃝Author(s) 2022. CC BY 4.0 License. 4.1 Qualitative observations and interaction with SAR imagery The first difference between experts (E) and novices (N) was noticed right away, based on their reactions when they were shown the first SAR image. When a new SAR scene was shown, experts were first visually scanning the entire image fore few seconds followed by the 10 identification of the geographical region present in the SAR image: harbours, islands or other useful location information or features. Novices spent more time before they proceeded with the actual analysis. Even if novices recognized fast (in the first five seconds) that the image shown is a SAR image and eventually also the geographical area covered by it, they spent longer time before they started describing sea ice features. They also talked in a slower rate indicating confusion or lack of knowledge (difficulty in recognition of the sea ice features or lack of confidence). 15 Novices’ gaze was random at first, jumping fast from one sea ice area to another, including open water areas or areas with very thin/fresh ice. They seemed confused about what they should look for in the image. They also use more general terms for ice rather than specific ones (e.g., "some ice" or "thick ice" instead of fast ice) indicating non-familiarity with the region or with the ice charting terminology. Novices also made excessive use of zoom and pan tools available, but this did not help (difficulty in recognition of the sea ice features or lack of confidence). 15 Novices’ gaze was random at first, jumping fast from one sea ice area to another, including open water areas or areas with very thin/fresh ice. They seemed confused about what they should look for in the image. They also use more general terms for ice rather than specific ones (e.g., "some ice" or "thick ice" instead of fast ice) indicating non-familiarity with the region or with the ice charting terminology. Novices also made excessive use of zoom and pan tools available, but this did not help much, their answers being confusing and limited. In general, the two novice users focused their gaze especially onto the most 20 ob io s (salient) feat res in the images s ch as islands ship tracks or clear ice bo ndaries much, their answers being confusing and limited. 4.1 Qualitative observations and interaction with SAR imagery To 15 understand better the significance of viewed scale values, it is worth to mention that images viewed at the default scale would be seen completely, while an increased scale meant that some of the content could not be visible on the screen at all times. 4.1 Qualitative observations and interaction with SAR imagery In general, a higher scale (i.e., the smaller area viewed) corresponded with a higher number of change of view (COV) times. To see how different participants reacted in average per image, we 10 https://doi.org/10.5194/tc-2022-8 Preprint. Discussion started: 25 February 2022 c⃝Author(s) 2022. CC BY 4.0 License. computed the average COV rate (number of COV times per image), average zoom rate (AZ) and average view (AV) duration, see Table 4. Table 4. Viewing measures computed for all five SAR images Table 4. Viewing measures computed for all five SAR images User ACOV rate (%) AZ rate (%) AV duration (s) E1 5 2 108 E2 22 6 349 N1 49 15 111 N2 9 9 177 ACOV = Average change of view; AZ = Average zoom; AV = Average view. In average, the zoom rate for all SAR images is smaller for the experts with 2% and 6% while the novices have an AZ rate of 9 % and 15 %. Clearly the experts have a lower zoom rate which can be explained by the familiarity with the type of scenes viewed, experience and speed of execution. By looking at the ACOV rate, we notice that E1 and N2 stay below 10 %, followed 5 by the E2 at 22 %. N1 reached the highest score of 49%. This shows that N1 spends very little time for the actual analysis. The AV duration is the shortest for E1 and N1 with only 108 and 111 s, respectively followed by N2 with an AV of 177 s per image. E2 has the longest view duration of 349 s per image. The AV times include the identification of geographical elements in the images. Both experts keep a low AZ rate value, but E2 has a higher ACOV rate percentage of about four times larger than E1 In average, the zoom rate for all SAR images is smaller for the experts with 2% and 6% while the novices have an AZ rate of 9 % and 15 %. Clearly the experts have a lower zoom rate which can be explained by the familiarity with the type of scenes viewed, experience and speed of execution. By looking at the ACOV rate, we notice that E1 and N2 stay below 10 %, followed 5 by the E2 at 22 %. N1 reached the highest score of 49%. 4.1 Qualitative observations and interaction with SAR imagery This shows that N1 spends very little time for the actual analysis. The AV duration is the shortest for E1 and N1 with only 108 and 111 s, respectively followed by N2 with an AV of 177 s per image. E2 has the longest view duration of 349 s per image. The AV times include the identification of geographical elements in the images. Both experts keep a low AZ rate value, but E2 has a higher ACOV rate percentage of about four times larger than E1 and also the longest AV duration. 10 We found a difference in the way the two experts used the zoom. In average, E1 viewed the SAR scenes at a scale of 23 % in average, with a minimum scale of 15 % and a maximum of 28 %. E2 viewed the SAR scenes at a scale of 57 % in average, with a minimum of 15% and a maximum of 78 %. Here, we consider that a scale of 28% or lower corresponds to the default scale (DV) while higher values correspond to increased scales (ZV). Therefore, E1 viewed all the ice regions at the default scale, and also the longest AV duration. 10 We found a difference in the way the two experts used the zoom. In average, E1 viewed the SAR scenes at a scale of 23 % in average, with a minimum scale of 15 % and a maximum of 28 %. E2 viewed the SAR scenes at a scale of 57 % in average, with a minimum of 15% and a maximum of 78 %. Here, we consider that a scale of 28% or lower corresponds to the default scale (DV) while higher values correspond to increased scales (ZV). Therefore, E1 viewed all the ice regions at the default scale, with 23 % in average. E2 had a default viewing scale at 18 % in average and an increased viewing scale at 78 % in average. To 15 understand better the significance of viewed scale values, it is worth to mention that images viewed at the default scale would be seen completely, while an increased scale meant that some of the content could not be visible on the screen at all times. with 23 % in average. E2 had a default viewing scale at 18 % in average and an increased viewing scale at 78 % in average. 4.1.1 SAR image analysis strategies For E1, the identification of ice types in the SAR images was carried out in general from the most dominant or challenging ice area and slowly moving towards more easy to navigate areas. In most images he started the analysis with fast ice found 20 close to land, followed by very close ice or close ice, ice boundary which may include a brash ice barrier (an obstacle for ship nagivation). In all cases, most of the ice channels were identified with their connecting harbour locations. Open water and new ice were also identified during the visual inspection but E1 spent very little time over these easy to navigate areas. For E1, the identification of ice types in the SAR images was carried out in general from For E1, the identification of ice types in the SAR images was carried out in general from the most dominant or challenging In a different manner compared to E1, E2 started to identify first the open water and new ice areas and then moved to thicker ice areas (i.e. level ice, fast ice, close ice and very close ice). Similarly to E1, E2 also identified ice channels with 25 11 https://doi.org/10.5194/tc-2022-8 Preprint. Discussion started: 25 February 2022 c⃝Author(s) 2022. CC BY 4.0 License. their connecting harbours when present, brash ice barriers and other ice features. A high level of detail was preserved by E2, spending more time describing smaller scale features than E1. their connecting harbours when present, brash ice barriers and other ice features. A high level of detail was preserved by E2, spending more time describing smaller scale features than E1. Both experts provided estimates of ice concentration and ice thickness, but not in a consistent manner to be able to include those into our analysis.Interestingly, the analysts did not identify the same number of sea ice areas or features. E1 identified a total of 49 ice regions, from which 31 correspond to an ice charting category, 17 correspond to ice channels and three to brash ice barriers. E2 identified a total of 109 regions, some of which were looked at multiple times and at different scales. From these, 88 regions correspond to an ice charting category, 11 to ice channels, and also three to brash ice barriers. 5 4.2.1 Natural images By dividing the image content in object and background areas, we were able to compare the gaze data of all users while looking 10 at the natural images, see Table 5. Table 5. Fixation duration mean, standard deviation, fixation duration sum and the number of fixations are the computed gaze measures for the two categories of users while viewing natural images. The measures are computed over the foreground objects and background areas. Expertise Focus area N FDM (s) STD (s) TFD (s) E background 203 0.43 0.31 86.63 object 442 0.40 0.27 178.13 N background 75 0.33 0.14 25.06 object 296 0.43 0.35 128.63 FDM = Fixation Duration Mean; N = Number of fixations; STD = standard deviation; TFD = Total Fixation Duration. The results show that there are no major differences in the way participants inspect an image with an easy to recognize object. Their task was to identify the objects in the scenes and label them. All participants had similar values for the fixation duration mean with an average of 0.4 s for both object and background areas. The only significant difference between experts The results show that there are no major differences in the way participants inspect an image with an easy to recognize object. Their task was to identify the objects in the scenes and label them. All participants had similar values for the fixation duration mean with an average of 0.4 s for both object and background areas. The only significant difference between experts and novices was in the number of fixations, which was much higher for the experts. This can be due to the longer time of 15 analysis for the experts, spending on average 17 s of fixation per background image and 36 seconds per object image. Novices spent on average only 5 s per background image and 26 s per object image. and novices was in the number of fixations, which was much higher for the experts. This can be due to the longer time of 15 analysis for the experts, spending on average 17 s of fixation per background image and 36 seconds per object image. Novices spent on average only 5 s per background image and 26 s per object image. 15 and novices was in the number of fixations, which was much higher for the experts. 4.2.1 Natural images Discussion started: 25 February 2022 c⃝Author(s) 2022. CC BY 4.0 License. 4.2.1 Natural images This can be due to the longer time of 15 analysis for the experts, spending on average 17 s of fixation per background image and 36 seconds per object image. Novices spent on average only 5 s per background image and 26 s per object image. The higher number of fixations for the experts was mainly due to the longer time of analysis. This was in average 3.4 times more than the novices’ for the background areas and 1.4 times more over the objects. Longer viewing time is also carried with The higher number of fixations for the experts was mainly due to the longer time of analysis. This was in average 3.4 times more than the novices’ for the background areas and 1.4 times more over the objects. Longer viewing time is also carried with a greater amount of details given by users during analysis 20 The higher number of fixations for the experts was mainly due to the longer time of analysis. This was in average 3.4 times more than the novices’ for the background areas and 1.4 times more over the objects. Longer viewing time is also carried with a greater amount of details given by users during analysis. 20 During the analysis of the natural images, individual differences were observed amongst all four participants, but in overall the findings are consistent with existing work (Yarbus, 1967). more than the novices for the background areas and 1.4 times more over the objects. Longer viewing time is also carried with a greater amount of details given by users during analysis. 20 During the analysis of the natural images, individual differences were observed amongst all four participants, but in overall the findings are consistent with existing work (Yarbus, 1967). a greater amount of details given by users during analysis. 20 During the analysis of the natural images, individual differences were observed amongst all four participants, but in overall the findings are consistent with existing work (Yarbus, 1967). a greater amount of details given by users during analysis. 20 During the analysis of the natural images, individual differences were observed amongst all four participants, but in overall the findings are consistent with existing work (Yarbus, 1967). During the analysis of the natural images, individual differences were observed amongst all four participants, but in overall the findings are consistent with existing work (Yarbus, 1967). 12 https://doi.org/10.5194/tc-2022-8 Preprint. 4.2.2 Experts and SAR image sea ice classification They both seem to have looked at and analyzed the same areas they previewed during the Interestingly, experts’ gaze during image scan shows a preview of their gaze during analysis phase, except they have only fewer gaze points in each area. They both seem to have looked at and analyzed the same areas they previewed during the scanning phase, including the problematic areas (i.e., sea ice regions which were more difficult to classify and which were 15 not classified in agreement between each other). This means, that in the first few seconds of looking at a SAR image, experts already identify the main sea ice regions to classify. scanning phase, including the problematic areas (i.e., sea ice regions which were more difficult to classify and which were 15 not classified in agreement between each other). This means, that in the first few seconds of looking at a SAR image, experts already identify the main sea ice regions to classify. Table 6. Fixation measures computed for Expert users while inspecting the SAR images during first scan and n measures computed for Expert users while inspecting the SAR images during first scan and analysis phase User Gaze Activity FD (s) FDM (ms) STD (ms) N TFD (m) N NF E1 scan 5 391 193 14 0.45 70 - analysis 5 674 521 10 4.45 476 49 E2 scan 7 404 157 18 0.56 90 - analysis 8 630 479 13 14 1443 109 average values(n) are computed per area/feature; NF = Nr. of areas/features identified. During the analysis phase, experts focused at one ice area at a time, spending 1-3 fixations when switching between regions. In some cases, they were re-scanning the viewed scene or part of it in between two consecutive analysis segments. In some cases, in addition to the sea ice categories identified in the SAR images, both experts provided detailed information such as 20 estimates of sea ice thickness or concentration as well as hypothesis on the ice formation or melt Compared to E2 E1 spent During the analysis phase, experts focused at one ice area at a time, spending 1-3 fixations when switching between regions. In some cases, they were re-scanning the viewed scene or part of it in between two consecutive analysis segments. 4.2.2 Experts and SAR image sea ice classification When the SAR images were first shown to the analysts, they first looked at the image content before starting the verbal explanations (analysis). Consequently, we have divided the gaze data into scanning phase (before they start the analysis), and the analysis phase. During scanning phase, both experts required on average 0.4 s of fixation duration mean, same time as for the natural images. 5 This shows that while they’re getting familiar with the viewed scene, they don’t require additional cognitive effort. However, they required different amount of time for the image scan. E1 required on the average 5 s of fixation time per image while E2 required on the average 7 s. During the scanning phase, the analysts identified the type of image shown, geographical area, and even some of the ice features. Their gaze paths during the scanning phase show individual differences. For example, E1 During scanning phase, both experts required on average 0.4 s of fixation duration mean, same time as for the natural images. 5 This shows that while they’re getting familiar with the viewed scene, they don’t require additional cognitive effort. However, they required different amount of time for the image scan. E1 required on the average 5 s of fixation time per image while E2 required on the average 7 s. During the scanning phase, the analysts identified the type of image shown, geographical area, and even some of the ice features. Their gaze paths during the scanning phase show individual differences. For example, E1 seemed to have a denser gaze onto the regions with higher risk for navigation, while E2 had its gaze covering a wider range of 10 sea ice types and geographical regions. Figure 4 shows an example of gaze data (fixations) collected for both experts during the scan and analysis phases of the SAR image 2. seemed to have a denser gaze onto the regions with higher risk for navigation, while E2 had its gaze covering a wider range of 10 sea ice types and geographical regions. Figure 4 shows an example of gaze data (fixations) collected for both experts during the scan and analysis phases of the SAR image 2. Interestingly, experts’ gaze during image scan shows a preview of their gaze during analysis phase, except they have only fewer gaze points in each area. 4.2.2 Experts and SAR image sea ice classification In some cases, in addition to the sea ice categories identified in the SAR images, both experts provided detailed information such as 20 estimates of sea ice thickness or concentration, as well as hypothesis on the ice formation or melt. Compared to E2, E1 spent less time fixating on each SAR image while providing detailed information. On average, total fixation duration for E1 was about 62 s per SAR image and only 5 s per ice area identified, while E2 had in average 5 minutes of total fixation duration per image and 14 s per ice area identified. 20 cases, in addition to the sea ice categories identified in the SAR images, both experts provided detailed information such as 20 estimates of sea ice thickness or concentration, as well as hypothesis on the ice formation or melt. Compared to E2, E1 spent less time fixating on each SAR image while providing detailed information. On average, total fixation duration for E1 was about 62 s per SAR image and only 5 s per ice area identified, while E2 had in average 5 minutes of total fixation duration per image and 14 s per ice area identified. This is an interesting result, showing that the experts need only few seconds (5-14) of fixation time to be able to classify an 25 ice area. Also it underlines a difference in style of analysis. E2 spent in average about five times longer analyzing an image and This is an interesting result, showing that the experts need only few seconds (5-14) of fixation time to be able to classify an 25 ice area. Also it underlines a difference in style of analysis. E2 spent in average about five times longer analyzing an image and 13 https://doi.org/10.5194/tc-2022-8 Preprint. Discussion started: 25 February 2022 c⃝Author(s) 2022. CC BY 4.0 License. Figure 4. Gaze plots of the two experts, during visual scanning phase (left) and analysis phase (right) of the RadarSat-2 SAR image 2 acquired over the Åland Sea and the Northern Baltic Proper. Regions with higher priority in analysis are also highlighted. Here the gaze points are represented by circles of size corresponding to the fixation duration and have one color for each analyst (red for E1, blue for E2). Here, the time they spend scanning the image differs considerably (the most, compared with other SAR images). E1 spends only 5 s while E2 spends 18 s. 4.2.2 Experts and SAR image sea ice classification This is, however, an exceptional case for E2, whom expressed clearly that this particular image was more difficult to interpret. 14 https://doi.org/10.5194/tc-2022-8 Preprint. Discussion started: 25 February 2022 c⃝Author(s) 2022. CC BY 4.0 License. about three times longer analyzing an ice area than E1. These differences could be explained by the inequality between the two experts in terms of years of experience and of training. The more experienced analyst (E1) shows faster scanning and analysis times (see Table 6). about three times longer analyzing an ice area than E1. These differences could be explained by the inequality between the two experts in terms of years of experience and of training. The more experienced analyst (E1) shows faster scanning and analysis times (see Table 6). Looking further into the actual recognition of the ice types and features, we divided the data into segments corresponding to each ice type or feature identified by the two experts, following the definitions typically used in the Baltic sea ice charts (see 5 Table 1). 5 In addition to the ice categories, both experts identified other important features such as ice edge, brash ice barriers, ice channels, leads or ridges. We included ice channels and brash ice barriers along with the ice categories in our results, because these are clear and recognizable features and extremely relevant for the ice navigation. Also, these two ice features were identified more than once by both experts during analysis. 10 10 The ice types and features identified for each segment and their computed eye movement measures are presented in Table 7. The gaze measures reported here are given as average value per an ice category. The ice types and features identified for each segment and their computed eye movement measures are presented in Table 7. The gaze measures reported here are given as average value per an ice category. tion measures per ice category/feature computed for the two experts while inspecting the SAR images Table 7. average values are computed per viewed ice area; N - fixation density (number of fixations recorded in an ice area); NSAR = Nr. of SAR scenes; NF = Number of features / areas. 4.2.2 Experts and SAR image sea ice classification At the default scale, most difficult to recognize / classify ice categories for E2 were new ice, open water, very open ice, and very close ice, from highest to lowest, respectively. Overall, the largest difference between the FDM data of two experts analyzing different ice categories were found over close ice, new ice and level ice regions, see Figure 5. From all of the ice types identified, we found in average a FDM difference of 76 ms between the two experts, with an average FDM of 556 ms for E1 and 632 ms for E2. However, for E2 we included here the FDM values during both default view and increased scale view. If we take into account only values at the default scale for E2, the average FDM per ice category identified increases to 720 ms and the difference between the experts increasing from 70 to 164 ms. This means that for E2 it was more 5 difficult to recognize / classify some features at the default scale, and by zooming-in, the recognition effort decreases. At the default scale, most difficult to recognize / classify ice categories for E2 were new ice, open water, very open ice, and very close ice, from highest to lowest, respectively. Overall, the largest difference between the FDM data of two experts analyzing different ice categories were found over close ice, new ice and level ice regions, see Figure 5. Figure 5. Average fixation duration mean with standard error (SEM) values recorded over recognized ice categories for experts. The ice categories are arranged based on the increasing ice concentration values from left to right. Figure 5. Average fixation duration mean with standard error (SEM) values recorded over recognized ice categories for experts. The ice categories are arranged based on the increasing ice concentration values from left to right. These values are higher than the values recorded while viewing natural images, indicatin These values are higher than the values recorded while viewing natural images, indicating a rise in the cognitive effort required for the identification and classification of more complex SAR features. The FDM values recorded for both analysts 10 vary between a minimum of 385 ms and a maximum of 956 ms, indicating that the level of complexity in perception for each ice category is different. 4.2.2 Experts and SAR image sea ice classification Fixation measures per ice category/feature computed for the two experts while inspecting the SAR images Participant identified ice category dwelltime (s) FDM (ms) STD (ms) N NSAR NF E1 open-water 4 721 654 8 3 7 very open ice 6 473 250 13 1 1 open ice 5 385 164 13 1 1 close ice 5 471 368 10 1 1 very close ice 9 638 517 16 3 5 new ice 5 454 236 10 4 4 level-ice 8 789 614 13 3 6 fast ice 8 520 276 17 2 3 brash ice barrier 6 482 240 12 3 3 ice channels 4 788 658 5 5 17 E2 open water 9 733 590 15 5 15 very open ice 7 485 170 17 1 3 open ice 6 498 260 11 2 5 close ice 16 956 755 32 2 3 very close ice 7 549 347 15 5 19 new ice 6 674 533 11 4 16 level ice 5 601 472 10 5 14 fast ice 11 562 389 16 5 13 brash ice barrier 10 578 349 18 2 3 ice channels 6 764 641 9 4 11 average values are computed per viewed ice area; N - fixation density (number of fixations recorded in an ice area); NSAR = Nr. of SAR scenes; Number of features / areas. average values are computed per viewed ice area; N - fixation density (number of fixations recorded in an ice area); NSAR = Nr. of SAR scenes; NF = Number of features / areas. 15 https://doi.org/10.5194/tc-2022-8 Preprint. Discussion started: 25 February 2022 c⃝Author(s) 2022. CC BY 4.0 License. From all of the ice types identified, we found in average a FDM difference of 76 ms between the two experts, with an average FDM of 556 ms for E1 and 632 ms for E2. However, for E2 we included here the FDM values during both default view and increased scale view. If we take into account only values at the default scale for E2, the average FDM per ice category identified increases to 720 ms and the difference between the experts increasing from 70 to 164 ms. This means that for E2 it was more difficult to recognize / classify some features at the default scale, and by zooming-in, the recognition effort decreases. 4.2.2 Experts and SAR image sea ice classification In general, higher FDM values were recorded during analysis of ice regions richer in features, and thus, potentially more challenging for navigation. In contrast, lower FDM values were recorded mostly over more uniform and easily navigable areas. For example, fast ice which grows close to the land and has homogeneous backscatter spatially is easily recognizable visually. Here, an average FDM value of 520 ms was recorded for E1 and 562 ms for E2. 15 required for the identification and classification of more complex SAR features. The FDM values recorded for both analysts 10 vary between a minimum of 385 ms and a maximum of 956 ms, indicating that the level of complexity in perception for each ice category is different. In general, higher FDM values were recorded during analysis of ice regions richer in features, and thus, potentially more challenging for navigation. In contrast, lower FDM values were recorded mostly over more uniform and easily navigable areas. For example, fast ice which grows close to the land and has homogeneous backscatter spatially is easily recognizable visually. Here, an average FDM value of 520 ms was recorded for E1 and 562 ms for E2. 15 The difficulty in interpretation of sea ice features in the SAR imagery is dependent on the level of expertise, training practices and personal style of analysis. This can also be seen from the slight variation of the FDM values recorded between the two experts over different ice categories or features. For example, E1 recorded the highest FDM value of 789 ms over level ice recognizable visually. Here, an average FDM value of 520 ms was recorded for E1 and 562 ms for E2. 15 The difficulty in interpretation of sea ice features in the SAR imagery is dependent on the level of expertise, training practices and personal style of analysis. This can also be seen from the slight variation of the FDM values recorded between the two experts over different ice categories or features. For example, E1 recorded the highest FDM value of 789 ms over level ice 16 https://doi.org/10.5194/tc-2022-8 Preprint. Discussion started: 25 February 2022 c⃝Author(s) 2022. CC BY 4.0 License. category, while E2 recorded the highest FDM value of 956 ms over close ice category. 4.2.2 Experts and SAR image sea ice classification This suggests that both had the same level of difficulty in identifying the ice channels feature. This can be due to the extremely small scale at which these features were viewed. Ice channels typically consist of very narrow lines indicating the ice-breakers routes that extend from one port to another. The presence of vessels along these routes may help analysts in the identification of such features. Here, longer fixations over the ice channels could be explained by the small size (i.e., one to several pixels) of the vessels which 15 need to be recognized. Yet, analysts need only few fixations (seven in average) to identify the ice channels. Somewhat in contrast to the ice channels, brash ice barriers are easier to identify because they lie at the boundary between ice cover and open water or ice free region. This is confirmed by the experts having recorded lower FDM values during brash ice barriers identification, such as 482 ms for E1 and 578 ms for E2. Here, they recorded a higher fixation density of 15 fixations in average. Both experts identified the same number of brash ice barriers (three) but they had different number of ice channels 20 identified (17 for E1 and 11 for E2). Contrary to our expectations, both experts had relatively high FDM values (721 ms for E1 and 733 ms for E2) over open water regions. This can be explained by the occasional presence of broken ice floes or rough sea ice conditions resulting in a mixture of very small and very high backscatter. in average. Both experts identified the same number of brash ice barriers (three) but they had different number of ice channels 20 identified (17 for E1 and 11 for E2). Contrary to our expectations, both experts had relatively high FDM values (721 ms for E1 and 733 ms for E2) over open water regions. This can be explained by the occasional presence of broken ice floes or rough sea ice conditions resulting in a mixture of very small and very high backscatter. Contrary to our expectations, both experts had relatively high FDM values (721 ms for E1 and 733 ms for E2) over open water regions. This can be explained by the occasional presence of broken ice floes or rough sea ice conditions resulting in a mixture of very small and very high backscatter. 4.2.2 Experts and SAR image sea ice classification The high FDM values recorded for E1 over level ice areas may be due to the fact that the analyst had more difficulty in estimating the overall thickness of the ice cover in those areas. For example, some of the level ice areas may be present within leads which seen at small scale may require longer time for interpretation. The lower FDM value of 601 ms recorded over level ice for E2, may be explained by the fact that this analyst had analyzed the SAR content in both default and increased scales. If true, than it can be assumed that not all 5 features can be easily recognized at lower scales, but seen larger they may require less cognitive effort for the classification. A direct consequence of lowering the effort of recognition may relate to a decrease in uncertainty in classification of a viewed ice region. On the other hand, the high FDM values recorded over close ice areas by E2 may be due to the difficulty in estimating the ice concentration, especially when the areas consist of a mixture of unknown ice thicknesses. For both experts, the lowest FDM values (constantly less than 500 ms) were recorded over open ice and very open ice 0 category, while E2 recorded the highest FDM value of 956 ms over close ice category. The high FDM values recorded for E1 over level ice areas may be due to the fact that the analyst had more difficulty in estimating the overall thickness of the ice cover in those areas. For example, some of the level ice areas may be present within leads which seen at small scale may require longer time for interpretation. The lower FDM value of 601 ms recorded over level ice for E2, may be explained by the fact that this analyst had analyzed the SAR content in both default and increased scales. If true, than it can be assumed that not all 5 that this analyst had analyzed the SAR content in both default and increased scales. If true, than it can be assumed that not all 5 features can be easily recognized at lower scales, but seen larger they may require less cognitive effort for the classification. A direct consequence of lowering the effort of recognition may relate to a decrease in uncertainty in classification of a viewed ice region. 4.2.2 Experts and SAR image sea ice classification On the other hand, the high FDM values recorded over close ice areas by E2 may be due to the difficulty in estimating the ice concentration, especially when the areas consist of a mixture of unknown ice thicknesses. For both experts, the lowest that this analyst had analyzed the SAR content in both default and increased scales. If true, than it can be assumed that not all 5 features can be easily recognized at lower scales, but seen larger they may require less cognitive effort for the classification. A direct consequence of lowering the effort of recognition may relate to a decrease in uncertainty in classification of a viewed ice region. On the other hand, the high FDM values recorded over close ice areas by E2 may be due to the difficulty in estimating the ice concentration, especially when the areas consist of a mixture of unknown ice thicknesses. For both experts, the lowest FDM l ( l l h 500 ) d d i d i FDM values (constantly less than 500 ms) were recorded over open-ice and very open-ice. 10 Interestingly, when identifying sea ice channels, both experts recorded an average FDM of 776 ms. This suggests that both had the same level of difficulty in identifying the ice channels feature. This can be due to the extremely small scale at which these features were viewed. Ice channels typically consist of very narrow lines indicating the ice-breakers routes that extend from one port to another. The presence of vessels along these routes may help analysts in the identification of such features. FDM values (constantly less than 500 ms) were recorded over open-ice and very open-ice. 10 Interestingly, when identifying sea ice channels, both experts recorded an average FDM of 776 ms. This suggests that both had the same level of difficulty in identifying the ice channels feature. This can be due to the extremely small scale at which these features were viewed. Ice channels typically consist of very narrow lines indicating the ice-breakers routes that extend from one port to another. The presence of vessels along these routes may help analysts in the identification of such features. FDM values (constantly less than 500 ms) were recorded over open ice and very open ice. 10 Interestingly, when identifying sea ice channels, both experts recorded an average FDM of 776 ms. 4.2.2 Experts and SAR image sea ice classification This slight increase in FDM values recorded for the default scale view may relate to the increase in difficulty in 15 ice type recognition when the SAR image is viewed at lower scale. Again, to show that dwell time is not necessarily related to the difficulty in the ice type recognition, here the computed average dwell time was 8 seconds per ice type for both default and increased scale view. 4.2.2 Experts and SAR image sea ice classification Both experts had the longest dwell time (i.e., the visual time spent classifying an ice category or feature) while looking at 25 the most feature rich areas, see Table 7. The dwell time per ice category or feature viewed varied between the two experts. E1 spent in average 9 seconds over very close ice and 8 seconds for both level ice and fast ice. E2 spent the longest dwell time of 16 seconds interpreting close ice category. E2 also recorded 11 seconds over fast ice region. Even though, fast ice may present in general a more uniform surface, in some cases the presence of islands, vessels and ice channels may keep the analysts’ gaze Both experts had the longest dwell time (i.e., the visual time spent classifying an ice category or feature) while looking at 25 the most feature rich areas, see Table 7. The dwell time per ice category or feature viewed varied between the two experts. E1 spent in average 9 seconds over very close ice and 8 seconds for both level ice and fast ice. E2 spent the longest dwell time of 16 seconds interpreting close ice category. E2 also recorded 11 seconds over fast ice region. Even though, fast ice may present in general a more uniform surface, in some cases the presence of islands, vessels and ice channels may keep the analysts’ gaze for longer in the area. Very close ice and fast ice regions were the two most dominant ice classes in all of the SAR images 30 viewed. In contrast, the shortest dwell times were recorded over areas that may be considered easy for navigation. E1 recorded the shortest dwell time of 4 s over open water, 5 s over open ice, new ice and close ice areas, and 6 s for very open ice areas. E2 recorded shortest dwell time of 5 s over level ice areas, followed by open ice and new ice areas for which he spent 6 s in average. However, E2 required as much as 7 s of dwell time over very open ice and very close ice regions. This may be 35 17 https://doi.org/10.5194/tc-2022-8 Preprint. Discussion started: 25 February 2022 c⃝Author(s) 2022. CC BY 4.0 License. 4.2.2 Experts and SAR image sea ice classification To see whether the increase of scale used by E2 affected his gaze and subsequently its difficulty in identifying and classifying To see whether the increase of scale used by E2 affected his gaze and subsequently its difficulty in identifying and classifying ice types in the SAR imagery, we compared the average FDM values recorded over the recognized ice categories during default 10 view (DV) and zoomed-in view (ZV) for all five SAR images. The average fixation duration mean computed during default scale is higher (7xx ms) than the one during increased scale view (5xx ms). Out of 88 sea ice types identified by E2, 37 ice regions were identified during default view with an average FDM of 720 ms recorded per ice type. 51 ice regions were identified during zoomed view with an average FDM of 551 ms ice types in the SAR imagery, we compared the average FDM values recorded over the recognized ice categories during default 10 view (DV) and zoomed-in view (ZV) for all five SAR images. The average fixation duration mean computed during default scale is higher (7xx ms) than the one during increased scale view (5xx ms). Out of 88 sea ice types identified by E2, 37 ice regions were identified during default view with an average FDM of 720 ms recorded per ice type. 51 ice regions were identified during zoomed view with an average FDM of 551 ms per ice region. This slight increase in FDM values recorded for the default scale view may relate to the increase in difficulty in 15 ice type recognition when the SAR image is viewed at lower scale. Again, to show that dwell time is not necessarily related to the difficulty in the ice type recognition, here the computed average dwell time was 8 seconds per ice type for both default and increased scale view. per ice region. This slight increase in FDM values recorded for the default scale view may relate to the increase in difficulty in 15 ice type recognition when the SAR image is viewed at lower scale. Again, to show that dwell time is not necessarily related to the difficulty in the ice type recognition, here the computed average dwell time was 8 seconds per ice type for both default and increased scale view. per ice region. 4.2.2 Experts and SAR image sea ice classification explained by the fact that both ice categories may consist of higher amount of features, either ridges, leads or cracks in the very close ice pack or a mixture of ice floes of different thicknesses in the very open ice regions. explained by the fact that both ice categories may consist of higher amount of features, either ridges, leads or cracks in the very close ice pack or a mixture of ice floes of different thicknesses in the very open ice regions. Interestingly, E1 identified only one close ice region which had similar gaze values as for the easy to navigate areas while spending also very little time analyzing it (5 s). In contrast, E2 identified three regions of close ice for which he also recorded the longest FDM and the highest number of fixations compared with all other ice categories identified. These differences may 5 have occurred due to the extra time spent analyzing the SAR images together with the increase in scale used by E2. Nonetheless, this result underlines the subjectivity and difference in perception and visual interpretation of the SAR image sea ice features when no additional information is available to the analysts. p g y y g ( ) , g the longest FDM and the highest number of fixations compared with all other ice categories identified. These differences may 5 have occurred due to the extra time spent analyzing the SAR images together with the increase in scale used by E2. Nonetheless, this result underlines the subjectivity and difference in perception and visual interpretation of the SAR image sea ice features when no additional information is available to the analysts. the longest FDM and the highest number of fixations compared with all other ice categories identified. These differences may 5 have occurred due to the extra time spent analyzing the SAR images together with the increase in scale used by E2. Nonetheless, this result underlines the subjectivity and difference in perception and visual interpretation of the SAR image sea ice features when no additional information is available to the analysts. 5 To see whether the increase of scale used by E2 affected his gaze and subsequently its difficulty in identifying and classifying ice types in the SAR imagery, we compared the average FDM values recorded over the recognized ice categories during default 10 view (DV) and zoomed-in view (ZV) for all five SAR images. 4.2.3 Discrepancies in ice information described by the two experts Beside the level of detail and duration of scanning and analysis of the presented SAR images, there are also discrepancies in 20 the ice information depicted and described by the two experts while looking at the same sea ice features in the SAR images. One of these examples is discussed here. During the analysis of the SAR image 5, E2 focused on an area (A-d), which seemed more difficult to analyze than other it. This segment of analysis, is deconstructed in Figure 6 where the gaze plots associated with each identified (or unidentified) 25 ice region are shown. First sub-segment (1) constitutes of 18 fixations recorded in 8 s. From these, 12 points are concentrated around an area with uniform backscatter and no distinct ice features, having a similar texture and contrast as the open-water area present in the southern Gulf of Finland. Here, the analyst notices an opening in the ice pack, but is unable to classify it as ice or open water due to lack of high contrast features. Then, he moves his attention to two more similar openings in vicinity (2), identified this 30 time as open ice in 7 s. At this moment, the analyst decided that these openings could belong to a larger area (3) that includes also the first identified opening, by virtually drawing its contour with the mouse cursor. Then, another polygon (4) is identified and shown with only five fixations in 4 s, polygon which was not classified nor well defined its contour. Lastly, the polygon due to lack of high contrast features. Then, he moves his attention to two more similar openings in vicinity (2), identified this 30 time as open ice in 7 s. At this moment, the analyst decided that these openings could belong to a larger area (3) that includes also the first identified opening, by virtually drawing its contour with the mouse cursor. Then, another polygon (4) is identified and shown with only five fixations in 4 s, polygon which was not classified nor well defined its contour. Lastly, the polygon 18 gure 6. RadarSat-2 SAR image 5 acquired over the Gulf of Finland. 4.2.3 Discrepancies in ice information described by the two experts Upper left corner: Original image; a) Zoomed-in view (130%) as s E2; b) gaze plot of E2 over an undefined sea ice area, the gaze plot here totals 33 seconds; this plot is divided in five sub-segment lows: 1 - the identification of an undefined area; 2 - identification of two smaller openings (open ice or very open ice); 3 - the contou e ice class region; 4 - the contour of another ice region; 5 - re-evaluated contour 3, identified as open ice or very open ice (3-4/10). c) ro ntours of each identified region, drawn by the authors. ps://doi.org/10.5194/tc-2022-8 eprint. Discussion started: 25 February 2022 Author(s) 2022. CC BY 4.0 License. Figure 6. RadarSat-2 SAR image 5 acquired over the Gulf of Finland. Upper left corner: Original image; a) Zoomed-in view (130%) as seen by E2; b) gaze plot of E2 over an undefined sea ice area, the gaze plot here totals 33 seconds; this plot is divided in five sub-segments as follows: 1 - the identification of an undefined area; 2 - identification of two smaller openings (open ice or very open ice); 3 - the contour of one ice class region; 4 - the contour of another ice region; 5 - re-evaluated contour 3, identified as open ice or very open ice (3-4/10). c) rough contours of each identified region, drawn by the authors. 3 is virtually re-drawn while becoming polygon 5, classified by the analyst as open ice or very open ice 3-4/10 during 10 s of analysis. 19 https://doi.org/10.5194/tc-2022-8 Preprint. Discussion started: 25 February 2022 c⃝Author(s) 2022. CC BY 4.0 License. By looking at these sequences of data, a clear difference in the gaze density of E2 occurred for the case when he is not able to classify an area (1) and for the cases when he does (2-5). A higher gaze density occurred while trying to classify region (1), while a lower gaze density was recorded for the other areas. The most comparable are sequence (1) and (2), where the analyst is classifying similar type of openings (open ice). Here, he spends about the same amount of time (8 and 7 s) but his gaze points have a wider spread over the region 2 which he identifies as open ice. 4.2.3 Discrepancies in ice information described by the two experts Region (3) and (5) are mostly constituted of fixations 5 during the analyst indicating the polygon’s boundary, which he identifies it with open ice or very open ice. In these cases, the points are distributed more equal and having similar duration. 5 Table 8. Fixation measures computed for E2 during analysis of the region with longer fixations, of the RADARSAT-2 SAR image show in Figure 6. Table 8. Fixation measures computed for E2 during analysis of the region with longer fixations, of the RADARSAT-2 SAR image show in Figure 6. Table 8. Fixation measures computed for E2 during analysis of the region with longer fixations, of the RADARSAT-2 SAR image show in Figure 6. segment dwell time (s) FDM (ms) STD (ms) N 1 8 442 325 18 2 7 396 201 17 3 4 329 102 13 4 4 271 100 13 5 10 360 119 28 On the other hand, by looking at the average fixation duration mean and standard deviation values over the same region (see table 8), we notice that the analyst recorded here the lowest values (in average 360 ms for FDM and 169 ms for STD) (see table 8), we notice that the analyst recorded here the lowest values (in average 360 ms for FDM and 169 ms for STD) compared with all other ice types identified in all SAR images viewed (in average 659 ms for FDM with 485 ms for STD and a 10 minimum of 549 ms for FDM and 347 ms for STD). This once again shows that when an area in SAR presents a high level of uniformity and lacks features with high informational value, the analyst will not spend longer fixations over that area trying to extract more information. This kind of result supports also our previously stated assumption that longer fixations correspond to those ice features rich in information that carry critical value to the analysis. On the contrary, E1 did not mention the A-d compared with all other ice types identified in all SAR images viewed (in average 659 ms for FDM with 485 ms for STD and a 10 minimum of 549 ms for FDM and 347 ms for STD). 4.2.3 Discrepancies in ice information described by the two experts This once again shows that when an area in SAR presents a high level of uniformity and lacks features with high informational value, the analyst will not spend longer fixations over that area trying to extract more information. This kind of result supports also our previously stated assumption that longer fixations correspond to those ice features rich in information that carry critical value to the analysis. On the contrary, E1 did not mention the A-d 10 region at all, nor spent any time gazing at it. Only few of E1’ gaze points fell onto this area, during the whole image inspection. 15 More precisely, we recorded only one fixation point during the DV scan of this SAR image, with a duration of only 332 ms and seven fixations during the ZV analysis, while describing the "consolidated ice or fast ice" region in vicinity. This can be explained by the fact that lack of features did not attract E1’ attention, therefore E1 spent extremely little time on it without even describing it. Long dwell time (33 s) over difficult area (Figure 6) shows that E2 was trying to find some relevant features that could help 20 to describe better the viewed ice covered area but short fixations together with verbal explanation reveal the lack of value-added features, leading to a final classification of the area which does not completely agree with the classification made by E1 nor the classification found in the official FIS ice chart. Long dwell time (33 s) over difficult area (Figure 6) shows that E2 was trying to find some relevant features that could help 20 to describe better the viewed ice covered area but short fixations together with verbal explanation reveal the lack of value-added features, leading to a final classification of the area which does not completely agree with the classification made by E1 nor the classification found in the official FIS ice chart. 20 igure 7. (a) An extract from the FIS ice chart (© Finnish Meteorological Institute) constructed with the RADARSAT-2 SAR im her data by two on duty analysts, other than the ones who participated in this study. Main sea ice boundaries drawn by E1 (b) a dependently of the eye movement data collection, using only the SAR image 5. Figure 7. 4.2.4 FIS ice chart vs. Experts Agreement is good over open water or ice free region in the south of GoF. However, disagreements are seen especially for ice types such as close ice, open ice, very open ice and level ice, where the analysts disagree. Not knowing the real sea ice conditions or the previous day situation, naturally explains these differences between experts’ analysis and the classification in 15 the FIS ice chart, especially for the lower ice concentration regions (open ice or less). However, in the case of open ice areas marked in the FIS ice chart, experts had contradictory interpretations, especially for the region close to the Estonian coast (low left side of Figure 6). E1 having marked the area as open water while E2 has marked the same area with very close ice. This is a considerable difference from the navigation point of view, since one expert is basically permitting independent navigation 4.2.3 Discrepancies in ice information described by the two experts (a) An extract from the FIS ice chart (© Finnish Meteorological Institute) constructed with the RADARSAT-2 SAR image 5 and other data by two on duty analysts, other than the ones who participated in this study. Main sea ice boundaries drawn by E1 (b) and E2 (c) independently of the eye movement data collection, using only the SAR image 5. Figure 7. (a) An extract from the FIS ice chart (© Finnish Meteorological Institute) constructed with the RADARSAT-2 SAR image 5 and other data by two on duty analysts, other than the ones who participated in this study. Main sea ice boundaries drawn by E1 (b) and E2 (c) independently of the eye movement data collection, using only the SAR image 5. 21 https://doi.org/10.5194/tc-2022-8 Preprint. Discussion started: 25 February 2022 c⃝Author(s) 2022. CC BY 4.0 License. 4.2.4 FIS ice chart vs. Experts To evaluate how the blind analysis of the SAR image sea ice features performed by the two experts would compare with the classification provided in the FIS ice chart, we asked the experts to draw the contours of the main ice regions and assign an ice class for each region in the SAR image 5. Their results are presented in Figure 7, together with the corresponding (next day) FIS ice chart (constructed by two analysts during their official duty with the SAR images used here as one of their several 5 information sources). To evaluate how the blind analysis of the SAR image sea ice features performed by the two experts would compare with the classification provided in the FIS ice chart, we asked the experts to draw the contours of the main ice regions and assign an ice class for each region in the SAR image 5. Their results are presented in Figure 7, together with the corresponding (next day) FIS ice chart (constructed by two analysts during their official duty with the SAR images used here as one of their several 5 information sources). The ice chart is showing the ice situation in the Gulf of Finland (GoF) contoured based on all sources of information available at that time. The region of frozen sea that separates the very thin ice and open water area from the fast ice in the northern part of GoF, is classified here as very close ice, rafted including two very open ice areas (same open ice areas were described by The ice chart is showing the ice situation in the Gulf of Finland (GoF) contoured based on all sources of information available at that time. The region of frozen sea that separates the very thin ice and open water area from the fast ice in the northern part of GoF, is classified here as very close ice, rafted including two very open ice areas (same open ice areas were described by E2) and also few small areas marked as level ice (purple color). There is also two open ice areas marked with yellow color. 10 When comparing the ice chart to the ice regions marked by the two experts, we notice a general agreement between the two, especially for the most dominant ice types, such as land fast ice and very close ice covering significant area along the coast. 4.3 Fixations statistics and their relation to SAR image complexity We studied the dependence of the fixation duration for the two experts as a function of SAR image complexity by computing the local edge and corner densities in the SAR images. Edge and corner points in the SAR imagery were detected by applying an edge and corner algorithm based on local binary patterns described in Karvonen (2013). Because the locations of the We studied the dependence of the fixation duration for the two experts as a function of SAR image complexity by computing the local edge and corner densities in the SAR images. Edge and corner points in the SAR imagery were detected by applying an edge and corner algorithm based on local binary patterns described in Karvonen (2013). Because the locations of the fixations are often not exactly at the target we studied the numbers of edge and corner points within two different circular 25 neighborhoods with radius of 20 or 50 SAR pixels from a fixation point. The fixation locations may not exactly match the centre of the region of interest because of possible measuring inaccuracies and also due to the fact that the human visual acuity at the foveal center corresponds to more than just one pixel at the centre of the region of interest, i.e., the foveal vision corresponds to approximately 2 degrees of the entire visual field (Duchowski, 2017). These comparisons were made using only the default scale images 30 fixations are often not exactly at the target we studied the numbers of edge and corner points within two different circular 25 neighborhoods with radius of 20 or 50 SAR pixels from a fixation point. The fixation locations may not exactly match the centre of the region of interest because of possible measuring inaccuracies and also due to the fact that the human visual acuity at the foveal center corresponds to more than just one pixel at the centre of the region of interest, i.e., the foveal vision corresponds to approximately 2 degrees of the entire visual field (Duchowski, 2017). These comparisons were made using only fixations are often not exactly at the target we studied the numbers of edge and corner points within two different circular 25 neighborhoods with radius of 20 or 50 SAR pixels from a fixation point. 4.3 Fixations statistics and their relation to SAR image complexity The fixation locations may not exactly match the centre of the region of interest because of possible measuring inaccuracies and also due to the fact that the human visual acuity at the foveal center corresponds to more than just one pixel at the centre of the region of interest, i.e., the foveal vision corresponds to approximately 2 degrees of the entire visual field (Duchowski, 2017). These comparisons were made using only the default scale images. 30 The results did not indicate very strong correlation between the fixation duration and the SAR image complexity. For the shorter fixation durations there was a large deviation in the image complexity. However, when divided into two duration categories of less than 500 ms and over 500 ms, the image complexity was about 10% higher for the longer duration category, 22 https://doi.org/10.5194/tc-2022-8 Preprint. Discussion started: 25 February 2022 c⃝Author(s) 2022. CC BY 4.0 License. measured both by number of edge points and corner points and for the both 20 and 50 pixel radius. We also noticed that for fixations longer than 2000 ms the complexity was always above a certain limit but for the shorter fixations the range of the image complexity was varying a lot below and above the limit value. measured both by number of edge points and corner points and for the both 20 and 50 pixel radius. We also noticed that for fixations longer than 2000 ms the complexity was always above a certain limit but for the shorter fixations the range of the image complexity was varying a lot below and above the limit value. We also analyzed the fixations statistics for each ice class looked at (the same sea ice cate We also analyzed the fixations statistics for each ice class looked at (the same sea ice categories presented previously in We also analyzed the fixations statistics for each ice class looked at (the same sea ice categories presented previously in Table 7) by the two experts. We computed the average and standard deviation of fixations duration for each ice class, and also 5 included the ratio of the total fixation time within each ice type segment and the ice type segment area. The segment area was defined by the convex polygon spanned by the segment points. The results of this analysis are shown in Table 9. 4.3 Fixations statistics and their relation to SAR image complexity and E2 to open water. This is a surprising result as open water typically does not contain much features, i.e. the SAR image 10 complexity for open water areas is typically low. However, distinguishing between thin or level ice and open water is often a difficult task and may require a lot of attention. 4.3 Fixations statistics and their relation to SAR image complexity It can be seen that in total E1 spent most time in recognizing ice channels and E2 in recognizing new ice. But when looking at the ratio of total fixation time to the segment area, the very open ice segments E1 seems to pay attention especially to very open ice and E2 to open water. This is a surprising result as open water typically does not contain much features, i.e. the SAR image 10 complexity for open water areas is typically low. However, distinguishing between thin or level ice and open water is often a difficult task and may require a lot of attention. y ( g p p y Table 7) by the two experts. We computed the average and standard deviation of fixations duration for each ice class, and also 5 included the ratio of the total fixation time within each ice type segment and the ice type segment area. The segment area was defined by the convex polygon spanned by the segment points. The results of this analysis are shown in Table 9. It can be seen that in total E1 spent most time in recognizing ice channels and E2 in recognizing new ice. But when looking at the ratio of total fixation time to the segment area, the very open ice segments E1 seems to pay attention especially to very open ice Table 7) by the two experts. We computed the average and standard deviation of fixations duration for each ice class, and also 5 included the ratio of the total fixation time within each ice type segment and the ice type segment area. The segment area was defined by the convex polygon spanned by the segment points. The results of this analysis are shown in Table 9. It can be seen that in total E1 spent most time in recognizing ice channels and E2 in recognizing new ice. But when looking at the ratio of total fixation time to the segment area, the very open ice segments E1 seems to pay attention especially to very open ice and E2 to open water. This is a surprising result as open water typically does not contain much features, i.e. the SAR image 10 complexity for open water areas is typically low. However, distinguishing between thin or level ice and open water is often a difficult task and may require a lot of attention. Table 9. Fixations statistics for the ice classes of the SAR image segments. 5 and the fixation duration images for the experts E1 and E2 corresponding to SAR1 are presented in Figure 8. It can be seen that 5 both E1 and E2 use relatively more time in the northern parts of the image with most of the sea ice. However, there also exist significant differences between E1’s and E2’s behaviors.The correlations between the quantities measuring image complexity (edge and corner densities) and the E1 and E2 segment-wise relative fixation duration were rather low, around 0.2, but still clearly positive. One evident reason for the low correlation and correspondence of the fixations duration with the image complexity is that 10 the fixation typically concentrate on the SAR segment boundaries, and thus often their location is within adjacent segments of the actual target segment. 10 Table 9. Fixations statistics for the ice classes of the SAR image segments. Table 9. Fixations statistics for the ice classes of the SAR image segments. Table 9. Fixations statistics for the ice classes of the SAR image segments. Expert N average STD rel time ice type E1 NA NA NA NA open-water 13 472.54 250.26 0.52 very open ice NA NA NA NA open ice 10 471.20 226.56 0.20 close ice 37 483.86 257.70 0.22 very close ice 12 484.08 231.25 0.21 new ice 52 719.06 733.47 0.18 level-ice 51 466.00 314.10 0.16 fast ice 13 606.77 400.93 0.20 brash ice barrier 19 894.89 1109.44 0.14 ice channels E2 151 663.48 619.91 0.31 open-water NA NA NA NA very open ice 25 492.72 278.92 0.12 open ice NA NA NA NA close ice 85 610.31 387.47 0.18 very close ice 41 820.98 1060.46 0.25 new ice 47 569.51 366.80 0.14 level-ice 104 568.10 510.36 0.28 fast ice 38 672.92 461.84 0.19 brash ice barrier 47 694.38 649.13 0.19 ice channels or the ice classes of the SAR image segments. Expert N average STD rel time ice type E1 NA NA NA NA open-water 13 472.54 250.26 0.52 very open ice NA NA NA NA open ice 10 471.20 226.56 0.20 close ice 37 483.86 257.70 0.22 very close ice 12 484.08 231.25 0.21 new ice 52 719.06 733.47 0.18 level-ice 51 466.00 314.10 0.16 fast ice 13 606.77 400.93 0.20 brash ice barrier 19 894.89 1109.44 0.14 ice channels E2 151 663.48 619.91 0.31 open-water NA NA NA NA very open ice 25 492.72 278.92 0.12 open ice NA NA NA NA close ice 85 610.31 387.47 0.18 very close ice 41 820.98 1060.46 0.25 new ice 47 569.51 366.80 0.14 level-ice 104 568.10 510.36 0.28 fast ice 38 672.92 461.84 0.19 brash ice barrier 47 694.38 649.13 0.19 ice channels 23 https://doi.org/10.5194/tc-2022-8 Preprint. Discussion started: 25 February 2022 c⃝Author(s) 2022. CC BY 4.0 License. We also performed a SAR segmentation applying the Iterative Cumulative Model (ICM) algorithm (Besag , 1986) and then computed the number of edge and corner pixels within each segment normalized by the segment area (in pixels). These ratios characterize the local complexity of the SAR image. These were then visually compared to the total duration of the fixations within each segment, also normalized by the segment size. Table 9. Fixations statistics for the ice classes of the SAR image segments. The resulting segmentation and the edge point, corner point images and the fixation duration images for the experts E1 and E2 corresponding to SAR1 are presented in Figure 8. It can be seen that 5 both E1 and E2 use relatively more time in the northern parts of the image with most of the sea ice. However, there also exist We also performed a SAR segmentation applying the Iterative Cumulative Model (ICM) algorithm (Besag , 1986) and then computed the number of edge and corner pixels within each segment normalized by the segment area (in pixels). These ratios characterize the local complexity of the SAR image. These were then visually compared to the total duration of the fixations within each segment, also normalized by the segment size. The resulting segmentation and the edge point, corner point images We also performed a SAR segmentation applying the Iterative Cumulative Model (ICM) algorithm (Besag , 1986) and then computed the number of edge and corner pixels within each segment normalized by the segment area (in pixels). These ratios characterize the local complexity of the SAR image. These were then visually compared to the total duration of the fixations within each segment, also normalized by the segment size. The resulting segmentation and the edge point, corner point images and the fixation duration images for the experts E1 and E2 corresponding to SAR1 are presented in Figure 8. It can be seen that 5 both E1 and E2 use relatively more time in the northern parts of the image with most of the sea ice. However, there also exist significant differences between E1’s and E2’s behaviors.The correlations between the quantities measuring image complexity (edge and corner densities) and the E1 and E2 segment-wise relative fixation duration were rather low, around 0.2, but still clearly positive. and the fixation duration images for the experts E1 and E2 corresponding to SAR1 are presented in Figure 8. It can be seen that 5 both E1 and E2 use relatively more time in the northern parts of the image with most of the sea ice. However, there also exist significant differences between E1’s and E2’s behaviors.The correlations between the quantities measuring image complexity (edge and corner densities) and the E1 and E2 segment-wise relative fixation duration were rather low, around 0.2, but still clearly positive. 5 Discussion and conclusions In this study we demonstrated for the first time that the eye tracking methodology can be used to identify sea ice regions or features in SAR images which are prone to human subjectivity, and therefore miss-classification. 15 While restricting our study to the Baltic Sea region, we asked two experts in the sea ice charting to look at a set of five RADARSAT-2 ScanSAR images and visually identify the sea ice types and features without any other sea ice information available to them. Experts were able to correctly classify the most dominant sea ice types in the viewed SAR images, such as very close ice and fast ice (i.e., large scale sea ice conditions). These are the two most restrictive ice categories for navigation, allowing passage only with the ice-breaker assistance. On the other hand, differences in classification occurred especially for 20 less restrictive ice classes such as close ice, level ice, open ice, very open ice and open water. While these areas are not restricted to independent navigation for lower ice class vessels, navigation in these ice conditions may be more challenging. Eye movement data measures such as the fixation duration mean (FDM), dwell time and fixation density were found to be extremely informative and directly related to the difficulty in interpretation of sea ice types or features viewed. We found allowing passage only with the ice-breaker assistance. On the other hand, differences in classification occurred especially for 20 less restrictive ice classes such as close ice, level ice, open ice, very open ice and open water. While these areas are not restricted to independent navigation for lower ice class vessels, navigation in these ice conditions may be more challenging. Eye movement data measures such as the fixation duration mean (FDM), dwell time and fixation density were found to be extremely informative and directly related to the difficulty in interpretation of sea ice types or features viewed. We found that FDM values of experts analyzing SAR images vary from 0.4 seconds (similar value was recorded for easy to recognize 25 objects in non-SAR stimuli) to as much as one second, depending on the features looked at. Higher FDM values were recorded especially over sea ice regions where experts had more difficulty in estimating the ice thickness. 5 Discussion and conclusions Individual differences between the two experts can be seen as slight increase in the gaze values computed for E2 in contrast t E1 F ll th t d f th t t l d th t E1 i f t d id tifi th i t d that FDM values of experts analyzing SAR images vary from 0.4 seconds (similar value was recorded for easy to recognize 25 objects in non-SAR stimuli) to as much as one second, depending on the features looked at. Higher FDM values were recorded especially over sea ice regions where experts had more difficulty in estimating the ice thickness. features in SAR images with less effort than E2. This result may be related to the higher expertise level for E1 and differences 30 in the ice charting training routines when compared to E2. Our data suggested that experts rely on changes in uniformity / homogeneity in an ice covered region to better understand its characteristics during the analysis. Thus, the more complex features an ice region presents (i.e., the more non homogeneous features in SAR images with less effort than E2. This result may be related to the higher expertise level for E1 and differences 30 in the ice charting training routines when compared to E2. Our data suggested that experts rely on changes in uniformity / homogeneity in an ice covered region to better understand its characteristics during the analysis. Thus, the more complex features an ice region presents (i.e., the more non homogeneous 24 https://doi.org/10.5194/tc-2022-8 Preprint. Discussion started: 25 February 2022 c⃝Author(s) 2022. CC BY 4.0 License. e 8. RADARSAT-2 SAR image 1 segmentation (a), edge density (b), corner density (c), ratio of the total fixation duration within nts normalized by the segment size for E1 (d) and for E2 (e). The color of the images indicates the relative amount of edges and co ents for (b) and (c). The value for (d) and (e) is scaled such that it is 100Tt/A, where Tt is the total time within a segment and e of the segment in pixels. gion is), the more attention and effort is required by experts to classify it. Their cognitive effort which often mixed effort of discriminating those features and associate them with characteristics of ice, can be identified by longer Figure 8. 5 Discussion and conclusions Their gaze also changed rapidly from one area to another, in many cases not concluding the analysis of an area with a clear ice definition. Global warming and the thinning of the Arctic sea ice will result in sea ice conditions that will be more challenging to analyze. In future, ice charts will contain more of lower ice categories and less of large consolidated ice areas than currently. Based on these facts, we conclude that manual ice charting requires more accurate classification of low ice concentration 20 regions which may facilitate independent navigation, i.e. open ice and very open ice. The subjectivity and the source of miss- classification should be measured and recorded, so that future ice charts will be not only more consistent, but more reliable for safe navigation in sea ice covered oceans. Based on these facts, we conclude that manual ice charting requires more accurate classification of low ice concentration 20 regions which may facilitate independent navigation, i.e. open ice and very open ice. The subjectivity and the source of miss- classification should be measured and recorded, so that future ice charts will be not only more consistent, but more reliable for safe navigation in sea ice covered oceans. 5 Discussion and conclusions RADARSAT-2 SAR image 1 segmentation (a), edge density (b), corner density (c), ratio of the total fixation duration within SAR segments normalized by the segment size for E1 (d) and for E2 (e). The color of the images indicates the relative amount of edges and corners in percents for (b) and (c). The value for (d) and (e) is scaled such that it is 100Tt/A, where Tt is the total time within a segment and A is the size of the segment in pixels. the region is), the more attention and effort is required by experts to classify it. Their cognitive effort which often mixed with visual effort of discriminating those features and associate them with characteristics of ice, can be identified by longer gaze 25 https://doi.org/10.5194/tc-2022-8 Preprint. Discussion started: 25 February 2022 c⃝Author(s) 2022. CC BY 4.0 License. duration or larger fixation mean over a feature or set of features. The number of salient features in an ice covered area, their scale and geographical spread are also important factors influencing the eye movements of experts in the SAR image analysis. This study is the first to suggest a method of identifying the areas of sea ice in the SAR imagery with the lowest confidence for the analysts that are subject to miss-classification. duration or larger fixation mean over a feature or set of features. The number of salient features in an ice covered area, their scale and geographical spread are also important factors influencing the eye movements of experts in the SAR image analysis. This study is the first to suggest a method of identifying the areas of sea ice in the SAR imagery with the lowest confidence for the analysts that are subject to miss-classification. Interaction with the SAR imagery is also an important aspect in the ice charting that we looked at. In this case, we have asked 5 also two novice analysts to join the experiment by visually interpreting the sea ice conditions in the same set of SAR images. In some cases users changed the scale of the viewed images several times during analysis. Novices rushed to increase the scale shortly after the images were displayed on the screen. At higher scales however, they still had difficulty in distinguishing between different ice types and features, but spending more time on panning and zooming the content. 5 Discussion and conclusions Interaction with the SAR imagery is also an important aspect in the ice charting that we looked at. In this case, we have asked 5 also two novice analysts to join the experiment by visually interpreting the sea ice conditions in the same set of SAR images. In some cases users changed the scale of the viewed images several times during analysis. Novices rushed to increase the scale shortly after the images were displayed on the screen. At higher scales however, they still had difficulty in distinguishing between different ice types and features, but spending more time on panning and zooming the content. 5 Experts analyzing a SAR frame focus especially on key ice features that are meaningful for the navigation, such as ice 10 edges, ridges, leads and ice channels, while spending very little time or none over areas with uniform backscatter. This is in line with previous research on expertise showing that experts focus on relevant information, extending this knowledge to the SAR imageinterpretation domain. In contrast, novices had difficulties recognizing sea ice features and in many cases their answers were confusing, unclear and required more assistive questions. Their lack of expertise also reflected in an unfocused Experts analyzing a SAR frame focus especially on key ice features that are meaningful for the navigation, such as ice 10 edges, ridges, leads and ice channels, while spending very little time or none over areas with uniform backscatter. This is in line with previous research on expertise showing that experts focus on relevant information, extending this knowledge to the SAR imageinterpretation domain. In contrast, novices had difficulties recognizing sea ice features and in many cases their answers were confusing, unclear and required more assistive questions. Their lack of expertise also reflected in an unfocused gaze, spending significant amount of time looking at islands or ice floes confused as islands, ice edges or even homogeneous 15 areas of open water. Their gaze also changed rapidly from one area to another, in many cases not concluding the analysis of an area with a clear ice definition. Global warming and the thinning of the Arctic sea ice will result in sea ice conditions that will be more challenging to analyze. In future, ice charts will contain more of lower ice categories and less of large consolidated ice areas than currently. areas of open water. 5.1 Limitations and Future work and to record the fixation coordinates in the original full-scale image to enable a thorough analysis with all the fixation data integrated in the same master image. and to record the fixation coordinates in the original full-scale image to enable a thorough analysis with all the fixation data integrated in the same master image. Furthermore, eye tracking data acquired during SAR image analysis could indicate the complicated ice areas where higher resolution data is required or when the information available is not sufficient for a reliable classification. As a hypothesis, long fixation duration are connected with larger uncertainties in the final ice charts. Because the ice charts at the moment lack uncertainty information, this is a very interesting topic that should be studied more. 5 The results presented here open new horizons for improving both manual and automatic analysis of SAR imagery for sea ice classification, but also for image classification in general, where the link between the observer and the automated method has not yet been established. Acknowledgements. Authors would like to thank Tobii AB (former Tobii Technology AB) company for providing the eye tracker and the 10 software for this study. Their software was also used in part for data analysis, and preparation of some of the examples shown. Authors would like to thank all the participants in the study for their contributions. Acknowledgements. Authors would like to thank Tobii AB (former Tobii Technology AB) company for providing the eye tracker and the 10 software for this study. Their software was also used in part for data analysis, and preparation of some of the examples shown. Authors would like to thank all the participants in the study for their contributions. Author contributions. 5.1 Limitations and Future work Work presented here is a first effort trying to understand the eye movements of a sea ice analyst looking at a SAR image. 25 The sample number in our study is low, and thus, the main findings are qualitative in nature. More quantitative and qualitative studies are required to better understand how sea ice parameters are visually interpreted by the experts and novices in the ice charting. Repeating this study with sea ice experts from several different organizations producing ice charts could reveal interesting insights into differences in the cultures of the SAR image analysis in these organizations. In general, we encourage the use of eye movement data in further studies to deepen this kind of knowledge and to understand the uncertainties introduced 30 by human analysts in the operational ice charting the use of eye movement data in further studies to deepen this kind of knowledge and to understand the uncertainties introduced 30 by human analysts in the operational ice charting. The aim of this study was to act as a proof of concept study. The deficiencies of the experiment design in this study can be improved based on the experience gained by this study for the related future research. More SAR data and more ice analysts are needed for a comprehensive study. And a more sophisticated software keeping account of all the zoom-ins, panning, cropping 26 https://doi.org/10.5194/tc-2022-8 Preprint. Discussion started: 25 February 2022 c⃝Author(s) 2022. CC BY 4.0 License. and to record the fixation coordinates in the original full-scale image to enable a thorough analysis with all the fixation da integrated in the same master image. Furthermore, eye tracking data acquired during SAR image analysis could indicate the complicated ice areas where high resolution data is required or when the information available is not sufficient for a reliable classification. As a hypothesi long fixation duration are connected with larger uncertainties in the final ice charts. Because the ice charts at the moment lac 5 uncertainty information, this is a very interesting topic that should be studied more. The results presented here open new horizons for improving both manual and automatic analysis of SAR imagery for se ice classification, but also for image classification in general, where the link between the observer and the automated metho has not yet been established. The authors declare that they have no conflict of interest. Author contributions. The concept of the study was conceived by AG. JV was one of the expert analysts mentioned in this study who helped with defining the research questions by providing insights into the ice charting practices and underlying existing challenges in the 15 ice charting process. JV also selected the SAR imagery and helped with the interpretation of the results. The data collection was performed by AG, who also conducted the analysis. ER and RB also helped with the data analysis. AG, ER, JV, JK and MM contributed to the interpretation of the results and drawing the conclusions. All authors contributed to the writing and editing of the text. 20 Competing interests. 20 Competing interests. The authors declare that they have no conflict of interest. 27 27 https://doi.org/10.5194/tc-2022-8 Preprint. Discussion started: 25 February 2022 c⃝Author(s) 2022. CC BY 4.0 License. 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JCOMM Expert Team on Sea Ice (2014) Sea-Ice Nomenclature: snapshot of the WMO Sea Ice Nomenclature WMO No. 259, volume 1 – Terminology and Codes; Volume II – Illustrated Glossary and III – International System of Sea-Ice Symbols) . Geneva, Switzerland, WMO-JCOMM, [121pp.] . (WMO-No. 259 (I-III)). DOI: https://doi.org/10.25607/OBP-1515 20 Zakhvatkina, N.; Smirnov, V.; Bychkova, I. Satellite SAR Data-based Sea Ice Classification: An Overview. Geosciences 2019, 9, 152. https://doi.org/10.3390/geosciences9040152 29

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The relationship between autonomy support and structure in early childhood nature-based settings: Practices and challenges

Learning environments research

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Citation for published version (APA): Arvanitis, A., Barrable, A., & Touloumakos, A. (2023). The relationship between autonomy support and structure in early childhood nature-based settings: Practices and challenges. Learning Environments Research. Advance online publication. https://doi.org/10.1007/s10984-023-09470-0 University of Dundee The relationship between autonomy support and structure in early childhood nature- based settings Arvanitis, Alexios; Barrable, Alexia; Touloumakos, Anna Arvanitis, Alexios; Barrable, Alexia; Touloumakos, Anna Arvanitis, Alexios; Barrable, Alexia; Touloumakos, Anna Document Version Publisher's PDF, also known as Version of record Citation for published version (APA): Arvanitis, A., Barrable, A., & Touloumakos, A. (2023). The relationship between autonomy support and structure in early childhood nature-based settings: Practices and challenges. Learning Environments Research. Advance online publication. https://doi.org/10.1007/s10984-023-09470-0 General rights Copyright and moral rights for the publications made accessible in Discovery Research Portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights. Take down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. Download date: 24. Oct. 2024 Download date: 24. Oct. 2024 Learning Environments Research https://doi.org/10.1007/s10984-023-09470-0 ORIGINAL PAPER Abstract From a Self-Determination Theory perspective, children are expected to grow according to innate tendencies in supportive environments, such as nature-based educational settings. The role of the practitioner is generally viewed as facilitative in these contexts. We report findings from a qualitative study with a sample of 18 UK-based outdoor educational prac- titioners. We asked them to respond to four hypothetical situations with differing levels of required intervention. Our purpose was to examine the extent of autonomy support and structure that they would hypothetically provide to the children in each of these different scenarios, and even uncover possible tensions in the application of autonomy support and structure. Our analysis suggests that, in situations of low interventional requirement, prac- titioners could understate structure and autonomy support and, in situations of high inter- ventional requirement, prioritize structure over autonomy support. The challenge for prac- titioners that was revealed in this research, as well its implications, are discussed. Keywords  Autonomy support · Motivation · Nature · Nature-based education · Self- determination theory · Structure Keywords  Autonomy support · Motivation · Nature · Nature-based education · Self- determination theory · Structure Keywords  Autonomy support · Motivation · Nature · Nature-based education · Self- determination theory · Structure The relationship between autonomy support and structure in early childhood nature‑based settings: Practices and challenges Alexios Arvanitis1   · Alexia Barrable2 · Anna Κ. Touloumakos3 Received: 25 August 2022 / Accepted: 11 April 2023 © The Author(s) 2023 1 Department of Psychology, University of Crete, Rethymno, Greece 2 School of Humanities, Social Sciences and Law, University of Dundee, Dundee, UK 3 Department of Psychology, Panteion University of Social and Political Sciences, Athens, Greece Structure An important aspect of educational support involves structure, that is information that is given by adults to children to help them achieve desired outcomes (Skinner & Belmont, 1993). In order to master a challenge, a child needs to understand the nature of an activ- ity, as well as the process of improvement, and to have a step-by-step improvement plan in mind. Relevant information offered by adults is likely to aid in that direction. When climbing trees, children can climb taller or smaller trees depending on their abilities. Prac- titioners can give guidance in how children can pick certain trees that are appropriate for this activity, and share information on what makes a good tree to climb. They can also design and communicate safety protocols, sometimes with the children, and offer feedback that can lead to improvement. While subtle, the processes described above, involving guid- ance, feedback and risk-assessment are processes of structure. Another process of structure would be one often seen in more traditional settings like primary schools, where the chil- dren are not allowed to climb trees at all. A third approach would be lacking structure, such as the case in which practitioners lead children to the forest or park and leave them on their own to do as they wish without any guidance. In this instance, there is potential for injury and other undesirable outcomes. The word that potentially comes to mind in this case is ‘chaos’. If structure lies on one end of the continuum, chaos lies at the other end. In many educational settings and learning environments, structure is provided by a cur- ricular framework and clear learning activities set out by the adult but, in nature-based education programs such as forest school or nature nurseries, the concept of structure takes on a different form, possibly less clear to the practitioner. There are indeed some settings that acknowledge a focus on National Curriculum targets, but most have much more holis- tic goals in mind, such as personal, social and emotional development, the enhancement of self-esteem (Harris, 2017) and the development of a connection to the natural world (Barrable & Booth, 2020). In providing structure, the challenge for the practitioner is to transform these holistic goals into a guiding framework that serves as a basis for communi- cating clear expectations and rules to the children, as well as offering sufficient supervision and constructive feedback. Introduction Early childhood practitioners and parents often find it difficult to provide guidance and direction to children. In a variety of everyday activities and situations, from eating healthy food to gaining useful knowledge and from staying safe to engaging in self-care, children will not necessarily follow instructions or adopt behaviors that they are asked to. The ques- tion then becomes one of how children can be motivated to do so. Self-determination the- ory (SDT; Ryan & Deci, 2017) is a motivational theory that argues that children have natu- ral tendencies to grow, master challenges and integrate experiences into a coherent self. * Alexios Arvanitis [email protected] 123456789) 1 3 Learning Environments Research Once it is accepted that children are naturally inquisitive and growing organisms, the ques- tion that was asked above can be rephrased to one of how adults can channel the children’s inherent tendencies in ways that best serve their interest. Once it is accepted that children are naturally inquisitive and growing organisms, the ques- tion that was asked above can be rephrased to one of how adults can channel the children’s inherent tendencies in ways that best serve their interest. In nature-based educational settings, such as nature nurseries and forest schools, chil- dren find themselves in an environment that poses various challenges and offers poten- tial for growth (Barrable, 2019; Barrable & Arvanitis, 2019; Harris, 2018). Pedagogical practices that make use of the affordances of the natural space can promote conditions for supporting the child’s innate tendencies (Barrable, 2020; O’Brien & Murray, 2007). Practi- tioners in this context are viewed as facilitators for child-led activities. However, there is no guarantee that every child will indeed grow within this type of educational setting without proper support by the practitioner. The challenge for the practitioner becomes greater when children in specific contexts cannot independently behave according to innate tendencies or when they engage in inappropriate behaviors. Our objective in this research project is to identify how practitioners deal with the specific challenge of supporting children in out- door settings by focusing on two basic concepts, structure and autonomy support, as well as their intricate relationship. Autonomy support Autonomy support is best understood through an SDT lens if it is distinguished from lack of interference or the promotion of independence (Arvanitis & Kalliris, 2017; Ryan, 1993). Children are not supported if they are encouraged to be independent, but only if they are supported in behaving in accordance with their interests and values. These interests and values lie at the core of autonomous behavior. Supporting children to act on their own does not necessarily enhance autonomy because what children do independently might not reflect their true interests and inherent tendencies. This concerns adults as well. For example, research on emerging adults’ well-being (Kins et al., 2009) indicates the posi- tive effect of dependent living with family (compared to independent living) so long as their living arrangement reflects their values and needs (Kins et al., 2009). With regard to children, autonomy can equally be supported when decisions are made by important others and when they make the decisions themselves (Bao & Lam, 2008). Therefore, it is con- ceivable for children, even emerging adults, to feel autonomous in states of dependence. It is also possible for children to feel pressured in states of independence. Practitioners need to attune to the children in their care to understand their motivational states and react accordingly. Autonomy support can be thought of in terms of stages during education (Reeve, 2016). At an initial stage, it is necessary to take the students’ perspective; at a middle stage, to vitalize inner motivational resources and offer rationale for an activity; at a later stage, to display patience, acknowledge negative affect and rely on informational, non-controlling language to engage the child in an activity. As can easily be deduced, autonomy support is by no means a passive form of non-interference but an active and purposeful effort by the educator that aims at tapping into children’s interests in order to constructively engage them in self-growing activities. In nature-based settings, practitioners are viewed as facilitators for child-led activities (Barrable, 2020) and are there to support the child’s innate tendencies. In forest school, an example of a nature-based learning environment, Knight (2009) writes that “the learning is play based and, as far as possible, child-initiated and child-led” (pp. 16–17). How prac- titioners understand what it means to support child-led learning is unclear. Structure Without such a framework—and in the absence of a curricular framework—communication with children can be restricted to a set of instructions that 1 3 1 3 Learning Environments Research simply ensure safety or to an even more relaxed ‘laissez-faire’ attitude that allows children to do as they wish. Understanding the range of structure practices in nature-based settings is one of the main aims of this study. simply ensure safety or to an even more relaxed ‘laissez-faire’ attitude that allows children to do as they wish. Understanding the range of structure practices in nature-based settings is one of the main aims of this study. Structure and autonomy support The relationship between structure and autonomy support is complicated. Initially, chaos (that results from adult inaction) cannot be autonomy supportive because it is dif- ficult in these situations for children to act according to their interests. As the level of structure improves, there are differing levels of autonomy support with which it can be combined with. Moreover, the two seem to have a linear relationship (Jang et al., 2010). As structure improves, so does autonomy support. Table 1 offers an example for a clas- sification of educational tactics according to different levels of autonomy support and structure, capturing how autonomy support becomes stronger as structure improves. Regarding autonomy support, we have relied on Reeve (2016) to construct three lev- els of autonomy support: (a) low, with no attunement to the child, (b) medium, with the child offering of feedback, and (c) high, with acknowledgement of conflict, offer- ing choice, use of autonomy-supportive language and positive feedback. Reeve offers a distinction with regard to autonomy support that helps to better understand these levels. On the one hand, autonomy support aims to deliver the curriculum in a need-satisfying way so that learning outcomes are better served. This aim is rather effortlessly achieved in nature-based settings where there is no official curriculum involved and nature becomes a primary source of learning and growth. On the other hand, Reeve argues that autonomy support involves forming a dialectical two-way relationship in which the practitioner is in sync with the children and both are mutually responsive to each other. The more the communication between practitioner and child becomes transactional, the more there is ground for helping the child grow according to innate tendencies, espe- cially when the child encounters difficulties. This is why high autonomy support in our classification involves two-way communication between the practitioner and the child. Although Reeve also discusses controlling educational practices, we have not included any controlling practices in our classification because of the pedagogical ‘child-led’ philosophy of nature-based educational settings. Regarding structure, we have three levels also: (a) low, with no information given to the child, (b) medium, with instructions of low informational value offered to the child and (c) high, with a guiding framework of high informational value offered to the child. What sets the guiding framework apart from the instructions of low informational value is its derivation from holistic goals of personal, emotional and social development of the child. Autonomy support Harris (2017) touches upon this point by explaining how some practitioners highlight ‘the challenge of negotiated learning’ as a delicate balance between allowing children to shape their activi- ties and more structured practice, led by the practitioner or the group. As such, autonomy support can be viewed differently by practitioners, from some believing that leaving a child alone is a part of child-led learning to others seeing their role as actively providing support that aims to tap into the children’s innate tendencies. According to SDT, the latter is better viewed as autonomy support and the former more likely promotes independence. As with structure, a main aim of this study was to understand more fully the range of autonomy support practices used in nature-based settings. 3 1 Learning Environments Research Participants We contacted established early childhood nature-based settings in England and Scotland through email to ask for our survey to be shared with staff. Overall, we had 18 practition- ers who responded to our call. Of those 11 were female, one was male and the rest did not respond to this question. Most practitioners had more than a year of experience in teaching in outdoor settings, with 12 having taught between one and four years, three between 5 and 10 years, and three with more than 10 years of experience in such settings. One practitioner had one year of experience and the rest did not respond to this question. All of the practi- tioners worked with children older than three years of age, and 84% worked with children between three and five years old. i Ethics approval for the data collection and data management was given by the School of Education and Social Work, University of Dundee (approval number E2018-21). All participants were anonymous and were fully informed of the purposes of the research, the data management systems were in place, and the right to withdraw prior to completion of the questionnaire was explained before giving consent. Structure and autonomy support It is manifested as a guidebook, rather than as seemingly isolated regula- tions, such as ‘don’t climb trees’ or ‘do as you please as long as you do not hurt others’. High structure provides sufficient information to the child in order to deal with personal, emotional and social challenges and is not restricted to simple rules of conduct within nature-based settings. We further propose that a richer structure can enhance autonomy support. For exam- ple, practitioners find it easier to mobilize inner motivational resources and offer choice if they have a guiding framework that they are communicating to the child rather than only instructions. Therefore there could be delicate improvements to autonomy support while structure becomes richer. This conceptual account could also provide an explanation for why autonomy support and structure have a linear relationship. The ideal combination of structure and autonomy support is based on two-way, autonomy-supportive communication involving a guiding framework for dealing with challenges in relation to personal, social and emotional development, as well as connection to the natural world. 1 3 1 Learning Environments Research Table 1   Example of classification of educational tactics according to different levels of autonomy support and structure Structure Autonomy support Low Medium High Low Non-interference Observation, emphasis on one-way communication (offer of feedback) Emphasis on two-way communication, (Acknowledge negative affect, use autonomy-supportive language, positive feedback) Medium Offer instructions Observation, emphasis on one-way communication (rationale of instructions, and offer of feedback) Emphasis on two-way communication, (Acknowledge conflict from instructions, use autonomy-supportive language, offer choice, positive feedback) High Present a guiding framework Observation, emphasis on one-way communication (rationale of guiding framework, conveyance of a sense of purpose and direction, offer of feedback) Emphasis on two-way communication, (Acknowledge conflict from guiding framework, conveyance of a sense of purpose and direction, use autonomy-supportive language, offer choice, posi- tive feedback) 1 3 Learning Environments Research Most educators, especially in nature-based and early childhood contexts, are qualified to understand the basic properties of structure and autonomy support and attempt to offer them. This does not mean that they offer them in every situation and in the best way pos- sible. Our purpose in this qualitative study was to examine how early childhood practition- ers working in outdoor settings (a) think about autonomy and structure (b) apply them in everyday scenarios and (c) behave according to different types of situations. Instrument A qualitative questionnaire was put together, with open-ended questions designed to elicit in-depth responses by practitioners. The qualitative questionnaire is a flexible, little-used method with great potential in social research (Braun et al., 2021). According to Braun et al. (2021), “qualitative surveys consist of a series of open-ended questions, crafted by a researcher and centered on a particular topic” (p. 641). In this way they provide the researcher with rich and complex responses (maintaining the strength of other qualitative approaches), while giving access to the participants’ experiences and narratives. At the same time, qualitative questionnaires have advantages over face-to-face methods, such as interviews or focus groups, in accessing sensitive topics, as well as potentially partially mitigating social desirability bias.i In this instance, we designed a qualitative questionnaire of three parts. In the first part, practitioners were asked about the sort of practices that they undertake within their set- ting, directly focusing on the manifestations of autonomy within early childhood outdoor settings. These results will be presented in a future paper. The second part consisted of the four scenarios outlined in Table 2, whose responses form the basis for this paper. Finally, in the third part of the questionnaire, participants were asked to give some demographic information about themselves, as well as some information concerning the type of setting and their experience teaching in outdoor settings. 1 3 Learning Environments Research Table 2   Hypothetical scenarios to which the participants were asked to respond Scenario Context Situation Scenario 1 Play The children are playing freely. One child is persistently playing on her own, preferring to collect sticks and building her own small shelter. How do you react? Scenario 2 Transition The children are getting ready for their lunch. One of the children is not willing to join in and wants to continue playing. How do you communicate with him? Scenario 3 A walk The group is scheduled to take a walk in another part of the woods. Some children don’t want to join. How do you deal with the situation? Scenario 4 Misbehavior An older child is acting in a way that is upsetting two of the younger children in the group. How do you deal with her? 1 3 Learning Environments Research Each scenario presented the practitioner with a realistic situation that could take place during a normal session within the setting. Procedure Having established relationships with several high-quality outdoor early childhood educa- tion settings throughout England and Scotland, we recruited practitioners through manag- ers at seven of those settings. A request was made to share the questionnaire with all of the practitioners at these settings—the low response rate was attributed by managers to time pressures for practitioners and the length of the questionnaire. Those who responded, how- ever, tended to give long and comprehensive answers. Instrument Participants were asked to respond in terms of how they would react in this particular situation. These scenarios were chosen as com- monly occurring and offering challenges to a practitioner within nature-based settings. Although these scenarios also can occur in indoor settings, the nature of an outdoor setting and the geography of it present a more challenging situation to the practitioner because of aspects such as geographical distance with the child or risks that can be present. Moreover, the design of the scenarios took into account the child-led pedagogical principles in for- est schools and other nature-based settings in order to present dilemmas for practitioners. These child-led principles would more readily conflict with a high need for intervention on the part of the practitioner, when there is a contextual requirement to ensure the safety and physical and emotional wellbeing of the child and others. The scenarios specifically offered different levels for the need for practitioner intervention. f The use of hypothetical scenarios such as these, or ‘vignettes’, can allow the participant to explore a situation in their own terms, but also to probe sensitive situations in a less per- sonal way (Barter & Renold, 1999). The purpose in this case was to examine the extent and the ways in which practitioners would use both autonomy support and structure in these hypothetical situations. Participant responses in the scenarios ranged from very short (two words) to several paragraphs (more than 600 words). Medium autonomy support/low structure Observation emerges as important in this instance, where practitioners leave the child to continue a chosen activity. Practitioners discuss the need to attune to the child and her inter- ests, but do not offer any input or information to the child. In all cases, the practitioners are available, observe and stay on hand to be able to provide support if the child needs them: Leave her to choose. If she wants to play alone, this is fine. Ensure an adult is acces- sible and attuned to the child. Monitor her interaction with her chosen activity, if appropriate engage her in discus- sion about her purposes. If she is flow and chatting to herself, listen. Scenario 1 In Scenario 1, practitioners reported responses that were autonomy supportive on a medium or high level (there is no low autonomy support response) and structure does not seem to be prioritized. In Table 3, we report the types of educational tactics that were iden- tified in the practitioners’ responses (marked by the ticks in the box). Below we discuss these responses in further detail. High autonomy support/low structure The focus in this case is on practitioners’ considerable efforts to understand the child and her interests, as well as on the accumulation of knowledge about the child’s habitual behav- ior, temperament, and current facial expressions; practitioners intend to engage in conver- sation to understand more about the child. As in the previous examples, offering structure (i.e. information of some sort) to the child is missing: […] that those children who crave time alone can have the freedom to take advantage of this. I would ask a leading question about what she’s doing without criticism then I would judge from her answer as to whether she may need help in socializing more of course it would depend to as to what is ’normal for her to. So I would speak with her teacher. No reason to intervene if I know this to be her/his abitual behaviour reflecting his/her temperament. If it is unusual I would observe other signs, as facial expression, and ask if everything is all right if in doubt. If I see this for some days I would tell other adults and the parents to know if they have some information on what’s happening. If something is wrong I would adress that and not tha fact tah he/she is playng alone. Data analysis We employed thematic analysis using a deductive ‘top-down’ approach. All participants’ responses were initially mapped on the two dimensions of autonomy support and structure drawing from SDT as portrayed in Table 1 and using three levels for each (low, medium, high). We used the template of Table 1 to map the responses before analyzing the content of these responses to approach the specific strategies that were employed by the practition- ers in each of the scenarios. Table 3   Classification of educational tactics according to different levels of autonomy support and structure in Scenario 1 Structure Autonomy support Low Medium High Low Medium High 1 3 Table 3   Classification of educational tactics according to different levels of autonomy support and structure in Scenario 1 Structure Autonomy support Low Medium High Low Medium High 1 3 1 Learning Environments Research Medium autonomy support/medium structure While attunement and attention to the cues of the child are still important here, practitioners in this scenario attempt to offer an initial interactional structure (but communication is still one-way). This takes place through either direct explorations with the child or inviting oth- ers to peripherally participate, if interested, or through parallel play as a means to interact: 3 3 Learning Environments Research Go and talk to her and ask what her ideas are with building het own shelter. I would chat with her to make sure she is happy and not feeling left out for any rea- son, then i would invoite the rest of the group to come and look at her amazing shel- ter, they may feel inspired to build their own and make it into a village, I would always follow the cues of the child. Scenario 2 Table 4 classifies the tactics that were reported by practitioners for Scenario 2. As in Sce- nario 1, practitioners mention educational tactics that are autonomy supportive (there is no low autonomy support response) but, in this case, structure is given a little more value (especially illustrated by medium autonomy support/high structure responses). More expla- nation is provided below. High autonomy support/high structure In this condition of high autonomy support and high structure, practitioners make con- siderable effort to attune to the child as well as to build a plan and offer structure for interacting with other children. This typically involves gaining insights and thoroughly understanding the emotions and intentions of the child who is playing alone through two- way communication and, moreover, looking to cater for the underlying need of children in a gentle way: Observe in the first instance. There’s not necessarily a problem with the child choos- ing to play by herself and I would see it as a positive thing depending on the context / history, etc. I would only intervene if after repeated observation I was of the view that this child was avoiding playing with other children out of fear / some other nega- tive experience or feeling. In that situation I might gently encourage the child towards the other children without forcing, being prepared to listen to feelings. I might also if appropriate and welcome join the child in her play to build connection / safety and to gain more insight. Medium autonomy support/low structure There is care to support autonomy in this case, expressed in terms of the need to ensure that there is time to accommodate activities as prioritized by the child—typically ensured through one-way supportive communication. There is no attempt to offer structure: Ensure he can play and have lunch when he is ready. Ensure he can play and have lunch when he is ready. Table 4   Classification of educational tactics according to different levels of autonomy support and structure in Scenario 2 Structure Autonomy support Low Medium High Low Medium High 1 3 Learning Environments Research Medium autonomy support/medium structure Balancing between some medium level of structure and autonomy support means that practitioners attempt to offer structure by offering instructions, namely, mentioning that it is time to have lunch, while at the same time, reassuring the children that they can return to play: Depending on the child, promise they can return to the play, or make a joke, pretend to chase and tickle them to distract them – it really depends on the child. Ask them to put everything together and explain we will leave it there and come back to it after lunch. Medium autonomy support/high structure In this case, providing structure becomes the dominant aspect in the responses. Practition- ers insist on following the principles of the guiding framework (in some cases expressed in the form of routines as in the example below), while they recognize the children’s interests; however, they do not go as far as engaging in two-way supportive interactions: Explain time – what’s happened – hand washing, toileting etc., and offer reassurance that play will be revisited. Reiterate the routine and meal times rules. Include child and give responsibility such as being a lunch time helper. Medium autonomy support/low structure The approach to tackle the case of the scenario for some practitioners allows the children to do what they ask for, and therefore provides medium autonomy support but no structure: Ensure the children who don’t wish to walk, can stay. Take only the children who want to go. Low autonomy support/low structure In this scenario, we encountered responses in which practitioners attempt neither to offer structure nor attune to students’ feelings. Instead, one practitioner proposed to engage in a mild form of teasing/punishment, a behavior that is by definition low in autonomy support, without really offering information to the child: Depending on staffing leave those children and then when we get back explain what a great time we had – next time they would come. High autonomy support/high structure for lunch would mean water gets in and ruins progress, then more likely to be flexible re lunch time if possible. If not possible, then use hand in hand tools—active listen- ing, empathising with their feelings, offer quality time to be with them during lunch and see how we can problem solve to help meet needs of individual and group. Scenario 3 Table 5 classifies the tactics reported by practitioners for Scenario 3. Compared with Sce- nario 1 and 2, practitioners mention potential responses that are more widely distributed across the spectrum. The nature of group VS individual interaction might have played a role in the practitioners proposing tactics that are not autonomy supportive. More details are provided below. High autonomy support/high structure In cases in which practitioners offer high autonomy support and high structure, they seem to make significant effort to empathize with the child and their needs while main- taining structure. The structure in this case does not entail a simple rule ‘now is time for lunch’, but rather guidance as to how children should operate within groups. The range of approaches discussed to maintain high autonomy support and structure include proposing compromises, offering reassurance that the children can return to their play next and expect big feelings, and helping children to work out how to meet their own and group needs—all entailing two-way communication: I would go over and join him for a moment in his play, coming down to his level, acknowledging what he’s doing and how much he’s enjoying it. After a few moments I would point out that it’s lunch time, asking him to join the other children. If he was reluctant I would stick with the limit, having empathy for his desire to play, but stay- ing with the fact that he’s part of the group and it’s lunch time now. Reassuring that there will be time to play after lunch. Expecting that big feelings might arise and being prepared to listen through them. I would go over and join him for a moment in his play, coming down to his level, acknowledging what he’s doing and how much he’s enjoying it. After a few moments Tell child that it’s lunch time and explore what is preventing child from coming for lunch. May be possible to reach some sort of negotiation with child. What is child doing? Is it something that is easy or hard to leave eg if making a dam and pausing 1 3 1 3 Learning Environments Research Table 5   Classification of educational tactics according to different levels of autonomy support and structure in Scenario 3 Structure Autonomy support Low Medium High Low Medium High for lunch would mean water gets in and ruins progress, then more likely to be flexible re lunch time if possible. If not possible, then use hand in hand tools—active listen- ing, empathising with their feelings, offer quality time to be with them during lunch and see how we can problem solve to help meet needs of individual and group. Medium autonomy support/medium structure Behaviors of medium autonomy support and medium structure in this instance mean that the practitioners exhibit flexibility and negotiate a plan while trying to maintain a structure of togetherness for the children: Maybe get them to link hands or have a blind trail. Encourage trust and interest in other children and the woods. Again, this would depend on staffing ratios, if it was viable to leave them to their own play, that would be ok, if it was going to affect the rest of the group, a discussion would need to be had to make it fair for everyone. If have staff so can separate group and still meet appropriate ratios, then that’s fine. Can do different things. If need to keep all children together, then need to explain why this is the case and hear their concerns. Low autonomy support/medium structure By contrast, the practitioners in the case presented in the excerpts below persist in the plan and use a majority rule or their own authority to convince children to go along, thereby offering the children medium structure but no real autonomy support: 1 3 1 Learning Environments Research Vote, majority choice decides or say we can have 5/10 more minutes nurse and then take the walk. Give them a task to carryout on the way (e.g. we have a colour of the week and we look for that colour or ask the to be a helper). Scenario 4 Table 6 classifies the tactics mentioned by practitioners in Scenario 4. In this scenario, practitioners mention responses that are even more widely distributed across the spectrum. Both dimensions of structure and autonomy support seem of equal importance. Details are discussed below. High autonomy support/high structure When looking at behaviors that are high in both structure and autonomy support, we find practitioners engaged in helping children achieve what seems to be interesting and impor- tant to them through deliberative and carefully planned collective action (through engaging them in two-way supportive communication): Divide adults into safe group one to stay one to hike, explain the value of moving as a group and ask the children to brainstorm what they may see along the way to encourage others, or do not go on a walk. Discuss if safe to split up. See if the children can negotiate a compromise. Lean my questioning towards a possible solution, but try to see if the children can come up with it. 1 3 Table 6   Classification of educational tactics according to different levels of autonomy support and structure in Scenario 4 Structure Autonomy support Low Medium High Low Medium High Table 6   Classification of educational tactics according to different levels of autonomy support and structure in Scenario 4 Structure Autonomy support Low Medium High Low Medium High Learning Environments Research Low autonomy support/medium structure In other cases, structure takes precedence over autonomy support. Practitioners choose to maintain some type of order through providing some (medium) structure but do not essentially provide autonomy support. In this case, there is evident lack of attunement to the children’s feelings and needs, even to the point of resorting to mild punishment: Leave for as long as possible and hope the children work it out themselves, but if the younger children are very upset and unable to stop the older child, explain the impact their behaviour is having (encourage the younger children to try to explain) then try to distract the older child with something interesting away from the younger children. Time out say if they continued with this misbehaviour there will be less time to do other play activities. Also ask the older child to be more caring to the younger ones. High autonomy support/low structure Potentially because of the level of the challenge that the children of this scenario face, practitioners might choose to abandon structure and focus only on autonomy support. This is expressed in terms of the practitioner interacting with and showing empathy to the child: Empathise, use prior knowledge of the child to support the child and her feelings. Accept her feelings by asking her how she is feeling and explaining that you under- stand. Medium autonomy support/medium structure When a medium level of autonomy support and structure is pursued by practitioners, talking with the children and attunement to their feelings complement structure in the form of social expectations and norms (e.g. ‘be nice to others’): Speak to her about how the other children might be feeling. Talk about how she could do or say things differently next time. Suggest that she asks the other chil- dren if there is something she could do to make them feel better. Discuss the scenario, allow child to be responsible for actions and possibly offer opportunities for older child to be given a caring role towards the younger children. 1 3 1 Learning Environments Research Medium autonomy support/high structure In other cases, practitioners talk about structure both in an attempt to explain what is acceptable or not and also in terms of consequences. This would be classified as low autonomy support but, when a practitioner (as in the excerpt below) makes an effort to convey choice, medium autonomy support is achieved: I believe in giving a warning first but as friendly as I am the children know they get one chance. So I would talk to the older child as I believe all behaviour has a cause and try to establish why they’re behaving in an unacceptable way I’d make it clear that this is their opportunity to change or their will be a consequence ie t minutes in base camp, then stages consequences ultimately leaving the session. But to me it’s important that the older child feels they have a choice. That choice would be limited to perhaps something like finding something else to do or having the consequence. The younger children should not be forgotten in this either and I would ensure they were ok. High autonomy support/medium structure In this case involving high autonomy support and medium structure, practitioners make considerable effort to engage children in two-way communication, when they attune to children, acknowledge emotional conflicts, and offer structure that takes on the form of social expectations: Inquire into her well being, if she is comfortable, has needs met, is bored, has unaddressed emotional state, resolve issues w leading questions, inquire if she’s bored and what she would like to do, make an activity suggestion that construc- tively develops observed misbehavior. Give her an opportunity to be away from the other children. Ask the younger ones for their feelings, discuss with the older one what you have seen, how they feel. Ask the older one how they feel. Listen and give it time. Don’t ask the older one to apologise to the younger ones, they should do so in their own time if they are ready. Discussion The purpose of our research was to examine the manifestations of autonomy support and structure in situations where outdoor education practitioners are called upon to deal with children who do not follow instructions or behave in line with educational and school norms. Our theoretical framework of Self-Determination Theory can potentially be of sig- nificant use in outdoor pedagogical child-led practices (e.g. Barrable & Arvanitis, 2019). i Children who do not follow the norms of the school might be doing so because they are drawing from their intrinsic tendencies and interests, or because of external and internal pressures. On the one hand, it is the job of the practitioner to be autonomy supportive, that is, to attune to the children, understand the motives for their behavior, acknowledge pos- sible conflicts, alleviate concerns, offer choice, and help them to see the value of the behav- ior in which they refuse to engage. All practitioners in our sample speak of doing this in one form or another. It is important to note that leaving children to do what they ask for is not necessarily supporting their autonomy, especially if their behavior is dictated by inter- nal or external pressures. Even if it is dictated by their interests, there are cases in which these should be put on hold for other beneficial activities or for avoiding harm to them- selves or others. On the other hand, it is also the job of the practitioner to provide structure, that is, contextual information, specific directions and guidance that children can use to navigate themselves in the setting in a predictable manner that ultimately helps in achiev- ing desirable outcomes, while also keeping everyone safe and healthy. Although structure and autonomy support have a linear relationship (Jang et al., 2010), this does not mean that high autonomy support goes hand-in-hand with high structure in every case or that this would be an easy task. Our purpose, therefore, was to see how practitioners can balance the two in different situations.i f Our scenarios had specific norms that needed to be followed: interaction with other chil- dren (Scenario 1), transition from play to eating lunch (Scenario 2), going on a group walk (Scenario 3), and behaving in an agreeable manner to other children (Scenario 4). All these behaviors are considered beneficial for children and persistent abstention might hinder val- ued educational outcomes. High autonomy support/high structure There is the case here too of practitioners making a considerable effort both to attune to the children and build structure that is appropriate. This translates into a number of strategies, from getting the whole story and dealing with all parties to building a group and dealing with unmet needs: We may find that we need to have better auditory monitoring of exchanges between children, group "camp" meetings - not to go over rules, but to share enough to create a more positive dynamic going into sessions, as part of forming a culture of shared objectives. All kinds of efforts build the group. Stories that illuminate how to express differences without domination, ultimatums or over-control, easy shared phrases, even small celebrations etc. After observing, take older child aside gently and ask what is happening from their perspective. If they have unmet needs, deal with these as priority. Eg cold, hungry, 1 3 Learning Environments Research upset, need to vent. It’s not possible to reason with someone who’s not in a place emotionally to hear what you are saying. Ask how they think the others feel re what happening and get their perspective. […] If the actions need a more immediate reac- tion eg the older child is hitting them, then intervene and separate immediately and deal with comforting the others (while radioing another staff member to see if they can possibly simultaneously provide support to the older child) and work together with all 3 children to get solution - possibly using restorative justice tools. Play lis- tening may also be helpful as a strategy. If the child’s behaviour is persistently upset- ting other children, then an additional support plan may be beneficial. Discussion What in essence was expected from practitioners was two-fold: (a) to provide some sense of guidance and information that would orient children toward valued activities or to prevent harm to themselves or others (structure), and (b) to attune to the children, acknowledge the conflict, and offer choice and flexibility (autonomy support). lfl Practitioners’ responses did not exhibit the same properties across all situations. Sce- narios 1 and 2 were perceived as not requiring structure, at least not to the extent that scenario 3 and, more so, scenario 4 required. In these scenarios, practitioners generally showed great care in providing autonomy support, probably because of the pedagogical 1 3 Learning Environments Research approaches used in nature-based settings which are often child-led (Barrable, 2019; Har- ris, 2018). However, this autonomy support seemed often to take the laissez-faire form of ‘let the child do as she asks’ without in-depth attunement to the child. This passive form of autonomy support does not necessarily benefit the child. Outdoor education practitioners might find it useful to keep in mind that autonomy support requires being actively involved even in situations that seem to be adequately led by the child. Sometimes educational goals require subtle interventions. approaches used in nature-based settings which are often child-led (Barrable, 2019; Har- ris, 2018). However, this autonomy support seemed often to take the laissez-faire form of ‘let the child do as she asks’ without in-depth attunement to the child. This passive form of autonomy support does not necessarily benefit the child. Outdoor education practitioners might find it useful to keep in mind that autonomy support requires being actively involved even in situations that seem to be adequately led by the child. Sometimes educational goals require subtle interventions. In scenario 3, our analysis revealed educational tactics that were less autonomy sup- portive and more forceful toward children. This is an interesting finding because a main difference with scenario 2 is that it involved a group of children rather than an individual child. We should take into account that using the power of majority or voted rules is a democratic process that finds acceptance among many children, but also it is found oppres- sive by some children, especially if they are often on the side of the minority. Discussion Practitioners’ care for autonomy support should find its way in managing the group of children by using group decision making in a way that acknowledges potential conflict and offers choice and flexibility.i l In scenario 4, our analysis identified responses scattered across the spectrum of auton- omy support and structure. In cases of misbehavior, some practitioners primarily leaned in the direction of understanding and helping the child who was misbehaving, whereas other practitioners focused on enforcing rules. The best of both worlds seemingly exists when children, in time, negotiate and actively endorse the rules necessary for their interaction under the supervision of the practitioner. From the wealth of the responses in our research, situations like misbehaving for which practitioner intervention is necessary, reveal the dif- ficulty in juggling autonomy support and structure at the same time. Conclusion In child-led outdoor educational settings, it might seem easy for practitioners to be auton- omy supportive to children. Structure might seem less significant. Our study examined sit- uations that need both autonomy support and structure to differing degrees. Practitioners’ responses revealed the intricate relationship between the two concepts, especially if the different requirements of situations are taken into account. Practitioners need to keep in mind that low-requirement intervention situations are not necessarily autonomy supportive and indeed could need structure, and that high-requirement intervention situations, while requiring structure, can often lead to overcontrolling the children. More research is needed into the effects of the different types of situations. Funding  Open access funding provided by HEAL-Link Greece. Declarations Conflict of interest  The authors declare no conflict of interest. Ethical approval  Ethical approval for the data collection and data management was given by the School of Education and Social Work, University of Dundee (approval number E2018-21). All participants were anony- mous and were fully informed of the purposes of the research, the data management systems in place and the right to withdraw prior to completion of the questionnaire before giving consent. 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, visit http://​creat​iveco​mmons.​org/​licen​ses/​by/4.​0/. Limitations and implications Our qualitative research focused on the completion of questionnaires presenting four situ- ations with the aim of examining how practitioners might respond to each of four differ- ent situations. Presenting the same set of scenarios in face-to-face interviews would have allowed an in-depth exploration and understanding of participants’ responses, which the completion of questionnaires could not grant. This is a limitation of the approach taken here. In addition, through this research, we offered a typology of structure/autonomy sup- port, but we did not offer a similar typology of situations. In particular, the underlying dimension of these situations was the contextual requirement for practitioner intervention. Our qualitative research focused on the completion of questionnaires presenting four situ- ations with the aim of examining how practitioners might respond to each of four differ- ent situations. Presenting the same set of scenarios in face-to-face interviews would have allowed an in-depth exploration and understanding of participants’ responses, which the completion of questionnaires could not grant. This is a limitation of the approach taken here. In addition, through this research, we offered a typology of structure/autonomy sup- port, but we did not offer a similar typology of situations. In particular, the underlying dimension of these situations was the contextual requirement for practitioner intervention. The guiding principle in progressing from Scenario 1 to Scenario 4 was that the require- ment for intervention for the practitioner became greater. There are two main take-away messages for practitioners based on this research: (a) a low requirement for intervention can lead to a passive educational stance that is not autonomy supportive and lacks structure and (b) a high requirement for intervention might bring forward an imposition of struc- ture that comes at the expense of autonomy support. In line with these points, practition- ers should keep in mind that there are different types of situations that require delicate uses of autonomy support and structure. However, this evidence is and should be treated as preliminary given the limitations imposed by the scenarios employed in this research (i.e. four scenarios presenting typical situations encountered in outdoor learning). To this end, further research is needed for creating a typology of situations appropriate for the study of structure and autonomy support in the context of outdoor learning, measuring the contex- tual requirement for intervention, and testing and expanding on the ideas outlined here. 3 3 Learning Environments Research References Arvanitis, A., & Kalliris, K. (2017). A self-determination theory account of self-authorship: Implications for law and public policy. Philosophical Psychology, 30(6), 763–783. https://​doi.​org/​10.​1080/​09515​089.​ 2017.​13073​33 Bao, X. H., & Lam, S. F. (2008). Who makes the choice? Rethinking the role of autonomy and relatedness in Chinese children’s motivation. Child Development, 79(2), 269–283. https://​doi.​org/​10.​1111/j.​1467-​ 8624.​2007.​01125.x Bao, X. H., & Lam, S. F. (2008). Who makes the choice? Rethinking the role of autonomy and relatedness in Chinese children’s motivation. 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Journal of Educational Psychology, 85(4), 571–581. https://​doi.​org/​10.​1037/​0022-​0663.​85.4.​571 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. 1 3 1 3

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The Effect of Obliquity of Slip in Normal Faults on Distribution of Open Fractures

Frontiers in earth science

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The Effect of Obliquity of Slip in Normal Faults on Distribution of Open Fractures Christoph von Hagke 1*†, Michael Kettermann 1, Nicolai Bitsch 1, Daniel Bücken 1, Christopher Weismüller 2 and Janos L. Urai 1 1 Institute of Structural Geology, Tectonics and Geomechanics, RWTH Aachen University, Aachen, Germany, 2 Inst N t t i d N t l H d RWTH A h U i it A h G 1 Institute of Structural Geology, Tectonics and Geomechanics, RWTH Aachen University, Aachen, Germany, 2 Institute of Neotectonics and Natural Hazards, RWTH Aachen University, Aachen, Germany Keywords: fault, mechanics, geometry, dilatant, analog modeling, Iceland, rift ORIGINAL RESEARCH published: 25 February 2019 doi: 10.3389/feart.2019.00018 ORIGINAL RESEARCH published: 25 February 2019 doi: 10.3389/feart.2019.00018 ORIGINAL RESEARCH published: 25 February 2019 doi: 10.3389/feart.2019.00018 ORIGINAL RESEARCH published: 25 February 2019 doi: 10.3389/feart.2019.00018 Edited by: Valerio Acocella, Università degli Studi Roma Tre, Italy Reviewed by: Daniele Trippanera, King Abdullah University of Science and Technology, Saudi Arabia Lorenzo Bonini, University of Trieste, Italy *Correspondence: Christoph von Hagke christoph.vonhagke@ emr.rwth-aachen.de †Present Address: Christoph von Hagke, Institute of Geology & Palaeontology, RWTH Aachen University, Aachen, Germany †Present Address: Christoph von Hagke, Institute of Geology & Palaeontology, RWTH Aachen University, Aachen, Germany Specialty section: This article was submitted to Structural Geology and Tectonics, a section of the journal Frontiers in Earth Science Structural Geology and Tectonics, a section of the journal Frontiers in Earth Science Received: 23 July 2018 Accepted: 30 January 2019 Published: 25 February 2019 Citation: von Hagke C, Kettermann M, Bitsch N, Bücken D, Weismüller C and Urai JL (2019) The Effect of Obliquity of Slip in Normal Faults on Distribution of Open Fractures. Front. Earth Sci. 7:18. doi: 10.3389/feart.2019.00018 Received: 23 July 2018 Accepted: 30 January 2019 Published: 25 February 2019 Close to surface, cohesive rocks fail in extension, which results in open fractures that can be several tens of meters wide, so-called massively dilatant faults. These open fractures make fault slip analysis in rifts challenging, as kinematic markers are absent. Faults in rifts often have oblique slip kinematics; however, how the amount of obliquity is expressed in the surface structure of massively dilatant faults remains enigmatic. Furthermore, the structures of oblique dilatant faults at depth is largely unconstrained. To understand the subsurface structures we need to understand how different obliquities of slip influence the surface structures and the corresponding structures at depth. We present analog models of oblique massively dilatant faults using different cohesive materials in a sandbox with adjustable basement fault slip obliquity from 0◦to 90◦. Experiments with different mean stress and material cohesion were run. Using photogrammetric 3D models, we document the final stage of the experiments and investigate selected faults by excavation. We show that fault geometry and dilatancy changes systematically with angle of obliquity. Connected open fractures occur along the entire fault to a depth of 6–8 cm, and as isolated patches down to the base of the experiments. Using the scaling relationship of our models implies that transition from mode-1 to shear fracturing occurs at depths of 250–450 m in nature. Our experiments show the failure mode transition is a complex zone and open voids may still exist at depths of at least 1 km. We apply our results to the dilatant faults in Iceland. We show that the relationship between angle of obliquity and average graben width determined on faults on Iceland matches experimental results. Similarly, fracture orientation with respect to fault obliquity as observed on Iceland and in our experiments is quantitatively comparable. Our results allow evaluation of the structure of massively dilatant faults at depth, where these are not accessible for direct study. Our finding of a complex failure mode transition zone has consequences for our understanding of fracture formation, but also influences our interpretation of fluid flow in rift systems such as magma ascent or flux of hydrothermal waters. INTRODUCTION Lithosphere-scale centrifuge models with K-feldspar powder representing the brittle upper crust, plasticine the lower crust, and a plasticine-silicone mixture the upper mantle show that the angle of obliquity changes fault geometries observed at the surface, and that dominant strike-slip forms at obliquities >45◦ (Agostini et al., 2009). These studies show how obliquity of slip influences rift geometry. However, we have little knowledge on fault geometries in the upper few 100 m of the crust, where rocks fail in extension. Particularly it remains enigmatic how obliquity of slip influences the distribution of open fractures at surface and depth. Oblique rifts form when the relative movement of two plate boundaries is not perpendicular to the rift trend. More than 70% of Earth’s divergent plate boundaries exhibit an oblique character (Woodcock, 1986; Philippon and Corti, 2016; Brune et al., 2018; Jeanniot and Buiter, 2018). Prominent examples include the Ethiopian rift, the Reykjanes Peninsula on Iceland, or the Mohns Ridge in the Arctic Ocean (e.g., Dauteuil and Brun, 1993; Grant and Kattenhorn, 2004; Corti, 2008). The influence of oblique spreading directions on fault geometries and orientations, as well as linkage processes and fault evolution, have been studied on crustal and lithospheric scale (Withjack and Jamison, 1986; Dauteuil and Brun, 1993; Clifton and Schlische, 2001, 2003; Clifton and Kattenhorn, 2006; Agostini et al., 2009; Brune, 2014; Zwaan et al., 2016; Zwaan and Schreurs, 2017). These studies show that obliquity of rifting influences geometry of structures at the surface, even though local stress reorientations may result in dip-slip motion along faults trending obliquely to extension direction (Morley, 2010; Corti et al., 2013; Philippon et al., 2015). Despite these efforts, we still know little about the detailed structures of these faults at depth, as they are commonly not accessible in the field. Furthermore, it remains poorly understood which structural features observed at the surface are characteristic for the amount of obliquity of slip. In this study, we investigate the influence of fault obliquity on fault evolution and geometries, focusing on the uppermost few 100 m of the rift system, where cohesive rocks fail in extension. This is vital, as understanding the geometry of open fracture distribution is essential for predicting fluid flux through the crust. INTRODUCTION Massively dilatant faults form in cohesive rocks under low differential stresses, mostly close to the surface but also at depth under high pore fluid pressure (e.g., van Gent et al., 2010; Holland et al., 2011). They are ubiquitous features occurring at a range of scales, with opening widths from mm-size to several tens of meters as found along rift zones. Dilatant faults and associated fractures form high-permeability corridors and thus are major pathways for fluids such as water, hydrocarbons, or magma (Ingram and Urai, 1999; Ferrill and Morris, 2003; Crider and Peacock, 2004; Faulkner et al., 2010; Trippanera et al., 2015; Kettermann et al., 2016). Consequently, dilatant faults are of great economic interest for water and geothermal energy supply (Jafari and Babadagli, 2011), geohazard assessment and geodynamics (Crone and Haller, 1991; Caine et al., 1996; Gudmundsson et al., 2001; Ehrenberg and Nadeau, 2005; Belayneh et al., 2006; Lonergan et al., 2007), or mineral deposits (Zhang et al., 2008). Dilatant fault systems occur at mid ocean ridges (Gudmundsson, 1987; Angelier et al., 1997; Wright, 1998; Friese, 2008; Sonnette et al., 2010; Trippanera et al., 2014), intra-plate volcanoes (Holland et al., 2006), continental rifts (Acocella et al., 2003; Acocella, 2014; Trippanera et al., 2015), but also in cemented carbonates and clastic sediments (McGill and Stromquist, 1979; Moore and Schultz, 1999; Ferrill and Morris, 2003; Lonergan et al., 2007; Wennberg et al., 2008; van Gent et al., 2010; Kettermann et al., 2015). Their internal structure has been studied using analog and numerical models (Abe et al., 2011; Holland et al., 2011; Hardy, 2013; Kettermann et al., 2016). These studies, in combination with field observations, show that distinctive features of dilatant faults include sub-vertical fault scarps, rotating hangingwall blocks, and tens of meters wide open fractures (e.g., Acocella et al., 2003; Grant and Kattenhorn, 2004). Conceptual models for 3D geometries of dilatant faults are based on outcrops and scaled models (e.g., McGill and Stromquist, 1979; Schultz-Ela and Walsh, 2002; Holland et al., 2006; Sonnette et al., 2010; Vitale and Isaia, 2014). Citation: von Hagke C, Kettermann M, Bitsch N, Bücken D, Weismüller C and Urai JL (2019) The Effect of Obliquity of Slip in Normal Faults on Distribution of Open Fractures. Front. Earth Sci. 7:18. doi: 10.3389/feart.2019.00018 February 2019 | Volume 7 | Article 18 Frontiers in Earth Science | www.frontiersin.org 1 Oblique Rifts and Massively Dilatant Faults von Hagke et al. Frontiers in Earth Science | www.frontiersin.org INTRODUCTION Analog and numerical models have been used to understand joint development, evolution of dilatant faults, and the influence of mechanical stratigraphy or pre-existing structures on surface geometries and fault processes (Clifton and Schlische, 2001; Seyferth and Henk, 2006; Schöpfer et al., 2007a,b; van Gent et al., 2010; Abe et al., 2011; Holland et al., 2011; Holohan et al., 2011; Hardy, 2013; Kettermann and Urai, 2015; Kettermann et al., 2015, 2016; Bonini et al., 2016). Most of these studies focus on dip-slip normal faults. Analog modeling of oblique rifts has shown that many aspects of the geometric complexity of these systems can be reproduced, including releasing and restraining bends, fault step- overs, antithetic faults, bookshelf structures, or fault lenses. Early analog experiments of oblique rifting with soft clay (a mixture of mostly kaolinite and quartz) showed that steeply dipping rift parallel strike-slip faults form at obliquities > 60◦(Withjack and Jamison, 1986). Lithosphere-scale models of oblique rifts with a layer cake of sand and silicone putty were used to establish first order geometries characterizing oblique rifts, such as mean fault trend differing from extension direction, common en- échelon pattern, or curved faults (Tron and Brun, 1991). Models including the asthenosphere represented by a honey syrup layer show that pull apart basins formed separately during early stages of rifting can still be recognized in the evolved unified rift (Mart and Dauteuil, 2000). These experiments were performed at obliquity angles of 15◦and 30◦. For highly oblique settings (60◦), Clifton et al. (2000) and Clifton and Schlische (2001) used scaled clay models to show that fault nucleation, growth and interaction influences the time evolution and geometry of faults in oblique rifts. Experimental results are comparable to structures observed on an obliquely rifting section of the Mid Atlantic Ridge outcropping on the Reykjanes Peninsula, SW-Iceland, including three sets of faults: rift-perpendicular right-lateral oblique-slip faults, rift-subparallel left-lateral oblique-slip faults and normal faults striking 20◦counter clockwise to the rift trend (Clifton and Schlische, 2001; Clifton and Kattenhorn, 2006). Here, we report results from analog models using cohesive powder as modeling material, representing the uppermost few km of basaltic crust. We build on results of Galland et al. (2006); February 2019 | Volume 7 | Article 18 2 Oblique Rifts and Massively Dilatant Faults von Hagke et al. Holland et al. (2006); Kettermann et al. (2016), and van Gent et al. (2010) and extend these to oblique slip kinematics. INTRODUCTION We show that there is a first order similarity between surface expression of models and features observed in nature such as wide open fractures, tilted blocks, or fissures (Figure 1). This supports the inference that experiments will also be representative for the deeper structure and provide insights into fault structures that are commonly not accessible in the field, and into the time-evolution of dilatant faults. However, whether the surface structures are not only qualitatively but quantitatively comparable remains to be tested. To this end we quantitatively analyze the surface structure of fault zones developed in the sandbox and formed at different obliquity angles. We then compare our observations with field data from southwest and northern Iceland and finally we provide a conceptual model of evolution of massively dilatant faults in cohesive rocks. rift shoulder. The box has no lateral walls and the sample is set up with natural slopes in strike direction. Because of the slopes, the 70 cm wide sandbox provides fault length of about 40 cm (Figure 2). An advantage of this setup is the experiment is not affected by friction of sidewalls influencing the evolving geometries. The box is driven by a geared motor to displacements of up to 70 mm. Our experiments are rate independent; we chose a speed of 3 mm/min so that fault evolution is captured in pictures taken in 10 s intervals. We use high-resolution digital single-lens reflex cameras to monitor the experiment. We use top- side- and front- view time lapse photography to create digital elevation models using “structure from motion” technique (Westoby et al., 2012), which allows for analyzing the structure of the fault zone in 3D. We use a vacuum cleaner to excavate parts of the fault zone from the top in order to look at the fault surface at resolutions much higher than by CT-scanning (Holland et al., 2006). This method has limitations: loose fragments of fault rock, bifurcations of the fault surface and parallel fault planes can produce structures which are difficult to interpret. Alternatively, we fill the open fractures with fine grained (<0.2 mm) sand to stabilize the structures. The experiments can then be sliced horizontally; however small open fractures and fractures which were not filled with sand may become damaged during excavation. Combining both excavation routines provides insights on the width of the fault zone and the distribution of open fractures down-dip. Experimental Setup To test the effect of obliquity on normal fault evolution we designed a new apparatus that allows for adjusting obliquity on a rigid basement fault in 15◦steps. We use a single basement fault dipping at 60◦, modeling structures at a single We performed a first set of seven experiments with hemihydrate powder thickness of 20 cm and obliquity angles of 0◦, 15◦, 30◦, 45◦, 60◦, 75◦, and 90◦. 0◦obliquity corresponds to a dip-slip experiment; 90◦corresponds to strike-slip faulting without a normal fault component. In a second set with a total of 20 experiments we used the same angles of obliquity but reduced powder thickness to 10 cm. We repeated the 10 cm hemihydrate experiments to test their reproducibility and to apply the different excavation methods. A third series was performed with a 10 cm thick layer of a 1:10 wt% hemihydrate-sand mix, also testing the seven different obliquities from 0 to 90◦. To analyze the results quantitatively, we use top view images of the time when the first visible fractures form, and images captured at 24 mm displacement. The latter is a stage where the fault has reached a mature phase, i.e., further extension is almost exclusively accommodated by widening of pre-existing fractures. Material Properties and Scaling Angle of the of basement fault has been set to 60◦. (B) In top view, the rift trend is parallel to the basement fault, obliquity ω is defined as the angle between dip direction of the basement fault and direction of plate movement (i.e., ω = 0◦for dip-slip, and ω = 90◦for strike-slip; a dip-slip fault has no or 0◦of obliquity). (C) Example picture of an experiment with 45◦obliquity. Note the box does not have sidewalls, and thus the fault is unencumbered by sidewall friction. FIGURE 3 | Combination of material characteristics in a cam-clay type plot. Normal stress is plotted on the x-axis, shear stress along the y-axis. Along the z-axis, pre-loading is plotted. Stippled green line represents tensile strength, stippled black line is cohesion. In a second experimental series, we use a 1:10 wt-% sand- powder mixture (1:10 mixture hereafter). This material has a 6.6 times lower cohesion than pure hemihydrate powder, representing weaker materials such as hyaloclastites, ashes, or scoria. In a second experimental series, we use a 1:10 wt-% sand- powder mixture (1:10 mixture hereafter). This material has a 6.6 times lower cohesion than pure hemihydrate powder, representing weaker materials such as hyaloclastites, ashes, or scoria. To characterize the materials, we measured densities and performed shear tests for normally consolidated and over- consolidated material, following the procedures of Holland et al. (2006) and van Gent et al. (2010). To characterize the tensile strength of cohesive powders at varying normal stresses, we constructed a tensile strength measuring setup similar to the Tensile Strength Tester (TST) of Schweiger and Zimmermann (1999). Table 1 shows the results of material characterization. Shear tests and tensile strength measurements can be combined in a Cam-Clay type model (Roscoe et al., 1963; van Gent et al., 2010). This plot (Figure 3) summarizes material properties, showing friction angle, tensile strength and cohesion as a function of over-consolidation. FIGURE 3 | Combination of material characteristics in a cam-clay type plot. Normal stress is plotted on the x-axis, shear stress along the y-axis. Along the z-axis, pre-loading is plotted. Stippled green line represents tensile strength, stippled black line is cohesion. mass is one to two orders of magnitude lower than the value for intact rock (Schultz, 1996). This is because consists not of a homogenous rock package but an intercalation of effusive and eruptive lavas of different origins, variable thicknesses, and different degrees of fracturing. Material Properties and Scaling p g Fine powders provide the cohesion and spatial resolution required to investigate dilatant fault processes (Walter and Troll, 2001). Holland et al. (2006) and Galland et al. (2006) REF presented the first experiments with full analog material characterization and thus a more complete understanding of the scaling relationships. Since then, different fine powders have been extensively used to model fractures in cohesive materials (Walter and Troll, 2001; Galland et al., 2006, 2015; Rodrigues et al., 2009; Gressier et al., 2010; van Gent et al., 2010; Holland et al., 2011; Trippanera et al., 2014). We use pure hemihydrate (CaSO4∗1/2 H2O) powder for our experiments, which is commercially available “Probau Baugips.” FIGURE 1 | Examples of massively dilatant faults in nature and experiment. (A) Thingvellir Fault in Iceland. For exact location see Figure 10. Red circle shows four geologists for scale. Top right shows sketch of fault geometry. The river flows on the hangingwall. Between hangingwall and footwall Massively Dilatant Faults (MDF) develop, which may be several tens of meters wide. These open fractures may be partly filled with sediments, water, ice or lava flows. Close the surface tilted blocks can form. (B) Experiment in hemihydrate powder shows open fractures and tilted blocks similar to the geometries observed in the field. Quantitative mapping of the geometry of analog examples may provide insights on the kinematics and deep structure of the system. February 2019 | Volume 7 | Article 18 Frontiers in Earth Science | www.frontiersin.org 3 Oblique Rifts and Massively Dilatant Faults von Hagke et al. FIGURE 2 | Experimental setup. (A) The box consists of a stable block and a hangingwall block that can move at different obliquities ranging from dip-slip to strike-slip. Angle of the of basement fault has been set to 60◦. (B) In top view, the rift trend is parallel to the basement fault, obliquity ω is defined as the angle between dip direction of the basement fault and direction of plate movement (i.e., ω = 0◦for dip-slip, and ω = 90◦for strike-slip; a dip-slip fault has no or 0◦of obliquity). (C) Example picture of an experiment with 45◦obliquity. Note the box does not have sidewalls, and thus the fault is unencumbered by sidewall friction. FIGURE 2 | Experimental setup. (A) The box consists of a stable block and a hangingwall block that can move at different obliquities ranging from dip-slip to strike-slip. Frontiers in Earth Science | www.frontiersin.org RESULTS on the angle of obliquity (Figure 5). In experiments with obliquities between 15 and 45◦, en-échelon structures are soon linked to form a through-going main fault. Both, underlapping and overlapping of fractures occur, leading to different linkage mechanisms. In most cases, fracture segments coalesce via hard linking of single underlapping fractures. Overlapping fractures develop into relay ramps that often breach during progressive deformation. Underlapping fault- parallel fractures often continue to grow parallel to the basement fault until they link with another fault segment along strike (see Supplementary Videos). En-èchelon fractures in the experiments with high obliquity (>60◦) eventually also link to form a through-going fault, but linkage occurs after 5–10 mm of displacement (Figure 5; Supplementary Videos). En-échelon fractures form not only during early stages of deformation, but also develop later in the graben center (Figure 5). If orientated approximately perpendicular to the direction of relative movement of the hanging wall, dilatancy of these fractures increases gradually during the experiment. Generally, orientation, curvature, location as well as amount of en-échelon fractures control main fault evolution as individual segments link during progressive deformation. For all experiments, we provide time-lapse videos in the digital appendix. All experiments have the same boundary conditions, that is a 60◦dipping basement fault and no sidewalls along strike the fault. They differ in the amount of obliquity. Here, we first report results of hemihydrate powder experiments, Structures for the two sets of 10 and 20 cm powder thickness are very similar, why it makes sense to discuss them jointly. We then compare these with results from 1:10 mixture experiments, before reporting (semi-) quantitative results of all experiments. Material Properties and Scaling Consequently, 1 cm of the hemihydrate represents ∼50 m of basalt (Table S1). This value matches observations in the field, where scarps of that height are common (Figure 1). To determine the scaling factor, the density and strength of Icelandic basaltic rocks were used, as these rocks represent the natural prototypes of the experiments. Using the scaling relationships of Hubbert (1937) and the cohesion and density of pure hemihydrate powder, 1 cm in the model would represent ∼500–2,800 m of intact basalt, depending on chemistry or degree of weathering (see Table 1 for material properties and Supplementary Data Sheet 1 for detailed discussion and scaling calculations). However, for modeling large-scale systems, bulk rock strength must be considered. Cohesion of basaltic rock Strength of the 1:10 mixture is 6.6 times weaker than the hemihydrate strength. This is similar to the strength contrast between intact basalt and weathered basalt or hyaloclastite or scoria. Thus, the 1:10 mixture is suitable to model weak rock as commonly outcropping in Iceland (Figure 1). 1:10 mixture is an alternative to crashed quartz sand, which is another material commonly used to model cohesive rocks (Galland et al., 2015). Details on material characterization and scaling are provided in the Supplementary Data Sheet 1. February 2019 | Volume 7 | Article 18 Frontiers in Earth Science | www.frontiersin.org 4 Oblique Rifts and Massively Dilatant Faults von Hagke et al. Hemihydrate Powder Experiments Surface Structures En-échelon fractures are the earliest structures that develop in all but pure dip slip experiments after ∼2 mm of displacement (Figure 4). They develop above the basement fault, as well as above future antithetic faults. At low obliquities (0◦and 15◦), orientation of the earliest fractures is largely parallel to the basement fault. At obliquities of 30◦and larger, en- échelon fractures form. Their orientation is variable, depending FIGURE 4 | Compilation of mapped initial fractures. With increasing obliquity, fracture orientation changes progressively. (A–G) Obliquity increases from 0 to 90◦; fractures mapped after 2 mm displacement. These early fractures control geometries of mature faults. Experiments with 20 cm hemihydrate thickness. Black arrows denote movement direction of the lower plate. FIGURE 4 | Compilation of mapped initial fractures. With increasing obliquity, fracture orientation changes progressively. (A–G) Obliquity increases from 0 to 90◦; fractures mapped after 2 mm displacement. These early fractures control geometries of mature faults. Experiments with 20 cm hemihydrate thickness. Black arrows denote movement direction of the lower plate. February 2019 | Volume 7 | Article 18 5 Frontiers in Earth Science | www.frontiersin.org Oblique Rifts and Massively Dilatant Faults von Hagke et al. RE 5 | Top view of final stages of 20 cm powder experiments and schematic cross section perpendicular to basement fault strike. (A–G) Obliquity increases from 0◦. Same experiments as shown in Figure 4. Pictures were taken after 24 mm of deformation. This is a stage where the fault has reached a mature phase, i.e., extension is almost exclusively accommodated by widening of pre-existing fractures. At obliquities between 0 and 60◦a graben forms between the main fault e antithetic fault on the hangingwall. At low obliquities, the entire fault shows dilatancy, whereas at higher obliquities dilatancy is restricted to releasing bends. how final fault geometries are influenced by orientation of early fractures (Figure 4). Red circles denote tilted blocks. Cross sections show vertical throw ated with fault movement (dv), and average widths of grabens. FIGURE 5 | Top view of final stages of 20 cm powder experiments and schematic cross section perpendicular to basement fault strike. (A–G) Obliquity increases from 0 to 90◦. Same experiments as shown in Figure 4. Pictures were taken after 24 mm of deformation. This is a stage where the fault has reached a mature phase, i.e., further extension is almost exclusively accommodated by widening of pre-existing fractures. Fault Plane and 3D Structure Fault Plane and 3D Structure To determine dilatancy at depth and to inspect individual fault surfaces, we excavated two experiments with 15◦and 60◦fault obliquity (Figure 6). It was only possible to excavate powder experiments, as the 1:10 mixture immediately collapsed and filled all open fractures. Open fractures exist down to depths of 6– 8 cm, and can be linked to the surface or are found below collapsed tilted blocks. Dilatant fractures are interconnected along the entire fault in experiments with low obliquities. In the graben center, open fractures are observed down to a depth of 10 cm. Generally, the amount and size of dilatant fractures decreases with depth. Smaller fractures are rapidly filled with debris, particularly in areas where tilted blocks collapse. The deepest cavities of up to 12 cm have been preserved at antithetic faults. Dilatancy close to the surface is much lower as compared to the main fault, and thus more easily filled at shallow depths. This prevents loose material from reaching the lower parts. Furthermore, because tilted blocks are rare, the amount of debris falling into the fault is relatively small. Consequently, permeability of the antithetic faults may be higher at depth as compared to the main fault, both in 15◦and 60◦ obliquity powder experiments. While rubble fillings partly clog the entire main fault, antithetic faults show connected cavities along strike (Figure 6). During progressive deformation, fractures step into the footwall, incorporating individual blocks of the main fault into the graben structure. This results in formation of tilted blocks and breached relay structures and thus decreasing fault sinuosity (defined as the curvilinear length of the fault divided by the linear distance between two points). This is most pronounced in experiments of 30–60◦obliquity. This is surprising, as intuitively this effect may be expected to be highest for experiments with highest obliquities. However, in experiments of 75–90◦obliquity, R-shears instead of early en-échelon fractures dominate final geometry (Figure 5; Supplementary Videos). Generally, in pure powder experiments, the final geometry of the main fault is established after 24 mm displacement. Because the fault dip in the model is steeper than basement fault dip, antithetic faults develop, resulting in graben structures. Antithetic faults are prominent features that are observed in all experiments except in 75◦-90◦powder. Antithetic faults form commonly after 4–10 mm displacement (see Supplementary Videos). Consequently, total displacements and apertures are smaller, ranging between 1 and 5 mm. Frontiers in Earth Science | www.frontiersin.org Hemihydrate Powder Experiments Surface Structures At obliquities between 0 and 60◦a graben forms between the main fault and the antithetic fault on the hangingwall. At low obliquities, the entire fault shows dilatancy, whereas at higher obliquities dilatancy is restricted to releasing bends. Note how final fault geometries are influenced by orientation of early fractures (Figure 4). Red circles denote tilted blocks. Cross sections show vertical throw associated with fault movement (dv), and average widths of grabens. February 2019 | Volume 7 | Article 18 Frontiers in Earth Science | www.frontiersin.org 6 Oblique Rifts and Massively Dilatant Faults von Hagke et al. Influence of strike-slip motion is most pronounced in all 75◦and 90◦obliquity experiments, where a set of R-shears forms at an angle between 15◦and 30◦to the underlying basement-fault. Because of linkage of en-échelon fractures during progressive deformation, fault geometries resemble a zig-zag pattern (Figure 5). Long R-Shears in experiments with obliquity of 75◦and 90◦, result in large wavelength of the structure (Figures 5F,G; Figure S3). restraining bends, dilatancy is negligible. This effect becomes most pronounced at obliquities of 75 and 90◦. In these experiments, open fractures are isolated. These fractures remain open during the entire experiments, as no tilted blocks form. Where restraining and releasing bends are less well-developed, the fault is open along its entire length. Fault Plane and 3D Structure At antithetic faults, tilted blocks are rare. Resulting graben geometries, sizes and formation mechanism differ between the individual experiments. Grabens do not form in strike-slip experiments and are rare in 75◦experiments. At low obliquities, narrow and deep grabens form, as observed in the 0◦10 and 20 cm powder experiments (Figure 5). With increasing obliquity, dilatancy along the main fault is often restricted to releasing bends at the surface, separated by intensively sheared zones. Consequently, dilatancy close to the surface becomes smaller with increasing obliquity. Releasing bends can remain open at depth as due to absence of tilted blocks little debris is filling them. This results in the (somewhat counterintuitive) general trend that faults with intermediate to high obliquities show dilatancy at greater depths than less oblique faults and a high vertical permeability. In all experiments, the area fraction of dilatancy decreases with depth. p p g Dilatancy along the main fault has been observed in all experiments with hemihydrate powder (Figure 5). The amount and localization of dilatancy depends on obliquity, sinuosity, cohesion of the material, and presence or absence of tilted blocks. Tilted blocks are typical features formed at the model surface. They are features developing at the hangingwall and tilting toward the graben center. In high obliquity experiments, tilted blocks are bound to transtensional areas (releasing bends) and are absent in strike-slip experiments. Tilted blocks form in different amounts and sizes, show different amount of tilting as well as variable degrees of fragmentation. There is no clear relationship between amount of tilt and amount of displacement. In many low-obliquity experiments, the blocks rotate back toward the hangingwall during late stages of deformation or collapse into dilatant faults. They always dip approximately at 90◦to the basement fault; we observe no variation of block orientation with increasing obliquity. In powder experiments, dilatant faults particularly fill where tilted blocks collapse. Where no tilted blocks form, wide dilatant fractures are present, and may be stable during the entire experiment. Excavated fault planes show slickensides on the surfaces of shear fractures, tracing the moving direction of the hangingwall block (Figure 7). Slickensides are observable in all experiments. In strike slip experiments, they are observed from close to the surface down to the bottom of the experiment. In dip-slip experiments, they only occur at depths >5 cm, as closer to the surface only dilatant fractures occur. Fault Plane and 3D Structure Vacuum cleaning results in more rugged surface as opposed to slicing, but small open fractures are unencumbered by the excavation process. Similar to (B), the area fraction of open gaps is larger at the antithetic fault, as the main fault is filled with debris of collapsing tilted blocks. FIGURE 6 | Excavated experiments. Two excavation techniques have been applied to visualize open fractures at depth. First, open fractures were filled with dyed sand, and the experiment was sliced from top to bottom. (A) Top view on surface structures of experiment with 10 cm powder thickness and 15◦obliquity. Down-thrown block at bottom, arrows indicate direction of movement. (B) Excavated experiment. Blue sand filled the open fractures at depth. At 3.5 cm wide open fractures exist at depth. Tilted blocks at the hangingwall collapsed and reduce area fraction of open gaps at the main fault. The antithetic fault remains open. (C) Top view of powder experiment with 20 cm powder thickness and 60◦obliquity. (D) Surface excavated with vacuum cleaner. Vacuum cleaning results in more rugged surface as opposed to slicing, but small open fractures are unencumbered by the excavation process. Similar to (B), the area fraction of open gaps is larger at the antithetic fault, as the main fault is filled with debris of collapsing tilted blocks. are mainly restricted to a narrow band in the 1:10 mixture experiments. In experiments with 30◦obliquity and higher, underlapping fault segments do not link during early stages of the experiment. Instead, they continue to grow subparallel to each other, forming overlapping fault segments that are subsequently linked by the formation of a relay. During late stages of the experiments, these relays are breached and a through-going main fault forms (Figure 8, see also Supplementary Videos). Generally, in pure powder experiments, the final geometry of the main fault is established after 2 mm displacement. In 1:10 mixture experiments geometries change until 20 mm displacement. FIGURE 7 | Slickensides on an excavated fault surface of a hemihydrate experiment. Slickensides are best visible in the encircled area, and absent in dilatant parts of the fault. FIGURE 7 | Slickensides on an excavated fault surface of a hemihydrate experiment. Slickensides are best visible in the encircled area, and absent in dilatant parts of the fault. Similar to powder experiments, in 1:10 mixture experiments grabens develop between main and antithetic fault except at high obliquities. Frontiers in Earth Science | www.frontiersin.org Fault Plane and 3D Structure Size of the slickenside planes is variable along strike individual slip planes, and may range between few millimeters to centimeters. Sand-Powder Mixture Experiments Results from 1:10 mixture experiments, scaled to weaker rocks such as hyaloclastites or scoria, can be compared to powder experiments (Figure 8). Generally, the 1:10 mixture experiments display similar surface structures as observed in experiments with hemihydrate powder only. These include the formation of Increase of obliquity changes the amount and location of dilatant parts along the main fault. The higher the angle of obliquity, the more dilatant releasing bends develop. In February 2019 | Volume 7 | Article 18 7 Oblique Rifts and Massively Dilatant Faults von Hagke et al. FIGURE 6 | Excavated experiments. Two excavation techniques have been applied to visualize open fractures at depth. First, open fractures were filled with dyed sand, and the experiment was sliced from top to bottom. (A) Top view on surface structures of experiment with 10 cm powder thickness and 15◦obliquity. Down-thrown block at bottom, arrows indicate direction of movement. (B) Excavated experiment. Blue sand filled the open fractures at depth. At 3.5 cm wide open fractures exist at depth. Tilted blocks at the hangingwall collapsed and reduce area fraction of open gaps at the main fault. The antithetic fault remains open. (C) Top view of powder experiment with 20 cm powder thickness and 60◦obliquity. (D) Surface excavated with vacuum cleaner. Vacuum cleaning results in more rugged surface as opposed to slicing, but small open fractures are unencumbered by the excavation process. Similar to (B), the area fraction of open gaps is larger at the antithetic fault, as the main fault is filled with debris of collapsing tilted blocks. FIGURE 6 | Excavated experiments. Two excavation techniques have been applied to visualize open fractures at depth. First, open fractures were filled with dyed sand, and the experiment was sliced from top to bottom. (A) Top view on surface structures of experiment with 10 cm powder thickness and 15◦obliquity. Down-thrown block at bottom, arrows indicate direction of movement. (B) Excavated experiment. Blue sand filled the open fractures at depth. At 3.5 cm wide open fractures exist at depth. Tilted blocks at the hangingwall collapsed and reduce area fraction of open gaps at the main fault. The antithetic fault remains open. (C) Top view of powder experiment with 20 cm powder thickness and 60◦obliquity. (D) Surface excavated with vacuum cleaner. Fault Plane and 3D Structure Antithetic faults form generally after 4–10 mm displacement. The graben in 1:10 mixture experiments shows a decrease in graben width with increasing obliquity at lower obliquities as compared to powder experiments (Figure 9). At higher obliquities (>45◦) 1:10 mixture experiments also show restraining and releasing bends, but apertures are much smaller than in the pure powder experiments and the open parts are less connected. The 75◦1:10 mixture experiment shows a distinct shear zone in its center without any opening (Figure 8). In summary, weaker material shows similar structural elements but observed geometries are often less pronounced than in powder experiments (e.g., smaller tilted blocks, narrow graben). Often features occur only transiently (Supplementary Videos), as sidewalls loose cohesion. Even small vibrations during the experiment can lead to a total disintegration of structures. cliffs, tilted blocks, (breached) relays, dilatant faults, antithetic faults, local areas of extension and compression as well as en- échelon faults and R-shears. We observe the strain is more localized in a narrow band around the basement fault trace. The influence of obliquity on the geometry and curvature of the antithetic fault is much smaller as compared to the hemihydrate powder. Generally, dilatant parts in the 1:10 mixture experiment are quickly filled as the sidewalls rapidly loose cohesion. Like in the powder experiments, en-échelon fractures are the first observable structures at the surface. However, while in powder experiments fractures form over a large area they February 2019 | Volume 7 | Article 18 Frontiers in Earth Science | www.frontiersin.org 8 Oblique Rifts and Massively Dilatant Faults von Hagke et al. FIGURE 8 | Comparison between experiments with different powder thickness and material strength. (A–C) Experiments with 0◦obliquity. In 20 (Continu FIGURE 8 | 10 cm powder experiments, open fractures form on both sides of a central graben between main and antithetic fault. Tilted blocks from that may collapse and fill parts of the fault with rubble. In weaker material (1:10 mixture) open fractures are rapidly filled with material collapsing from the sidewalls (C). Vertical cliffs form, but open fractures are only sustained close to the surface. (D–F) Experiments with 45◦obliquity. A shallow and wide graben forms in the 20 cm powder experiment; the influence of early en-échelon fractures is visible in the hangingwall. Dilatant fractures at the surface are less continuous and narrower as compared to the experiments with 0◦obliquity. Fault Plane and 3D Structure In the 10 cm and 1:10 mixture experiments, a narrow graben forms (F). Rotated and collapsed blocks oriented parallel to the basement fault are visible in all experiments. Dilatant fracturing in the 1:10 mixture experiments is much lower than in powder experiments. (G–I) Experiments with 75◦obliquity. Typical strike-slip structures occur and fault geometry is dominated by orientation of R-shears. Dilatancy in powder experiments is most pronounced at releasing bends. Width of the fault zone decreases in the 1:10 mixture experiments (I). (Semi-) Quantitative Analysis ( ) y For quantitative analysis, we determine dilatancy, graben width, fracture distribution, sinuosity (defined as the ratio of the curvilinear length of the fault trace divided by fault length), and the shape and distribution of tilted blocks along strike the fault. Results of quantitative analysis are provided in Figure 9. Graben width correlates with obliquity (Figure 9A). With increasing obliquity, grabens first tend to become slightly wider before decreasing at obliquities >45◦for 10 cm powder experiments, and obliquities >60◦for 20 cm powder experiments, respectively. Expectedly graben width is zero for 90◦obliquity, i.e., strike slip faulting without normal fault component (Figure 9). To characterize fracture orientation, we report mean azimuth with respect to the basement fault and length. Orientation of early fractures in dip slip experiments is dominantly parallel to the basement fault, as expected. With increasing obliquity en-échelon fractures form and the strike of the fractures changes progressively, until it stabilizes at an angle of 15 ± 5◦with respect to the basement fault (Figure 9B). At final stage of deformation and zero obliquity (dip-slip deformation), fractures longer than 6 mm are oriented parallel to the basement fault. Smaller fractures occur in all directions. With increasing obliquity, fewer small fractures are observed, particularly in the graben, and their orientation is more similar to that of the main fault. Strike of fractures progressively changes with increasing obliquity, to a deviation of ∼20◦from the basement fault in strike slip experiments (Figure 9C). This is consistent with expected orientations of R-Shears. In all experiments, the area between main and antithetic fault is progressively more fractured during the experiments. Therefore, mean fracture length does not change during the experiments. FIGURE 8 | Comparison between experiments with different powder hickness and material strength. (A–C) Experiments with 0◦obliquity. In 20 and A general decrease in fault dilatancy with increasing obliquity can be observed in all experiments (Figures 9D,E). The thickness of the powder layer does not change measured dilatancy, as it depends on slip of the basement fault (as opposed to graben width, which correlates with layer thickness). Similarly, dilatancy measured in pure powder experiments is comparable to values in 1:10 mixture experiments, and depends on the amount of horizontal displacement on the fault. However, dilatancy determined at the surface is always higher than the FIGURE 8 | Comparison between experiments with different powder thickness and material strength. (A–C) Experiments with 0◦obliquity. (Semi-) Quantitative Analysis In 20 and (Continued) February 2019 | Volume 7 | Article 18 Frontiers in Earth Science | www.frontiersin.org 9 Oblique Rifts and Massively Dilatant Faults von Hagke et al. FIGURE 9 | Quantitative evaluation of all experiments. (A) Average graben width (i.e., the distance between the main fault and the antithetic fault) for experiments 10 and 20 cm powder thickness and 1:10 mixture experiments as a function of obliquity. Graben width is constant or slightly increasing for low to medium obliquit and drops to zero at high obliquities. No graben forms in strike-slip experiments. Faults (A,C) are natural examples from Iceland (Figure 13). (B) Orientation of init fractures for different obliquities as observed in experiments, as measured after 2 mm of displacement. Progressive change of strike direction can be seen until ∼4 after which the orientation is roughly constant at an angle of 15 ± 5◦with respect to the basement fault. Red markers represents fracture orientation observed at evolving fault in Iceland. (C) Orientation of fractures as observed after 24 mm of displacement in experiments. Mean strike values are variable, but a general trend increasing obliquity can be inferred. The values measured in our experiments are similar to values obtained from soft clay models (Withjack and Jamison, 1986) (D) examples of dilatancy as determined in 10 cm powder experiments. Top view of experiments, blue areas are dilatant parts. (E) Quantification of changes of dilatancy with increasing obliquity. The general decreasing trend can be seen in all experiments. Dilatancy scales with horizontal displacement on the basement fa (black dots). (F) Number of tilted blocks plotted vs. fault obliquity. A general decrease is observed. The size of tilted blocks remains constant for low obliquities, bu decreases slightly when obliquity is larger than 45◦(G). This trend is less apparent for 1:10 mixture experiments. (H) Fault sinuosity as a function of obliquity. horizontal displacement at the basement fault This is an exciting Closely linked to the amount of dilatancy is the numb FIGURE 9 | Quantitative evaluation of all experiments. (A) Average graben width (i.e., the distance between the main fault and the antithetic fault) for experiments with 10 and 20 cm powder thickness and 1:10 mixture experiments as a function of obliquity. Graben width is constant or slightly increasing for low to medium obliquities and drops to zero at high obliquities. No graben forms in strike-slip experiments. Frontiers in Earth Science | www.frontiersin.org (Semi-) Quantitative Analysis Faults (A,C) are natural examples from Iceland (Figure 13). (B) Orientation of initial fractures for different obliquities as observed in experiments, as measured after 2 mm of displacement. Progressive change of strike direction can be seen until ∼45◦, after which the orientation is roughly constant at an angle of 15 ± 5◦with respect to the basement fault. Red markers represents fracture orientation observed at an evolving fault in Iceland. (C) Orientation of fractures as observed after 24 mm of displacement in experiments. Mean strike values are variable, but a general trend with increasing obliquity can be inferred. The values measured in our experiments are similar to values obtained from soft clay models (Withjack and Jamison, 1986) (D) examples of dilatancy as determined in 10 cm powder experiments. Top view of experiments, blue areas are dilatant parts. (E) Quantification of changes of dilatancy with increasing obliquity. The general decreasing trend can be seen in all experiments. Dilatancy scales with horizontal displacement on the basement fault (black dots). (F) Number of tilted blocks plotted vs. fault obliquity. A general decrease is observed. The size of tilted blocks remains constant for low obliquities, but decreases slightly when obliquity is larger than 45◦(G). This trend is less apparent for 1:10 mixture experiments. (H) Fault sinuosity as a function of obliquity. Closely linked to the amount of dilatancy is the number of tilted blocks along the main fault (Figure 9F). We counted the number and measured sizes of tilted blocks in 10 cm powder, 20 cm powder and 1:10 mixture experiments. The number of tilted blocks decreases with increasing obliquity. 1:10 mixture experiments show consistently more tilted blocks as compared horizontal displacement at the basement fault. This is an exciting observation, as it implies it is not straightforward to determine the amount of fault slip by measuring dilatancy. As observed in the sliced experiments, dilatancy deceases with increasing obliquity, as it is successively more concentrated in releasing bends in strike slip setting. February 2019 | Volume 7 | Article 18 Frontiers in Earth Science | www.frontiersin.org 10 Oblique Rifts and Massively Dilatant Faults von Hagke et al. the fault) form to a depth of 5–9 cm in hemihydrate powder. Down to this depth no slickensides were observed. Below this, in the hybrid fracture zone, open fractures occur together with slickensided patches. (Semi-) Quantitative Analysis Using our scaling relationship, the failure mode transition in the Iceland basalts occurs at depths of 250–450 m, possibly down to 1.5–2 km for stronger intact rock (see details on scaling in the Supplementary Data Sheet 1). Considering a tensile strength of 6 MPa and a mean density of 2,400 kg/m3, Acocella et al. (2003) calculate the depth of failure mode transition to occur between 260 and 780 m, which is similar to our results. Depth of open fractures are comparable to estimates of Gudmundsson (1992), Acocella et al. (2003), Grant and Kattenhorn (2004), or Hardy (2013), who predict open fractures in basalts to occur down to depths of up to 800 m, c. 500 m, and several hundred meters, respectively, depending on tensile strength and rock density. to pure powder experiments. This is because the lower cohesion of the material results in fractionation of the blocks during the experiment. The size of tilted blocks is constant for low obliquities, but decreases at obliquities >45◦. This trend is less apparent for 1:10 mixture experiments; size of tilted blocks in 1:10 mixture experiments is lower than in powder experiments. Blocks are largest at low obliquities and in 20 cm powder experiments (Figure 9G). During progressive deformation, tilted blocks are successively disintegrated. Consequently, the volume of open voids at the final stage of deformation is lower the more tilted blocks formed. We determined sinuosity (defined as the ratio of the curvilinear length of the fault and its length) of the main fault in all experiments from mapping fault traces (Figure 9H). In powder experiments with 10 cm powder thickness, fault sinuosity increases for obliquities from 0 to 60◦, followed by a decrease. This pattern evolves because early fractures and their linkage influence sinuosity (see also above). In low obliquity experiments, early fractures are oriented approximately parallel to the basement fault, resulting in a main fault with low sinuosity after early fracture linkage. With increasing obliquity, en-échelon fractures form, progressively deviating from basement fault strike (Figure 9B). Linkage leads to successively higher sinuosity of the mature fault. At obliquities of 60◦and higher, initial fractures are crosscut by the later main fault, resulting in a sinuosity decrease. In experiments with 20 cm powder thickness, the decrease occurs already at 45◦; for 1:10 mixture experiments, sinuosity of experiments with low obliquity is higher than observed in powder experiments. (Semi-) Quantitative Analysis Despite these general trends, considerable variation of sinuosity can be observed in the repeated experiments (Figure 9H). This is because local material heterogeneities influence fault geometry. This implies that for individual faults, sinuosity is not always a good measure to determine fault obliquity. In summary, our analyses show the angle of obliquity influences fault geometry systematically. Parameters particularly indicative for obliquity are orientation of initial fractures, graben width, dilatancy at the surface and at depth, and number of tilted blocks. In this study, we focus mostly on dilatant structures, as other parameters have been studied earlier (see e.g., Acocella, 2014 for a recent review). In our hemihydrate powder models, dilatant jogs still exist at the base of the box, i.e., at a depth of 20 centimeters, independent of fault kinematics. For intact basalt rock, this suggests that in dilatant jogs in normal faults may exist down to depths of ∼5 km and possibly deeper. Weak materials such as hyaloclastite or scoria are not able to sustain open fractures, at these depths. Consequently, depending on the material involved in the faulting process, as well as its degree of fragmentation and weathering, different depths of failure mode transition may occur in nature. Interlayering of weak and strong materials, as often observed in rift systems, will result in a complex failure mode transition zone that can be spread out over hundreds of meters (Smart and Ferrill, 2018). The observation of dilatancy is consistent with findings of earlier studies, using hemihydrate powder and a dip-slip experimental setup (Holland et al., 2006; van Gent et al., 2010; Kettermann et al., 2016). This study shows additionally that obliquity influences the location of dilatant patches at depth and at the surface. While dip-slip experiments show a continuous connection of open fractures at the surface, strike-slip experiments show open fractures only in releasing bends. Average dilatancy at the surface reduces approximately linearly with increasing obliquity for the same slip vector (Figure 9E). In dip slip experiments, tilted blocks form, which are later disintegrated and fill the open fractures with rubble. With increasing obliquity, smaller tilted blocks form, and they have not been observed in our strike slip experiments. Hence, the open fractures are not filled with collapsing material. Frontiers in Earth Science | www.frontiersin.org Analog Models p g Our models build on previous work that showed how obliquity influences rift geometries at crustal to outcrop scale. Discrete element models by Deng et al. (2018) show how fracture patterns at the surface are affected by a pre-existing weakness. Based on analog models, Withjack and Jamison (1986) predict maximum extension direction at oblique rifts (which is perpendicular to orientation of early en-échelon fractures) is a function of the angle between rift trend and displacement of the rift walls. This relationship has been corroborated in clay models and field data in Iceland (Clifton and Schlische, 2003), and is also observed in our experiments (Figures 9B,C). Furthermore, it has been suggested that fault sinuosity depends on obliquity, and reaches a maximum at 60◦obliquity (Clifton et al., 2000). A general observation in our experiments is that the geometry of the fault is strongly affected by the orientation of the initial fractures. Location, orientation and densitiy of en-échelon fractures then affects the formation of tilted blocks, graben geometry, and fault sinuosity in the massively dilatant zone. The location of initial fractures is strongly dependent on local heterogeneities in the model material, and this has also been seen in DEM models (Abe et al., 2011). In the natural prototype, this corresponds to the state of jointing in the basalt (Holland et al., 2011). This implies that much of the variability of fractures is controlled by heterogeneities in the model material. In our experiments, the deepest open fractures that can be followed along strike (i.e., the massively dilatant part of February 2019 | Volume 7 | Article 18 11 Oblique Rifts and Massively Dilatant Faults von Hagke et al. ∼30% obliquity to spreading direction of the mid ocean ridge (Gudmundsson, 1987; Grant and Kattenhorn, 2004; Clifton and Kattenhorn, 2006; Villemin and Bergerat, 2013). The northern and western rift zones are dominated by axial spreading (Angelier et al., 1997; Friese, 2008; Sonnette et al., 2010; Hjartardóttir et al., 2012). In Thingvellir, located in the western rift zone, the rift axis trends approximately normal to the plate spreading direction at 30◦. Similarly, the Krafla fissure swarm and the Theistareykir fissure swarm of the northern volcanic zone are also dominated by axial rift. Faults observed in Iceland show different amounts of dilatancy, ranging from several tens of meters to cm scale. Oblique Faults on Iceland The results of our study may be applied to faults on Iceland located on the Mid Atlantic Ridge. Iceland is the only place on Earth where a mid ocean ridge is exposed above sea level, atop the extensional plate boundary separating the North American plate and the Eurasian plate. The reason for the aerial exposure of the Mid Atlantic Ridge in Iceland is the presence of a mantle plume, the Icelandic hotspot (Kaban et al., 2002). This mantle plume is the cause for increased volcanic activity and a wider, more complex deformation zone compared to oceanic plate boundaries where no plume activity occurs, as the relative movement of the plate boundary with respect to the Icelandic hotspot leads to rift jumps and unstable boundaries (Einarsson, 2008). To quantitatively compare our analog models with nature, we focused on the Northern and Western Rift Zones, which offer exquisite outcrop conditions and good accessibility to faults (Figure 11). We used unmanned aerial vehicles to photograph the faults and successively create a digital elevation model at 5– 15 cm resolution (Figure 12). In total, we cover an area of roughly 12 km². Details on data gathering and DEM building are provided in the Supplementary Data Sheet 1. These high-resolution data sets enable us to characterize fault geometries in the models at the decimeter scale. The average graben width represents the mean distance between the bounding faults, measured along scanlines between the manually mapped graben boundaries in intervals of 1 m orthogonally to the average fault strike. Angles of obliquity are calculated from the opening width and horizontal displacement of associated features along the fault. The strike values for the faults and en-échelon fractures were calculated as linear directional mean from the vectors of the fault traces. The mid-Atlantic ridge breaks up into a series of more of less oblique segments on the Icelandic main land (Figure 10). Zones of active rifting in Iceland are indicated by increased volcanic activity. Some of the rifting segments are purely divergent and characterized by normal faulting and fissuring. This type of fracturing and volcanism is observed in the Northern Volcanic Zone, and in two sub-parallel rift zones in South Iceland, the Western and Eastern Volcanic Zones, respectively. Analog Models Vertical depths of dilatant fractures measurable in the field can be >40 m, before the open fractures are filled with water, aeolian sand, or rubble (Figure 11). Vertical walls show lava flows with columnar joints and different degrees of fracturing, and weathering intensity varies. Particularly for large openings it is challenging to correlate geometries at the surface. Climbing into the open fractures allows correlations in deeper sections. Along all faults, tilted blocks occur, with lengths ranging from few to several hundreds of meters. As in our analog models, collapsing of the blocks into the open fractures can be observed. Relays and breached relays are common. Similar to our analog models, antithetic faults can be observed, with less vertical throw and dilatancy as compared to the main fault. In the Western Rift Zone, newly developing faults exist, showing en-échelon fractures as observed in our models. 1:10 mixture experiments may be compared to mechanically weaker rock, such as hyaloclastite, ignimbrites or tufflayers exposed in Iceland. These rocks are capable of supporting vertical cliffs but are much more prone to weathering. In weathered parts, no tilted blocks are preserved, and dilatant sections are often filled with rubble. This agrees with findings of our study (Figure 9H). However, our experiments show that fault sinuosity is variable, and depends on location of early fractures, their position with respect to the basement fault, and fault linkage processes. Particularly early fracture localization depends on small scale heterogeneities, and fault linkage processes do not depend on obliquity but on the local stress field and its variation (e.g., Mansfield and Cartwright, 2001; Clifton and Kattenhorn, 2006), as well as on the amount of stretching (Acocella et al., 2005); see Fossen and Rotevatn (2016) for a review. Sinuosity values obtained in our experiments vary between 1.05 and 1.16. For clay experiments; Clifton et al. (2000) obtained values between 1.4 and 2.26. The higher values are a result of two linking fault populations, whereas in our experiments sinuosity mostly depends on linkage of en-échelon fractures. As observed in our experiments and reported from field data (e.g., MacDonald et al., 1979; Kureth and Rea, 1981), sinuosity of the fracture zone in transform settings controls formation of releasing and restraining bends along the fault, which in turn influence dilatancy at depth. Frontiers in Earth Science | www.frontiersin.org February 2019 | Volume 7 | Article 18 Oblique Faults on Iceland Other segments of the rift are transform zones characterized by strike-slip movement, such as the South Iceland Seismic Zone connecting the Western and Eastern Volcanic Zones, and the Tjörnes Fracture Zone in the North of Iceland. Additionally, oblique segments such as the Reykjanes Volcanic Belt are characterized by interlinked volcanism and strike-slip faulting (e.g., Einarsson, 2008; Savry and Cañón-Tapia, 2014). On Iceland dilatant faults similar to those observed along all mid ocean ridges are spectacularly exposed (e.g., Bubeck et al., 2017), Figure 11. It has been shown that these dilatant faults control fluid pathways and geothermal anomalies (Walter et al., 2018). We chose three faults, where geometries of dilatant fractures on both sides can be correlated to determine obliquity. Fault A is located in the Krafla Fissure Swarm (Kelduhverfiare) of the Northern Rift Zone, near the Asbyrgi canyon. Faults B and C are on the Reykjanes Peninsula in the Western Rift Zone. Fault B belongs to the Reykjanes and Fault C to the Vogar fissure swarms, respectively. Five en-échelon fractures were identified along fault A (in Asbyrgi), intersecting the main fault at an angle of 25◦. Two antithetic faults are present, and the respective graben widths are The Reykjanes Peninsula is situated in the south-western most part of Iceland (Figure 10). It is the direct prolongation of the Reykjanes ridge, i.e., the region where the mid-Atlantic ridge hits land. It is the only onshore oblique rift segment of the mid-Atlantic ridge, with the plate boundary oriented at February 2019 | Volume 7 | Article 18 12 Oblique Rifts and Massively Dilatant Faults von Hagke et al. FIGURE 10 | Geological map of Iceland including main fault strucutres and volcanic belts. RR, Reykjanes Ridge; RP, Reykjanes Peninsula; RVB, Reykjanes Volcanic Belt; WVZ, EVZ, and NVZ, Western, Eastern and Northern Volcanic Zone; TFZ, Tjörnes Fracture Zone; Thv, Thingvellir. Geological map modified after Jóhannesson (2014). Rift zones from Thordarson and Larsen (2007), GPS data from Árnadóttir et al. (2008), stress data from Heidbach et al. (2016). FIGURE 10 | Geological map of Iceland including main fault strucutres and volcanic belts. RR, Reykjanes Ridge; RP, Reykjanes Peninsula; RVB, Reykjanes Volcanic Belt; WVZ, EVZ, and NVZ, Western, Eastern and Northern Volcanic Zone; TFZ, Tjörnes Fracture Zone; Thv, Thingvellir. Geological map modified after Jóhannesson (2014). Rift zones from Thordarson and Larsen (2007), GPS data from Árnadóttir et al. (2008), stress data from Heidbach et al. (2016). Frontiers in Earth Science | www.frontiersin.org Oblique Faults on Iceland Field impressions show exceptional outcrop conditions of the rift system, allowing for high-resolution mapping of structures close to the surface. Apertures of open fractures at the surface range from few cm to several tens of meters, as for instance observed at the famous world heritage site of Thingvellir. Correlating both sides of the open fracture is commonly possible at fractures of widths of 10 meters or less. Fractures are partly filled with rubble. Image 4 was taken with an unmanned aerial vehicle. Note the tilted block in foreground of image 4. FIGURE 11 | Field sites on Iceland that may be compared to our analog models. Field impressions show exceptional outcrop conditions of the rift system, allowing for high-resolution mapping of structures close to the surface. Apertures of open fractures at the surface range from few cm to several tens of meters, as for instance observed at the famous world heritage site of Thingvellir. Correlating both sides of the open fracture is commonly possible at fractures of widths of 10 meters or less. Fractures are partly filled with rubble. Image 4 was taken with an unmanned aerial vehicle. Note the tilted block in foreground of image 4. decrease of dilatancy with depth, partly because of collapsing tilted blocks. Strike-slip faults do show a much weaker trend, and dilatant patches occur close to the surface and at depth. Acocella et al., 2003; Trippanera et al., 2015). The open fractures are partly filled with debris from collapsing tilted blocks. In strike-slip kinematics, dilatancy is constrained to releasing bends. This results in lower connectivity of open fractures at the surface. This model may be applied to Iceland. We show that faults are dilatant to depths of several hundred meters. At depths below 450 m transition to shear fracturing occurs, and more intermittent dilatant fractures may exist at depths of at least one kilometer. Fractures may be well-connected at depth, and large caves may exist. At faults with an oblique component such as observed in the Northern Rift Zone and the Reykjanes Peninsula open fractures also exist at deep levels. However, their fracture network is less well-connected. Partly the faults may be more permeable at depth, as rubble fillings are less common. These findings extend our previous knowledge on the fault systems in Iceland and other rift zones (Figure 14). Oblique Faults on Iceland with results from analog models (Figure 9). This suggests that structures are not only qualitatively but also quantitatively comparable, and thus the structures we observe at depth in our analog models are also present in the faults on Iceland. That surface structures in the field and the models are quantitatively comparable suggests structures at depth observed in the models are also present in nature. Figure 13 shows a conceptual model of geometries at faults with different obliquities, summarizing robust features of the models. In dip slip deformation, tilted blocks form and dilatancy occurs at the main and the antithetic fault. Early fractures are oriented parallel to the main fault. Tilted blocks may collapse, filling open fractures. Because at antithetic faults tilted blocks are rare, open fractures remain open. Below a depth of ∼6–8 cm, the volume of open fractures decreases and they are often not connected. This depth of open fractures is consistent with predictions from the Mohr-Coulomb diagram and natural data from fractured rocks (Gudmundsson, 1992; 285 m for the proximal and 363 m for the distal fault. To quantify obliquity, we inspected geometries of the adjacent fault-plane traces and connected matching features. Fault A has an angle of obliquity of 41◦(Figure 12). (The general extension direction in the area is orthogonal.) The 33◦average strike direction of Fault B has been calculated from a vector from fault tip to fault tip of the analyzed segment. The initial en-échelon fractures were mapped as straight lines from fault tip to fault tip, from which the mean strike direction of all en-échelon fractures was calculated. Average en-échelon direction is 47◦. Thus, the en- échelon fractures are oriented in an acute angle of 13.5◦to the main fault. For fault C (Vogar) the mean graben width of 422 m has been measured from the main fault in the SE toward the next antithetic fault (Figure 12). The fault traces have been mapped and projected toward the adjacent fault trace to illustrate this fault has no oblique slip component. Measurements of graben width and orientation of early fractures are in good agreement February 2019 | Volume 7 | Article 18 13 Oblique Rifts and Massively Dilatant Faults von Hagke et al. FIGURE 11 | Field sites on Iceland that may be compared to our analog models. Frontiers in Earth Science | www.frontiersin.org Oblique Faults on Iceland Close to the surface and down to depths of at least 450 m but as deep as the critical depth of ∼800 m (Gudmundsson, 1992; Acocella et al., 2003), exclusively mode one fractures form. Below this, the failure mode transition to shear fracturing occurs. Our experiments show this is a complex zone in which locally open fractures Open fractures are present also at depths greater than predicted by standard mechanical models, reaching the base of our experiments. This implies the failure mode transition from dilatant to shear fracturing is a complex zone that may extend to depths of at least 1 km. Open fractures at depth are not connected along strike, but are separated by shear fractures. In our analog models this is witnessed by slickensides on the fault surfaces. During oblique slip early fractures are oriented at an angle to the main fault, which is increasing with increasing obliquity. This results in increasing sinuosity and possibly a decrease of connectivity of open fractures. Tilted blocks are smaller with increasing obliquity and are absent in strike-slip experiments. This influences the distribution of open fractures because collapsing tilted blocks fill open voids at depth. Releasing bends close to the surface and at depth can remain open where less tilted blocks are present. Dip-slip faults feature a pronounced February 2019 | Volume 7 | Article 18 14 Oblique Rifts and Massively Dilatant Faults von Hagke et al. FIGURE 12 | High resolution digital elevation models of the selected field sites where obliquity can be determined. Fault (A) shows two antithetic faulty that can be used for determining graben width. Insets 1 and 2 show how both fracture surfaces can be linked. Fault (B) shows a series of early fractures and their angle to the main fault. Early fractures are indicative for fault obliquity. Fault (C) shows no obliquity, as determined by matching the respective fracture surfaces on both sides. FIGURE 12 | High resolution digital elevation models of the selected field sites where obliquity can be determined. Fault (A) shows two antithetic faulty that can be used for determining graben width. Insets 1 and 2 show how both fracture surfaces can be linked. Fault (B) shows a series of early fractures and their angle to the main fault. Early fractures are indicative for fault obliquity. Fault (C) shows no obliquity, as determined by matching the respective fracture surfaces on both sides. Frontiers in Earth Science | www.frontiersin.org Oblique Faults on Iceland to the dike system that accommodates rift extension, as has been suggested by field studies in different rift systems (Nobile et al., 2012; Phillips et al., 2017), as well as and analog models (Trippanera et al., 2014; Galland et al., 2015). Modeling of dike intrusion into cohesive powders shows that surface fractures propagate from top to bottom, growing toward the upward- propagating dike (Abdelmalak et al., 2012). Strain analysis of the experiments show that at the tip of the dike shear bands form which connect to the surface fault during later stages of the experiments (Abdelmalak et al., 2012). Field evidence for these processes is rare, as outcrops are very limited. In Iceland, few outcrops of the deeper section of the rift system exists on the east coast of the Trollaskagi Peninsula in North Iceland (Gudmundsson, 2000). Here, primarily dikes and normal faults have been observed. In some areas the faults are only observed in the shallow part of the lithological column may be maintained to depths of at least 1 km, however in dilatant jogs. The deepest possible occurrence of open fractures has yet to be determined. This depth is not only controlled by mechanical stratigraphy resulting from interlayering of lava flows and ash and scoria layers; lava flows themselves have different strengths depending on their degree of fracturing and jointing. Furthermore, the occurrence of open fractures at depth depends on fluid pressure (Townend and Zoback, 2000); in cohesive carbonates dilatant open fractures have been observed at depths of several kilometers, associated with high fluid pressures (Hilgers et al., 2006; Grobe et al., 2018). We are not aware of fluid pressure data from Iceland measured at depths >1 km and consequently as of now the depth below which open fractures can be maintained remains speculative. Below the complex zone of failure mode transition only shear fracturing can occur. Ultimately, the faults are connected February 2019 | Volume 7 | Article 18 15 Oblique Rifts and Massively Dilatant Faults von Hagke et al. FIGURE 13 | Generic model of the observed structures depending on obliquity. (A) Dip slip deformation. A graben develops and tilted blocks form. Dilatancy is large close to the surface and decreases with depth. Fractures strike roughly parallel to the basement fault. In the graben, fractures perpendicular to the fault form occasionally. Sinuosity of the main fault is low. Open fractures are connected along the entire fault. Oblique Faults on Iceland (B) Oblique slip deformation. Graben is less wide, and tilted blocks are smaller. Sinuosity is high and influenced by early en-échelon fractures. Dilatancy occurs close to the surface and at depth. Red lines show slickensides on the fault surface. (C) Strike-slip faulting. No graben is present, and dilatancy is bound to Riedl Shears and releasing bends. This leads to potentially higher dilatancy at depth as compared to dip-slip faulting. FIGURE 13 | Generic model of the observed structures depending on obliquity. (A) Dip slip deformation. A graben develops and tilted blocks form. Dilatancy is large close to the surface and decreases with depth. Fractures strike roughly parallel to the basement fault. In the graben, fractures perpendicular to the fault form occasionally. Sinuosity of the main fault is low. Open fractures are connected along the entire fault. (B) Oblique slip deformation. Graben is less wide, and tilted blocks are smaller. Sinuosity is high and influenced by early en-échelon fractures. Dilatancy occurs close to the surface and at depth. Red lines show slickensides on the fault surface. (C) Strike-slip faulting. No graben is present, and dilatancy is bound to Riedl Shears and releasing bends. This leads to potentially higher dilatancy at depth as compared to dip-slip faulting. FIGURE 14 | (A) Conceptual model of the results applied to a rift (not to scale). In the uppermost section down to a depth of up to 800 m only dilatant fractures occur in cohesive rocks. Below this critical depth, transition to shear fracturing can be observed. Dilatant parts of the fault are isolated patches. Location of these patches depends on the heterogeneity and mechanical stratigraphy of the rock column. At greater depths elevated pore pressures may result in Mode 1 fracturing. The depth below which only shear fracturing can occur is unknown. If extension is large enough the surface fracture connects with the dike system at depth and may guide magma ascent. This can result in fissure eruptions and lava flows filling the MDF (B). FIGURE 14 | (A) Conceptual model of the results applied to a rift (not to scale). In the uppermost section down to a depth of up to 800 m only dilatant fractures occur in cohesive rocks. Below this critical depth, transition to shear fracturing can be observed. Dilatant parts of the fault are isolated patches. Oblique Faults on Iceland Location of these patches depends on the heterogeneity and mechanical stratigraphy of the rock column. At greater depths elevated pore pressures may result in Mode 1 fracturing. The depth below which only shear fracturing can occur is unknown. If extension is large enough the surface fracture connects with the dike system at depth and may guide magma ascent. This can result in fissure eruptions and lava flows filling the MDF (B). REFERENCES Árnadóttir, T., Geirsson, H., and Jiang, W. (2008). Crustal deformation in Iceland: plate spreading and earthquake deformation. Jökull 58, 59–74. Abdelmalak, M. M., Mourgues, R., Galland, O., and Bureau, D. (2012). 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SUPPLEMENTARY MATERIAL The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/feart. 2019.00018/full#supplementary-material Supplementary Videos 1–15 | Videos show top view of the experiments with hemihydrate powder (10 and 20 cm thickness), and experiments with 1:10 mixture for comparison. Slip of basement fault is dextral. Supplementary Videos 1–15 | Videos show top view of the experiments with hemihydrate powder (10 and 20 cm thickness), and experiments with 1:10 mixture for comparison. Slip of basement fault is dextral. FUNDING • Dilatancy determined at the surface is higher than horizontal displacement of the basement fault. This study is part of the project MDF: The structure and evolution of near-surface massively dilatant faults funded by the German Science Foundation (DFG). Grant number: 316167043. These findings have implications for our understanding of the connectivity of massively dilatant faults at depth. Main fault lateral connectivity at depth is decreasing with increasing obliquity as dilatancy is increasingly bound to small transtensional areas formed along the main fault. ACKNOWLEDGMENTS These observations can be applied to faults in active rift systems such as Iceland. In addition to being qualitatively comparable, detailed mapping of outcrops in Iceland shows that quantitatively, structures are comparable, too. Consequently, our results allow prediction of the geometry of the structure of massively dilatant faults at depth, where these are not accessible for direct study. The finding that open fractures may possibly occur at depths of at least 1 km for strong rocks (even if only as isolated patches) has consequences for our understanding of geothermal systems. Open fractures form major fluid pathways, and knowing their distribution at depth is vital for fluid flux modeling studies. Daniele Trippanera, Lorenzo Bonini and a third reviewer are thanked for constructive comments that helped improve the manuscript. We thank Valerio Acocella for fruitful discussions and editorial handling. CONCLUSIONS (Acocella and Trippanera, 2016). These may represent areas where the connection between the feeder dike and fracture has not yet been established, or where rifting was fast enough that a new dike formed before fracture and dike connected. However, as soon as the dike and fracture connect, dike propagation will be guided by the fracture (Magee et al., 2016). Our models suggest that dike propagation a within the failure mode transition zone may be guided by occurrence of open fractures in the subsurface (Figure 14). This may help understanding how dikes propagate through the fractured crust. Additionally these findings may help guiding geothermal exploration in the area, as the fracture pattern influences fluid flux. In this study, we studied the influence of fault obliquity on fault evolution and geometry in extensional settings, using cohesive powders. Our analog models offer a quantitative evaluation of how changes in the kinematic boundary conditions influences fault zone evolution in 3D. We conclude from our analog models that evolution of fault geometry, formation of tilted blocks, graben geometry and fault sinuosity are prescribed already during the earliest stages of deformation and a result of progressive linkage of early fractures. The most important aspect of this study is the variability of dilatant fractures developed during different slip obliquity. In particular our models show that February 2019 | Volume 7 | Article 18 Frontiers in Earth Science | www.frontiersin.org 16 Oblique Rifts and Massively Dilatant Faults von Hagke et al. AUTHOR CONTRIBUTIONS • Dilatancy changes progressively with increasing obliquity. Low fault obliquities are characterized by long open fractures with large apertures close to the surface and smaller dilatant fractures at depth, often filled with rubble. CvH, MK, and JU conceived of the study, DB performed material tests with input from MK, JU, and CvH. NB performed experiments including fault analysis with input from other authors. CW produced and interpreted DEMs with input from MK and CvH. CvH and MK wrote the manuscript with input from other authors. All authors contributed to interpretation and discussion of the results. This is publication #1 of the MDF project. • High fault obliquity results in dilatancy localized close to the surface, but more, deeper reaching vertical open conduits in releasing bends as compared to lower obliquities. • Deepest open fractures occur at antithetic faults, as absence of tilted blocks and smaller opening width and displacement reduces the amount of available debris. REFERENCES World stress map 2016. 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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. Thordarson, T., and Larsen, G. (2007). Volcanism in Iceland in historical time: volcano types, eruption styles and eruptive history. J. Geodyn. 43, 118–152. doi: 10.1016/j.jog.2006.09.005 Townend, J., and Zoback, M. D. (2000). How faulting keeps the crust strong. Geology 28, 399–402. doi: 10.1130/0091-7613(2000)28<399:HFKTCS>2.0.CO;2 February 2019 | Volume 7 | Article 18 Frontiers in Earth Science | www.frontiersin.org 19

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Exogenously applied GA3 promotes plant growth in onion by reducing oxidative stress under saline conditions

Tarım bilimleri dergisi/Ankara Üniversitesi Ziraat Fakültesi tarım bilimleri dergisi

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1. Introduction Onion (Allium cepa L.) is a biennial crop, and it belongs to Alliacea family (Adamicki & Kepka 1974). It is the one of most popular vegetables in the daily diet. Onion is an important commercial crop for the economy of Pakistan (FAO 2012). Salinity has adverse effect on growth and production of agricultural crops (Munns & Tester 2008). Studies showed that due to the saline toxicity every aspect of physiological and biochemical of plant is effect. (Khan & Panda 2008). Plants face two basic problems under saline conditions. Firstly, excessive salt lower down the osmotic potential in soil solution which reduces uptake of water in plant. Secondly, maximum Na+ and Cl ̶ ions uptake diverts the absorption rate of essential minerals and ascribe toxicity to plants (Tester & Davenport 2003). Specific ion toxicities damaged the tissues of transpiring leaves which occur due to boron, sodium, and chloride accumulation. This accumulation of adverse ions may reduce protein synthesis, photosynthesis, and inactivate enzymes, and also damaged chloroplasts as well as other plant organelles (Taiz & Zeiger 2002). Onion lacks tap root system and root hairs. Most of the root system is confined within top 20-25 cm of soil. Plant growth rate is half of other vegetables such as cauliflower and cabbage (Brewster 1994). Onion is more vulnerable to salinity than other vegetables, particularly the seedling emergence stage (Brewster 1994). Threshold electrical conductivity (EC) level for onions is 1.2 dS m-1 at 25 °C and each unit change in EC cause about 16% reduction in yield (Allen et al. 1998). Gibberellic acid (GA3) affects production by increasing the stem length and internodes of onion plant. It restricts senescence by change in lipid peroxidation and adjusts high level of cellular antioxidants like superoxide dismutase and catalase (Dhindsa et al. 1982). It also promotes the growth of the plant, promote cell division and cell extension (Olszewski et al. 2002; Ubeda et al. 2009). Sarkar et al. (2018) reported that GA3 at 60 mg L-1 increased bulb weight over the control under normal conditions. GA enhances the water use efficiency of tomato plants at low salinity level by reducing stomatal resistance (Maggio et al. 2010). So, in the present study, it was hypothesized that GA3 can alleviate salt stress in onion. ABSTRACT Onion (Allium cepa L.) is a biennial crop of high commercial value in Pakistan. Onion is considered as salt sensitive plant species. The present investigation was carried out to investigate the effect of salinity on onion and its alleviation through exogenously applied gibberellic acid (GA3; 100 mg L-1). Foliar application of GA3 (100 mg L-1) was applied on onion seedlings grown under three levels (0, 2 or 4 dS m-1) of salinity after 45 days of sowing. Results revealed that growth parameters and total soluble Keywords: Allium cepa, PGRs, Saline stress, Antioxidative response, Yield © Ankara University, Faculty of Agriculture ARTICLE INFO Research Article Research Article Research Article Corresponding Author: Muhammad Awais GHANI, E-mail: [email protected] Received: 18 September 2019 / Revised: 15 December 2019 / Accepted: 16 December 2019 / Online: 31 May 2021 protein (TSP) contents declined with increase in soil salinity level. While, antioxidant enzyme activities (CAT, SOD and POD) were increased with salinity. However, exogenously applied GA3 significantly enhanced the plant growth and TSP in onion seedlings. Interestingly, CAT, SOD and POD concentration decreased with GA3 application which depicts stress alleviation in saline stressed onion plants due to GA3. It was concluded that the growth of onion could be enhanced to some extent by the application of GA3 under salinity stress. Journal of Agricultural Sciences (Tarim Bilimleri Dergisi) 2021, 27 (2) : 122 - 128 DOI: 10.15832/ankutbd.621546 Journal of Agricultural Sciences (Tarim Bilimleri Dergisi) J Agr Sci-Tarim Bili e-ISSN: 2148-9297 jas.ankara.edu.tr Exogenously Applied GA3 Promotes Plant Growth in Onion by Reducing Oxidative Stress Under Saline Conditions Muhammad Awais GHANİa* , Amina MUSHTAQa , Khurram ZİAFa , Basharat ALİb , Muhammad Muzammil JAHANGİRa , Rashad Waseem KHANa , Imran KHANb , Muhammad AZAMa , Anam NOORa aInstitute of Horticultural Sciences, University of Agriculture, Faisalabad 38040, PAKİSTAN bDepartment of Agronomy, University of Agriculture, Faisalabad 38040, PAKİSTAN Journal of Agricultural Sciences (Tarim Bilimleri Dergisi) 2021, 27 (2) : 122 - 128 DOI: 10.15832/ankutbd.621546 Journal of Agricultural Sciences (Tarim Bilimleri Dergisi) J Agr Sci-Tarim Bili e-ISSN: 2148-9297 jas.ankara.edu.tr Exogenously Applied GA3 Promotes Plant Growth in Onion by Reducing Oxidative Stress Under Saline Conditions Muhammad Awais GHANİa* , Amina MUSHTAQa , Khurram ZİAFa , Basharat ALİb , Muhammad Muzammil JAHANGİRa , Rashad Waseem KHANa , Imran KHANb , Muhammad AZAMa , Anam NOORa aInstitute of Horticultural Sciences, University of Agriculture, Faisalabad 38040, PAKİSTAN bDepartment of Agronomy, University of Agriculture, Faisalabad 38040, PAKİSTAN Journal of Agricultural Sciences (Tarim Bilimleri Dergisi) 2021, 27 (2) : 122 - 128 DOI: 10.15832/ankutbd.621546 Journal of Agricultural Sciences (Tarim Bilimleri Dergisi) J Agr Sci-Tarim Bili e-ISSN: 2148-9297 jas.ankara.edu.tr 2021, 27 (2) : 122 - 128 Journal of Agricultural Sciences (Tarim Bilimleri Dergisi) Journal of Agricultural Sciences (Tarim Bilimleri Dergisi) DOI: 10.15832/ankutbd.621546 Exogenously Applied GA3 Promotes Plant Growth in Onion by Reducing Oxidative Stress Under Saline Conditions Muhammad Awais GHANİa* , Amina MUSHTAQa , Khurram ZİAFa , Basharat ALİb , Muhammad Muzammil JAHANGİRa , Rashad Waseem KHANa , Imran KHANb , Muhammad AZAMa , Anam NOORa aInstitute of Horticultural Sciences, University of Agriculture, Faisalabad 38040, PAKİSTAN bDepartment of Agronomy, University of Agriculture, Faisalabad 38040, PAKİSTAN ARTICLE INFO 2. Material and Methods The Phulkara genotype which is local variety was selected for this experimental study. Seeds were obtained from Punjab Food Corporation Faisalabad, Pakistan. The experiment was carried out in the vegetable experimental area of Institute of Horticultural Sciences (IHS), University of Agriculture Faisalabad, Pakistan in the year 2018 and 2019. Seed sowing was done in soil pots containing 10 kg soil. Before the sowing of onion seeds, the electrical conductivity (EC) of the soil from representative area was calculated and compared to the results of real EC dS m-1 of soil. Then artificially salinity was caused by adding the measured quantity of salt (NaCl) in soil and mixed well according to get the desired salinity dS m-1. The calculations were calculated according to the Rayment & Higginson (1992) method. Measured quantity of salt was added to the soil and mixed well by the soil mixer to get the homogeneous mixture. Then the experimental area was cleaned and layered with polythene tunnel sheet to avoid the leaching of salt in other soil. Inorganic fertilizers were supplied properly at the rate of 50 kg ha-1 K as potassium sulphate, 50 kg ha-1 of N as urea and 80 kg ha-1 of P as di-ammonium phosphate. The six treatments were maintained as; (T0) control; (T1) GA3: GA3100 mg L-1; (T2) salinity level: 2 dS m-1; (T3) salinity level: 2dS m-1 + Gibberellic acid: GA3100 mg L-1; (T4) salinity level: 4 dS m-1; (T5) salinity level: 4 dS m-1 + Gibberellic acid: GA3 100 mg L-1. The foliar spray of GA3 (100 mg L-1) was applied after 45 days of sowing days. Onion plants were harvested after 135 days of GA3 application and different growth parameters were analyzed. Samples were saved in -20 °C for biochemical analysis. 1. Introduction Therefore, the present work was taken up under field conditions to determine the impact of different level of salinity on growth of onion and alleviation of stress by GA3. Ghani et al. - Journal of Agricultural Sciences (Tarım Bilimleri Dergisi), 2021, 27(2): 122-128 2.2. Germination percentage 2.2. Germination percentage Data was recorded for germination on daily basis after one week of sowing seeds till 14 days. Germination percentage was then calculated by using the following formula: Germination percentage = no.of germinated seeds Total no of seeds sown×100 Germination percentage = no.of germinated seeds Total no of seeds sown×100 2.1. Growth parameters The plant height was calculated by the scale from the tip of that plant to the base on ground. The length of the leaf blade was measured from the base of leaf of observing plant to the tip. It was done by using a scale meter. Evacuated plants were washed with clean water, straightened and after that its root length was estimating by utilizing a tape meter in centimeters and the average was taken for each replicate. The diameter of onion bulb was calculated at right angles to longitudinal axis at the widest form of the bulb of arbitrarily chosen plants in each plot by using veneer caliper (model 141) (Demisie & Tolessa 2017). Plants chosen from each treatment were harvested at the end of the experiment. Roots were removed from that plants then they were washed with water to expel the dirt and soil. Then root weight was measured by using weighing balance. Root dry weight was estimated by putting the sample in oven at 65 ºC for 72 hr to dry the samples. At that point, weight was ascertained by adjust and the normal mean for each sample was figured. 2.2. Germination percentage 3. Results and Discussion 3. Results and Discussion Data about growth parameters (plant height, root fresh and dry weight, leaf blade length, root length, bulb diameter and germination percentage) of treated and untreated plants of onion seedlings are shown in Table 1. The foliar spray of GA3 100 mg L-1 showed significant effect on plant height, root fresh and dry weight, leaf blade length, root length, bulb diameter and germination percentage under salinity (Table 1). Previously, Ali et al. (2015) also found that application of GA3 has positive impact on growth and yield of onion. However, salinity stress reduced the growth parameters as compared to the control conditions (El-Shaieny 2015; Nasri et al. 2017). As the salinity increased, there was a gradual decline in all growth parameters of onion (Stia-Baba et al. 2010). Similarly, results showed that when salinity level increased the plant growth declined as compared to control (Table 1). The decrease in plant growth in saline stress might be as a result of that salinity removes the potassium ions via plant roots, which generates physiological discrepancy because potassium ion is essential for the synthesis of proteins and metabolism (Chen et al. 2007). However, exogenously applied GA3 enhanced all the growth parameters under salinity stress as compared to their respective controls (Table 1). Similarly, Chauhan et al. (2019) reported that GA3 enhanced the plant growth under the salinity condition. It might be due to the effect of GA3 which partially diminishes the toxic effect of salinity by increasing anti-oxidative, vigor, accumulation of osmolytes, and enzyme activities (Neelambari et al. 2018). Another study reported that decline in growth of plant under saline stress because of osmotic stress (Hakim et al. 2010). 3. Results and Discussion Table 1- Effect of Gibberellic acid (GA3, 100 mg L-1) and salinity (0, 2, 4 ds m-1) on plant height, leaf blade length, root length, bulb diameter, root fresh weight, root dry weight and germination percentage of onion (phulkara variety) plants Treatments Plant height (cm) Leaf blade length (cm) Root length (cm) Bulb diameter (cm) Root fresh weight (g) Root dry weight (g) Germination percentage (GP%) Control 46.7± 1.10ab 38.6± 1.37b 12.5± o.77ab 3.8± 0.52b 56.6± 2.71b 6.43± o.41b 77.7± 3.2b GA3 50.4± 1.15a 47.7± 1.08a 14.3± 0.72a 5.5± 0.33a 68.1± 2.11a 8.33± 0.31a 88.8± 3.2a 2 dS m-1 43.7± 0.92b 36.3± 1.21bc 11.6± 0.50b 3.5± 0.21b 41.5± 2.06d 4.03± 0.42c 66.6± 3.2c 2 dS m-1 + GA3 45.1± 1.82b 37.9± 1.19bc 12.3± 0.70ab 3.7± 0.29b 46.9± 1.62c 4.8± 0.34c 83.3± 3.2ab 4 dS m-1 34.7± 1.99c 33.6± 1.35c 7.4± 0.81c 2.5± 0.28c 33.6± 2.19e 2.9± 0.55d 44.4± 3.2d 4 dS m-1 + GA3 44.8± 1.23b 36.7± 1.55bc 11.8± 0.40b 3.3± 0.08b 36.2± 2.97e 3.1± 0.34d 75.9± 4.8bc Each data values are represented as mean and +SD of three replications and different lower case letters are representing the significant difference between treatments and same lower-case letters represent the no significant difference by according to LSD test (P≤0.05) able 1- Effect of Gibberellic acid (GA3, 100 mg L-1) and salinity (0, 2, 4 ds m-1) on plant height, leaf blade bulb diameter, root fresh weight, root dry weight and germination percentage of onion (phulkara va Gibberellic acid (GA3, 100 mg L-1) and salinity (0, 2, 4 ds m-1) on plant height, leaf blade length, root lengt r, root fresh weight, root dry weight and germination percentage of onion (phulkara variety) plants Each data values are represented as mean and +SD of three replications and different lower case letters are representing the significant difference between treatments and same lower-case letters represent the no significant difference by according to LSD test (P≤0.05) Results of biochemical assays depict that salinity stress significantly have increased the activity of antioxidant enzymes (CAT, POD and SOD) as compared to control conditions (Figures 1-3). When the salinity was increased then the activity CAT, POD and SOD was increased maximum in onion. While foliar application of GA3 further enhanced the activity of CAT, POD and SOD under salinity stress as compared to their respective control. 2.3. Biochemical analysis 2.3. Biochemical analysis Fresh samples were collected from the experimental area and stored at -20 °C. All the samples were crushed in 50 mL of 100 mM sodium phosphate (pH 7) buffer containing 0.5% (w/v) polyvinyl pyrrolidone and 1 mM ascorbic acid and homogenize mixture was prepared. After preparation of mixture sample were placed at 4 °C for 5 min. The collected mixture was filter by using filter paper and centrifuged it for 15 min at 5000 RPM and the supernatant was collected. Peroxidase (POD) was determined by using the method of (Onsa et al. 2004). In this method 4-methylecatechol was used as a substrate. To check the activity of POD reaction mixture was prepared by using 4-methylcatechol, 10 mM sodium phosphate buffer, 5 mM H2O2 and 500 μL of total volume of 3 mL crude extract of the sample at room temperature. By using spectrophotometer absorption was measured at 420 nm. Superoxide dismutase (SOD) activity was determined by using the method of (Kumar et al. 2012). Reaction mixture was prepared by using 0.2 mM EDTA, 12 mM L-methionine, 50 mM buffer of sodium phosphate (pH 7) .10 uM riboflavin, 50 μM NBT and 100 μL of final volume of 3 mL of the crude extracted sample. The reaction mixture was incubated into the white light for 15min at room temperature. After incubation of 15 min absorption was observed at 560 nm by using spectrophotometer. Catalase (CAT) activity was measured by using the method of Aebi 1983. To calculate the CAT activity spectrophotometer was used. The reaction mixture was prepared by using 30 mM H2O2 100 mM sodium phosphate buffer (pH 7) and 100 uL crude extract of sample by volume of 3 mL. Absorption was calculated at 240nm at room temperature in spectrophotometer. 123 Ghani et al. - Journal of Agricultural Sciences (Tarım Bilimleri Dergisi), 2021, 27(2): 122-128 The soluble protein (TSP) was measured according to Coomassie Brilliant Blue G-250 Staining Method (Sedmak & Grossberg 1977). 2.4. Statistical analysis 2.4. Statistical analysis All experiments were conducted in triplicate with two factorial randomized complete block design (RCBD) and all results were expressed as the average ± standard error of the measurements. Statistix 8.1 software was used for statistics. 3. Results and Discussion The basic function of POD in plants is to break down the hydrogen peroxide (H2O2) which is very toxic and reactive element (Botella et al. 1994). Saline stress condition might be causing the univalent reduction in O2− which produce hydrogen peroxide. In the salt stress condition, the level of H2O2 was reduced which was the damage of plant defence system due to the higher concentration of POD. Sancho et al. (1996) also stated same consequences in his study and recognized this to variations in the mechanical characteristics of cell wall which in turn, might be connected to the salinity adjustment mechanism. The enhancement of POD with GA3 under salinity stress (Figure 1) might be due to that GA3 increases the gibberellins which stimulates the decrease in hydrolytic enzymes and sugars (Mathew & Murray 1968). GA3 have competition with saline conditions via improving the membrane permeability of plant cell and adjust the level of nutrients in cell. Eventually this leads to increase the growth, and GA3 also has induced physiochemical variations which are responsible for the influence of salt tolerance (Amal & Mohamed 2014). 124 Ghani et al. - Journal of Agricultural Sciences (Tarım Bilimleri Dergisi), 2021, 27(2): 122-128 Figure 1- Effect of Gibberellic acid (GA3, 100 mg L-1) and salinity (0, 2, 4 dS m-1) on peroxidase activity (POD) of onion seedlings. Each data values are represented as mean and +SD of three replications and different lower-case letters are representing the significant difference between treatments and same lower-case letters represents the no significant difference by according to LSD test (P≤0.05) c c b d a c 0 2 4 6 8 10 12 14 16 control GA3 2 dS m-1 2 dS m- 1+GA3 4 dS m-1 4 dS m- 1+GA3 Peroxidase activity (U mg-1) Treatments m-1 c c b d a c 0 2 4 6 8 10 12 14 16 control GA3 2 dS m-1 2 dS m- 1+GA3 4 dS m-1 4 dS m- 1+GA3 Peroxidase activity (U mg-1) Treatments m-1 Figure 1- Effect of Gibberellic acid (GA3, 100 mg L-1) and salinity (0, 2, 4 dS m-1) on peroxidase activity (POD) of onion seedlings. 3. Results and Discussion Each data values are represented as mean and +SD of three replications and different lower-case letters are representing the significant difference between treatments and same lower-case letters represents the no significant difference by according to LSD test (P≤0.05) Superoxide dismutase (SOD) is an important antioxidant which scavenges the reactive oxygen species (ROS) and it is activated under stress conditions. This is the reason that SOD enhanced in saline stress condition (Figure 2). It gives the initial line of protection against the noxious effects of stress. SOD eliminates superoxide radicals by catalyzing the dismutation of superoxides and reduces it to peroxide which is also oxidized by another antioxidant POD (Gill & Tuteja 2010). Foliar spray of GA3 increased SOD activity to some extent because it increased the gibberellins which stimulate the decrease in hydrolytic enzymes and sugars (Mathew & Murray 1968). The SOD decreased in saline treated plants by foliar treatment of GA3 (100 mg L-1) as compared to control (Figure 2). It is due to that foliar application of GA3 improves the membrane of plant cell and adjusts the level of nutrients in the cells (Chakrabarti & Mukharji 2003). Eventually this leads to decrease in SOD, and GA3 induces physiochemical variations which are responsible for the influence of salt tolerance (Amal & Mohamed 2014). c c b d a c 0 5 10 15 20 25 30 35 40 45 control GA3 2 dS m-1 2 dS m- 1+GA3 4 dS m-1 4 dS m-1+GA Super oxide dismutase (U mg-1) Treatments 2 dS m-1 +GA3 Figure 2- Effect of Gibberellic acid (GA3, 100 mg L-1) and salinity (0, 2, 4 dS m-1) on superoxide dismutase activity (SOD) of onion seedlings. Each data values are represented as mean and +SD of three replications and different lower-case letters are representing the significant difference between treatments and same lower-case letters represents the no significant difference by according to LSD test (P≤0.05) Catalases (CAT) also plays important role in plant during the saline stress condition. During the saline stress condition, the CAT level increased as compared to control (Figure 3). 3. Results and Discussion Eyidogan & Oz (2007) observed that CAT level was increased in the c c b d a c 0 5 10 15 20 25 30 35 40 45 control GA3 2 dS m-1 2 dS m- 1+GA3 4 dS m-1 4 dS m-1+GA Super oxide dismutase (U mg-1) Treatments 2 dS m-1 +GA3 Figure 2- Effect of Gibberellic acid (GA3, 100 mg L-1) and salinity (0, 2, 4 dS m-1) on superoxide dismutase activity (SOD) of onion seedlings. Each data values are represented as mean and +SD of three replications and different lower-case letters are representing the significant difference between treatments and same lower-case letters represents the no significant difference by according to LSD test (P≤0.05) Catalases (CAT) also plays important role in plant during the saline stress condition. During the saline stress condition, the CAT level increased as compared to control (Figure 3). Eyidogan & Oz (2007) observed that CAT level was increased in the 125 Ghani et al. - Journal of Agricultural Sciences (Tarım Bilimleri Dergisi), 2021, 27(2): 122-128 leaves of C. arietinum under saline conditions. In stress conditions, isoforms of CAT presents on different chromosomes which regulate independently (Scandalias 1990). CAT helps the plants to fight against oxidative stress that improves the plant growth (Polidoros & Scandalios 1999). Exogenous application of GA3 improved the membrane permeability of plant cell and adjusts the level of nutrients in cell. This leads to decrease in CAT and also GA3 induce physiochemical variations which are responsible for the influence of salt tolerance (Chakrabarti & Mukharji 2003; Amal & Mohamed 2014). leaves of C. arietinum under saline conditions. In stress conditions, isoforms of CAT presents on different chromosomes which regulate independently (Scandalias 1990). CAT helps the plants to fight against oxidative stress that improves the plant growth (Polidoros & Scandalios 1999). Exogenous application of GA3 improved the membrane permeability of plant cell and adjusts the level of nutrients in cell. This leads to decrease in CAT and also GA3 induce physiochemical variations which are responsible for the influence of salt tolerance (Chakrabarti & Mukharji 2003; Amal & Mohamed 2014). 3. Results and Discussion e d b c a b 0 5 10 15 20 25 30 35 40 45 50 control GA3 2 dS m-1 2 dS m-1+GA3 4 dS m-1 4 dS m-1+GA3 Catalase activity (U mg-1) Treatments Figure 3- Effect of Gibberellic acid (GA3, 100 mg L-1) and salinity (0, 2, 4 dS m-1) on catalase activity (CAT) of onion seedlings. Each data values are represented as mean and +SD of three replications and different lower-case letters are representing the significant difference between treatments and same lower-case letters represents the no significant difference by according to LSD test (P≤0.05) Total soluble proteins (TSP) has been observed lower when the plants are grown under saline toxic soils (Zhang et al. 2009) me proteins which have defensive mechanism, stimulated the plant growth under salinity and tolerate against salt stress haei et al. 2008). Significant effects of GA3 and NaCl and its interaction was observed on TSP contents in onion (Figure 4) P contents firstly increased at low salinity levels with GA3 and then the contents of TSP decreased at high concentrations of nity level (Figure 4). The similar results were also found by Jiao et al. (2019) in caster bean as the concentration of GA3 eased the TSP content first increased and then decreased e d b c a b 0 5 10 15 20 25 30 35 40 45 50 control GA3 2 dS m-1 2 dS m-1+GA3 4 dS m-1 4 dS m-1+GA3 Catalase activity (U mg-1) Treatments Figure 3- Effect of Gibberellic acid (GA3, 100 mg L-1) and salinity (0, 2, 4 dS m-1) on catalase activity (CAT) of onion seedlings. Each data values are represented as mean and +SD of three replications and different lower-case letters are representing the significant difference between treatments and same lower-case letters represents the no significant difference by according to LSD test (P≤0.05) Total soluble proteins (TSP) has been observed lower when the plants are grown under saline toxic soils (Zhang et al. 2009), some proteins which have defensive mechanism, stimulated the plant growth under salinity and tolerate against salt stress (Aghaei et al. 2008). Significant effects of GA3 and NaCl and its interaction was observed on TSP contents in onion (Figure 4). TSP contents firstly increased at low salinity levels with GA3 and then the contents of TSP decreased at high concentrations of salinity level (Figure 4). 3. Results and Discussion The similar results were also found by Jiao et al. (2019) in caster bean as the concentration of GA3 increased, the TSP content first increased and then decreased. Total soluble proteins (TSP) has been observed lower when the plants are grown under saline toxic soils (Zhang et al. 2009), some proteins which have defensive mechanism, stimulated the plant growth under salinity and tolerate against salt stress (Aghaei et al. 2008). Significant effects of GA3 and NaCl and its interaction was observed on TSP contents in onion (Figure 4). TSP contents firstly increased at low salinity levels with GA3 and then the contents of TSP decreased at high concentrations of salinity level (Figure 4). The similar results were also found by Jiao et al. (2019) in caster bean as the concentration of GA3 increased, the TSP content first increased and then decreased. 126 Figure 4- Effect of Gibberellic acid (GA3, 100 mg L-1) and salinity (0, 2, 4 dS m-1) on SOD, POD, CAT, soluble protein, and proline content of onion seedlings. Each data values are represented as mean and +SD of three replications and different lower-case letters are representing the significant difference between treatments and same lower-case letters represents the no significant difference by according to LSD test (P≤0.05) b a d c e de 0 1 2 3 4 5 6 7 8 9 Control GA3 2 dS m-1 2 dS m-1+GA3 4 dS m-1 4 dS m-1+GA3 Total soluble protien (U mg-1) Treatment Figure 4- Effect of Gibberellic acid (GA3, 100 mg L-1) and salinity (0, 2, 4 dS m-1) on SOD, POD, CAT, soluble protein, and proline content of onion seedlings. Each data values are represented as mean and +SD of three replications and different lower-case letters are representing the significant difference between treatments and same lower-case letters represents the no significant difference by according to LSD test (P≤0.05) 126 Ghani et al. - Journal of Agricultural Sciences (Tarım Bilimleri Dergisi), 2021, 27(2): 122-128 4. Conclusions Our findings revealed that the salinity stress significantly reduced plant growth parameters in onion seedlings. However, exogenously applied GA3 significantly enhanced the plant growth and by reducing oxidative stress. Interestingly, a decline was observed in antioxidant enzyme activities (SOD, POD and CAT) with the application of GA3 in saline stress conditions. Although these activities were increased to maximum level at salinity stress alone treatment. It is concluded from our study that we can enhance the growth of onion plant in saline stress with the foliar application of GA3. Less is known about the mechanism of GA3 in onion under salinity stress. Hence, to check the mechanism and role of GA3 under saline conditions in different plant species more study is required. References References Acta Physiologiae FAO (2012) Th St t f F d d A i lt R t i d i 2012 f htt // f / bli ti / f /2012 Eyidogan F & OZ M T (2007). Effect of salinity on antioxidant responses of chickpea seedlings. Acta Physiologiae Plantarum 29: 485-493 FAO (2012). The State of Food and Agriculture. Retrieved in 2012 from http://www.fao.org/publications/sofa/2012/en/ Gill S S & Tuteja N (2010) Reactive oxygen species and antioxidant machinery in abiotic stress tolerance in crop plants Plant Physiology Eyidogan F & OZ M T (2007). Effect of salinity on antioxidant responses of chickpea seedlings. Acta Physiologiae Plantarum 29: 485-493 FAO (2012). The State of Food and Agriculture. Retrieved in 2012 from http://www.fao.org/publications/sofa/2012/en/ Eyidogan F & OZ M T (2007). Effect of salinity on antioxidant responses of chickpea seedlings. Acta Physiologiae Plantarum 29: 485-493 FAO (2012). The State of Food and Agriculture. Retrieved in 2012 from http://www.fao.org/publications/sofa/2012/en/ y g ( ) y p p g y g FAO (2012). The State of Food and Agriculture. 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dbEMT: an epithelial-mesenchymal transition associated gene resource

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dbEMT: an epithelial-mesenchymal transition associated gene resource received: 05 September 2014 accepted: 28 April 2015 Published: 23 June 2015 Min Zhao1,*, Lei Kong2,*, Yining Liu1,* & Hong Qu2 As a cellular process that changes epithelial cells to mesenchymal cells, Epithelial-mesenchymal transition (EMT) plays important roles in development and cancer metastasis. Recent studies on cancer metastasis have identified many new susceptibility genes that control this transition. However, there is no comprehensive resource for EMT by integrating various genetic studies and the relationship between EMT and the risk of complex diseases such as cancer are still unclear. To investigate the cellular complexity of EMT, we have constructed dbEMT (http://dbemt.bioinfo- minzhao.org/), the first literature-based gene resource for exploring EMT-related human genes. We manually curated 377 experimentally verified genes from literature. Functional analyses highlighted the prominent role of proteoglycans in tumor metastatic cascades. In addition, the disease enrichment analysis provides a clue for the potential transformation in affected tissues or cells in Alzheimer’s disease and Type 2 Diabetes. Moreover, the global mutation pattern of EMT-related genes across multiple cancers may reveal common cancer metastasis mechanisms. Our further reconstruction of the EMT-related protein-protein interaction network uncovered a highly modular structure. These results illustrate the importance of dbEMT to our understanding of cell development and cancer metastasis, and also highlight the utility of dbEMT for elucidating the functions of EMT- related genes. The epithelial-mesenchymal transition or transformation (EMT) refers to the change of epithelial cells to mesenchymal cells, which are critical for tissue development and initiation of metastasis for cancer progression1–3. Epithelial and mesenchymal cells differ in phenotype as well as function. Epithelial cells often closely connect to one another by cellular junctions such as tight junctions. In addition, they often show apico-basal polarity and polarization of the actin cytoskeleton. In contrast, mesenchymal cells link to each other by focal points and lack cellular polarization. During the EMT process, epithelial cells usu- ally lose their tight cell connection and cellular polarity. As a result, a small number of epithelial cells can become migratory and invasive properties to transform into mesenchymal cells. These morphological and cellular changes during EMT have profound impacts on development, fibrosis and wound healing, and cancer progression2,4.hi p g The importance of EMT was first recognized in embryonic development5,6. It is well acknowledged that EMT is associated with the initiation of placenta formation in the early stage of embryogenesis. www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports Scientific Reports | 5:11459 | DOI: 10.1038/srep11459 Results i Functional enrichment analysis revealed fundamental role of proteoglycans in EMT. Gene-set enrichment analyses have been widely used to help biologists in the analysis and interpretation of the results from any interesting gene list9. In this article, the implemented hypergeometric tests were adapted to characterize whether an input gene-set had a different frequency of annotation pairs unlikely by chance, given the background genes sizes. The expected frequency was further obtained for each of the functional terms. Using protein-coding genes in the human as background, we identified 62 statistically significant enriched pathways (Table  1) and 28 diseases (Table  2) associated with the 377 genes in dbEMT. The enriched functional pathways are mainly related to cancer pathways such as TGF-beta signalling, prote- oglycans in cancer, colorectal cancer, and pancreatic cancer (Table 1). In addition to cancer-related path- ways, the genes were also highly enriched in cell surface interaction such as non-integrin membrane-ECM interaction, extracellular matrix organization, focal adhesion, and adhesion junction. These results show that multiple membrane-related signaling events are related to EMT, especially proteoglycans in cancer. Specifically, there are 65 genes in our dbEMT that are reported to relate to proteoglycans in cancer with a corrected P-value of 5.95E-11. Proteoglycan content and distribution are markedly altered during cancer progression10. The specific structure of proteoglycan allows it to interact with both the ligands and recep- tors that regulate cancer pathogenesis. Therefore proteoglycan, as well as glycosaminoglycans, often have important function in the tumor metastatic cascade by modulating key downstream signaling mediators such as epidermal growth factor receptor, insulin growth factor receptor, estrogen receptors, and Wnt members11. In summary, the level of complexity of cell surface signaling systems involved in EMT arises from their fundamental roles in response to microenvironment regulation. Enrichment diseases on the 377 EMT-related genes. As a fundamental role on cell destination, it is not surprising that the EMT-related genes are consistently related to a number of complex diseases. However, the enrichment analysis of an EMT-related gene does not reveal if EMT has contributed to a specific disease. It does allow to test the risk factor of EMT in relevant diseases. The enrichment analysis on a few disease-related databases has confirmed that the 377 genes are related to a broad-spectrum of human cancers such as (Table 2). In total, 277 genes are related to various cancers. www.nature.com/scientificreports/ mesenchymal cells to form the heart mesoderm1. In vertebrates, epithelial and mesenchymal cells are the basic tissue units. During embryogenesis, epithelial cells of neuroectoderm may change to mesenchymal neural crest cells. As a result, these migratory mesenchymes can dissociate from neural folds and differ- entiate to specific cell types after travelling to specific target regions in the embryo. pi ypt g pi g g y Recently, the relationship between EMT and cancer has become increasingly clear from numerous studies7. EMT can assist the invasion during initiation of cancer metastasis. Cancer cells in primary sites can lose cell connection which increases their invasive properties, and they can enter the bloodstream through intravasation. Subsequently, these circulating tumor cells (CTCs) can colonize at new metastatic sites by forming micro-metastases. In addition, there is increasing evidence that shows that cells under- going EMT have stem cell-like properties8. These cellular processes conferred by EMT are harmful for the patient not only because they enable the cancer cells to circulate with bloodstream but also because they reflect the properties of stemness that promote proliferation of cancer cells4. yl p p p p Although essential in our understanding of the development of complex diseases, the genetic and biological information is scattered in the literature. To the best of our knowledge, there has been no sys- tematic review of this literature to help us understand the molecular mechanisms underlying initiation of metastasis as it relates to cancer progression and the associated development processes. A comprehensive gene resource is needed to generate a complete molecular picture for EMT and associated diseases. In this study, we present the database dbEMT, the first literature-based EMT-related gene resource that serves as a reference dataset for understanding the cellular mechanisms of EMT-related processes such as initiation of metastasis for cancer progression. The resultant gene list in dbEMT and containing addi- tional functional and genetic information will be a valuable resource for the EMT research community. Moreover, our systematic pathway and disease enrichment analyses reveal that the EMT-related genes enriched in multiple signal events are involved with many cancers and developmental processes. We believe that, dbEMT is the first example of an integrated and comprehensive gene resource to help elucidate the relationship between EMT and development and cancers. This database could have pro- found implications for the diagnosis, treatment and prevention of EMT-related diseases such as cancer metastasis. dbEMT: an epithelial-mesenchymal transition associated gene resource After EMT, trophoectoderm cells are able to invade the endometrium and appropriate placenta placement; this enhances the exchange of gas and nutrients to the embryo. In addition, EMT is also involved in gastrulation during the later stage of embryogenesis. In this process, the cells that undergo EMT are able to ingress in the primitive streak of amniotes. In higher chordates, the mesenchymal cells that changed from the epithelial cells of primitive streak can migrate to forge the somites and interact with neural crest 1School of Engineering, Faculty of Science, Health, Education and Engineering, University of the Sunshine Coast, Maroochydore DC, Queensland, 4558, Australia. 2Center for Bioinformatics, State Key Laboratory of Protein and Plant Gene Research, College of Life Sciences, Peking University, Beijing 100871, P.R. China. *These authors contributed equally to this work. Correspondence and requests for materials should be addressed to H.Q. (email: [email protected]) Scientific Reports | 5:11459 | DOI: 10.1038/srep11459 1 www.nature.com/scientificreports/ www.nature.com/scientificreports/ www.nature.com/scientificreports/ Pathway Adjusted P-values* KEGG pathway   Proteoglycans in cancer 5.95E-11   Pathways in cancer 1.21E-09   Colorectal cancer 1.95E-07   Pancreatic cancer 5.05E-07   MicroRNAs in cancer 5.81E-07   Endometrial cancer 1.84E-06   Prostate cancer 5.90E-06   Chronic myeloid leukemia 1.66E-05   Bladder cancer 1.78E-05   TGF-beta signaling pathway 2.13E-05   Focal adhesion 4.17E-05   Adherens junction 4.36E-05   Hippo signaling pathway 6.08E-05   ErbB signaling pathway 0.000171393   Melanoma 0.000188448   Renal cell carcinoma 0.000211855   Thyroid cancer 0.000558926   Acute myeloid leukemia 0.000746089   Glioma 0.001053132   Prolactin signaling pathway 0.002672626   Non-small cell lung cancer 0.002846076   Hepatitis B 0.004685088   HIF-1 signaling pathway 0.005619905   VEGF signaling pathway 0.0089538 Reactome pathway   Non-integrin membrane-ECM interactions 5.21E-06   Extracellular matrix organization 9.03E-06   Signaling by SCF-KIT 1.30E-05   Signaling by ERBB4 6.35E-05   Syndecan interactions 0.000155583   Signaling by FGFR in disease 0.000170943   GAB1 signalosome 0.000186436   Fc epsilon receptor (FCERI) signaling 0.000491729   Signaling by FGFR 0.000517638   Downstream signal transduction 0.000517638 Table 1. The statistically significant enriched pathways of EMT-related genes in core dataset from different pathway databases. *Adjusted P-values: the P-values of the hypergeometric test were corrected by Benjamini-Hochberg multiple testing correction. supported by AD-specific biomarkers, which were selectively detected only in the affected AD brain12. The potential role of EMT in T2D was also found recently13. Although the direct association of AD and T2D with EMT has not reported. The study in adult human exocrine pancreatic cells revealed that the EMT could impact on dedifferentiation of ductal cells during the regeneration of type 2 diabetic pancreatic tissues. Therefore, further data mining on our dbEMT may provide a clue about a potential transformation in affected tissues or cells, including changes of upstream signaling and other cell surface interactions during the AD/T2D disease. supported by AD-specific biomarkers, which were selectively detected only in the affected AD brain12. The potential role of EMT in T2D was also found recently13. Although the direct association of AD and T2D with EMT has not reported. The study in adult human exocrine pancreatic cells revealed that the EMT could impact on dedifferentiation of ductal cells during the regeneration of type 2 diabetic pancreatic tissues. Therefore, further data mining on our dbEMT may provide a clue about a potential transformation in affected tissues or cells, including changes of upstream signaling and other cell surface interactions during the AD/T2D disease. Prioritizing the important genes in EMT and their mutational landscape in pan-cancer genomics data. The molecular basis that underlies EMT is unclear because of its high cellular com- plexity. Results i The cancers mainly occur in the digestive and urinary systems, including colorectal, pancreatic, endometrial, prostate, blad- der, melanoma, renal cell, thyroid, oral, gastric, ovary, leukemia, cholangiocarcinoma, choriocarcinoma. Although many reports suggest that EMT is associated with cancer invasion and metastasis, there is no comparison of EMT across cancers. It is possible that the underlying common molecular mechanism between EMT and any cancers are the same. As shown in Fig. 1, there are 27 EMT-related genes in both colorectal cancer and pancreatic cancer. However, only 18 of these are common to both: SMAD2, SMAD3, SMAD4, PIK3CA, MAP2K1, TP53, AKT1, BRAF, TGFBR1, RAF1, TGFB3, TGFB2, MAPK3, MAPK1, MAPK8, KRAS, RAC1, TGFB1. It is interesting to note that the 337 EMT-related genes are over-represented in the Alzheimer’s dis- ease (AD) and type 2 diabetes (T2D). The possible association of EMT with Alzheimer’s disease is also Scientific Reports | 5:11459 | DOI: 10.1038/srep11459 2 www.nature.com/scientificreports/ www.nature.com/scientificreports/ Pathway Adjusted P-values* KEGG disease   Cancers 2.29E-07   Cancers of the digestive system 3.52E-07   Cancers of the urinary system and male genital organs 6.52E-06   Gastric cancer 2.64E-05   Penile cancer 0.000250093   Cancers of the breast and female genital organs 0.000314144   Hepatocellular carcinoma 0.000375659   Cholangiocarcinoma 0.002488127   Head and neck cancers 0.003815715   Pancreatic cancer 0.003867987   Skin cancers 0.005592227   Choriocarcinoma 0.00804457 GAD database   Prostate cancer 9.82E-06   Breast cancer 3.02E-05   Colorectal cancer 3.43E-05   Lung cancer 0.000106932   Stomach cancer 0.000271348   Bladder cancer 0.001245754   Kidney cancer 0.00169854   Ovarian cancer 0.003580256   Alzheimer's disease 0.004749989   Melanoma 0.004782684   Diabetes, type 2 0.005049578   Colorectal neoplasms 0.008608511   Endometrial cancer 0.009991705   Overall effect 0.009991705 FunDO database   Oral cancer 0.002189338   Endometriosis 0.003400183 Table 2. Significant enriched pathways in the reconstructed EMT-specific network. *Adjusted P-values: the P-values of the hypergeometric test were corrected by Benjamini-Hochberg multiple testing correction. Pathway Adjusted P-values* KEGG disease   Cancers 2.29E-07   Cancers of the digestive system 3.52E-07   Cancers of the urinary system and male genital organs 6.52E-06   Gastric cancer 2.64E-05   Penile cancer 0.000250093   Cancers of the breast and female genital organs 0.000314144   Hepatocellular carcinoma 0.000375659   Cholangiocarcinoma 0.002488127   Head and neck cancers 0.003815715   Pancreatic cancer 0.003867987   Skin cancers 0.005592227   Choriocarcinoma 0.00804457 GAD database   Prostate cancer 9.82E-06   Breast cancer 3.02E-05   Colorectal cancer 3.43E-05   Lung cancer 0.000106932   Stomach cancer 0.000271348   Bladder cancer 0.001245754   Kidney cancer 0.00169854   Ovarian cancer 0.003580256   Alzheimer's disease 0.004749989   Melanoma 0.004782684   Diabetes, type 2 0.005049578   Colorectal neoplasms 0.008608511   Endometrial cancer 0.009991705   Overall effect 0.009991705 FunDO database   Oral cancer 0.002189338   Endometriosis 0.003400183 Table 2. Significant enriched pathways in the reconstructed EMT-specific network. *Adjusted P-values: the P-values of the hypergeometric test were corrected by Benjamini-Hochberg multiple testing correction. Table 2. Significant enriched pathways in the reconstructed EMT-specific network. *Adjusted P-value the P-values of the hypergeometric test were corrected by Benjamini-Hochberg multiple testing correctio specific genes functions related to EMT. However, the big map for the EMT and the relative importance for EMT-related genes have not been systematically evaluated. Using the gene ranking tool Endeavour, we obtained the top ten ranked genes involved inCTNNB1, SMAD3, HIF1A, TGFB1, ZEB2, EGFR, CDH1, ILK, ZEB1, TWIST1 (Table 3). Not surprisingly, majority of these top ranked genes are involved in the key pathway of EMT, such as the “Pathways in cancer”, “Adherens junction”, “Focal adhesion”, and “TGF-beta signaling pathway”. www.nature.com/scientificreports/ Traditional characterization of candidate genes in individual studies often focuses on verifying Scientific Reports | 5:11459 | DOI: 10.1038/srep11459 3 Scientific Reports | 5:11459 | DOI: 10.1038/srep11459 www.nature.com/scientificreports/ g g p y Although EMT-related genes have been demonstrated to have abnormal gene expression or other functional relevance to cancer, the systematic examination of the genetic variants has not been con- ducted. These are useful for users to identify potential cancer metastasis genes for further screening. As shown in Fig. 2, the top 100 ranking EMT-related genes have tremendous mutations in cancers. It is interesting that the 100 genes are 98.9% mutated in both head/neck carcinomas (276 individuals) and lung squamous cell carcinomas (176 individuals). In 234 uterine cancer samples, there were more single nucleotide mutations for the patients. The most striking point is that 95.2% of the lung squamous cell carcinoma population from an analysis of 40 patients at the Johns Hopkins . with only single nucleotide mutations. The same observation can be found in colorectal cancer from the Genentech dataset (90.3% in 65 individuals) and esophagus cancer (88.4% in 129 individuals). In contrast, the three studies on medulloblastoma (MBL) only show less than 20% of mutation frequency in the designed case population. This suggests that MBL may have distinct metastasis mechanisms that are different from other cancers. Scientific Reports | 5:11459 | DOI: 10.1038/srep11459 4 www.nature.com/scientificreports/ www.nature.com/scientificreports/ www.nature.com/scientificreports/ f h l h d d l d d ff Figure 1. The highlighted mutated EMT-related genes in colorectal and pancreatic cancers. (a) The KEGG signaling map for colorectal cancer; (b) The KEGG signaling map for pancreatic cancer. The genes with pink color in two maps are detected in our dbEMT; the remaining green genes are human genes not in our dbEMT." Figure 1. The highlighted mutated EMT-related genes in colorectal and pancreatic cancers. (a) The KEGG signaling map for colorectal cancer; (b) The KEGG signaling map for pancreatic cancer. The genes h k l d d db h h Figure 1. The highlighted mutated EMT-related genes in colorectal and pancreatic cancers. (a) The KEGG signaling map for colorectal cancer; (b) The KEGG signaling map for pancreatic cancer. The genes with pink color in two maps are detected in our dbEMT; the remaining green genes are human genes not in our dbEMT." To further explore the common and distinct EMT-related somatic variants in different cancer types, we mapped the 377 EMT-related genes to the mutation data of TCGA pan-cancer14. To focus on the functional variants, we excluded non-sense and silent mutations. The remaining contains mis-senses, splicings, frameshift SNVs and INDELs. As shown in the Fig. 3, majority of EMT-related somatic variants Scientific Reports | 5:11459 | DOI: 10.1038/srep11459 5 www.nature.com/scientificreports/ GeneSymbol (Ranked) Global Rank P-value1 Global Rank ratio2 CTNNB1 5.62E-10 0.00291 SMAD3 7.17E-10 0.00581 HIF1A 3.34E-09 0.00872 TGFB1 8.90E-09 0.0116 ZEB2 1.06E-08 0.0145 EGFR 2.41E-08 0.0174 CDH1 4.24E-08 0.0203 ILK 5.13E-07 0.0233 ZEB1 1.04E-06 0.0262 TWIST1 7.58E-06 0.0291 SNAI2 8.77E-06 0.032 EPAS1 2.11E-05 0.0349 SMAD2 2.59E-05 0.0378 SNAI1 0.000157 0.0407 SMAD4 0.000247 0.0436 IGF1R 0.000723 0.0465 ERBB2 0.000819 0.0494 MET 0.00133 0.0523 AKT1 0.00191 0.0552 SMAD7 0.00313 0.0581 Table 3. The top 20 ranked EMT-related genes. 1Represent the probability that a candidate gene would GeneSymbol (Ranked) Global Rank P-value1 Global Rank ratio2 CTNNB1 5.62E-10 0.00291 SMAD3 7.17E-10 0.00581 HIF1A 3.34E-09 0.00872 TGFB1 8.90E-09 0.0116 ZEB2 1.06E-08 0.0145 EGFR 2.41E-08 0.0174 CDH1 4.24E-08 0.0203 ILK 5.13E-07 0.0233 ZEB1 1.04E-06 0.0262 TWIST1 7.58E-06 0.0291 SNAI2 8.77E-06 0.032 EPAS1 2.11E-05 0.0349 SMAD2 2.59E-05 0.0378 SNAI1 0.000157 0.0407 SMAD4 0.000247 0.0436 IGF1R 0.000723 0.0465 ERBB2 0.000819 0.0494 MET 0.00133 0.0523 AKT1 0.00191 0.0552 SMAD7 0.00313 0.0581 Table 3. The top 20 ranked EMT-related genes. 1Represent the probability that a candidate gene would obtain these ranks by chance. 2The global ranking ratio from Endeavour. Scientific Reports | 5:11459 | DOI: 10.1038/srep11459 Table 3.  The top 20 ranked EMT-related genes. 1Represent the probability that a candidate gene would obtain these ranks by chance. 2The global ranking ratio from Endeavour. www.nature.com/scientificreports/ To evaluate the statistical significance, we checked each randomly selected gene Scientific Reports | 5:11459 | DOI: 10.1038/srep11459 7 www.nature.com/scientificreports/ Figure 4. The heatmap for the 35 genes whose expression are increasing from stage III to stage IV in TCGA ovarian cancer samples. Figure 4. The heatmap for the 35 genes whose expression are increasing from stage III to stage IV in TCGA ovarian cancer samples. set whether the number of genes with meaning mutations was more than the actual number of mutated genes in our top 100 EMT related-genes. Using the number of randomly selected node sets with more mutated genes as input, we calculated the empirical P-values (See Methods section). As shown in Table S2, all the 19 empirical P-values are all less than 0.01, which means the top 100 EMT related genes are highly mutated in the 19 pan-cancer datasets. set whether the number of genes with meaning mutations was more than the actual number of mutated genes in our top 100 EMT related-genes. Using the number of randomly selected node sets with more mutated genes as input, we calculated the empirical P-values (See Methods section). As shown in Table S2, all the 19 empirical P-values are all less than 0.01, which means the top 100 EMT related genes are highly mutated in the 19 pan-cancer datasets. Highly expressed and mutated EMT genes during stage III to stage IV in ovarian cancer. As the EMT often occurs during the cancer metastasis, we only extracted those ovarian cancer samples related to stage III to stage IV, where metastasis transition occurs. The aim of this analysis was to identify those EMT-related genes whose expression is increasing between Stage III to stage IV. As shown in the Fig. 4, there are 35 mutated genes having comparatively high expression in average in ovarian cancer (student t test, P-values <  0.05). Multiple genes (MUC1615, POSTN16) have been reported to be related to ovarian cancer metastasis or cell movement. Our analysis reveals a similar relationship between peri- ostin (POSTN) and EMT of ovarian cancer. Some of genes (e.g. MMP7) were reported to have potential biomarker role in ovarian cancer, but not for EMT process17. Those genes might be used as biomarkers for the ovarian cancer EMT-related process, especially, the strong different expression of periostin from stage III to IV which may indicate the ovarian cancer EMT process. www.nature.com/scientificreports/ Figure 2. The mutational landscape for the top 100 EMT-related genes in multiple cancers. Figure 2. The mutational landscape for the top 100 EMT-related genes in multiple cancers. Figure 2. The mutational landscape for the top 100 EMT-related genes in multiple cancers. Scientific Reports | 5:11459 | DOI: 10.1038/srep11459 6 www.nature.com/scientificreports/ Figure 3. The shared somatic variants related to EMT across 19 cancer types. The length of circularly arranged segments is proportional to the total variants in each cancer type. The ribbons connecting different segments represent the number of shared variants between cancer types. The three outer rings are stacked bar plots that represent relative contribution of other cancer types to the cancer type’s totals. Figure 3 The shared somatic variants related to EMT across 19 cancer types The length of circularly Figure 3. The shared somatic variants related to EMT across 19 cancer types. The length of circularly arranged segments is proportional to the total variants in each cancer type. The ribbons connecting different segments represent the number of shared variants between cancer types. The three outer rings are stacked bar plots that represent relative contribution of other cancer types to the cancer type’s totals. are shared between cancers. For instance, it is interesting that Uterine Corpus Endometrioid Carcinoma (UCEC) has many overlapping variants with colon and rectal adenocarcinoma (COADREAD) and sug- gests that the three cancers might have similar processes related to EMT. Or is simply because of the closeness of two organs. The similar comparison can be applied to other cancers. In summary, not all the cancers share the same set of EMT-related mutations. Considering both Figs 2 and 3, it is concluded that different cancers may have different mutation numbers and contents for EMT-related genes. According to the mutation frequency, some driver mutations occurred in some of critical genes, including MUC16 (1720 mutations), TP53 (1113 mutations), PTEN (1113 mutations), PIK3CA (291 mutations), NF1 (288 mutations), VCAN (274 mutations), ATM (236 mutations) and LAMA1 (230 mutations) in all the pan-cancer samples. These driver mutations in EMT-related pathway may induce the cancer cells in the progressive direction during EMT. To further test whether these top 100 EMT related-genes are mutated more frequently than expected by chance, we conducted 1000 resamplings to choose 100 mutated genes from 19 pan-cancer data. www.nature.com/scientificreports/ www.nature.com/scientificreports/ Figure 5. Reconstructed EMT map using protein-protein interaction data . (A) The 335 genes in orange are genes from the core dataset in our dbEMT. The remaining 36 genes in red are linker genes that bridge the 335 genes; (B) the degree distribution; (C) the short path length frequency; (D) the correlation between closeness centrality and the number of neighbors. Figure 5. Reconstructed EMT map using protein-protein interaction data . (A) The 335 genes in orange are genes from the core dataset in our dbEMT. The remaining 36 genes in red are linker genes that bridge the 335 genes; (B) the degree distribution; (C) the short path length frequency; (D) the correlation between closeness centrality and the number of neighbors. reveals the EMT-related genes are highly connected to each other and form a high density cellular mod- ular. Further network topological analysis also indicates that most molecules in our map were closely t d O l 80 d ith t i d d t d Thi th j it f d reveals the EMT-related genes are highly connected to each other and form a high density cellular mod- ular. Further network topological analysis also indicates that most molecules in our map were closely connected. Only 80 nodes were without in-degree and out-degree. This means the majority of nodes can communicate with each other in short steps. The degrees of all nodes in our reconstructed EMT map follow a power law distribution P(k) ~ k-b, where P(k) is the probability that a molecule has connections with other k molecules and b is an exponent with an estimated value of 0.998. This means that our EMT map is different from all the human PPI network where most of nodes were sparsely connected with exponent b as 2.919. This feature made the shortest path distribution for the entire network was a smaller number (2 to 4), and means that about 53% of the node communication can be reached only by three steps (Fig. 5C). This observation is also confirmed from the relation between closeness and the number of neighbors. As shown in Fig. 5D, the nodes with higher closeness are more likely to have more neighbors and vice versa. With high modularity, the hub nodes in this network may have prominent roles as common connections to mediate information transduction in the short path. www.nature.com/scientificreports/ In total, there are 13 genes with at least 50 connections and these are: SRC (99), EGFR (69), PTK2 (65), PRKCA (64), MAPK1 (64), AKT1 (61), JAK2 (58), CTNNB1 (57), ERBB2 (57), FGFR1 (56), MAPK3 (53), TP53 (50), MAPK14 (50). All 13 genes are from our literature-based gene set; none are linker genes. In summary, our reconstructed map not only discovers multiple paths related to a few known signaling pathways but also provides a broader picture for the highly modular structure of the different previously unconnected signaling pathways. www.nature.com/scientificreports/ The reconstructed EMT-related protein-protein interaction network exhibits a highly modu- lar structure. In the past decade, the knowledge of the cellular pathway for metabolism and signaling transduction have been summarized in a few popular biological pathway data such as the KEGG path- way. Recently, the Pathway Commons database combined all the popular pathway databases to provide gene-gene functional interaction pairs and these are useful for further pathway reconstruction18. In our study, we utilized reliable public data sources and constructed a more comprehensive cellular map for EMT. The reconstructed EMT contains 371 genes and 2357 gene-gene interactions is based on current evidence from known biological pathways (Fig. 5A). Of the 371 nodes, 335 of them are from our curated 377 IQ-related genes. The remaining 36 are the novel genes that may potentially bridge the EMT-related gene to fully implement their cellular function. The majority of curated EMT-related genes are linked to each other in a highly modular structure. This not only support the accuracy of our data, but it also 8 Scientific Reports | 5:11459 | DOI: 10.1038/srep11459 www.nature.com/scientificreports/ Conclusion dbEMT is constructed as a free database and analysis server to enable users to rapidly search and retrieve summarized EMT-related genes. The comprehensive functional enrichment analyses reveal that EMT-related genes are enriched in multiple signal events associated with development and cancers. Important questions should now be directed towards integration of various signaling pathways to initial EMT events. The dbEMT is freely available at http://dbemt.bioinfo-minzhao.org/. Scientific Reports | 5:11459 | DOI: 10.1038/srep11459 9 www.nature.com/scientificreports/ Figure 6. Pipeline for collection, expansion and annotation of EMT-related genes. Figure 6. Pipeline for collection, expansion and annotation of EMT-related genes. Methods Extensive literature search for EMT-related genes and gene curation. To obtain a precise EMT-related gene list with experimental evidence, we performed our literature search and curation as the following four steps (Fig. 6). Firstly, we first performed an extensive literature query against PubMed (on Dec 19th, 2013) using complex expression: ("Epithelial-mesenchymal transition"[Title/Abstract] OR "Epithelial mesenchymal transition"[Title/Abstract] OR "EMT"[Title/Abstract]) AND (("genome-wide association study" [Title/Abstract] OR "genome wide association study" [Title/Abstract]) OR ("gene"[- Title/Abstract] AND ("association"[Title/Abstract] OR "microarray" [Title/Abstract] OR "expression" [Title/Abstract] OR "linkage" [Title/Abstract] OR "proteomics" [Title/Abstract] OR "genetic" [Title/ Abstract] OR "metabolomics" [Title/Abstract] OR "copy number variation" [Title/Abstract] OR "idi- opathic" [Title/Abstract] OR "hereditable" [Title/Abstract] OR "family" [Title/Abstract] OR "mouse model" [Title/Abstract] OR "animal model" [Title/Abstract] OR "microRNA" [Title/Abstract] OR "muta- tion" [Title/Abstract] OR "SNP" [Title/Abstract] OR "drug" [Title/Abstract] OR "transporter" [Title/ Abstract]))). Secondly, 1507 abstracts were retrieved and grouped by the “Related Articles” function in Entrez system. Thirdly, we then extracted the EMT-related description from the grouped abstracts. Those sentences related to EMT were manually read to extract the gene names and organism information with experimental evidence. Lastly, the extracted candidate gene name and organism information were manually checked. Finally, the 377 Entrez human Gene IDs with high confidence were collected as core EMT-related genes. This core gene list will be regularly updated based on newly published literature. Biological functional annotations and database construction. To present the biological func- tion involved and over-represented in our 377 EMT-related genes, we retrieved comprehensive func- tional information from public resources. The basic gene information and sequences are included and crosslinked to the NCBI Entrez gene20, UniProt21, Ensembl22 and Gene Ontology23. The mRNA expres- sion profiling data from both normal and tumor tissues are imported from BioGPS24. To obtain compre- hensive pathway-related information, we annotated the EMT-related genes using transporter substrate database25, BioCyc26, KEGG Pathway27, rate-limiting enzyme database28 PANTHER29, PID Curated30, pathway localization database31, PID Reactome32,33. The involved diseases were incorporated from GAD (gene association database)34, KEGG Disease35, Fundo36,37, NHGIR38, as well as OMIM20. In addition, the original EMT-related literature references in the NCBI PubMed database are hyperlinked to each Scientific Reports | 5:11459 | DOI: 10.1038/srep11459 10 www.nature.com/scientificreports/ gene. A semi-automatic annotation pipeline is implemented to integrate functional information from Gene annotation39, Gene Ontology annotation, HPRD/BIND/BioGRID interaction annotation, KEGG LIGAND/BioCarta signaling event annotation40,41, and OMIM annotation using Perl Script and Swiss knife module42–45. Methods To obtain updated relevant publications in future, we constructed an automatic literature searching terms using My NCBI tool, which will return matched articles every two weeks. We will consider using the Entrez literature similarity to cluster the newly available articles to facilitate the curation. In addition, to keep pace with fast generation of cancer genomic data, we have implemented an automatic system to import functional information from a variety of public data sources, which can help us integrate more annotations quickly. Once the data content is updated, the web interface will be updated accordingly annually. Gene set enrichment analysis. Throughout the paper, the functional enrichment analysis from KEGG, Reactome and other disease databases for each gene set were identified by KOBAS46. In these enrichment analyses, all the human protein-coding genes were set as background to calculate statisti- cal significance. In addition, the Benjamini-Hochberg multiple testing corrected P-values for enriched annotations were adopted based on hypergeometric test by using KOBAS. Finally, the enriched human pathways with corrected P-values less than 0.01 were identified as over representative annotations for each gene set. Gene ranking using Endeavour and cancer mutation landscape. We performed a gene prioriti- zation using the Endeavour web server47 to help the user prioritize all 377 genes in dbEMT. Endeavour utilizes multiple dimensional data to rank the input genes, including gene expression, regulatory infor- mation, functional annotations, sequence features, and literature mining data. It begins by extracting fea- tures about the genes that are known to play a role in the EMT. In this study, we compiled a training gene list that included 12 genes (TGFB1, SNAI1, CDH1, ZEB1, SNAI2, TWIST1, SMAD3, ILK, HIF1A, EGFR, ZEB2, and CTNNB1) with at least eight PubMed abstracts to train the ranking model. In the second stage, the ranking model was used to prioritize the remaining 365 genes using multiple genomic data. Finally, Endeavour combined all the rankings to a global ranking for all the candidate EMT-related genes using order statistics. In total, 209 valid human genes were ranked (Table S1). The top 100 ranking of EMT related-genes are input into the cBio portal to obtain a mutation pattern across multiple cancers48. Empirical resampling for evaluating the single nucleotide mutations of EMT related-genes across multiple cancers. To determine whether the SNV somatic mutation rate of top 100 EMT related-genes are relatively higher than expected, we conducted a total of 1000 resamplings. Methods To this aim, we first downloaded all the SNV somatic mutations in MAF format across 19 TCGA tumor-types (https:// www.synapse.org/#!Synapse:syn1729383)49. During each resampling process, 100 human genes were ran- domly selected from 20059 human genes with SNV somatic mutations. To provide insight into biological meaning, we only focused on somatic mutations containing mis-senses, splicings, frameshift SNVs and INDELs instead of non-sense and silent mutations. We repeated this random process 1000 times. Next, we counted the number of randomly selected node sets (N) whose number of mutated genes was more than the actual number of mutated genes in the top 100 EMT related-genes. Finally, we calculated the empirical P-value using N/1000 to represent the significance of how the top 100 EMT related-genes are highly mutated in certain cancers. Based on this empirical resampling approach, the empirical P-values for the relationship between the top 100 EMT related-gene set and cancers are all less than 0.01 (Table S2). Gene expression analysis in ovarian cancer. To explore the gene expression changes during cancer metastasis, we focused on a gene expression profile with 489 high-grade serous OVC samples produced by using three gene expression microarray platforms (Affymetrix Exon 1.0 array, Agilent 244 K whole genome expression array, and Affymetrix HT-HG-U133A array)50. To present a unified gene expression, all the three datasets were normalized and the expression values were calculated for each sample and gene on each platform separately. After subtracting the mean value across samples for the same gene, the expression values were divided by the standard deviation across samples to obtain the relative gene expression scores. Finally, the relative expression data from three platforms was integrated into a unified data set of 11,864 genes using a factor analysis model without batch effects51–53. The processed final gene expression data was downloaded from the TCGA website in a format of matrix, in which one row for each gene and one column for each of the sample (https://tcga-data.nci.nih.gov/docs/publications/ov_2011/). g p p g g p From the prepared gene expression matrix of ovarian cancer, we extracted the expression values of the mutated EMT related-genes in stage III and IV. In total, there are 65 EMT related-genes with somatic mutations in ovarian cancer. Next, we extracted all the 65 gene expression profiles from 381 stage III samples and 79 stage IV samples of ovarian cancer. Scientific Reports | 5:11459 | DOI: 10.1038/srep11459 www.nature.com/scientificreports/ Figure 7. Web interface of dbEMT. (A) The basic information in each EMT-related gene page; (B) Query interface for text search; (C) BLAST search interface for comparing query against all sequences in dbEMT; (D) Browser interface for genes in top 10 enriched pathways, top 10 enriched diseases and shared cytobands. Figure 7. Web interface of dbEMT. (A) The basic information in each EMT-related gene page; (B) Query interface for text search; (C) BLAST search interface for comparing query against all sequences in dbEMT; (D) Browser interface for genes in top 10 enriched pathways, top 10 enriched diseases and shared cytobands. Reconstructing a protein-protein interaction network related to EMT genes. To present the underlying biological mechanisms related to EMT genes, we extracted a protein-protein interact- ing relation between 377 EMT-related genes with the remaining human genes. To this end, we col- lected a non-redundant human interactome based on all the known protein-protein interactions using PathCommons, containing 3629 nodes and 36034 protein-protein links. It is noteworthy that the col- lected protein-protein interactions are from pathway databases (HumanCyc, NCI signaling pathway database, Reactome, and KEGG), which have biological significance. The final interactome represents pathway-based gene–gene interaction links. To extract a sub-network related to the 377 EMT-related genes of interest, we used the similar approach implemented in our previous study54. In this algorithm, all input genes were mapped to the human interactome, which was used to produce a sub-network with input genes connected by their shortest path.t p g y p In spite of its high complexity, the biological network often follows a few simple rules that may relate to its function55. Generally, the topological characters may give more clues to reveal the potential function for a network. To decompose the reconstructed interactome from our 377 EMT-related genes, topological analyses were conducted using the NetworkAnalyzer plugin in Cytoscape 2.8 (Fig. 5B–D)56. Often the amount of connections at each node was represented as degree in a network55. Finally, close- ness centrality was used to reveal the shortest steps for one node to reach another55. The final network visualization was performed using Cytoscape56. Web interface development. The reliable open source relational database MySQL was used to store all the data and annotations in dbEMT on a Linux server. The CGI Web-based interface to the database is implemented using Perl. Methods We determined the gene expression changes of the transition between stage III and IV using the student test. Finally, we found 35 genes with paired t-test P-values less than 0.05. All the statistic tests and heatmap generations were using R programing environ- ment (R Development Core Team 2008). Scientific Reports | 5:11459 | DOI: 10.1038/srep11459 11 www.nature.com/scientificreports/ www.nature.com/scientificreports/ Using the Perl CGI module and JavaScript technology, web pages for each gene in the database are generated. In dbEMT, all the genes are annotated comprehensively with hyperlinks to their original data resources (Fig. 7A). Gene expression in various normal tissues and cancer samples Scientific Reports | 5:11459 | DOI: 10.1038/srep11459 12 www.nature.com/scientificreports/ are represented in a colored bar chart (Fig. 5A). In addition, the extensive literature evidence associated with EMT genes are also complied and highlighted with keywords related to EMT or diseases. g p g g y Our dbEMT provides a user-friendly web interface to perform text query (Fig. 7B), or to run a sequence similarity search (BLAST) against the nucleotide and protein sequences in dbEMT (Fig. 7C). In the text-based query page, six different powerful input forms are provided for the Entrez Gene ID, pathway and disease information, genomic location, literature evidence, and gene expression range in normal/cancer samples. Additionally, a quick text search for GeneID, gene symbol, and gene alias is at the top right of each page, and is convenient for a user to obtain any data in the database, especially literature-based annotations. Furthermore, users can browse the data in dbEMT in a variety of ways, including significantly enriched pathway, related disease, reported linkage region, and chromosome number (Fig. 7D). For each related KEGG pathway, the marked chart is provided to highlight the entire related EMT-related gene. Finally, for any advanced study, dbEMT provides all downloadable gene anno- tation and sequence information in a plain text format for all the collected 377 genes related to EMT. References l h y j 15. Theriault, C. et al. MUC16 (CA125) regulates epithelial ovarian cancer cell growth, tumorigenesis and metastasis. Gynecol Oncol 121, 434–443 (2011). 6. Gillan, L. et al. Periostin secreted by epithelial ovarian carcinoma is a ligand for alpha(V)beta(3) and alpha(V)beta(5) integrin and promotes cell motility. Cancer Res 62, 5358–5364 (2002). , y p g p ( ) ( ) p ( ) ( ) g and promotes cell motility. Cancer Res 62, 5358–5364 (2002). 17 Schummer, M et al Evaluation of ovarian cancer remission markers HE4, MMP7 and Mesothelin by comparison to the and promotes cell motility. Cancer Res 62, 5358 5364 (2002). 17. Schummer, M. et al. Evaluation of ovarian cancer remission markers HE4, MMP7 and Mesothelin by comparison to the established marker CA125 Gynecol Oncol 125 65 69 (2012) p y 7. Schummer, M. et al. Evaluation of ovarian cancer remission markers HE4, MMP7 and Mesothelin by comparison to th established marker CA125. Gynecol Oncol 125, 65–69 (2012). established marker CA125. Gynecol Oncol 125, 65–69 (2012). y 18. Cerami, E. G. et al. Pathway Commons, a web resource for biological pathway data. Nucleic Acids Res 39, D685–690 (2011 18. Cerami, E. G. et al. Pathway Commons, a web resource for biological pathway data. Nucleic 18. Cerami, E. G. et al. Pathway Commons, a web resource for biological pathway data. Nucleic Acids Res 39, D685–690 (2011). 19 Jin Y Turaev D Weinmaier T Rattei T & Makse H A The evolutionary dynamics of protein protein interaction networks 18. Cerami, E. G. et al. Pathway Commons, a web resource for biological pathway data. Nucleic Acids Res 39, D685–690 (2011). 19. 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Integrated genomic analyses of ovarian carcinoma. Nature 474, 609–615 (2011). 51. Zhao, M., Sun, J. Acknowledgmenth g This work was supported by the National Natural Science Foundation of China (No. 31171270) and the research start-up fellowship of university of sunshine coast to MZ. g This work was supported by the National Natural Science Foundation of China (No. 31171270) and the research start-up fellowship of university of sunshine coast to MZ. Scientific Reports | 5:11459 | DOI: 10.1038/srep11459 References l 5 K hi M G t S F i hi M T b M & Hi k M KEGG f t ti d l i f l l t h 35. Kanehisa, M., Goto, S., Furumichi, M., Tanabe, M. & Hirakawa, M. KEGG for representation and analysis of molecular networks involving diseases and drugs. Nucleic Acids Res 38, D355–360 (2010). g g , ( ) 6. Osborne, J. D. et al. Annotating the human genome with Disease Ontology. BMC Genomics 10 Suppl 1, S6 (2009). Scientific Reports | 5:11459 | DOI: 10.1038/srep11459 13 Additional Information upplementary information accompanies this paper at http://www.nature.com/srepihi Supplementary information accompanies this paper at http://www.nature.com/srepihi Competing financial interests: The authors declare no competing financial interests. How to cite this article: Zhao, M. et al. dbEMT: an epithelial-mesenchymal transition associated gene resource. Sci. Rep. 5, 11459; doi: 10.1038/srep11459 (2015). This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Com- mons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/ Scientific Reports | 5:11459 | DOI: 10.1038/srep11459 14

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Trial for the Prevention of Depression (TriPoD) in final-year secondary students: study protocol for a cluster randomised controlled trial

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Trial for the Prevention of Depression (TriPoD) in final-year secondary students: study protocol for a cluster randomised controlled trial Trial for the Prevention of Depression (TriPoD) in final-year secondary students: study protocol for a cluster randomised controlled trial Yael Perry1*, Alison L. Calear2, Andrew Mackinnon3, Philip J. Batterham2, Julio Licinio4, Catherine King1, Noel Thomsen1, Jan Scott5, Tara Donker6, Sally Merry7, Theresa Fleming7, Karolina Stasiak7, Aliza Werner-Seidler1 and Helen Christensen1 © 2015 Perry et al. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Abstract Background: Evidence suggests that current treatments cannot fully alleviate the burden of disease associated with depression but that prevention approaches offer a promising opportunity to further reduce this burden. Adolescence is a critical period in the development of mental illness, and final school examinations are a significant and nearly universal stressor that may act as a trigger for mental health difficulties such as depression. The aim of the present trial is to investigate the impact of SPARX-R, an online, gamified intervention based on cognitive behavioural principles, on the prevention of depression in secondary school students before their final examinations. Methods/Design: Government, independent and Catholic secondary schools in New South Wales, Australia, will be recruited to participate in the trial. All students enrolled in their final year of high school (year 12) in participating schools will be invited to participate. To account for possible attrition, the target sample size was set at 1600 participants across 30 schools. Participating schools will be cluster randomised at the school level to receive either SPARX-R or lifeSTYLE, an attention-controlled placebo comparator. The control intervention is an online program aimed at maintaining a healthy lifestyle. The primary outcome will be symptoms of depression, and secondary outcomes will include symptoms of anxiety, suicidal ideation and behaviours, stigma and academic performance. Additional measures of cost-effectiveness, as well as process variables (e.g., adherence, acceptability) and potential predictors of response to treatment, will be collected. Consenting parents will be invited to complete measures regarding their own mental health and expectations for their child. Assessments will be conducted pre- and post-intervention and at 6- and 18-month follow-up. Primary analyses will compare changes in levels of depressive symptomatology for the intervention group relative to the attention control condition using mixed-effects model repeated-measures analyses to account for clustering within schools. Discussion: This is the first trial of a universal depression prevention intervention delivered to school students in advance of a specific, significant stressor. If found to be effective, this program may offer schools a new approach to preparing students for their final year of schooling. Trial registration: Australian New Zealand Clinical Trials Registry identifier: ACTRN12614000316606. Registered 25 March 2014. * Correspondence: [email protected] 1Black Dog Institute, University of New South Wales, Hospital Road, Randwick, Sydney, NSW 2031, Australia Full list of author information is available at the end of the article STUDY PROTOCOL Open Access Trial for the Prevention of Depression (TriPoD) in final-year secondary students: study protocol for a cluster randomised controlled trial Yael Perry1*, Alison L. Calear2, Andrew Mackinnon3, Philip J. Batterham2, Julio Licinio4, Catherine King1, Noel Thomsen1, Jan Scott5, Tara Donker6, Sally Merry7, Theresa Fleming7, Karolina Stasiak7, Aliza Werner-Seidler1 and Helen Christensen1 TRIALS Perry et al. Trials (2015) 16:451 DOI 10.1186/s13063-015-0929-1 STUDY PROTOCOL Open Access Trial for the Prevention of Depression (TriPoD) in final-year secondary students: study protocol for a cluster randomised controlled trial Yael Perry1*, Alison L. Calear2, Andrew Mackinnon3, Philip J. Batterham2, Julio Licinio4, Catherine King1, Noel Thomsen1, Jan Scott5, Tara Donker6, Sally Merry7, Theresa Fleming7, Karolina Stasiak7, Aliza Werner-Seidler1 and Helen Christensen1 TRIALS Perry et al. Trials (2015) 16:451 DOI 10.1186/s13063-015-0929-1 Perry et al. Trials (2015) 16:451 DOI 10.1186/s13063-015-0929-1 TRIALS Background With regard to diagnosis of depressive disorders, targeted interventions reduced diagnoses post- intervention (risk difference = −0.07) and at 3–9-month follow-up (risk difference = −0.06) relative to no inter- vention. Universal programs were also found to be ef- fective immediately following the intervention and 3–9 months later (risk difference = −0.12 and −0.19, respect- ively). Similar results were found for depressive symptoms, with both targeted and universal interventions demonstrat- ing evidence of efficacy post-intervention (standardised mean difference = −0.31 and −0.10, respectively) and at follow-up (standardised mean difference = −0.22 and −0.09, respectively). Despite mixed evidence, the finding that universal pro- grams may indeed be effective is important, given that they may also be easier to implement and are less stig- matising [17, 18]. Universal programs also have the added advantage of providing a foundation of skills upon which subsequent targeted programs can build [17]. Given that there is some support for universal programs but that this approach may not be effective in all cir- cumstances, there is a clear case for further investigation of this kind of prevention. It is important, however, to determine the specific parameters within which universal prevention interventions are most likely to succeed. One potentially useful approach is to conduct universal inter- ventions in advance of a stressor that is experienced uni- versally and predictably. Despite evidence indicating the overall effectiveness of prevention of depression [9], it remains unclear exactly which of the various prevention approaches is superior, for whom prevention programs are most effective, and under what circumstances the programs should be deliv- ered. Universal prevention approaches are applied to whole populations, without regard to individual risk, whereas selective and indicated approaches target high-risk individ- uals on the basis of the presence of existing risk factors and subthreshold symptoms, respectively. Reviews in the litera- ture have generally found larger effect sizes for targeted interventions. For example, data derived from a systematic review of school-based depression prevention programs found indicated programs to be the most effective [10]. Six of ten indicated trials demonstrated significant differences between the intervention and control conditions at post- test, with effect sizes ranging from 0.25 to 1.35. In con- trast, only 9 of 23 universal interventions found significant effects over the control condition, though the range of effect sizes in these trials was similar to those in the indicated trials (d = 0.30–1.40). Background reduction) to occur [11]. However, a key problem with the indicated approach in mental health is that indi- viduals who are not yet symptomatic are not targeted in the intervention (see [12]). This means that individ- uals who may ultimately progress to develop symptoms will be missed. In contrast, by definition, universal preven- tion approaches provide greater scope and catchment of individuals who may be on the trajectory towards develop- ing depression. By 2030, depression is predicted to be the second leading cause of disease burden worldwide [1]. Currently, in young people aged 10–24 years, it carries the greatest burden of disease [2]. Andrews et al. [3] calculated that only 13 % of this burden can be averted by available treatments and that, even with improved coverage, clinician competence and adherence, only 36 % of the burden of depression could potentially be alleviated using current knowledge and ther- apies. Indeed, Australian national data on service provision and access to treatment have demonstrated a considerable improvement over the previous two decades, with no discernible effect on prevalence, which, if anything, has in- creased [4]. This leaves the majority of the burden of major depression unaddressed. Further, relapse and recurrence of depression are frequent. More than 80 % of individuals with major depressive disorder (MDD) experience multiple epi- sodes [5], with each additional episode predicting a greater likelihood of future recurrence [6], even after successful treatment [7, 8]. Given the potential chronicity of this dis- order, intervening before the onset of a first episode repre- sents a unique opportunity to change a lifelong trajectory of depression. A recent meta-analysis of 32 randomised controlled trials indicated a 21 % reduction in incidence of depression following the implementation of prevention programs [9]. Although there are some limitations to this analysis, prevention approaches represent a potential crit- ical pathway to decreasing depression rates and the associ- ated burden of this disorder. From an empirical perspective, whereas three recent uni- versal prevention trials failed to find significant effects [13– 15], a Cochrane review of 39 targeted (including both se- lective and indicated) and 31 universal psychological and educational prevention interventions for young people found evidence to support both targeted (number needed to treat [NNT] = 14) and universal programs (NNT = 8) [16]. Abstract Keywords: Adolescents, CBT, Computerised CBT, Depression, eHealth, Prevention, RCT, School * Correspondence: [email protected] 1Black Dog Institute, University of New South Wales, Hospital Road, Randwick, Sydney, NSW 2031, Australia Full list of author information is available at the end of the article © 2015 Perry et al. 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. Perry et al. Trials (2015) 16:451 Page 2 of 17 Background It should be noted, however, that it is easier to demonstrate suc- cess in indicated programs because individuals are selected on the basis of symptoms, meaning that there is greater opportunity for change (typically symptom Rates of depression begin to increase at 12–13 years of age, but the growth in incidence is continuous, with new cases of depression emerging at a similar rate through- out adolescence [18]. In determining the point at which a preventive intervention might be optimally delivered, it is notable that stressful life events act as predictors and candidate causal factors in the development of depres- sion [19]. Final examinations represent a significant stres- sor for most adolescents. More specifically, in cultures where significant emphasis is placed on university Page 3 of 17 Perry et al. Trials (2015) 16:451 entrance examinations in the final year of school, students tend to spend much more time doing schoolwork and less time in discretionary activities and to have more negative affective states across daily activities and higher rates of depression, relative to cultures without this focus on ex- aminations [20]. Indeed, Kouzma and Kennedy [21] noted that the main sources of stress reported by final-year stu- dents in Australia were school-related. Amongst these stressors, examinations and academic outcomes were endorsed most strongly (see also [22–24]). Further, more than 40 % of year 12 students in Australia reported symp- toms of depression, anxiety and stress that fall outside normal ranges [25]. In extreme cases, examination stress has even been linked to suicidal ideation, behaviour and completion [26, 27]. Because stress can lead to social and emotional difficulties, which can then interfere with aca- demic achievement, programs that help students to man- age stress before examinations may be beneficial, in terms of both improving mental health and enhancing academic performance [28]. school settings, though the authors acknowledged that online delivery of programs is becoming increasingly popular. With regard to setting, schools are an ideal location to deliver mental health interventions, not only because young people spend more time in these institutions than any other, but also because of the integral role schools play in students’ social, academic, cognitive, emotional and behavioural development [31]. Further, the provision of mental health interventions in schools (in contrast to providing students access to these interventions inde- pendently) necessarily increases uptake and adherence [32]. Background From cost-effectiveness, engagement and delivery perspectives, online technology is an advantageous means by which to deliver programs to students en masse [4]. Further, automated online programs guarantee fidelity because the information cannot be distorted or adapted during implementation of the program, nor does it rely on personal delivery [33]. These programs also have the benefit of avoiding a number of practical difficulties, such as the need for expensive face-to-face input from health professionals [34] or time-consuming teacher training (see, e.g., [14]). Therefore, delivering a univer- sal, school-based CBT program to adolescents before a major stressor has the potential to prevent the onset of depression. To date, the potential for prevention has not been examined in the context of school-based stressors. How- ever, researchers in one study examined the effect of a prevention program in a group of medical students in the United States [29]. Participants (mean age 27.6 years) were allocated to receive either MoodGYM, an online cognitive behavioural intervention, or an attention con- trol program before their first-year internship. Com- pared with the control group, interns who completed the intervention were almost four times less likely to experience an episode of depression during their intern- ship (13.0 % vs. 34.8 %). This finding suggests that pre- vention programs delivered before an anticipated period of increased stress may protect against the development of depression. p The present SPIRIT-compliant [35] protocol (see Table 1) describes the methodology for a cluster randomised con- trolled trial designed to test the effectiveness of SPARX-R, an online, CBT-based, gamified intervention designed to prevent the development of depression in students’ final year of secondary studies (known as the Higher School Certificate [HSC]). SPARX was designed as a treatment intervention for depression. For the purpose of the current study, SPARX will be revised for use as a preventive inter- vention, known as SPARX-R. SPARX has been shown to be non-inferior to treatment as usual (primarily individual face-to-face therapy) in depressed help-seeking adolescents [36], for whom use of the program resulted in clinically significant reductions in depressive symptoms. An online active control program (lifeSTYLE) focused on maintaining a healthy lifestyle will serve as an attention-controlled com- parator. Background In addition to evaluating the relative impact on depressive symptoms, in the trial we will also explore the impact of SPARX-R on a range of secondary outcome vari- ables, including academic performance, anxiety and suicidal ideation, as well as parental, process and cost-effectiveness variables. In terms of the most effective approaches to preven- tion interventions, one of the key factors to consider is the therapeutic orientation of the program. Hetrick and colleagues [30] recently conducted an exploratory meta- analysis, specifically examining program content, based on the trial data included in Merry et al.’s 2011 Cochrane review [16]. Of the 50 intervention arms examined, 38 were classified as based purely on cognitive behavioural therapy (CBT), 4 reported data on interpersonal psychotherapy (IPT) interventions and 8 were classified as ‘other’ in orien- tation. Results indicate not only that CBT is the most studied type of intervention for depression prevention but also that there is evidence to support its effective- ness in reducing the risk of developing a depressive disorder and reducing depressive symptoms both post- intervention and at follow-up. Mixed evidence was found for IPT, and other interventions were not found to be effective. The majority of programs in the review were delivered face-to-face to groups of adolescents in A secondary aim of the trial is to determine the role of individual differences in response to both stress and the intervention, as these are likely to impact outcomes of interest. These variables (which may either increase vulner- ability or act as protective factors) include family history of depression, social support [37], mastery [38], hopelessness Perry et al. Background Trials (2015) 16:451 Page 4 of 17 Table 1 Items from the World Health Organisation dataset Data category Information Primary registry and trial identifying number Australian New Zealand Clinical Trials Registry identifier: ACTRN12614000316606 Date of registration in primary registry 25 March 2014 Secondary identifying number U1111-1154-5917 Sources of monetary or material support National Health and Medical Research Council Primary sponsor Black Dog Institute, University of New South Wales Secondary sponsors Contact for public queries YP, HC Contact for scientific queries YP, HC Public title Trial for the Prevention of Depression (TriPoD) Scientific title Inoculating final-year high school students: effectiveness of a universal prevention program for depression in adolescence Country of recruitment Australia Health conditions or problems studied Depression, anxiety and suicidal ideation and behaviour Interventions Active comparator: SPARX-R, an online, interactive game based on the principles of cognitive behavioural therapy designed to prevent depression in young people Placebo comparator: lifeSTYLE, an online program providing interactive content on a range of general health and well-being topics Key inclusion and exclusion criteria Inclusion criteria: male and female final-year secondary school students at participating schools Exclusion criteria: none Study type Study type: interventional Allocation: randomised Intervention model: parallel assignment Primary purpose: prevention Date of first enrolment February 2015 Target sample size 1600 Recruitment status Recruiting Primary outcome Symptom levels of depression Key secondary outcomes Symptoms of anxiety, academic performance, suicidal ideation and behaviour, and stigma Symptoms of anxiety, academic performance, suicidal ideation and behaviour, and stigma also anticipate that depressive symptoms for SPARX-R participants will be lower than those in the lifeSTYLE group at 6- and 18-month follow-up. For secondary out- comes, we expect that, relative to the lifeSTYLE group, participants in the SPARX-R group will perform signifi- cantly better in their HSC and demonstrate reduced anxiety, suicidal ideation and/or behaviour and stigma following the intervention and at follow-up. We antici- pate that SPARX-R will have an effect on the parents of students allocated to this group, reducing their own symptom levels at 6 months, relative to parents of students in the attention control group. Finally, we pre- dict that SPARX-R will be more cost-effective than life- STYLE, owing to fewer days out of role (e.g., studying, working), better quality of life and less use of health services at 6- and 18-month follow-up. Background [39], ruminative response style [40], neuroticism [41], be- longingness and burdensomeness [42], disturbances in the sleep–wake cycle (see [43]), expressed emotion [44] and cortisol levels [45]. In addition to these factors, genetics are implicated in the onset of depression, both alone and in combination with environmental variables (see, e.g., [46]). Therefore, we also plan to examine a range of genetic markers to determine if, and in what way, genetics may interact with outcomes. Collection of cortisol and gen- etic data will be subject to approval of relevant govern- ing ethics bodies. Hypotheses With regard to the primary outcome measure, we pre- dict that, relative to participants in the attention control condition, students who are assigned to the SPARX-R condition will demonstrate lower levels of depressive symptoms immediately following the intervention. We For those in the SPARX-R group, we predict that a posi- tive response to the prevention program will be associated Page 5 of 17 Perry et al. Trials (2015) 16:451 cohort (which we expect will comprise primarily inde- pendent and Catholic schools), pending ethical approval. with the following variables at baseline: absence of family history of depression, high social support, high mastery, low rumination, low neuroticism and/or negative affect, low hopelessness, low thwarted belongingness, low bur- densomeness, low sleep disturbance, unimpaired sleep– wake patterns, low expressed emotion, low cortisol levels and lack of genetic vulnerability factors. The 2015 cohort will comprise students from selective government secondary schools. Although study-related stress is a widely experienced phenomenon, academically gifted students may be particularly at risk of experien- cing stress-related mental health difficulties during the final examination period, given the high rates of perfec- tionism in this group, as well as the pressure to succeed from parents, schools, the media and students them- selves (see, e.g., [47]). Perfectionism has been shown to be a strong predictor of prospective stress in the context of an academically demanding end-of-semester period [48]. Further, the increased academic workload and con- siderable number of hours spent studying by students in a demanding education program is likely to compound this pressure and act as an additional source of stress [49]. Indeed, high-stakes examinations can lead to a range of negative outcomes in high-achieving students, including anxiety, depression, school absence, helpless- ness and ulcer [21, 50]. Thus, the characteristics of stu- dents in selective high schools make these schools a particularly relevant target for the present trial. It should be noted that some government schools have both se- lective and comprehensive streams. All students at these partially selective high schools will be eligible to partici- pate; however, streaming status will be recorded, and school type will be accounted for in analyses. Methods/Design Trial design This study is a cluster randomised controlled superiority trial with two parallel arms, consisting of an experimen- tal condition and an attention-matched control condi- tion. We will employ an adaptive design, such that accumulating data will be used to decide if or how to modify the study as it continues. To facilitate this design and to stagger recruitment, the trial will take place in two stages across consecutive years, with each stage be- ginning at the start of the academic calendar (February 2015 and February 2016). The intervention phase will last 5 weeks with a follow-up period of 18 months. Re- sults from the 2015 cohort will be subject to an interim analysis to determine preliminary effects and determine whether any modifications are required in the conduct of the trial in 2016. Feasibility of continuation will also be assessed at this stage in terms of student and teacher acceptability of the intervention and any implementation issues that may arise during the first year of the trial. Participants All adolescents enrolled in their final year of high school in participating schools will be invited to participate. The age range at baseline is 16–18 years. Owing to the universal nature of the study, there are no exclusion cri- teria. Students are free to receive additional support from health care professionals while participating in the trial, and this will be assessed and accounted for in sec- ondary analyses. This trial has received ethical approval from the New South Wales Department of Education and Training, the University of New South Wales, the University of Melbourne, Australian National University and South- ern Adelaide Clinical Human Research Ethics Commit- tees. Recruitment of schools will not be conducted until all local approval has been obtained. Any ad- verse events will be reported to the director of the Black Dog Institute and to the relevant human research ethics committees. Recruitment A flowchart outlining recruitment, randomisation and participation in this trial is provided in Fig. 1. Setting h Thirty secondary schools across the state of New South Wales will be recruited to participate in the trial. School principals will be notified about the trial in writing and invited to allow their schools to partici- pate. Research personnel may also meet with princi- pals and/or teachers in person to provide information about the study and answer any questions. Once prin- cipals formally agree to their schools’ participating in the trial, parents and students will be informed about the trial. The trial will be conducted in government, independent and Catholic secondary schools in New South Wales, Australia. Government schools in New South Wales are classified as either comprehensive or selective. Compre- hensive schools accept enrolments of all students in the surrounding area, whereas selective high schools use an entry examination to enrol students with superior aca- demic ability. The first stage of the trial will be con- ducted in government schools in metropolitan Sydney. Ethical approval to collect biological data in government schools was not granted. Therefore, measures of cortisol and genetic vulnerability will be collected only in the 2016 Assignment to the intervention arm will occur at the school level. All students will complete the intervention, Perry et al. Trials (2015) 16:451 Page 6 of 17 Fig. 1 Planned flow of participants through the Trial for Prevention of Depression cluster randomised controlled trial Fig. 1 Planned flow of participants through the Trial for Prevention of Depression cluster randomised controlled trial forms directly. Parents will be asked to either return a hard copy of the signed consent form to the school via their child or send a scanned or photographed copy via email. Parents will also be invited to participate in a parent survey by providing their email address and con- sent in an optional section of the consent form. In- formed consent will be obtained from each participant, and students will be asked to return their signed consent forms to the school. In addition to participating in the study, students will have the option to provide consent for their school to provide their final examination results to the trial researchers and for the Trial for the Prevention of Depression (TriPoD) investigators to contact them in the which will be integrated into the curriculum at each par- ticipating school. Sample size p Depression has an estimated annual prevalence of 8.3 % in adolescents [12], and between 8 % and 20 % of young people in community samples develop depression over an 18-month period [51–53]. On the basis of the age of the current sample, together with the timing of the intervention (before a major, universal stressor), we ex- pect that onset rates will be high (up to 15 %). Calcula- tions of required sample size for the primary outcome measure were based on detecting a post-intervention effect size of 0.20 in combined analyses of data collected from 2015 and planned 2016 cohorts. This estimate is based on previous depression prevention research using an online intervention [54]. Although the expected effect is small, it reflects the universal nature of this interven- tion. Power was set at 0.80, α = 0.05 (two-tailed) and a correlation of 0.5 assumed between baseline and end- point scores. To allow for possible clustering effects (i.e., participants from the same school having characteristics and outcomes more alike than between schools), a de- sign effect [55] was calculated assuming an intraclass correlation coefficient (ICC) of 0.02 and an average class size of 25. The estimate of the ICC was derived from a previous Australian school-based study that found a non- significant ICC of 0.02 [54]. The estimated sample size was 1166. The target size sample size was set at 1600, or 800 students per condition, to accommodate an attrition rate of approximately 20 % (see [56]). This translates to approximately 50 students (or 2 classes) per school in 30 schools. An interim analysis of students in the 2015 co- hort will involve approximately 300 students per arm. Under the assumptions outlined above, there will be 80 % power to detect an effect size of 0.40 standard deviations between groups. We will use the method developed and implemented by Carter and Hood [58] to ensure balance between intervention arms. This enumerates all possible divisions of the available schools into two groups and calculates a balance index [59] for each of these groupings. The 10 most balanced groupings will be retained after excluding any grouping resulting in a deviation of more than 10 % in predicted individual participant sample size between inventions. The allocation schedule will be selected at random from the groupings retained. Randomisation Each participating school (cluster) will be randomised to complete the SPARX-R or lifeSTYLE program. School- level randomisation will be used to avoid contamination between conditions, for the ecological validity of provid- ing the intervention at the cluster level and for practical convenience. A statistician not involved in the implemen- tation of the trial will randomly allocate schools using the allocation sequence outlined below. The identity of schools will be concealed from the statistician during this process. Using student email addresses provided by the schools, all students will be emailed a unique identification code before commencing the trial, which will allow them to register to access their allocated program via an online platform. This secure platform was designed specifically to facilitate the delivery of online interventions in a re- search context and can be configured to meet the needs of specific trials. For TriPoD, both of the interventions, as well as the research questionnaires, can be accessed through this platform. Those students without parental or personal consent will still register and access the pro- gram through the research platform; however, only those with consent will be given access to the research ques- tionnaires as well. Students who do not have consent will complete an innocuous filler task while their peers are completing the research questionnaires. Randomisation in cluster randomised trials is prob- lematic because the number of clusters involved is usu- ally relatively small. In large, individually randomised trials, balance in the makeup of each arm is achieved as a function of the numbers involved. Balance between arms can be achieved by stratification or minimisation [57]. Stratification can accommodate only a limited num- ber of balancing variables, and minimisation is based on incremental assignment of participants to groups. In TriPoD, the number of potentially relevant variables to be balanced between arms is quite large (e.g., sex, number of enrolled students, Index of Community Socio-Educational Advantage school index, language background other than English), and all available sites will be enumerated before commencement of the trial. Achieving approximately equal numbers of participants in each arm is also an aim of the allocation design, as this will determine the study’s power in the absence of substantial clustering effects. Accordingly, this attribute of each school will be used not to estimate the degree of balance, but rather to screen potential grouping after balanced combinations have been selected. Setting h Consent will be sought from students and parents only with regard to the research component of the interventions (i.e., the completion of outcome mea- sures and collection of other data). To maximise consent rates, information and consent forms will be distributed during the final term of the preceding school year. School administrators or class teachers will be provided with parental and student information and consent forms to pass on to all students enrolled in Year 12 the following academic year. Alternatively, schools may prefer to pro- vide parents and students with consent forms at parent information sessions or to email parents the consent Perry et al. Trials (2015) 16:451 Page 7 of 17 Page 7 of 17 future to inform them about any follow-up studies that may be conducted. Procedures d l Immediately before the baseline assessment, during an allo- cated class period, school teachers will provide students with a URL that will take them to the trial registration page. Students will use their unique identification code to register at this page and set up a personal password. Students will also be required to provide a personal email address and mobile telephone number so that they can be contacted during the follow-up period after they have left school. SPARX-R uses the same content as SPARX but is framed as a preventive intervention. For example, in the original version, participants are told that ‘SPARX was made to help young people who feel down or depressed’. In contrast, the SPARX-R introduction informs participants that ‘this version of SPARX was made to help young people who are having hassles and feeling down, stressed or angry a lot of the time. Even if you are doing fine, SPARX-R can help strengthen your skills for dealing with problems when they do come along’. Participants using SPARX-R choose and personalise an avatar and are led through the program by a virtual guide character who provides context and instruc- tion and relates the content of the program to the partici- pant’s real-life experiences. The participant navigates through a series of challenges and obstacles within a fantasy world that has been overrun by gloomy, negative, automatic thoughts. The student’s mission is to restore balance in the game world. The program has seven modules (levels), each of which takes approximately 20–30 minutes to complete. The modules cover the following topics: (1) finding hope, (2) being active, (3) dealing with strong emotions, (4) over- coming problems, (5) recognising unhelpful thoughts, (6) challenging unhelpful thoughts and (7) bringing it all together. SPARX-R will be delivered to participants via the internet on desktop computers in school classrooms. Students with parental and personal consent will complete questionnaires on three occasions during class time (pre-intervention, post-intervention and 6- month follow-up), as well as an additional 18-month follow-up questionnaire once they have graduated from secondary school. The timing of assessments was de- signed such that the 6-month follow-up will be con- ducted just before the trial HSC examination period, one of two major examination periods during the final year of school. All assessments will be conducted via the online plat- form. Interventions SPARX-R The experimental intervention in this trial is SPARX-R, a revised version of SPARX, which was originally devel- oped using an unguided, interactive fantasy game format that provides CBT skills to treat mild to moderate symp- toms of depression in help-seeking adolescents. SPARX resulted in a decline in symptoms of depression and was shown to be non-inferior to treatment as usual, which con- sisted primarily of face-to-face therapy with a counsellor, general practitioner or clinical psychologist [36]. In this Perry et al. Trials (2015) 16:451 Perry et al. Trials (2015) 16:451 Page 8 of 17 trial, response rates and treatment satisfaction did not differ between groups, nor did symptoms of depression, anxiety, hopelessness or quality of life, at post-intervention and 3- month follow-up. Further, remission rates were higher in the SPARX group. A pragmatic randomised controlled trial in a group of young people excluded from mainstream edu- cation also found SPARX to be superior to waitlist control with regard to reducing symptoms of depression [60]. Atti- tudes towards SPARX have been predominantly positive, with youth workers in New Zealand, young people alien- ated from mainstream schooling and rural Australian youth endorsing the program as a viable alternative to traditional therapeutic interventions [61–63]. Indeed, students in alter- native education felt that SPARX should be offered to all adolescents in courses like their own, as they felt that pro- viding the intervention at an indicated or targeted level might be stigmatising and that all could benefit from the lessons provided [64]. strategies or techniques to prevent or manage mental health difficulties. The program also includes seven mod- ules (approximately 25 minutes each) and covers the following topics: (1) independence, (2) participating in your community, (3) work skills, (4) mobile phone safety and hygiene, (5) healthy skin, (6) sustainable eating and (7) maintaining a healthy home environment. Each mod- ule includes information about the specified topic as well as interactive activities such as quizzes, myth busters, videos and scenarios that students can reflect on and re- spond to. As with SPARX-R, the intervention is delivered to students in school classrooms via the online platform on desktop computers. Procedures d l Teachers will direct students to log on to the platform during the first three assessments, and partici- pants will be emailed a link to log on directly from their personal computer on the final assessment occasion. All students will complete their allocated intervention (SPARX-R or lifeSTYLE) over the course of approxi- mately 5–7 weeks in school via the online platform. Schools will timetable the intervention sessions to fit in with their respective schedules, but they will be advised to space sessions out so that students have sufficient time to reflect on the material presented in each mod- ule. On each assessment occasion, students who do not complete their assessments during the scheduled class time (owing to absence or other conflicting duties) will be asked to complete the questionnaires in their own time. A reminder email will be sent 1 week after the scheduled assessment, and a final reminder will be sent another week later to any student who still has not completed the scheduled assessment. Trial researchers will monitor completion of assessments and will remain Measures The administration schedule for each of the assessment measures described below is provided in Table 2. Secondary outcome measures y Screen for lifetime presence of a major depressive episode or manic episode Screen for lifetime presence of a major depressive episode or manic episode Screening questions from the Structured Clinical Inter- view for DSM-IV disorders–Mood module [68] were included to provide an indication of a lifetime presence of a major depressive episode or manic episode. Mood, anhedonia, mania and irritability are assessed, and par- ticipants respond by answering ‘yes’ or ‘no’ to whether they have ever had prolonged experiences of each symptom (2 weeks for mood and anhedonia, 1 week for mania and irritability). lifeSTYLE lifeSTYLE is an adaptation of an interactive, online pro- gram that was originally developed as a control interven- tion for a previous trial targeting adults with suicidal thoughts [65]. The format and structure of the program have been retained, but the content has been adapted to suit the needs of a younger group. The aim of the inter- vention is to provide an engaging and useful resource for young people that matches the intervention in terms of duration and attention but without providing any Page 9 of 17 Perry et al. Trials (2015) 16:451 Perry et al. Trials (2015) 16:451 Spence Children’s Anxiety Scale in close contact with participating schools to ensure that school staff encourage assessment and intervention completion by all participating students. Students will no longer be able to access or complete assessments 1 month after their school’s scheduled assessment date. Spence Children’s Anxiety Scale The Spence Children’s Anxiety Scale is a 44-item meas- ure comprising 6 subscales. The scale was designed to measure the severity of children’s and adolescents’ anx- iety symptoms based broadly on DSM-IV criteria for anxiety disorders [69]. Respondents rate the degree to which they experience each symptom on a 4-point fre- quency scale, ranging from 0 (never) to 3 (always). Only items on the Social Phobia and Generalised Anxiety subscales will be administered in the present study. The scale has demonstrated high internal consistency and satisfactory test-retest reliability [18]. It was also reported to show both convergent [70] and divergent [18] validity. Predictor variables Schuster Social Support Scale The Schuster Social Support Scale is a 15-item measure of social support used to examine an individual’s social relationships with others and the associated impact on their emotional functioning. Each item is rated on a 4- point scale from 0 (not at all) through to 3 (all the time). The scale has demonstrated acceptable reliability coeffi- cients [75]. For the present study, 10 items from 2 sub- scales which measure support from friends and family will be used. Primary outcome measure Major Depression Inventory The Major Depression Inventory is a 12-item self-report measure of depressive symptoms [66]. The items of the MDI evaluate the presence and duration of depressive symptoms according to criteria of both the International Classification of Diseases, 10th revision (ICD-10), and the Diagnostic and Statistical Manual of Mental Disor- ders, Fourth Edition (DSM-IV) [67]. Participants rate the degree to which they have experienced each of the 10 symptoms over the preceding 2 weeks on a 6-point scale, ranging from 0 (at no time) to 5 (all of the time). The MDI has acceptable sensitivity and specificity for the diagnosis of depression according to the ICD-10 and DSM-IV [66]. To accord with DSM criteria and evaluate functional impairment, we included an extra question on the MDI asking participants to indicate (on the same scale) the degree to which their depressive symptoms in- terfered with activities of daily living over the previous 2-week period. Youth Risk Behavior Survey The Youth Risk Behavior Survey (YRBS) was designed to assess health risk behaviours among school students in years 9–12. For the purposes of the present study, the YRBS will be abbreviated to three items concerning sui- cidal thoughts, plans and attempts over the preceding month, for which participants will provide a yes-or-no answer. Endorsement of any of these items will trigger the trial’s participant risk management protocol (see below). Studies have shown that the suicidality items demonstrate both substantial reliability [71] and good convergent and divergent validity in high school samples [72]. Depression Stigma Scale The Depression Stigma Scale (DSS) is an 18-item meas- ure that assesses personal and perceived stigma towards depression. In the present study, only the nine-item per- sonal stigma subscale will be used. These items require participants to rate how strongly they personally agree with a statement about depression (e.g., ‘people with depression are unpredictable’) on a 5-point Likert scale ranging from 0 (strongly disagree) to 4 (strongly agree). The sum of each of the items yields a total stigma score, where higher scores indicate greater stigma. The per- sonal stigma subscale of the DSS has been shown to have moderate internal consistency in an adolescent sample [73, 74]. Pearlin Mastery Scale The Pearlin Mastery Scale is a widely used instrument designed to measure participants’ perceived sense of mastery over life outcomes. Respondents are required to rate how strongly they agree or disagree with a list of seven statements on a scale from 1 (strongly disagree) to 4 (strongly agree). The total scores range from 7 to 28, with higher ratings indicative of a higher level of self- mastery, or control of the forces that affect their lives. Australian Tertiary Admission Rank Trials (2015) 16:451 Page 10 of 17 Pearlin Mastery Scale The scale has shown good construct and predictive val- Table 2 Assessment administration schedule Assessment Construct Baseline Post-intervention 6 months 18 months Other Primary outcome MDI Depression symptoms X X X X Secondary outcomes ATAR Academic performance Collected via schools after final examination results are released SCAS Anxiety symptoms X X X X YRBS Suicidal ideation X X X X DSS-P Personal stigma X X X X Predictor variables SSSS Social support X PMS Mastery X X X RRS Rumination X X X PID-5-BF Personality traits X HPLS-Brief Hopelessness X INQ Belongingness/burdensomeness X ACSS Capability X ISI Sleep disturbance X MEQr Sleep–wake patterns X PS/PC Expressed emotion X Cost-effectiveness variables TiC-P Service use X X Questionnaire Days out of role X X AQoL Quality of life X X Process variables Questionnaire Expectation of success X Intervention data Adherence Collected automatically via SPARX-R and lifeSTYLE Questionnaire Acceptability, usability X IIAM Reasons for dropout X Parent outcomes PHQ-9 Depression X X GAD-7 Anxiety X X ACSS Acquired Capability for Suicide Scale; AQoL Assessment of Quality of Life; ATAR Australian Tertiary Admission Rank; DSS-P Depression Stigma Scale–Personal Stigma subscale; GAD-7 Generalized Anxiety Disorder 7-item scale; HPLS Hopelessness Scale for Children; IIAM Internet Intervention Adherence Measure; INQ Interpersonal Needs Questionnaire; ISI Insomnia Severity Index; MDI Major Depression Inventory; MEQr Reduced version of the Morningness Eveningness Question- naire PS/PC Perceived Sensitivity/Perceived Criticism scale; PHQ-9 Patient Health Questionnaire-9; PID-5-BF Personality Inventory for DSM-5; Brief Form; PMS Pearlin Mastery Scale; RRS Ruminative Response Scale; SCAS Spence Children’s Anxiety Scale; SSSS Schuster Social Support Scale; TiC-P Trimbos/iMTA ques- tionnaire for Costs associated with Psychiatric illness; YRBS Youth Risk Behavior Survey Collected automatically via SPARX-R and lifeSTYLE ACSS Acquired Capability for Suicide Scale; AQoL Assessment of Quality of Life; ATAR Australian Tertiary Admission Rank; DSS-P Depression Stigma Scale–Personal Stigma subscale; GAD-7 Generalized Anxiety Disorder 7-item scale; HPLS Hopelessness Scale for Children; IIAM Internet Intervention Adherence Measure; INQ Interpersonal Needs Questionnaire; ISI Insomnia Severity Index; MDI Major Depression Inventory; MEQr Reduced version of the Morningness Eveningness Question- naire PS/PC Perceived Sensitivity/Perceived Criticism scale; PHQ-9 Patient Health Questionnaire-9; PID-5-BF Personality Inventory for DSM-5; Brief Form; PMS Pearlin Mastery Scale; RRS Ruminative Response Scale; SCAS Spence Children’s Anxiety Scale; SSSS Schuster Social Support Scale; TiC-P Trimbos/iMTA ques- tionnaire for Costs associated with Psychiatric illness; YRBS Youth Risk Behavior Survey ACSS Acquired Capability for Suicide Scale; AQoL Assessment of Quality of Life; ATAR Australian Tertiary Admission Rank; DSS-P Depression Stigma Scale–Personal Stigma subscale; GAD-7 Generalized Anxiety Disorder 7-item scale; HPLS Hopelessness Scale for Children; IIAM Internet Intervention Adherence Measure; INQ Interpersonal Needs Questionnaire; ISI Insomnia Severity Index; MDI Major Depression Inventory; MEQr Reduced version of the Morningness Eveningness Question- naire PS/PC Perceived Sensitivity/Perceived Criticism scale; PHQ-9 Patient Health Questionnaire-9; PID-5-BF Personality Inventory for DSM-5; Brief Form; PMS Pearlin Mastery Scale; RRS Ruminative Response Scale; SCAS Spence Children’s Anxiety Scale; SSSS Schuster Social Support Scale; TiC-P Trimbos/iMTA ques- tionnaire for Costs associated with Psychiatric illness; YRBS Youth Risk Behavior Survey ACSS Acquired Capability for Suicide Scale; AQoL Assessment of Quality of Life; ATAR Australian Tertiary Admission Rank; DSS-P Depression Stigma Scale–Personal Stigma subscale; GAD-7 Generalized Anxiety Disorder 7-item scale; HPLS Hopelessness Scale for Children; IIAM Internet Intervention Adherence Measure; INQ Interpersonal Needs Questionnaire; ISI Insomnia Severity Index; MDI Major Depression Inventory; MEQr Reduced version of the Morningness Eveningness Question- naire PS/PC Perceived Sensitivity/Perceived Criticism scale; PHQ-9 Patient Health Questionnaire-9; PID-5-BF Personality Inventory for DSM-5; Brief Form; PMS Pearlin Mastery Scale; RRS Ruminative Response Scale; SCAS Spence Children’s Anxiety Scale; SSSS Schuster Social Support Scale; TiC-P Trimbos/iMTA ques- tionnaire for Costs associated with Psychiatric illness; YRBS Youth Risk Behavior Survey The scale has shown good construct and predictive val- idity (see [76]) and good internal consistency [77]. Australian Tertiary Admission Rank Participants’ academic performance will be assessed by means of their estimated Australian Tertiary Admission Rank (ATAR) scores. With students’ consent, schools will provide researchers with students’ HSC results, from which an estimate of each student’s ATAR score will be calculated using a standard online ATAR calculator. Perry et al. Collected automatically via SPARX-R and lifeSTYLE Hopelessness Scale for Children–Brief The Hopelessness Scale for Children (HPLS)–Brief is a 5- item measure adapted from the HPLS [84]. Participants indicate their agreement with statements by circling ‘true’ or ‘false’, and responses are numerically scored. Higher scores reflect greater hopelessness or negative expecta- tions about the future. Internal reliability and test-retest reliability were both found to be reasonable for the short- ened version of the HPLS and comparable to the full 17- item version [85]. Perceived Sensitivity/Perceived Criticism Scale The Perceived Sensitivity/Perceived Criticism (PS/PC) scale is a very brief measure designed to rapidly assess levels of criticism from relatives as perceived by the participant [44]. The measure is designed to assess how critical the respondent perceives their relative to be and how sensitive the respondent is to this criticism. In the present study, we will ask specifically about how critical students perceive their mother and father to be and how sensitive they are to this criticism. The measure contains two single items, the first for perceived criticism and the second for perceived sensitivity to criticism, which are both rated on a scale from 1 (not at all critical/sensitive) to 10 (very critical/sensitive indeed). The PS/PC scale Ruminative Response Scale–short version The Ruminative Response Scale–short version is a 10- item, self-report questionnaire in which participants reflect upon the causes, meanings and consequences of their negative mood states. Participants are asked to rate how well the statements reflect what they generally do Perry et al. Trials (2015) 16:451 Page 11 of 17 of thwarted belongingness and perceived burdensome- ness. The scale was evaluated to have good internal consistency [86] and construct validity [87]. when feeling sad, blue or depressed on a 4-point Likert scale from 1 (almost never) to 4 (almost always). Higher scores indicate a greater degree of rumination about negative feelings and experiences. The short version has been shown to be a reliable measure of rumination that is less confounded by depression than the original ver- sion [78]. The scale has good construct validity [79] and convergent and discriminant validity [80]. It also has adequate internal consistency (α = 0.77 and 0.72 for the brooding and reflection subscales, respectively) and test- retest stability [78]. Personality Inventory for DSM-5 Brief Personality Inventory for DSM-5 Brief The Personality Inventory for DSM-5, Brief Form (PID- 5-BF), is a 25-item self-rated personality trait assess- ment scale developed by the American Psychiatric Association’s Personality Disorders Work Group [81]. It measures the five proposed DSM-5 personality trait do- mains, including negative affect, detachment, antagon- ism, disinhibition and psychoticism, with each domain composed of five items. The scale asks respondents to rate how well the item describes themselves generally on a 4-point scale from 0 (very false or often false) to 3 (very true or often true). Overall scores range from 0 to 75, with higher scores indicating greater personality dysfunction. The scale has demonstrated good conver- gent validity with other widely used personality mea- sures [82]. Further, though originally developed for adults, the PID-5 was also shown to be a reliable tool for measuring personality pathology within adolescent popu- lations, with good reliability reported for personality trait domains and strong construct validity [83]. Insomnia Severity Index The Insomnia Severity Index is a brief instrument with seven items that assess the nature, severity and impact of insomnia upon the responder. Questions assess both day- and night-time aspects of insomnia over the pre- ceding 2 weeks and the associated interference with nor- mal functioning. Items are rated using a 5-point Likert scale, from 0 (no problem) through to 4 (very severe problem). The scale has previously been shown to have excellent internal consistency [89] and good test-retest reliability, as well as face and content validity [90]. Reduced Morningness-Eveningness Questionnaire The Reduced Morningness-Eveningness Questionnaire (MEQr) is a self-report measure consisting of 5 ques- tions (reduced from the original version containing 19 items) designed to explore participants’ sleep and waking rhythms and habits. Known as a circadian questionnaire, the aim of the MEQr is to uncover the individual’s particular biological rhythm. The stability and external validity of the reduced questionnaire have been reported previously [91, 92]. Acquired Capability for Suicide Scale The Acquired Capability for Suicide Scale is a five-item self-report questionnaire that aims to measure an indi- vidual’s capability to enact lethal self-injury. Questions assess fear of death and the pain associated with dying. Items are rated on a scale from 0 (not at all like me) to 4 (very much like me), where higher scores are thought to be more indicative of one’s capacity for lethal self- injury. The scale has shown good convergent and dis- criminant validity [86] and high internal consistency in previous studies [88]. Interpersonal Needs Questionnaire The shortened measure of the original 25-item Interper- sonal Needs Questionnaire (INQ) contains 15 items designed to assess the extent to which participants feel like a burden on the people in their lives (i.e., perceived burdensomeness) and the extent to which they feel disconnected from others (i.e., thwarted belongingness). Statements are rated using a 7-point Likert scale in terms of how accurately they reflect participants’ recent feelings. After coding, higher scores reflect greater levels Page 12 of 17 Perry et al. Trials (2015) 16:451 has been shown to reliably predict outcome for bipolar disorder [93]. estimates ranging from 0.73 to 0.84 [97]. There is also some evidence to suggest that the AQoL is more sensitive than other health-related quality of life instruments to health states [98]. Acceptability of the intervention h At the post-intervention assessment, participants will be asked a range of questions designed to assess the accept- ability and usability of the intervention they completed. These questions will measure how understandable and useful the program was, whether participants perceived that it affected their behaviour and whether they would recommend the program to others. Participants will also be provided with a list of techniques or skills specific to the program they completed (SPARX-R or lifeSTYLE) and will be asked which of these skills they have tried since completing the program and how helpful they were. These questions are also based on those used in previous research [99]. Trimbos/iMTA questionnaire for Costs associated with Psychiatric Illness The Trimbos/iMTA questionnaire for Costs associated with Psychiatric Illness questionnaire was designed to assess the costs of psychiatric illness incurred within the health system, as well as the economic burden associated with production loss [94]. In the present study, partici- pants will be asked to recall details about their consulta- tions with health professionals that have occurred over the past 6 months, including clinic visits, appointments, telephone consultations and house calls. The self-report version of the questionnaire consists of 13 items, each re- quiring a yes-or-no answer and, if relevant, specification of the number of times each particular consultation occurred. Days out of role This brief questionnaire, administered at 6- and 18-month follow-up, consists of four questions assessing the number of days an individual is unable to attend to (or has some difficulty managing) normal activities (e.g., attending school, university or work) because of mental health problems. Questions were adapted from core items from the World Health Organization Disability Assessment Scale [95] to focus specifically on mental health. Assessment of Quality of Life in four dimensions Assessment of Quality of Life in four dimensions The Assessment of Quality of Life in four dimensions (AQoL-4D) [96] is a standardised, self-report instrument designed to measure health-related quality of life across five domains: illness, independent living, social relation- ships, physical senses and psychological well-being. Each domain consists of three items; however, the illness sub- scale is not used for scoring purposes and therefore will not be collected during this trial. Some minor language adaptations were made to better suit the adolescent population (e.g., examples of household tasks were chan- ged from ‘cleaning the house’ and ‘cooking’ to ‘cleaning your room’ and ‘helping with meals’). These adaptations were based on language in the AQoL-6D, a 20-item ado- lescent version of the same scale. The AQoL-4D has dem- onstrated sound psychometric properties, with reliability Process variables Expectation of success At the pre-intervention assessment, participants will be asked a number of questions to determine their expect- ation of success associated with the intervention they are about to complete. These questions will assess (1) beliefs about internet treatments in general and (2) pref- erence for trial condition. These questions are based on those used in previous research [99]. Cortisol and genetics Cortisol data can be collected via hair strands in class- rooms, and the procedure is non-invasive, painless, sim- ple and quick. Saliva samples will also be collected in classrooms using standardised protocols. These data will be used for genetic analysis. Both types of biological data will be collected from students enrolled in the 2016 cohort, pending ethical approval. Adherence to the intervention Participants’ adherence to their allocated intervention will be assessed using automated data collection through the SPARX-R and lifeSTYLE programs. For each partici- pant, these data will include total number of visits to the site, average length of each visit and number of modules completed. Generalized Anxiety Disorder 7-item scale The Generalized Anxiety Disorder 7-item scale is a widely used self-report anxiety scale which consists of seven items designed to measure the severity of symptoms of general- ised anxiety disorder [103]. Participants rate individual statements on a 4-point frequency scale from 0 (not at all sure) to 3 (nearly every day) as they pertain to the previous 2 weeks. Individual item scores are summed to produce an overall score between 0 and 21, with higher scores indica- tive of greater anxiety. The scale was reported by its devel- opers to have good reliability in addition to strong criterion, construct, factorial and procedural validity [103]. Participant risk management protocol All student participants will be given a contact sheet with the name and contact details of their school counsellor, as well as contact information for other counselling and support services (local and online). In addition, on each assessment occasion, participants’ responses will be monitored using an automated alert system that identifies severe mental health concerns, defined as endorsement of one or more items on the YRBS. Participants will be asked if they have experi- enced serious suicidal ideation or engaged in suicidal behaviour in the previous month. Endorsement of any item will trigger an alert whereby trial researchers will be notified immediately, prompting initiation of the study’s risk management protocol. The researcher will confidentially communicate the student’s name to the student’s school counsellor so that the counsellor can contact the student to offer immediate support. School counsellors of all participating schools will be briefed in advance about this risk management procedure. Students will also be provided with a popup alert via the online platform indicating that one or more of their responses suggest they may be distressed and encouraging them to seek support and/or use the con- tacts provided on their contact sheet. An email pro- viding the same message will also be sent to their nominated email account. Models for binary outcomes analogous to the primary analysis approach will be used to compare the preva- lence rates (MDD status) and other dichotomous out- comes between the two treatment arms at the endpoint and other occasions of measurement. Relative and abso- lute risk reduction, NNT (see [105]) and other relevant indices will be calculated for these outcomes and under- taken separately for participants who did not meet criteria for caseness at pre-test to assess the effect of the intervention on incidence. In analyses of scaled second- ary variables, we will use methods comparable to those of the primary analysis. Subsidiary complier and related analyses will estimate the efficacy of SPARX-R in partici- pants who completed modules from the SPARX-R pro- gram to produce a clinical impact (estimated to be four or more modules) [36]. In exploratory analyses, we will examine the effects of moderators and mediators of treatment (see [106]). Reasons for non-adherence Participants who are designated as non-completers of their allocated program will be asked to complete a brief questionnaire assessing their reasons for non-adherence to the program. These items are adapted from the Inter- net Intervention Adherence Measure [100], which was originally developed in the context of a paediatric enco- presis intervention. The current items are similar to the adapted version of this measure developed by Farrer and colleagues [101] for use with an internet-based interven- tion for depression. Farrer et al.’s version of this measure includes two additional categories of reasons for non- adherence which may be relevant to the present trial: (1) engagement issues and (2) disease-specific issues (per- taining to depression). Page 13 of 17 Perry et al. Trials (2015) 16:451 Patient Health Questionnaire-9 The Patient Health Questionnaire-9 (PHQ-9) is a self- administered nine-item depression screening and diagnos- tic tool based on DSM-IV criteria. The scale assesses the nine depression symptom criteria for frequency of occur- rence during the previous 2 weeks, with scores ranging from 0 (not at all) to 3 (nearly every day). The psychomet- ric quality of the PHQ-9 has been established [102]. Analysis Primary analyses will be undertaken on an intention to treat basis, including all participants as randomised, regardless of treatment actually received. Effectiveness of SPARX-R will be established using a planned contrast of change from pre-test to the post-intervention in the active compared with placebo condition on the MDI within a mixed-effects model repeated-measures analysis [104]. School will be included in analyses as a random effect to evaluate and accommodate clustering effects. Variables used in determining allocation balance will be evaluated and retained in analyses where they are signifi- cant or quasi-significant. An unconstrained variance–co- variance matrix will model within-individual dependencies. Transformation of scores, including categorisation, may be undertaken to meet distributional assumptions and accom- modate outliers. Contrasts comparing change on the MDI from pre-test to other occasions of measurement will be undertaken as secondary analyses. Parent outcomes Institute. Data collected routinely through the SPARX-R program will be passed to the online platform via an appli- cation programming interface. Data will be coded using a unique identification code. A list of emails associated with each identification code will be kept separately from the outcome database for the sole purpose of matching data to an individual participant in case of risk. Data will be stored securely on the university server, and only appro- priate members of the TriPoD research team will have access to data collected in the context of this trial. All research personnel not involved with the day-to-day man- agement of the trial will remain blinded to intervention allocation. Discussion In the present trial, we will investigate the effectiveness of the SPARX-R intervention in the prevention of de- pression in young people. To our knowledge, this is the first trial of a universal prevention intervention delivered to school students in advance of a specific, significant exogenous stressor. Should it be shown to be effective, SPARX-R may offer an accessible, cost-effective, scalable and youth-friendly option for delivering a wide-scale prevention program in schools. Additional file Additional file 1: Completed SPIRIT 2013 checklist of recommended items to address in a clinical trial protocol and related documents. (DOC 121 kb) Data collection and management Interim analysis of outcomes of the 2015 cohort will be undertaken following the methods described above. Although the sample available will be of limited size, the All self-report data for the present trial will be collected via the online platform designed for and by the Black Dog Perry et al. Trials (2015) 16:451 Page 14 of 17 Page 14 of 17 universal intervention within a selective (high-risk) sam- ple. Nevertheless, we intend to continue this study in non-selective schools following the initial stage of the trial, which will allow for comparison across different school types and students. outcome of this analysis will be used to guide the second stage of the trial, planned for 2016. Should there be clear evidence of lack of effect, based point estimates of effect- iveness and associated confidence intervals, consideration may be given to abandoning second-stage recruitment or seeking possible modifications of the intervention. Dissemination h l Twenty-three selective or partially selective high schools were approached to participate in the trial between August and October, 2014. Fourteen of the schools agreed to participate; however, four schools subsequently withdrew from the study. The final sample for the 2015 cohort consisted of two partially selective and eight selective government secondary schools. The 2015 co- hort of schools completed their baseline assessments in February, 2015, followed by their respective online pro- grams. All post-intervention assessments were com- pleted by April, 2015 and six-month follow-up data was collected in August, 2015. Educational outcomes (HSC results) and 18 month follow-up data will be collected in 2016 for this cohort. This trial is registered with the Australian New Zealand Clinical Trials Registry (ACTRN12614000316606). All protocol amendments will be listed on this registry. A lay-language summary of key findings will be provided to participating schools at the conclusion of the trial, and results will be disseminated to the scientific commu- nity via publications and conference presentations. This trial will be reported in accordance with the World Health Organisation Statement on Public Disclosure of Clinical Trial Results [107]. No restrictions have been imposed on the dissemination of the data by funders or any other party. Competing interests Th ll l The focus on selective high schools in the first stage of this study has both advantages and limitations. In terms of advantages, the nature of the sample ensures that we are delivering the intervention to young people who may be particularly at risk of developing mental health diffi- culties caused by increased rates of stress, achievement orientation and perfectionism [47–49]. In contrast, this may limit the generalizability of our findings, as selective schools provide a unique sample of youth. Indeed, one may argue that the first year of the trial is in fact a The intellectual property rights for SPARX are owned by the University of Auckland. In the event of commercialisation, SM, TF and KS could derive financial benefit from its operation. The other authors declare that they have no competing interests. The intellectual property rights for SPARX are owned by the University of Auckland. In the event of commercialisation, SM, TF and KS could derive financial benefit from its operation. The other authors declare that they have no competing interests. Abbreviations ACSS: Acquired Capability for Suicide Scale; AQoL: Assessment of Quality of Life; ATAR: Australian Tertiary Admission Rank; CBT: Cognitive behavioural therapy; DSM-IV: Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition; DSS-P: Depression Stigma Scale–Personal Stigma subscale; GAD- 7: Generalized Anxiety Disorder 7-item scale; HPLS: Hopelessness Scale for Children; HSC: Higher School Certificate; ICC: Intraclass correlation coefficient; ICD-10: International Classification of Diseases, 10th revision; IIAM: Internet Intervention Adherence Measure; INQ: Interpersonal Needs Questionnaire; IPT: Interpersonal Psychotherapy; ISI: Insomnia Severity Index; MDD: Major Depressive Disorder ; MDI: Major Depression Inventory; MEQr Reduced Morningness-Eveningness Questionnaire NNT: Number needed to treat; PS/ PC: Perceived Sensitivity/Perceived Criticism scale; PHQ-9: Patient Health Questionnaire-9; PID-5-BF: Personality Inventory for DSM-5, Brief Form; PMS: Pearlin Mastery Scale; RRS: Ruminative Response Scale; SCAS: Spence Children’s Anxiety Scale; SSSS: Schuster Social Support Scale; TiC-P: Trimbos/ iMTA questionnaire for Costs associated with Psychiatric illness; TriPoD: Trial for the Prevention of Depression; YRBS: Youth Risk Behavior Survey. Although there is evidence to support the use of SPARX as a treatment for depression, its use as a preventive inter- vention has not yet been evaluated. Further, although previous qualitative studies have found the intervention to be feasible and acceptable in other samples (including students in alternative education and youth in rural Australia), the feasibility of this program in a school con- text and, in particular, with academically selective students is not known. In addition to acceptability information collected from students, staff feedback questionnaires will be provided to teachers who helped facilitate the trial to determine their perspectives on the feasibility of the intervention. References Am J Psychiatry. 2000;157(2):229–33. 32. Lillevoll KR, Vangberg HC, Griffiths KM, Waterloo K, Eisemann MR. Uptake and adherence of a self-directed internet-based mental health intervention with tailored e-mail reminders in senior high schools in Norway. 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Building a Brand: A Social Interaction Perspective

International Journal For Multidisciplinary Research

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Introduction Brand building is a much debated topic. But many are still unsure as to exactly what are the different stages of this process, especially when it comes to practice. Most of this uncertainty can be ascribed to two factors. The first one is that not all are clear as to what exactly is a brand. As Kapferer (2004) puts it, each expert comes up with his or her own definition of brand or nuances of definition. The second reason is that this process - like most effects of communication - is something that happens in the mind of the target audience and hence is likely to be intangible and difficult to measure. This paper takes up the former factor first. Abstract There is a proliferation of brands in the country. Companies after companies are launching products, trying to build them into brands, some succeeding, and some coming up short. It is a topic of hot discussion in all academic and even industrial circles. But there is still a gap when it comes to the proper understanding of the term brand as well as brand building. Different people perceive it in different ways. Consequently what are all the steps which forms the process of brand building still remains under debate. In this article we go through different literature to discuss what exactly is meant by branding and also the different stages of the brand building process based on a social interaction perspective. Keywords: Marketing, Brand Building, Advertising, Social Interaction Building a Brand: A Social Interaction Perspective Nimal C N1, Manju M Mathew2 1Associate Professor, Department of Management Studies, Adi Shankara Institute of Engineering & Technology, Kalady. 2Assistant Professor, Department of Management Studies, Adi Shankara Institute of Engineering & Technology, Kalady Brand as a Mark It has another clear, well-known, established definition: "a name, term, design, symbol, or any other feature that identifies one seller's good or service as distinct from those of other sellers" (Bennett 1988). This definition is the official one of the American Marketing Association (2007) and is found in virtually every leading introductory marketing textbook (Kotler et al, 2005, McCarthy et al 1984, Schoell et al 1992, Aaker, 1991) with minor variations. But there is a problem with such definitions. It only reaches up to a certain extent and no further. These describe the brand as an identity and even as a legal instrument. Brand is a legal statement of ownership (Crainer, 1985). Oxford dictionary (2009) has defined brand as “a particular sort or class of goods, as indicated by the trade mark on them”. An identity system aids as a memory shortcut and reduces risk. Developing an identity not only differentiates and protects against competitors, but also enables a firm to gain economic advantage (Fomburn and Shanly, 1990). If the above definitions are the ultimate then why are those products that are sold at the roadside pavements and the local Mandis or Sandhais called unbranded products? Examined closely these sometimes have beautiful packaging, with names like Pepsi Baniyans or Reliance Watches. Many of them have logo units, mascots and other identity elements that will make a seasoned graphic designer proud. These products can be easily identified and differentiated from the others of the same ilk. So there is more to a brand than those factors mentioned above. Brand as an Image The brand alternatively has been defined as an image in the consumer’s mind (Martineau, 1959). This image includes both its functional and psychological attributes. Further the brand image is explained as everything people associate with a brand (Newman, 1957). Another way to define a brand is that a brand is a consumer’s idea of a product (Pitcher, 1985). Brand as a Personality Many researchers have defined brand as a symbolic personality that users value beyond functional utility (Blackston, 1992; Arnold, 1992; Goodyear, 1993). While choosing among competing brands, consumers assess the fit between the personalities (perceived) of the brands and the personality they wish to project (Zinkhan et al.,1996), maybe consciously or subconsciously. Personality and values are inter-related (Gutman,1982), with personality being a sub part of value sets. Brand personality could primarily be the net result of the firm’s communication, whilst image is the way consumers perceive the brands personality (Plummer, 1985). Personality is a prerequisite for a relationship between consumers and brands (Duboff, 1986; Woodward, 1991). Brand: Definitions A brand has been defined in a number of ways. The origin of the word can be seen from the definition “To burn a distinctive mark into or upon with a hot iron, to indicate quality, ownership, etc., or to mark as infamous (as a convict).” (Webster’s 1913 Dictionary). Ancient Mesopotamian and Greek cultures made considerable use of symbols and names to identify or designate their offerings, which were mostly ointments, pots, wines, or metals. (Sarkar and Singh, 2005). In the US, cattle used to be similarly marked so that it could be distinguished from other people's herds if it became mixed up. (Ward, 2003). The oldest generic brand in continuous use in India is the herbal medicine known as Chyawanprash, which is consumed for its alleged health benefits and credited to the revered Rishi (seer) named Chyawan (Mahabharata). Volume 5, Issue 4, July-August 2023 IJFMR23045228 1 Brand Building Considering the above definition of the brand how can a brand be built? What are the steps in brand building? There are different approaches towards brand building. According to Kapferer, (1998, p. 46 (7), branding building is a holistic process. Giving a product or service a name and indicating to the outside world that it has been inscribed with an organization's logo and identity do not constitute branding. The deliberate strategy of market segmentation and product differentiation forms the basis of brands. Aaker (2002) enumerates a six step process of brand building. According to him exposure to different promotion first creates an awareness about the brand. Next the consumer acquires knowledge about the brand attributes and benefits. This in turn develops a perception of the brand, which later converts to emotion and feelings towards the brand. Linking with peers and group norms follows, and further by another reminder or incentive for brand trial. Social Interaction Perspective In this paper we look at brand building based on a social interaction perspective. So what is social interaction theory? Social interaction theory is concerned about how people interact in social settings. This theory emphasizes the importance of face-to-face interactions and the role of symbols, language, and gestures in shaping human behavior and society (Mead, 1934). This knowledge of social interaction can be crucial when communicating with your potential or current customers. And further branding. This theory can be applied to any social network, not just social media—any platform that connects people. From the literature it was found that the brand building process based on the social interaction perspective could be summarized into three important stages. Brand as a Composite Summarizing the above definitions a brand ‘is the feelings, images or ideas that come to mind on seeing or hearing the name, term, sign or symbol which identifies a seller’s good or service as different from those of competition.’ It is the emotional pay-off. The motivational button that catalyses the brand decision in the user’s heart and mind. In the earlier definition AVT is the brand, as different from Brooke Bond or Tata Tea but in this definition it also includes the perception of strength and consistent taste of the tea, the pictures of the rolling tea estates of Kerala, the white colour pouch pack of AVT Premium, the Shop Boards with the AVT name across South India, all images, cues or feelings that come to mind in association with the name AVT. Brand as Relationships A brand relationship is a logical extension of the brand personality (Blackston, 1992) with the consumers having a relationship with them if a brand can be personified. (Kapferer, 1992). Consumers' decisions are influenced by personal and cultural values (Franzen et al., 2008). Clark (1987) remarks consumers find value in the brand, in its heritage, in their personal experience with it and how it reflects what the individual stands for. Sheth et al. (1991) define brands as value systems. In sum, building a brand implies something intangible in the mental sphere. And hence more nebulous. Volume 5, Issue 4, July-August 2023 IJFMR23045228 2 rnational Journal for Multidisciplinary Research (IJFMR) E-ISSN: 2582-2160 ● Website: www.ijfmr.com ● Email: editor@ijfm Similarly the first step in the brand building process is making people aware of the brand. This involves getting the people to remember the brand name then associate the name with the product. For this the choice of the name is very important. It has to be easy to pronounce and easy to remember. Research says a three syllable word is best on both counts. ITC is easier on the tongue and ear than Indian Tobacco Company. Ideally the word should have some connection with the product in terms of the features or the usage of the product. Good Knight and Snuggies are two examples that prove the point. But having something like tobacco in the name is restricting the option of launching products in entirely independent categories. If it is a word that is there in the dictionary there is nothing like it as there won’t be any misunderstandings. The logo unit and the letter style also goes a long way in the identification process. The logo or emblem is a symbol which denotes that particular brand or company. It should be simple and can represent something that the company stands for as that would give greater remembrance. The logo unit includes a particular font type or way in which the name is written along with the logo. The font type used will depend on the kind of product. It would usually be serious for a product like a bank, frivolous for products for kids or state-of-the-art for a beauty product. Should be unique but at the same time readable. Any and all communication should ideally use the same font, many companies go to the extent of keeping similar layouts and fonts. The house colour like the red and blue of Pepsi makes the communication memorable and help the brand stand out in a crowd. Some companies use a mascot, like the Amul Baby which helps them establish a clear connect with their target audience. Once these which are called the identity factors are set all kinds of awareness media are used to communicate the same. With continuous bombardment from all quarters including POPs like posters, danglers, stickers and streamers and outdoor media like glow signs, shop boards, hoardings, wall paintings, bus shelters or on telephone poles this process doesn’t take much time. rnational Journal for Multidisciplinary Research (IJFMR) Companies may use auto tops or the backs of buses also to create awareness. Mainline advertising in TV, newspapers and magazines is good at creating reach but not cost effective as there is usually a lot of spillover. In the brand life cycle it starts from the introductory stage and extends a fair way into the growth stage as well. Stage 1 Creating a Recognition A product can be a physical good, service, place, institution, person or idea as per Kotler (2005). These in turn can also become brands. So a person like Amitabh Bachan or Narayana Murthy can be built up into a brand just like a product or service based brand. Conversely how a brand is built imitates the way social relationships develop. How does social relationships develop? The first and may be the only thing someone does when he or she meets somebody for the first time and get introduced to that person is remember their name. Still he or she is able to identify the person at the next encounter. According to Levinger (1983) this phase in relationship development is called Acquaintanceship. However Knapp (1978) terms this initiating where individuals make their first impression on each other. Volume 5, Issue 4, July-August 2023 IJFMR23045228 3 3 rnational Journal for Multidisciplinary Research (IJFMR) E-ISSN: 2582-2160 ● Website: www.ijfmr.com while both parties look for areas of shared experience or interest. This is the build-up stage, according to Levinger (1983). At this point, the relationship genuinely starts to develop. People begin to trust one another and stop being strangers. As opposed to people from diverse backgrounds and distinct ambitions, those with similar interests and backgrounds tend to get along better. Each person starts imputing certain character and personality to the other. Similarly each touch point adds up to certain images that you have of the brand. The baseline or tagline of the brand is a positioning statement. To stand out from the crowd the brand differentiates itself from others based on the product or some other features. This then is burned into the minds of the target audience as per Ries et al (1993). For example, while Dunlop has throughout been Dunlop and always ahead, MRF has been the tyre with muscle. These then become the core values of the brand in terms of usage, benefits or features associated with it. This also in turns produces some images, feelings and ideas in the minds of the consumers or target audience. And just like in the case of people start imputing personality to the brand. Stage 3 Appearance of the Connect The constant inundation with the same message from different touch points and the resultant imputing certain character to the brand result in feelings towards it, positive or negative. These feelings of the customer create brand liking and the much vaunted brand loyalty. According to Levinger (1983), this marks the conclusion of the second stage of the relationship's growth or "build up stage." He asserts that this phase of a relationship is frequently characterised by two people being intimate, passionate, and feeling for each other. This stage is referred to as intensifying by Knapp (1978). The two people will carry out more experimentation at this stage to see if there is emotional attachment and affection shared by both. While this may be conscious or sub conscious in the case of people in the case of brands the development of the positive or negative feeling by the customer is more at the sub conscious or unconscious level. They also bring certain ideas and images to the mind when you hear the particular name spoken. When you hear the name Tatas you immediately think of something credible, dignified, traditional, sober and gentlemanly while a Pepsi brings with it an image of youth, enthusiastic, but impertinent, slightly irrational and irresponsible. These in turn produces feelings of warmth and trust in one case and vigour and vitality in the other. These feelings give you an edge whenever the customer thinks about buying that particular product. Brand liking ensures that the customer will still think positively of the brand even when things go wrong once or twice. Once the awareness, association and brand liking is established these have to be carefully maintained over the years. Any dissonance will result in confusion in the minds of the customer and affects the brand negatively. Ramifications of the study Ramifications of the study The model of brand building based on the stages can be simply outlined as below. This simple model should help the marketer with a simple architecture for building his brand. In case of big companies there are a number of people and resources whose main concern is building the brand. But in the case of smaller entities they have limited resources and either the marketing manager or the Recognition Associations Connect The model of brand building based on the stages can be simply outlined as below. Stage 2 Developing an Association In a social situation after one or two meetings with a person you are able not only to identify him but differentiate him from another similar person or person with similar sounding name. According to Knapp (1978), experimenting is the second phase in the formation of a social relationship. The phase of experimentation is when people start talking about themselves in order to get to know one another. Details are collected from mutual friends and acquaintances as well as family members. Any published material about the person is also perused with interest. In case of brand a customer comes in contact with it in many different ways. Each of these points of contact is called a touch point. Neslin et al (2006) defines touch points as a customer contact point or a medium through which the firm and the customer interact. Many of these are discrete encounters. A touch point need not create a reciprocal interaction as in a TV or press advertisement. They may not even get into contact directly as in word of mouth. A customer using a product and gaining or not gaining a satisfaction out of it is also an encounter that is a touch point. Knapp (1978) goes on to say that during this phase, the participants explore and obtain a sense of the relationship as well as one another. In this phase, communication often takes the shape of small talk IJFMR23045228 Volume 5, Issue 4, July-August 2023 IJFMR23045228 4 4 References: 1. Aaker, David (1991). Managing Brand Equity: Capitalizing on the Value of a Brand Name. New York: The Free Press. 1. Aaker, David (1991). Managing Brand Equity: Capitalizing on the Value of a Brand Name. New York: The Free Press. 2. Aaker, David. (2002) A, Building Strong Brands, Simon & Schuster UK Ltd., London. 3. American Marketing Association, AMA (2007). Definition of Brand, AMA Dictionary Retrieved from http://www.marketingpower.com/_layouts/Dictionary.aspx?dLetter=B 3. American Marketing Association, AMA (2007). Definition of Brand, AMA Dictionary Retrieved from http://www.marketingpower.com/_layouts/Dictionary.aspx?dLetter=B 4. Arnold, David (1992). The handbook of brand management. Century Business, The Economist Books. 4. Arnold, David (1992). The handbook of brand management. Century Business, The Economist Books. 5. 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Conclusion Thus building a brand is a focused and integrated process that goes through three distinct stages although they are overlapping to some extent. It requires patience and determination to stick to the processes as well as come out with the same idea in your communication again and again. Always the tendency is there to do things in a different way, and deviate from the well-worn path, especially when it comes to the creative souls in the advertising agencies. But if it can be done it will be well worth the effort. International Journal for Multidisciplinary Research (IJFMR) E-ISSN: 2582-2160 ● Website: www.ijfmr.com ● Email: [email protected] owner himself has to take care of the branding among numerous other equally important and maybe more urgent matters. So if a checklist can be created out of this model with the activities at each stage delineated rather than mentioning abstract concepts and vague dos and don’ts it will be useful for the smaller organisations especially MSMEs who are strapped for fund, personnel and other resources. Connect This simple model should help the marketer with a simple architecture for building his brand. In case of big companies there are a number of people and resources whose main concern is building the brand. But in the case of smaller entities they have limited resources and either the marketing manager or the Volume 5, Issue 4, July-August 2023 IJFMR23045228 5 References: Gaski, Some Troublesome Definitions of Elementary Marketing Concepts--Have You Ever Looked at It This Way? in 1995 AMA Winter Educators' Conference: Marketing Theory and Applications, Vol. 6, D.W. Stewart and N.J. Vilcassim, eds. (Chicago: American Marketing Association, 1995). Volume 5, Issue 4, July-August 2023 IJFMR23045228 6 E-ISSN: 2582-2160 ● Website: www.ijfmr.com ● Email: [email protected] 15. Kapferer, Jean-Noel (1992). Strategic Brand Management, Kogan Page, London. 16. Kapferer, J. N. (1995). Brand confusion: empirical study of a legal concept. Psychology & Marketing, 12 (3), pp.551-68. 17. Kapferer, J.N. (1998): Strategic Brand Management, 2nd edition, Kogan Page Ltd., London 18. Kapferer, J.N. (2004). The New Strategic Brand Management: Creatingand Sustaining Brand Equity Long Term. Kogan Page, London 19. Knapp, M. L. (1978). Social Intercourse: From Greeting to Goodbye. Allyn and Ba L. (1978). Social Intercourse: From Greeting to Goodbye. Allyn and Bacon. 20. Kotler, Philip, Wong, Veronica, Saunders, John and Armstrong, Gary (2005), Pearson Prentice Hall, Fourth European Edition. 21. Krishna-Dwaipayana Vyasa, Mahabharata, Vana Parva, Sections 122–125, Gutenberg Press, 2013. 22. Levinger G (1983). Development and change. In Kelly HH (ed.). Close relationships. New York: W.H. Freeman and Company. pp. 315–359. 23. Martineau, Pierre (1959). Sharper focus for the corporate image. Harvard Business Review, 3S, 1, pp.49-58. 24. McCarthy, Jerome E, Perreault, William D, Jr. Basic Marketing: A Managerial Approach, Irwin Series in Marketing, 1984. 25. Mead, G.H. (1934). Mind, Self, and Society from the Standpoint of a Social Behaviorist. University of Chicago Press: Chicago. 26. Neslin, Scott A., Dhruv Grewal, Robert Leghorn, Venkatesh Shankar, Marije L. Teerling, Jacquelyn S. Thomas and Peter C. Verhoef (2006), “Challenges and Opportunities in Multichannel Customer Management,” Journal of Service Research, 9 (2), 95–112. 27. Newman, Joseph W. (1957). Motivation research and marketing management. Norwood: The Plimpton Press 28. Pitcher, AE. (1985). The role of branding in International advertising. International Journal of Advertising, 4, pp. 241-246. 29. Plummer J T (1985). How personality makes a difference. Journal of Advertising Research. 24 (6):27-31. 30. Ries, Al, Trout, Jack, 22 Immutable Laws of Marketing, 1st Edition, Harper Business, 1993 31. Schoell, William, F, Guiltinan, Joseph, E, Marketing: Contemporary Concepts and Practices, Allyn & Bacon, 1992. 32. Sheth, J., Newman, B. and Gross, B. (1991). Why we buy, what we buy: a theory of consumption values. Journal of Business Research, 22, pp. 159-70. 33. Ward, Faye E, The Cowboy at Work, Dover Publications Inc, New York, 2003 34. Wells, William, Burnett, John, Moriarty, Sandra, Advertising – Principles and Practice, 5th Edition, Prentice Hall India, New Delhi, 2002. 35. Woodward, Stephen (1991). Competitive Marketing. In Cowley, Don. ed. Understanding Brands by 10 people who do. Kogan Page, London. pp. 119-134. 36. Zinkhan, G., Haytko, D. and Ward, A. (1996). Self-concept theory. E-ISSN: 2582-2160 ● Website: www.ijfmr.com ● Email: [email protected] Journal of Marketing Communication, 2(1), pp. 1-19. Volume 5, Issue 4, July-August 2023 IJFMR23045228 7

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Supplementary Figure 1 from HER2 Isoforms Uniquely Program Intratumor Heterogeneity and Predetermine Breast Cancer Trajectories During the Occult Tumorigenic Phase

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Supplementary File 4. MaXFISH coregistry of sequenced tumors. A portion of tumors used for scRNAseq were MaXFISH coregistered (top) with H&E (middle) and IHC for Krt14 (bottom) on serial sections. All scale bars are 1mm. 0393-2 0393-4 TU1 TU4 TU3 TU2 0165B 0167B MT1 MT2 MT3 MT4 Supplementary File 4. MaXFISH coregistry of sequenced tumors. A portion of tumors used for scRNAseq were MaXFISH coregistered (top) with H&E (middle) and IHC for Krt14 (bottom) on serial sections. All scale bars are 1mm. 0393-2 0393-4 TU1 TU4 TU3 TU2 0165B 0167B MT1 MT2 MT3 MT4 0167B 0165B 0393-4 Supplementary File 4. MaXFISH coregistry of sequenced tumors. A portion of tumors used for scRNAseq were MaXFISH coregistered (top) with H&E (middle) and IHC for Krt14 (bottom) on serial sections. All scale bars are 1mm.

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Contribution to the Siberian species of Ichneumon LINNAEUS (Hymenoptera, Ichneumonidae, Ichneumoninae)

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©Biologiezentrum Linz, Austria; download unter www.zobodat.at ©Biologiezentrum Linz, Austria; download unter www.zobodat.at Matthias RIEDEL A b s t r a c t : In this study, new distributional records from Siberia are presented for 42 Ichneumon species. Of these, six taxa are reported from the Eastern Palaearctic region for the first time: Ichneumon acuticornis THOMSON, 1896, Ichneumon exilicornis WESMAEL, 1857, Ichneumon fulvicornis GRAVENHORST, 1829, Ichneumon karpathica HEINRICH, 1951, Ichneumon minutorius DESVIGNES, 1856 and Ichneumon veressi (KISS, 1915). The females of 20 new species are described and illustrated: Ichneumon altaicurtulus nov.sp., Ichneumon berlovi nov.sp., Ichneumon brevipunctatus nov.sp., Ichneumon breviscopatus nov.sp., Ichneumon genator nov.sp., Ichneumon granulatus nov.sp., Ichneumon hakassiacus nov.sp., Ichneumon inquinatops nov.sp., Ichneumon lanceolator nov.sp., Ichneumon mandibulatus nov.sp., Ichneumon mesonotator nov.sp., Ichneumon nigrostigmaticus nov.sp., Ichneumon orientopodius nov.sp., Ichneumon paravafer nov.sp., Ichneumon paravulnerator nov.sp., Ichneumon pseudemancipatus nov.sp., Ichneumon rufolateralis nov.sp., Ichneumon sayanicus nov.sp., Ichneumon scopator nov.sp. and Ichneumon thyridiator nov.sp. One new synonym is established: Ichneumon jakovlevi KOKUJEV, 1904 is a new synonym of Ichneumon cessator MÜLLER, 1776. In addition, a determination key for the females is given for all known Siberian species of Ichneumon LINNAEUS. K e y w o r d s : Ichneumonidae, Ichneumon, new species, Siberia, Eastern Palaearctic region. Contribution to the Siberian species of Ichneumon LINNAEUS (Hymenoptera, Ichneumonidae, Ichneumoninae) Matthias RIEDEL 1510 The Siberian fauna of Ichneumon is by far less well studied. Several publications, mainly from the first half of the last century, had addressed species of the genus Ichneumon (WOLDSTEDT 1881, KOKUJEV 1904, 1913, 1927, ROMAN 1914, 1927, MEYER 1930, UCHIDA 1926, 1935, HEINRICH 1931). More recently, HEINRICH (1978, 1980) has described several new Ichneumon species from the Eastern Palaearctic region improving our knowledge of this difficult genus. KOKUJEV´s Siberian species were revised by RASNITSYN (1984), the material described by Roman and Heinrich was included in the revision of HILPERT (1992). Since I was not able to verify the material mentioned from Siberia by WOLDSTEDT, MEYER and UCHIDA yet, some of their citations of Siberian species which are included in the key might be incorrect in the light of the modern revisions. In the last years, I could study some Siberian Ichneumon material from my own collec- tion (coll. RIEDEL) as well as from the Zoological Staatssammlung München (ZSM) in Munich/Germany, the Natural History Museum (NHM) in London/Great Britain and the Senckenberg Deutsches Entomologisches Institut (SDEI) in Müncheberg/Germany. The material contains many species of the genus Ichneumon which are described as new here. However, due to the limited material, one can still expect a large number of Eastern Palaearctic species remaining undescribed yet. Material and Methods For this study, only females were used for determination and description since a correct correlation of sexes was rarely possible with the available material (see Introduction). For the descriptions below, the nomenclature proposed by HEINRICH (1966-1968) for body structures of the Ichneumoninae was used except that the area posteromedia (of Heinrich) is named area petiolaris here. The distributional records were taken from the revision of HILPERT (1992) and the catalogue of YU et al. (2016). For the measurements the following relations were used: Length of 1st flagellomere was measured in lateral view (length without anellus); width of gena and eye were measured from lateral; length and width of hind femur in lateral view. For the punctuation of body parts the following definitions were used: very scattered – distance of punctures >2x their diameter; scattered – distance 1-2x their diameter; rather dense - distance about as their diameter; dense – diameter of punctures larger than their distance. The following index was applied: OOD - distance of lateral ocellus to eye in relation to its diameter. For the measurements an Olympus SZX 7 stereo microscope with dividing eyepiece was used. The figures were taken with an Olympus SC 30 CCD-camera using the AnalySIS getIT and Helicon Focus Pro softwares and processed with the Microsoft Office Picture manager. Ichneumon acuticornis THOMSON, 1896 M a t e r i a l e x a m i n e d : East Siberia, Baikal lake, 10 km southwest of Vydrino, Snezlinaya river valley, 1♀ 6-10.viii.2001, leg. Berlov (coll. Riedel). Introduction The genus Ichneumon LINNAEUS is a very large genus of the Hymenopteran family Ichneumonidae, subfamily Ichneumoninae which are all known as parasitoids of differ- ent Lepidoptera. Although the distribution of this genus is worldwide (YU et al. 2016), the highest diversity of this genus seems to be found in the Holarctic regions. Due to a strong sexual dimorphism, a correlation of both sexes is often difficult and sometimes questionable in this genus and could only be solved by further rearing studies and/or molecular analysis in many cases. Ther Western Palaearctic species of Ichneumon have recently been revised and described by HILPERT (1992) who was able to include several Eastern Palaearctic species in his monographic revision. HILPERT accepted 126 valid Ichneumon species from Europe which have been described as females - together with some taxa where only males were described so far. 1510 Ichneumon altaicurtulus nov.sp. (figs 19, 63) T y p e m a t e r i a l : Holotype: ♀ "12-15 VII 2001, 2000 m, Nura vill., NE Altai Mt. rng., S Kirgizstan, leg. Osipov" (coll. Riedel). T y p e m a t e r i a l : Holotype: ♀ "12-15 VII 2001, 2000 m, Nura vill., NE Altai Mt. rng., S Kirgizstan, leg. Osipov" (coll. Riedel). D e s c r i p t i o n : Body length 11 mm. Antenna slightly lanceolate, with 33 flagello- meres; 1st flagellomere 1.8x longer than wide, 7th flagellomere square, widest flagello- meres c.0.6x as long as wide; preapical flagellomere 0.75x as long as wide. Temple slightly and roundly narrowed behind eye. OOD 1.5. Gena 1.0x as wide as eye, with scattered punctures in ventral half. Malar space c.1.0x as long as width of mandibular base and 0.8x as long as 1st flagellomere. Genal carina complete, not interrupted ventral- ly. Hypostomal carina slightly elevated. Mesosoma covered with brownish hairs. Mesoscutum densely punctate, lateral field with rather dense punctures centrally. Mesopleuron and metapleuron coarsely striate-punctate; coxal carina present. Scutellum flat, about as long as wide, with fine very scattered punc- tures. Area superomedia rectangular, about as long as wide; costula absent. Area petio- laris without lateral carina. Hind coxa densely punctate, without scopa. Hind femur 3.7x longer than wide, with scattered punctures basally and in ventral 1/3. Hind tibia without denticular spurs apico-externally. Tarsomeres slender, 3rd mid tarsomere 1.9x longer than wide. Metasoma strongly oxypygous, hypopygium short. Postpetiolus strongly widened, lateral field 0.7x as wide as the median one, median field with rather coarse regular aciculation. 2nd tergite 0.8x as long as wide. Gastrocoelus impressed, with several ridges; thyridium slightly oblique, c.0.8x as wide as the interval. 2nd and 3rd tergites densely punctate and finely rugose medially. Ovipositor sheath distinctly extending behind metasomal apex. Color: Black. Scapus, pedicel and flagellomeres 1-12 yellowish-red. Mandible except teeth, clypeus largely and facial and frontal orbits narrowly reddish. Collare, hind edge of pronotum and tegula red. Scutellum yellow. 2nd and 3rd tergites yellow, gastrocoelus reddish, 3rd tergite with wide basal (1/3 of its length) and narrow apical blackish bands. 5th to 7th tergite with very wide and large yellow stripes covering the whole dorsal area. 1511 D i s t r i b u t i o n : Known from the Western Palaearctic region, new record for the Eastern Palaearctic region. List of species M a t e r i a l e x a m i n e d : East Siberia, Baikal lake, 10 km southwest of Vydrino, Snezlinaya river valley, 1♀ 6-10.viii.2001, leg. Berlov (coll. Riedel). M a t e r i a l e x a m i n e d : East Siberia, Baikal lake, 10 km southwest of Vydrino, Snezlinaya river valley, 1♀ 6-10.viii.2001, leg. Berlov (coll. Riedel). 1511 Ichneumon berlovi nov.sp. (figs 1, 21, 45, 64) T y p e m a t e r i a l : Holotype: ♀ "Siberia, Irkutsk region, 5 km E Pivovarkha, 16 vii 2007, leg. Berlov" (coll. Riedel). T y p e m a t e r i a l : Holotype: ♀ "Siberia, Irkutsk region, 5 km E Pivovarkha, 16 vii 2007, leg. Berlov" (coll. Riedel). D e s c r i p t i o n : Body length 9.5 mm. Antenna filiform, with 32 flagellomeres; 1st flagellomere 2.4x longer than wide, 8th or 9th flagellomeres square, widest flagellomeres about square; preapical flagellomere 0.94x as long as wide. Temple distinctly and round- ly narrowed behind eye. OOD 1.4. Frons with superficial and dense punctures, granulate, ± opaque. Face and clypeus densely punctate and granulate. Gena 1.1x as wide as eye, with fine striation dorsally and rather dense punctures ventrally. Malar space 1.6x as long as width of mandibular base and 0.95x as long as 1st flagellomere. Genal carina com- plete, not interrupted ventrally. Hypostomal carina narrow. Mesosoma covered with brownish hairs. Mesoscutum densely punctate and granulate, opaque. Mesopleuron completely rugose-punctate (including speculum); metapleuron coarsely rugose-punctate; coxal carina present. Scutellum moderately elevated, about as long as wide, with rather dense punctures. Propodeum rather short. Area superomedia slightly elevated above surrounding areas, hexagonal, 1.35x wider than long; costula weakly present. Area petiolaris bordered by lateral rugae in apical 2/3. Hind coxa dense- ly punctate and granulate, without scopa. Hind femur 3.8x longer than wide, with very superficial punctures, granulate. Hind tibia with c.6 external denticular spurs. Tarsi not enlarged, 3rd mid tarsomere 1.6x longer than wide. Metasoma oxypygous; hypopygium short. Postpetiolus strongly widened, median field c.2x wider than the lateral one, with regular aciculation; lateral field rugose-striate. 2nd tergite 0.68x as long as wide. Gastrocoelus narrow, with fine ridges; thyridium transverse and very wide, 1.8x wider than the interval. 2nd and 3rd tergites very densely punctate and granulate, ± opaque; 2nd tergite with fine longitudinal rugosity medially. Ovipositor sheath slightly extending behind metasomal apex. Color: Black. Rings on flagellomeres 6-10 and roundish spots on 6th and 7th tergites ivory. Tegula and small central spot on scutellum reddish. Postpetiolus and 2nd tergite reddish, 3rd tergite reddish-brown. Coxae and trochanters black; legs otherwise yellowish-red; fore femur in basal 2/5 and mid and hind femora mainly dark brown; distal tarsomeres brownish apically. Wings hyaline, pterostigma yellowish. Ichneumon altaicurtulus nov.sp. (figs 19, 63) Coxae and trochanters black; legs otherwise reddish-yellow; hind femur black, its basal 1/3 and hind trochantellus red; hind tibia and tarsus yellow; apical 0.2 of hind tibia and tips of hind tarsomeres reddish. Wings with yellowish tint, pterostigma yellow. T a x o n o m i c a l r e m a r k : This species belongs to group G 1 sensu HILPERT (1992) and runs to I. caedator GRAVENHORST in his key. It differs by the slenderer hind femur and yellow hind tibia and hind tarsus with reddish tips. The new taxon also resem- bles Ichneumon curtulus KRIECHBAUMER and can be differentiated by its slenderer legs and different color pattern of 3rd tergite. However, this species might represent a subspecies of Ichneumon curtulus which have been previously reported from the Western Palaearctic region, Kazakhstan and Iran (YU et al. 2016). 1512 Ichneumon berlovi nov.sp. (figs 1, 21, 45, 64) T a x o n o m i c a l r e m a r k : This species belongs to group I of HILPERT (1992) and runs to I. memorator WESMAEL in his key. It is closely related to I. thyridiator nov.sp. and differs by the square central flagellomeres, wider area superomedia and red hind tibia and scutellum. T y p e m a t e r i a l : Holotype: ♀ "1-5 vii 2003, 2000 m, SW Tannu-Ola Mt reg., Sogly vill., SW Tuwa reg., S Siberia, leg. Vastchenko" (coll. Riedel). Ichneumon brevipunctatus nov.sp. (figs 2, 20, 22, 46, 39) T y p e m a t e r i a l : Holotype: ♀ "1-5 vii 2003, 2000 m, SW Tannu-Ola Mt reg., Sogly vill., SW Tuwa reg., S Siberia, leg. Vastchenko" (coll. Riedel). D e s c r i p t i o n : Body length 9 mm. Antenna almost filiform, with 34 flagello- meres; 1st flagellomere 1.3x longer than wide, c. 3rd flagellomere square, widest flagel- lomeres c.0.65x as long as wide; preapical flagellomere 0.7x as long as wide. Temple moderately and roundly narrowed behind eye. OOD 1.4. Frons rather densely punctate, smooth between punctures. Face and clypeus with scattered punctures, smooth between 1513 punctures. Gena wide, 1.3x as wide as eye, with very scattered punctures in ventral half. Malar space 1.0x as long as width of mandibular base and 1.7x as long as 1st flagello- mere. Genal carina complete, not interrupted ventrally. Hypostomal carina narrow. punctures. Gena wide, 1.3x as wide as eye, with very scattered punctures in ventral half. Malar space 1.0x as long as width of mandibular base and 1.7x as long as 1st flagello- mere. Genal carina complete, not interrupted ventrally. Hypostomal carina narrow. Mesosoma covered with brownish hairs. Mesoscutum rather densely punctate, lateral field centrally with an almost smooth area with only very scattered punctures. Mesopleu- ron coarsely punctate, in frontal half with scattered punctures; metapleuron densely rugose-punctate, partly striate in frontal third; coxal carina present. Scutellum slightly elevated, about as long as wide, with fine scattered punctures. Area superomedia rectan- gular, 1.2x longer than wide; costula absent. Area petiolaris without lateral border. Mid and hind coxae with very scattered punctures, almost completely smooth apico-ventrally, hind coxa without scopa. Hind femur 3.3x longer than wide, punctate and finely granu- late, with very scattered punctures basally and in ventral 1/2. Hind tibia with c.2 denticu- lar spurs apico-externally. Fore and mid tarsomeres moderately enlarged, 3rd mid tarso- mere c.1.3x longer than wide. Metasoma strongly oxypygous, hypopygium short. Postpetiolus strongly widened, medi- an field with very fine and superficial aciculation. 2nd tergite very wide, 0.62x as long as wide. Gastrocoelus impressed, with several ridges; thyridium small, slightly oblique, c.0.5x as wide as the interval. 2nd and 3rd tergites with fine dense punctures, 2nd tergite with longitudinal rugosity medially. Ovipositor sheath slightly extending behind meta- somal apex. Color: Black. Scapus and flagellomeres 1-12 red, pedicel blackish. Head completely black. Ichneumon brevipunctatus nov.sp. (figs 2, 20, 22, 46, 39) Mesosoma black; collare and tegula reddish. 2nd and 3rd tergites reddish-brown, with diffuse blackish suffusion; 6th tergite with small yellow apical spot, 7th tergite with narrow yellow stripe medially. Coxae and trochanters black; fore coxa frontally and fore trochanter apically reddish. Legs otherwise red; hind femur black, in basal 1/10 and apical 1/4 reddish. Wings with slightly yellowish tint, pterostigma yellow. T a x o n o m i c a l r e m a r k : The species belongs to group E 3 sensu HILPERT (1992) and runs to Ichneumon mordax KRIECHBAUMER in his key. It differs by widened fore and mid tarsomeres, black head without reddish color pattern, black scutellum and a different color pattern of metasoma. Ichneumon breviscopatus nov.sp. (figs 23, 40, 65) Ichneumon breviscopatus nov.sp. (figs 23, 40, 65) T y p e m a t e r i a l : Holotype: ♀ "Russia: Primorskiy kray, Samarka 70 km N Chuguyevka, 44.43 N 134.12 E, 30.v.1993, 200 m, leg. A. Taeger" (SDEI) D e s c r i p t i o n : Body length 15 mm. Antenna slightly lanceolate, with 41 flagello- meres; 1st flagellomere 1.9x longer than wide, 8th flagellomere square, widest flagello- meres 0.62x as long as wide; preapical flagellomere 0.8x as long as wide. Temple slight- ly and roundly narrowed behind eye. OOD 1.4. Occiput and frons densely and coarsely rugose-punctate. Face coarsely rugose-punctate. Clypeus with scattered punctures, smooth and shining between punctures. Gena widened ventrally, 1.2x as wide as eye, with rather dense punctures in ventral 1/2. Malar space 1.0x as long as width of mandibular base and 1.0x as long as 1st flagellomere. Genal carina complete ventrally; hypostomal carina narrow. Mesosoma covered with brownish hairs. Mesoscutum densely rugose-punctate and ± 1514 opaque. Mesopleuron with dense punctures, striate caudally. Metapleuron coarsely rugose-punctate; coxal carina present. Scutellum almost flat, wider than long, with rather dense punctures. Area superomedia hexagonal, about as long as wide; costula absent. Hind coxa coarsely and finely punctate, with small and slightly elevated scopa. Hind femur 3.7x longer than wide, densely punctate. Hind tibia with 1-2 denticular spurs apico-externally. Tarsomeres slender, 3rd mid tarsomere c.1.5x longer than wide. opaque. Mesopleuron with dense punctures, striate caudally. Metapleuron coarsely rugose-punctate; coxal carina present. Scutellum almost flat, wider than long, with rather dense punctures. Area superomedia hexagonal, about as long as wide; costula absent. Hind coxa coarsely and finely punctate, with small and slightly elevated scopa. Hind femur 3.7x longer than wide, densely punctate. Hind tibia with 1-2 denticular spurs apico-externally. Tarsomeres slender, 3rd mid tarsomere c.1.5x longer than wide. Metasoma oxypygous, hypopygium slightly elongate. Postpetiolus strongly widened, lateral field coarsely striate-punctate, c.0.5x as wide as the median one, median field with fine regular aciculation. 2nd tergite 0.82x as long as wide. Gastrocoelus moderately impressed, with ridges; thyridium slightly oblique, c.1.0x wider than the interval. 2nd and 3rd tergites very finely rugose-punctate, not distinctly striate medially. Ovipositor sheath slightly extending behind metasomal apex. Color: Black. Ring on flagellomeres 8-14 and round spots on 6th and 7th tergites ivory. Narrow frontal orbit and scutellum yellow. Ichneumon caedator GRAVENHORST, 1829 M a t e r i a l e x a m i n e d : Far East Russia, Primorskii Krai, Lazovski Zapovednik, c. 170 km east Vladivostok, Korpad, N 43°17.16´ E 134°07.09´, 506 m, 1♂ 14.vii.-4.viii.2001, Malaise trap 483, leg. M. Quest (NHM). D i s t r i b u t i o n : Palaearctic, known from Mongolia, new record for Far East Russia. D i s t r i b u t i o n : Palaearctic, known from Mongolia, new record for Far East Russia. Ichneumon breviscopatus nov.sp. (figs 23, 40, 65) 2nd and 3rd tergites red; 3rd tergite diffusely infuscate basally and apically. Legs black; fore and mid femora apically, fore and mid tibiae frontally and fore tarsus frontally reddish; hind tibia diffusely dark reddish medio- externally. Wings with slight yellowish tint, pterostigma yellow-red. T a x o n o m i c a l r e m a r k : The new species runs to group E 1 sensu Hilpert. It is closely related to Ichneumon tuberculipes WESMAEL which might also have the 2nd and 3rd tergites ± reddish, but differs by the narrow hypostomal carina, complete genal carina, densely punctate hind femur and shorter 1st flagellomere. Ichneumon caedator GRAVENHORST, 1829 Ichneumon confundor HEINRICH, 1978 M a t e r i a l e x a m i n e d : South Siberia, Southwestern Tannu-Ola mountain range, Sogl village, southwest Tuwa region, 2000 m, 1♀ 1-5.vii.2003, leg. Vastchenko (coll. Riedel). T a x o n o m i c a l r e m a r k : This specimen slightly differs from the description of the holotype (HEINRICH, 1978: 27-28) by the less extensive red coloration: clypeus, malar space and gena black; inner orbits narrowly red. Mesoscutum completely black, subtegular ridge black; 3rd tergite red, with black transverse bands in basal 1/5 and apical 1/5. Otherwise a typical specimen. D i s t r i b u t i o n : Southern Siberia and Far East Russia. D i s t r i b u t i o n : Southern Siberia and Far East Russia. Ichneumon exilicornis WESMAEL, 1857 M a t e r i a l e x a m i n e d : Southwest Siberia, Orenburg region, Ilek surrounds, 1♀ 1- 5.viii.2005, leg. Osipov (coll. Riedel). D i s t r i b u t i o n : Known from the Western Palaearctic region, new record for Siberia. 1515 yellow scutellum. I have not seen the type of Ichneumon jakovlevi KOKUJEV, but from the description given by RASNITSYN (1984: 794) who reported the slender basal flagel- lomere, this var. of I. cessator seems to be conspecific with Ichneumon jakovlevi KOKUJEV which had also been found in the Irkutsk area. yellow scutellum. I have not seen the type of Ichneumon jakovlevi KOKUJEV, but from the description given by RASNITSYN (1984: 794) who reported the slender basal flagel- lomere, this var. of I. cessator seems to be conspecific with Ichneumon jakovlevi KOKUJEV which had also been found in the Irkutsk area. D i s t r i b u t i o n : Palaearctic region, known from Siberia (KOKUJEV 1904, MEYER 1933). Ichneumon formosus GRAVENHORST, 1829 M a t e r i a l e x a m i n e d : South Siberia, East Sayan mountains, near Mondy village, 1400 m, 1♀ 4.vi.1983, leg. Berlov (coll. Riedel). D i s t r i b u t i o n : Palaearctic region, known from Siberia (WOLDSTEDT 1881). Ichneumon fulvicornis GRAVENHORST, 1829 M a t e r i a l e x a m i n e d : South Siberia, Southwest Tannu-Ola mountain range, Southwes Tuwa region, Sogly surrounds, 2000 m, 1♀ 1-5.vii.2003, leg. Vastchenko (coll. Riedel). T a x o n o m i c a l r e m a r k : Antenna with 32 flagellomeres, structure and colo otherwise as described for the European material (HILPERT 1992). D i s t r i b u t i o n : Known from the Western Palaearctic region, new for Siberia. Ichneumon extensorius LINNAEUS, 1758 M a t e r i a l e x a m i n e d : East Siberia, Lake Baikal near Anghasolka (overwintering), 35♀♀ 6.iv.2007, leg. Berlov (coll. Riedel). D i s t r i b u t i o n : Palaearctic region, known from Siberia (KOKUJEV 1904). Ichneumon cessator MÜLLER, 1776 syn. nov. Ichneumon jakovlevi KOKUJEV, 1904: 81 syn. nov. Ichneumon jakovlevi KOKUJEV, 1904: 81 1♀ M a t e r i a l e x a m i n e d : East Siberia, Irkutsk region, Shelehov environment, 1♀ 2.viii.2005, leg. A. Shavrin (coll. Riedel). D e s c r i p t i o n : Body length 14.5 mm. Antenna with 43 flagellomeres, bristle- shaped; 1st flagellomere 3.3x longer than wide, c.14th flagellomere square, widest flagel- lomeres 0.87x as long as wide, preapical flagellomere 1.15x longer than wide. Malar space 0.7x as long as 1st flagellomere. Area superomedia 0.75x as long as wide, widest apically. Hind femur 4.4x longer than wide, with scattered punctures in ventral 1/5. D e s c r i p t i o n : Body length 14.5 mm. Antenna with 43 flagellomeres, bristle- shaped; 1st flagellomere 3.3x longer than wide, c.14th flagellomere square, widest flagel- lomeres 0.87x as long as wide, preapical flagellomere 1.15x longer than wide. Malar space 0.7x as long as 1st flagellomere. Area superomedia 0.75x as long as wide, widest apically. Hind femur 4.4x longer than wide, with scattered punctures in ventral 1/5. Color: Black. Antenna with diffuse reddish-yellow ring on flagellomeres 8-12. Scutellum and stripes on 6th and 7th tergites cream-yellow. Coxae, trochanters and fore and mid trochantelli black; legs otherwise red; hind tibia infuscate in apical 1/3; hind tarsus blackish. Pterostigma reddish. T a x o n o m i c a l r e m a r k : The available specimen is structurally similar to European material of Ichneumon cessator MÜLLER. It differs only by the completely 1515 Ichneumon genator nov.sp. (figs 3, 24, 41, 47, 66) M a t e r i a l e x a m i n e d : Holotype: ♀ "1-5 vii 2003, 2000 m, SW Tannu-Ola Mt rng, Sogly vill, SW Tuwa reg., S Siberia, leg. Vastchenko" (coll. Riedel). Paratype: ♀ "E Siberia, lake Baikal, Baikalo-Lenskiy reg., Cordon Onkholoi, 19.vii.2005, leg. Berlov" (coll. Riedel). 1516 Males unknown. T a x o n o m i c a l r e m a r k : The new species runs to group G 4 sensu HILPERT. It is closely related to Ichneumon mandibulatus nov.sp., but can be differentiated by the widely interrupted genal carina, longer malar space, wider scutellum, stouter hind femur and larger number of denticular spurs on hind tibia. 1516 D e s c r i p t i o n : Body length 16 mm. Antenna stout and slightly lanceolate, with 36 flagellomeres; 1st flagellomere 1.6x longer than wide, 4th flagellomere square, widest flagellomeres 0.58x as long as wide; preapical flagellomere 0.65x as long as wide. Temple slightly and roundly narrowed behind eye. OOD 1.4. Occiput and frons densely and coarsely rugose-punctate, finely granulate. Central face with coarse transverse striae, face otherwise punctate, facial orbit with scattered punctures. Clypeus large and wide, with rather dense punctures, smooth between punctures. Mandible thick, with parallel sides or slightly widened apically, teeth blunt, lower tooth much smaller than the upper one. Gena strongly widened ventrally, 1.4x as wide as eye, with scattered punctures in ventral half. Malar space 1.4x as long as width of mandibular base and 1.5x as long as 1st flagellomere. Genal carina widely interrupted ventrally; hypostomal carina narrow. Mesosoma covered with brownish hairs. Mesoscutum densely rugose-punctate but shining. Mesopleuron and metapleuron coarsely striate-punctate; coxal carina present. Scutellum slightly elevated, 1.4x wider than long, with rather dense punctures. Area superomedia square; costula absent. Area petiolaris without lateral border. Hind coxa coarsely punctate, without scopa or ventro-internal edge (but with scattered punctures apico-ventrally in paratype). Hind femur 2.9-3.0x longer than wide, densely punctate. Hind tibia with c.12 denticular spurs externally, mainly in apical 1/2. Tarsomeres moder- ately widened, 3rd mid tarsomere c.1.35x longer than wide. Metasoma strongly oxypygous, hypopygium short. Postpetiolus strongly widened, lateral field punctate, c.0.6x as wide as the median one, both fields with rather strong regular aciculation. 2nd tergite 0.68x as long as wide. Gastrocoelus impressed, with ridges; thyridium slightly oblique, c.0.7x wider than the interval. 2nd and 3rd tergites very finely rugose-punctate, not distinctly striate medially. Ovipositor sheath slightly extending behind metasomal apex. Color: Black. Ring on flagellomeres 8/9-13/14, scutellum and small round spots on 6th and 7th tergites ivory. Mandible except teeth, side of clypeus and frontal orbit red; red spot on frontal orbit widened dorsally and reaching lateral ocellus. Collare and hind edge of pronotum, tegula, subtegular ridge and spot on prescutellar carina red. 2nd and 3rd tergites completely red or 3rd tergite with narrow black basal band (in paratype). Coxae, trochanters and femora black; tibiae and tarsi reddish; hind tibia darkened in apical ¼- 1/5, hind tarsomeres completely red or with darkened tips. Wings with slightly yellowish tint, pterostigma brown. Males unknown. M a t e r i a l e x a m i n e d : East Siberia, Lake Baikal near Anghasolka (overwintering), 10♀♀ 6.iv.2007, leg. Berlov (coll. Riedel). D i s t r i b u t i o n : Palaearctic region, known from Siberia (MEYER 1933). M a t e r i a l e x a m i n e d : East Siberia, Lake Baikal near Anghasolka (overwintering), 10♀♀ 6.iv.2007, leg. Berlov (coll. Riedel). D i s t r i b u t i o n : Palaearctic region, known from Siberia (MEYER 1933). Ichneumon granulatus nov.sp. (figs 4, 25, 48, 67) M a t e r i a l e x a m i n e d : Holotype: ♀ "Russia, S Siberia, Hakassia reg., W. Sagan mts, 2400 m, Sallyghem-Taiga reg., 5 vii 2004, leg. Osipov" (coll. Riedel). D e s c r i p t i o n : Body length 9.8 mm. Antenna slightly lanceolate, with 37 flagel- lomeres, 1st flagellomere 1.6x longer than wide, 7th flagellomere square, widest flagello- meres 0.63x as long as wide, preapical flagellomere 0.78x as long as wide. Temple moderately and roundly narrowed behind eye. Frons and face densely punctate and granulate. Clypeus with rather dense punctures. Gena 1.25x wider than long, with rather dense punctures ventrally. Malar space 1.3x longer than width of mandibular base and 1.3x longer than 1st flagellomere. Genal carina complete, hypostomal carina slightly elevated. Mesosoma covered with brownish hairs. Mesoscutum densely punctate, median field finely granulate. Mesopleuron and metapleuron densely punctate and distinctly granu- late; coxal carina present. Scutellum almost flat, slightly wider than long, with scattered punctures. Area superomedia hexagonal, 1.2x wider than long; costula weakly deve- loped, at least medially. Area petiolaris without lateral carina. Hind coxa coarsely punc- tate and granulate, with rather long brownish hairs, but without scopa. Hind femur 3.8x longer than wide, with scattered punctures in ventral third. Hind tibia with c.4-5 denticu- lar spurs apico-externally. Tarsomeres slender, 3rd mid tarsomere c.1.5x longer than wide. Metasoma strongly oxypygous, hypopygium short. Postpetiolus strongly widened, median field c.1.9x wider than the lateral one, with fine aciculation and some punctures, lateral field densely punctate. 2nd tergite 0.69x as long as wide. Gastrocoelus impressed, with ridges; thyridium slightly oblique, c.0.6x as wide as the interval. 2nd and 3rd tergites densely punctate and granulate, ± opaque, without longitudinal rugae medially. Oviposi- tor sheath distinctly extending behind metasomal apex. Color: Black. Scapus except narrow brownish base, pedicel and flagellomeres 1-14 red; flagellomeres 10-12 with a yellow stripe dorsally, following flagellomeres blackish. Head and mesosoma black; collare, tegula and scutellum reddish. 2nd and 3rd tergites red, 2nd tergite diffusely infuscate centrally, 3rd tergite with black band in apical third. 6th and 7th tergites with roundish ivory spots apically. All coxae and trochanters and fore and mid trochantelli black. Hind trochantellus and legs red. Hind femur with blackish stripe centrally-dorsally (half as long as the femur). Wings with slightly brownish tint. Pterostigma reddish. Males unknown. Ichneumon gracilentus WESMAEL, 1845 Ichneumon gracilentus WESMAEL, 1845 M a t e r i a l e x a m i n e d : East Siberia, Lake Baikal near Anghasolka (overwintering), 10♀♀ 6.iv.2007, leg. Berlov (coll. Riedel). D i s t r i b u t i o n : Palaearctic region, known from Siberia (MEYER 1933). D i s t r i b u t i o n : Palaearctic region, known from Siberia (MEYER 1933 1517 Ichneumon granulatus nov.sp. (figs 4, 25, 48, 67) T a x o n o m i c a l r e m a r k : This species belongs to the group G 4 of HILPERT (1992) and runs to I. pilulicornis HEINRICH in his key. It differs by the complete genal carina, slightly elevated hypostomal carina and wide malar space. M a t e r i a l e x a m i n e d : East Siberia, 10 km East of Irkutsk, 1♀ 25.ix.2004, leg. Berlov (coll. Riedel). Ichneumon haglundi HOLMGREN, 1864 M a t e r i a l e x a m i n e d : East Siberia, 10 km East of Irkutsk, 1♀ 25.ix.2004, leg. Berlov (coll. Riedel). D i s t r i b u t i o n : Palaearctic region, known from Siberia (ROMAN 1904). D i s t r i b u t i o n : Palaearctic region, known from Siberia (ROMAN 1904). Ichneumon hakassiacus nov.sp. (figs 5, 26, 49, 68) Ichneumon hakassiacus nov.sp. (figs 5, 26, 49, 68) M a t e r i a l e x a m i n e d : Holotype: ♀ "Russia, S Siberia, Hakkasia, W Sayan Mts, Aradan Mt rng, Buyba, 25.viii.1990, leg. V. Gromenko" (coll. Riedel). Paratype: ♀ with same labels (coll. Riedel). D e s c r i p t i o n : Body length 9.5 mm. Antenna moderately lanceolate, with 34 flagellomeres; 1st flagellomere 1.6x longer than wide, c. 7th flagellomere square, widest flagellomeres c.0.61x as long as wide; preapical flagellomere 0.8x as long as wide. Temple moderately and roundly narrowed apically. OOD 1.3. Frons densely rugose- punctate and granulate. Face densely punctate and granulate; clypeus with dense punc- tures basally and with scattered punctures in apical 1/2, smooth between punctures. Gena 1.35x as wide as eye, with dense punctures and fine striation dorsally and scattered punc- tures ventrally. Malar space 1.3x as long as width of mandibular base and 1.2x as long as 1st flagellomere. Genal carina complete, not interrupted ventrally. Hypostomal carina narrow. Mesosoma covered with brownish hairs. Mesoscutum densely punctate and granulate, opaque; lateral field opaque, but with shining central area. Mesopleuron and metapleuron densely rugose-punctate, partly finely striation; coxal carina present. Scutellum slightly elevated, slightly wider than long, with rather dense punctures. Area superomedia rectangular, with slightly rounded sides, c.1.6x wider than long; costula present as short stub. Area petiolaris without lateral carina. Hind coxa densely punctate and granulate, without scopa. Hind femur 3.8-4.0x longer than wide, with superficial punctures, smoothened in ventral half. Hind tibia with c.6 denticular spurs apico-externally. Tarso- meres slender, 3rd mid tarsomere 1.7x longer than wide. Metasoma strongly oxypygous, hypopygium short. Postpetiolus strongly widened, medi- an field not distinctly separated, c.1.9x wider than the lateral one, both with fine acicula- tion. 2nd tergite 0.78x as long as wide. Gastrocoelus strongly impressed, with fine ridges; thyridium large, slightly oblique, c.1.5x wider than the interval. 2nd and 3rd tergites very densely punctate and granulate, ± opaque, without longitudinal rugosity medially. Ovipositor sheath slightly extending behind metasomal apex. Color: Black. Flagellomeres 1-8 red, flagellomeres 9-12 reddish-yellow, following flagellomeres black. Lateral field of mesoscutum, hind edge of pronotum, tegula and scutellum red (in paratype also collare and postscutellum). Median field of postpetiolus and 2nd and 3rd tergites red; 6th and 7th tergites with roundish ivory spots. Ichneumon hakassiacus nov.sp. (figs 5, 26, 49, 68) Coxae and trochanters black; legs otherwise red; distal tarsomeres ± brownish apically. Wings almost hyaline, pterostigma yellow. Males unknown. T a x o n o m i c a l r e m a r k : This species belongs to group I sensu HILPERT (1992) and runs to I. stigmaticus ZETTERSTEDT ín his key. It differs by its red hind femur, black frontal orbit, shorter 1st flagellomere and wider area superomedia. Ichneumon inops HOLMGREN, 1880 Ichneumon inops HOLMGREN, 1880 M a t e r i a l e x a m i n e d : South Siberia, southwest Tannu-Ola mountain range, Southwest Tuwa region, Sogly surrounds, 2000 m, 1♀ 1-5.vii.2003, leg. Vastchenko (coll. Riedel). D i s t r i b u t i o n : Palaearctic region, known from Siberia (KOKUJEV 1904, MEYER 1933). Ichneumon haglundi HOLMGREN, 1864 M a t e r i a l e x a m i n e d : East Siberia, 10 km East of Irkutsk, 1♀ 25.ix.2004, leg. Berlov (coll. Riedel). D i s t r i b u t i o n : Palaearctic region, known from Siberia (ROMAN 1904). 1518 Ichneumon inquinatops nov.sp. (figs 6, 27, 50, 69) M a t e r i a l e x a m i n e d : Holotype: ♀ "S Siberia, Hakassia reg., Sallyghen-Taiga mt. reg., 2400 m, 5 vii 2005, leg. Osipov" (coll. Riedel). M a t e r i a l e x a m i n e d : Holotype: ♀ "S Siberia, Hakassia reg., Sallyghen-Taiga mt. reg., 2400 m, 5 vii 2005, leg. Osipov" (coll. Riedel). D e s c r i p t i o n : Body length 12 mm. Antenna filiform, with 40 flagellomeres; 1st flagellomere 1.8x longer than wide, c.6th flagellomere square, widest flagellomeres c.0.8x as long as wide; preapical flagellomere 0.78x as long as wide. Temple moderately narrowed apically. OOD 1.7. Frons densely rugose-punctate. Face coarsely punctate and finely granulate, centrally with coarse transverse striae; clypeus with scattered punctures, smooth between punctures. Gena 1.3x as wide as eye, with scattered punctures in ventral half and fine striation dorsally. Malar space 1.35x as long as width of mandibular base and 1.25x as long as 1st flagellomere. Genal carina complete, not interrupted ventrally. Hypostomal carina narrow. Mesosoma covered with brownish hairs. Mesoscutum densely punctate, median field finely granulate, lateral field smooth between punctures. Mesopleuron and metapleuron densely rugose-punctate; coxal carina present. Scutellum slightly elevated, slightly wider than long, with scattered punctures. Area superomedia slightly trapezoid, widest basally, 1.1x wider than long; costula absent. Area petiolaris without lateral carina. Hind coxa densely punctate and granulate, without scopa. Hind femur 3.8x longer than wide, with scattered punctures in ventral 1/5. Hind tibia with 4-5 denticular spurs apico-externally. Tarsomeres slender, 3rd mid tarsomere 1.55x longer than wide. Metasoma strongly oxypygous, hypopygium short. Postpetiolus strongly widened, median field 1.6x wider than the lateral one, with fine regular aciculation; lateral field with coarser striae. 2nd tergite 0.65x as long as wide. Gastrocoelus deeply impressed, with some strong ridges; thyridium transverse, c.0.6x as wide as the interval. 2nd and 3rd tergites densely and finely punctate, 2nd tergite with fine longitudinal rugosity medially. Ovipositor sheath distinctly extending behind metasomal apex. Color: Black. Scapus ventrally and flagellomeres 1-7 reddish-brown, flagellomeres 9-13 darker red, following flagellomeres black. Palps ochreous. Rings on flagellomeres 8-13, complete scutellum and narrow stripes on 6th and 7th tergites ivory. Mandible except teeth, collare, and hind edge of pronotum and tegula red. Metasoma black, 2nd to 3rd tergites with some diffuse dark reddish tint. Ichneumon inops HOLMGREN, 1880 M a t e r i a l e x a m i n e d : South Siberia, southwest Tannu-Ola mountain range, Southwest Tuwa region, Sogly surrounds, 2000 m, 1♀ 1-5.vii.2003, leg. Vastchenko (coll. Riedel). 1519 Ichneumon inquinatops nov.sp. (figs 6, 27, 50, 69) Coxae and trochanters black; legs otherwise red; fore and mid femora basally and hind femur black except red basal 1/10. Wings with slightly brownish tint, pterostigma reddish. Males unknown. T a x o n o m i c a l r e m a r k : This species belongs to the group F of HILPERT (1992) and runs to I. inquinatus WESMAEL in his key. It differs by a higher number of flagellomeres, slenderer 1st flagellomere and completely red hind tibia and tarsus. M a t e r i a l e x a m i n e d : South Siberia, Hakassia region, West Sayan mountains 2400 m, Sallyghem-Taiga region, 1♀ 5.viii.2004, leg. Osipov (coll. Riedel). T a x o n o m i c a l r e m a r k : The available ♀ is smaller (body length 10 mm, antenna with 36 flagellomeres) than the European specimens, but is otherwise typical. Ichneumon lanceolator nov.sp. (figs 7, 28, 51, 70) M a t e r i a l e x a m i n e d : Holotype: ♀ "1-5 vii 2003, 2000 m, SW Tannu-Ola Mt rng, Sogly vill, SW Tuwa reg., S Siberia, leg. Vastchenko" (coll. Riedel). M a t e r i a l e x a m i n e d : Holotype: ♀ "1-5 vii 2003, 2000 m, SW Tannu-Ola Mt rng, Sogly vill, SW Tuwa reg., S Siberia, leg. Vastchenko" (coll. Riedel). D e s c r i p t i o n : Body length 10.2 mm. Antenna very stout and strongly lanceolate, with 35 flagellomeres; 1st flagellomere 1.1x longer than wide, 2nd flagellomere square; widest flagellomeres c.0.5x as long as wide; preapical flagellomere 0.9x as long as wide. Temple slightly and roundly narrowed behind eye. OOD 1.6. Frons coarsely rugose- punctate. Face centrally with dense punctures, smooth between punctures; facial orbit and clypeus with scattered punctures. Gena 1.1x as wide as eye, with rather dense punc- tures in ventral 1/2. Malar space 1.15x as long as width of mandibular base and 1.25x as long as 1st flagellomere. Genal carina complete, not interrupted ventrally. Hypostomal carina narrow. Mesosoma covered with brownish hairs. Mesoscutum densely punctate, lateral field centrally with scattered punctures. Mesopleuron densely punctate, smooth between punc- tures. Metapleuron coarsely rugose-punctate; coxal carina present. Scutellum slightly elevated, wider than long, with scattered punctures. Area superomedia rectangular, 1.5x wider than long; costula absent. Area petiolaris without lateral border. Mid coxa with scattered punctures ventrally, hind coxa with dense punctures, without scopa. Hind femur 3.3x longer than wide, with very scattered punctures in ventral 1/3. Hind tibia with c.9 denticular spurs apico-externally. Fore and mid tarsomeres moderately enlarged, 3rd mid tarsomere c.1.3x longer than wide. Metasoma strongly oxypygous, hypopygium short. Postpetiolus strongly widened, lateral field 0.5x as wide as the median one, median field with fine and very superficial acicula- tion. 2nd tergite very wide, 0.63x as long as wide. Gastrocoelus impressed, with weak ridges. Thyridium small, transverse, c.0.5x as wide as the interval. 2nd and 3rd tergites with fine and very dense punctures, finely granulate, but without longitudinal rugosity medially. Ovipositor sheath slightly extending beyond metasomal apex. Color: Black. Mandible except teeth, side of clypeus, facial and frontal orbits, scapus, pedicel and flagellomeres 1-10 red. Collare and hind edge of pronotum, tegula and scutellum red. Postpetiolus apically and 2nd and 3rd tergites completely red. Ichneumon karpathica HEINRICH, 1951 M a t e r i a l e x a m i n e d : South Siberia, Hakassia region, West Sayan mountains 2400 m, Sallyghem-Taiga region, 1♀ 5.viii.2004, leg. Osipov (coll. Riedel). M a t e r i a l e x a m i n e d : South Siberia, Hakassia region, West Sayan mountains 2400 m, Sallyghem-Taiga region, 1♀ 5.viii.2004, leg. Osipov (coll. Riedel). T a x o n o m i c a l r e m a r k : The available ♀ is smaller (body length 10 mm, antenna with 36 flagellomeres) than the European specimens, but is otherwise typical. D i s t r i b u t i o n : Known from Europe, new record for Siberia. 1520 Ichneumon lanceolator nov.sp. (figs 7, 28, 51, 70) 6th and 7th tergites with rather large ivory spots medially. Coxae and trochanters black, fore and mid coxae frontally and all trochanters apically red. Legs otherwise red; hind femur black centrally, red in basal 0.1 and apical 0.2. Wings with slight yellowish tint, pterostigma yellow. Males unknown. Ichneumon lautatorius DESVIGNES, 1856 M a t e r i a l e x a m i n e d : Far East Russia, Primorskij Kray, Ryazanovska, 14 km southwest of Slavyanka, N 42°48´ E 131°12´, 50 m, 3♀♀ 16.vi.1993, leg. Taeger (SDEI); Far East Russia, Primorskij Kray, Samarka, 70 km north of Chuguyerka, 200 m, N 44°43´ E 134°12´, 1♀ 30.v.1993, leg. Taeger (SDEI); Far East Russia, Primorskii Krai, Lazovski Zapovednik, c. 170 km east of Vladivostok, Ta-Chingousa, N 43°01.07´ E 134°07.46´, 0 m, 1♀ and 12♂♂ 31.viii- 16.ix.2001, sandy coast, Malaise trap 495, leg. M. Quest (NHM). T a x o n o m i c a l r e m a r k : The Eastern Palaearctic specimens differ from the European material by yellow-reddish hind femur and tibia which are slightly infuscate apically. The ♀ from Samarka has a basal ivory band on 7th tergite. D i s t r i b u t i o n : Widespread in Eurasia. Ichneumon magistratus HILPERT, 1992 M a t e r i a l e x a m i n e d : Far East Russia, Primorskij Kray, Vladivostok: Sedanka, N 32°05´ E 131°53´, 1♀ 20.vi.1993, leg. Taeger (SDEI); Far East Russia, Primorskij Kray, Ryazanovska, 14 km Southwest of Slavyanka, N 42°48´ E 131°12´, 50 m, 1♀ 16.vi.1993, leg. Taeger (SDEI). D i s t r i b u t i o n : Known from Siberia (HILPERT 1992). Males unknown. T a x o n o m i c a l r e m a r k : This species belongs to group G 4 sensu HILPERT, but differs from all known species in this group by the stout and strongly lanceolate flagellum and the red color pattern of basal antenna, scutellum and legs. It resembles Ichneumon grandicornis THOMSON (group E) due to the structure of flagellum, but dif- fers by the red color pattern, absent scopa of hind coxa, stouter metasoma and wider area superomedia. 1521 Males unknown. T a x o n o m i c a l r e m a r k : Due to the edge on hind coxa, the new species belongs to group E 4 sensu HILPERT (1992) and runs to Ichneumon rufigena KRIECHBAUMER in his key. It differs by the parallel-sided thick mandible, trapezoid area superomedia and black head. If one neglects the form of hind coxa, it runs to group G 4 sensu HILPERT (1992) and here to Ichneumon jugicola HEINRICH in his key. It can be differentiated by the form of mandible and the ventrally widened gena. rugosity medially. Ovipositor sheath slightly extending behind metasomal apex. rugosity medially. Ovipositor sheath slightly extending behind metasomal apex. Color: Black. Flagellomeres 5-13 with reddish-yellow ring. Mandible centrally, collare and tegula partly red. Scutellum ivory. 2nd and 3rd tergites red; 5th tergite with a small and 6th and 7th tergites with larger ivory spots. Coxae and trochanters, mid trochantellus, basal 2/3 of mid femur and hind femur except narrow red base blackish. Legs otherwise red; hind tibia diffusely yellowish subbasally. Wings with yellowish tint, pterostigma reddish. Color: Black. Flagellomeres 5-13 with reddish-yellow ring. Mandible centrally, collare and tegula partly red. Scutellum ivory. 2nd and 3rd tergites red; 5th tergite with a small and 6th and 7th tergites with larger ivory spots. Coxae and trochanters, mid trochantellus, basal 2/3 of mid femur and hind femur except narrow red base blackish. Legs otherwise red; hind tibia diffusely yellowish subbasally. Wings with yellowish tint, pterostigma reddish. Males unknown. Ichneumon mandibulatus nov.sp. (figs 8, 29, 43, 52, 71) M a t e r i a l e x a m i n e d : Holotype: ♀ "S Siberia, Hakassia reg., Sallyghen-Taiga mt. reg., 2400 m, 10 vii 2005, leg. Osipov" (coll. Riedel). M a t e r i a l e x a m i n e d : Holotype: ♀ "S Siberia, Hakassia reg., Sallyghen-Taiga mt. reg., 2400 m, 10 vii 2005, leg. Osipov" (coll. Riedel). D e s c r i p t i o n : Body length 13.5 mm. Antenna stout and almost filiform, with 36 flagellomeres; 1st flagellomere 1.5x longer than wide, 4th flagellomere square, widest flagellomeres c.0.68x as long as wide; preapical flagellomere 0.65x as long as wide. Temple moderately and roundly narrowed behind eye. Ocelli rather small, OOD 1.8. Occiput densely and coarsely punctate and granulate. Frons coarsely rugose-punctate. Face densely punctate, smooth between punctures. Clypeus with rather dense punctures, smooth between punctures. Mandible thickened, with parallel sides, teeth blunt, lower tooth much smaller than the upper one. Gena strongly widened ventrally, 1.5x as wide as eye, with scattered punctures in ventral 1/2. Malar space 1.0x as long as width of man- dibular base and 1.3x as long as 1st flagellomere. Genal carina complete, not interrupted ventrally; hypostomal carina narrow. Mesosoma covered with brownish hairs. Mesoscutum densely punctate, smooth between punctures. Mesopleuron and metapleuron coarsely striate-punctate; coxal carina present. Scutellum slightly elevated, about as wide as long, with scattered punctures. Area superomedia trapezoid, widest frontally, c.1.2x longer than wide; costula weakly indica- ted. Area petiolaris without lateral border. Hind coxa coarsely punctate, without scopa, but with a longitudinal blunt ventro-internal edge (as in Ichneumon stramentarius GRAVENHORST). Hind femur 3.4x longer than wide, with scattered punctures in ventral 1/3. Hind tibia with c.2 denticular spurs apico-externally. Tarsomeres slender, 3rd mid tarsomere c.1.55x longer than wide. Metasoma oxypygous, hypopygium short. Postpetiolus strongly widened, lateral field punctate and c.0.9x as wide as the median one, median field with fine regular acicula- tion. 2nd tergite c.0.6x as long as wide. Gastrocoelus rather weakly impressed, with several ridges; thyridium transverse, c.0.7x wider than the interval. 2nd and 3rd tergites with fine dense punctures, granulate and ± opaque; 2nd tergite with fine longitudinal 1522 Ichneumon mesonotator nov.sp. (figs 9, 30, 53, 72) Legs otherwise red; hind tibia diffusely yellow- ish subbasally; infuscate in apical 1/10; 1st to 3rd hind tarsomeres red with black tips, 4th and distal hind tarsomeres blackish. Wings with yellowish tint, pterostigma yellow. l k notum, mesoscutum completely, tegula and subtegular ridge red. Scutellum ivory. Lateral field of postpetiolus red; 2nd and 3rd tergites yellowish-red; 5th to 7th tergites with large ivory spots. Coxae and trochanters black; fore coxa frontally and mid and hind coxae apico-dorsally with reddish spots. Legs otherwise red; hind tibia diffusely yellow- ish subbasally; infuscate in apical 1/10; 1st to 3rd hind tarsomeres red with black tips, 4th and distal hind tarsomeres blackish. Wings with yellowish tint, pterostigma yellow. Males unknown. T a x o n o m i c a l r e m a r k : The species belongs to group C sensu HILPERT (1992) and runs to Ichneumon cynthiae KRIECHBAUMER in his key. It differs by the red coloration of basal antenna and mesoscutum and the dense pilosity of mid and hind coxae. From Ichneumon gracilicornis GRAVENHORST it can be differentiated by the red mesoscutum and shorter 1st flagellomere. Ichneumon minutorius DESVIGNES, 1856 M a t e r i a l e x a m i n e d : East Siberia, Lake Baikal near Anghasolka, overwintering unde bark, 1♀ 6.iv.2007, leg. Berlov (coll. Riedel). M a t e r i a l e x a m i n e d : East Siberia, Lake Baikal near Anghasolka, overwintering u bark, 1♀ 6.iv.2007, leg. Berlov (coll. Riedel). D i s t r i b u t i o n : Palaearctic region, new record for Siberia. D i s t r i b u t i o n : Palaearctic region, new record for Siberia. Ichneumon mesonotator nov.sp. (figs 9, 30, 53, 72) M a t e r i a l e x a m i n e d : Holotype: ♀ "1-5 vii 2003, 2000 m, SW Tannu-Ola Mt reg Sogly vill., SW Tuwa reg., S Siberia, leg. Vastchenko" (coll. Riedel) D e s c r i p t i o n : Body length 14 mm. Antenna moderately lanceolate, with 40 flagellomeres; 1st flagellomere 2.3x longer than wide, c.9th flagellomere square, widest flagellomere c.0.70x as long as wide; preapical flagellomere 0.9x as long as wide. Tem- ple moderately and almost linearly narrowed behind eye. OOD 1.6. Frons coarsely rugose-punctate. Face densely punctate, with fine granulation but shining. Clypeus with scattered punctures, smooth between punctures. Gena 1.1x as wide as eye, with rather dense punctures in ventral 1/2. Malar space 1.3x as long as width of mandibular base and 1.0x as long as 1st flagellomere. Genal carina complete, not interrupted ventrally. Hypostomal carina narrow. Mesosoma covered with brownish hairs. Mesoscutum densely punctate, smooth between punctures. Mesopleuron and metapleuron coarsely punctate, partly striate; coxal carina present. Scutellum slightly elevated, wider than long, with scattered punctures. Area superomedia slightly hexagonal, about as long as wide; costula absent. Area petiolaris bordered with irregular rugae in apical half. Mid and hind coxae with dense long pale brownish hairs; hind coxa with denser hairs almost forming an indistinct scopa apico- ventrally. Hind femur 3.9x longer than wide, densely punctate. Hind tibia with c.4 den- ticular spurs apico-externally. Tarsomeres not enlarged, 3rd mid tarsomere c.1.7x longer than wide. Metasoma strongly oxypygous, hypopygium short. Postpetiolus strongly widened, median field with weak aciculation and some coarse punctures. 2nd tergite 0.75x as long as wide. Gastrocoelus strongly impressed, with several ridges; thyridium large, trans- verse, c.1.4x wider than the interval. 2nd and 3rd tergites with fine dense punctures, both with longitudinal rugosity medially. Ovipositor sheath slightly extending behind meta- somal apex. Color: Black. Scapus, pedicel and flagellomeres 1-14 red, distal flagellomeres black. Mandible centrally red; narrow frontal orbit pale reddish. Collare and hind edge of pro- 1523 notum, mesoscutum completely, tegula and subtegular ridge red. Scutellum ivory. Lateral field of postpetiolus red; 2nd and 3rd tergites yellowish-red; 5th to 7th tergites with large ivory spots. Coxae and trochanters black; fore coxa frontally and mid and hind coxae apico-dorsally with reddish spots. Ichneumon orientopodius nov.sp. (figs 11, 32, 55, 74, 82) M a t e r i a l e x a m i n e d : Holotype: ♀ "Far East Russia, Primorje reg., Anisimovka vill 290 m, N 43°10´07´´, E 132°46´10´´, 18.vi.2015, leg. Gromenko" (coll. Riedel). M a t e r i a l e x a m i n e d : Holotype: ♀ "Far East Russia, Primorje reg., Anisimovka vill., 290 m, N 43°10´07´´, E 132°46´10´´, 18.vi.2015, leg. Gromenko" (coll. Riedel). D e s c r i p t i o n : Body length 16 mm. Antenna almost filiform, with 43 flagello- meres; 1st flagellomere 2.1x longer than wide, c.8th flagellomere square, widest flagello- meres c.0.7x as long as wide; preapical flagellomere 0.8x as long as wide. Temple rather long, slightly narrowed behind eye. OOD 1.5. Occiput with transverse rugae; frons coarsely rugose-punctate. Face densely punctate, rugose-punctate centrally, facial orbit smooth between punctures. Clypeus with scattered punctures, smooth between punc- tures. Gena wide, 1.2x as wide as eye, with scattered punctures in ventral 1/2. Malar space 1.1x as long as width of mandibular base and 0.9x as long as 1st flagellomere. Genal carina complete, not interrupted ventrally. Hypostomal carina narrow. Mesosoma covered with brownish hairs. Mesoscutum densely punctate, smooth and shining between punctures. Mesopleuron coarsely punctate, coarsely striate in ventral 1/2. Metapleuron coarsely rugose-punctate; coxal carina present. Scutellum almost flat, wider than long, with scattered punctures. Area superomedia hexagonal, 1.2x wider than long; costula weakly present. Area petiolaris completely bordered by strong irregular rugae laterally. All coxae with dense long brownish hairs; hind coxa densely punctate and with small dense scopa presenting as a slightly elevated area. Hind femur 3.4x longer than wide, with dense punctures. Hind tibia with c.5 denticular spurs externally. All tarsomeres distinctly widened, 3rd mid tarsomere c.1.2x longer than wide. Metasoma semi-amblypygous, hypopygium moderately elongate (about as long as the length between its apical margin and the metasomal apex). Postpetiolus strongly widened, median field with fine regular aciculation. 2nd tergite 0.68x as long as wide. Gastrocoelus impressed, with several ridges; thyridium slightly oblique, c.0.75x as wide as the interval. 2nd and 3rd tergites with fine dense punctures, 2nd tergite not rugose medially. Ovipositor sheath slightly extending behind metasomal apex. Color: Black. Antenna reddish basally and darkened in apical 1/3, with yellowish rings on flagellomeres 7-13. Scutellum and wide spots on 6th and 7th tergites yellow. Ichneumon nigrostigmaticus nov.sp. (figs 10, 31, 54, 73) M a t e r i a l e x a m i n e d : Holotype: ♀ "1-5 vii 2003, 2000 m, SW Tannu-Ola Mt reg., Sogly vill., SW Tuwa reg., S Siberia, leg. Vastchenko" (coll. Riedel) M a t e r i a l e x a m i n e d : Holotype: ♀ "1-5 vii 2003, 2000 m, SW Tannu-Ola Mt reg., Sogly vill., SW Tuwa reg., S Siberia, leg. Vastchenko" (coll. Riedel) D e s c r i p t i o n : Body length 14 mm. Antenna slightly lanceolate, with 41 flagello- meres; 1st flagellomere 1.85x longer than wide, c.7th flagellomere square, widest flagel- lomeres c.0.75x as long as wide; preapical flagellomere 0.67x as long as wide. Temple moderately and roundly narrowed behind eye. Ocelli small, OOD 1.8. Frons densely rugose-punctate. Face densely rugose-punctate and granulate. Clypeus densely punctate, with some coarse striae in apical 1/2. Gena wide, 1.3x as wide as eye, densely punctate, but with a small ventral area with scattered punctures. Malar space 1.4x as long as width of mandibular base and 1.5x as long as 1st flagellomere. Genal carina complete, reaching the hypostomal one far from mandibular base (distance as long as width of mandibular base). Hypostomal carina narrow. Mesosoma covered with brownish hairs. Mesoscutum rather densely punctate, lateral field centrally with scattered punctures. Mesopleuron and metapleuron coarsely rugose- punctate; coxal carina absent. Scutellum slightly elevated, slightly longer than wide, with fine scattered punctures. Area superomedia about square; costula absent. Area petiolaris not bordered laterally. Hind coxa densely punctate, with rather long brownish hairs, but without scopa. Hind femur slender, 4.1x longer than wide, densely punctate, with scat- tered punctures in ventral 1/5. Hind tibia with 3-4 denticular spurs apico-externally. Tarsomeres slender, 3rd mid tarsomere c.1.7x longer than wide. Metasoma strongly oxypygous, hypopygium short. Postpetiolus strongly widened, median field not distinctly separated and c.1.6x wider than lateral field, with very fine and regular aciculation; lateral field punctate and with fine aciculation. 2nd tergite 0.8x as long as wide. Gastrocoelus strongly impressed, with several strong ridges; thyridium slightly oblique, c.0.8x as wide as the interval. 2nd and 3rd tergites with fine dense punc- tures and granulation, basal third of 2nd tergite finely rugose medially. Ovipositor sheath slightly extending behind metasomal apex. 1524 Color: Black. Flagellomeres 1-12 completely and mandible centrally red. Ichneumon nigrostigmaticus nov.sp. (figs 10, 31, 54, 73) Mesosoma black; collare, tegula and central spot on scutellum reddish. Lateral field of postpetiolus with reddish stripe, 2nd tergite completely red, 3rd tergite only laterally. 6th and 7th tergites diffusely reddish apically, without distinct pale spots. Coxae and trochanters black; legs otherwise red; fore and mid femora basally blackish; hind femur black, in basal 1/10 and apical 1/5 reddish. Wings with brownish tint, pterostigma black, paler proximally. M l k T a x o n o m i c a l r e m a r k : The species belongs to group G 4 sensu HILPERT (1992). It is characterized by the strongly sculptured clypeus, slender hind femur and characteristical coloration of metasoma. Ichneumon orientopodius nov.sp. (figs 11, 32, 55, 74, 82) T a x o n o m i c a l r e m a r k : The species belongs to group E 1 sensu HILPERT Ichneumon paravafer nov.sp. (figs 12, 33, 56, 75) M a t e r i a l e x a m i n e d : Holotype: ♀ "S Siberia, Hakassia reg., Sallyghen-Taiga mt. reg 2400 m, 5 vii 2005, leg. Osipov" (coll Riedel). D e s c r i p t i o n : Body length 13 mm. Antenna strongly lanceolate, with 40 flagel- lomeres; 1st flagellomere 1.5x longer than wide, 4th flagellomere square, widest flagello- meres c.0.5x as long as wide; preapical flagellomere 0.67x as long as wide. Temple moderately narrowed apically, slightly rounded. OOD 1.6. Frons densely rugose- punctate. Face coarsely punctate and finely granulate; clypeus with rather dense punc- tures, smooth between punctures. Gena 1.3x as wide as eye, with scattered punctures in ventral 1/2. Malar space 1.1x as long as width of mandibular base and 1.15x as long as 1st flagellomere. Genal carina complete, not interrupted ventrally. Hypostomal carina narrow. Mesosoma covered with brownish hairs. Mesoscutum densely punctate; median field finely granulate, lateral field with rather dense punctures centrally, smooth between punctures. Mesopleuron very densely punctate; metapleuron coarsely rugose-punctate; coxal carina present. Scutellum slightly elevated, slightly wider than long, with scattered punctures. Area superomedia slightly hexagonal, about as long as wide; costula absent. Area petiolaris without lateral carina. Hind coxa densely punctate and granulate, partly with less dense punctures externally, ± opaque, without scopa. Hind femur 3.6x longer than wide, with scattered punctures in ventral 1/2. Hind tibia and tarsus missing. Tarso- meres slender, 3rd mid tarsomere 1.6x longer than wide. Metasoma semi-amblypygous; hypopygium moderately elongate, about as long as the distance of its apical margin to metasomal apex. Postpetiolus strongly widened, median field 1.6x wider than the lateral one, with regular aciculation; lateral field aciculate. 2nd tergite 0.76x as long as wide. Gastrocoelus impressed, with fine ridges; thyridium slight- ly oblique, about as wide as the interval. 2nd and 3rd tergites densely punctate and granu- late, ± opaque; following tergites strongly shining; 2nd tergite with fine longitudinal rugosity medially. Ovipositor sheath slightly extending behind metasomal apex. Color: Black. Rings on flagellomeres 8-12, scutellum and equally sized spots on tergite 5-7 ivory. Collare, tegula, postpetiolus and 2nd tergite completely red, 3rd tergite with narrow reddish lateral margin. Coxae and trochanters black; legs otherwise red (hind tibia and tarsus missing). Wings with brownish tint, pterostigma yellowish-red. Males unknown. Ichneumon orientopodius nov.sp. (figs 11, 32, 55, 74, 82) 2nd and 3rd tergites reddish, with yellowish tint in apical parts; 3rd tergite with black basal band. Coxae and trochanters black; legs otherwise red; all tibiae yellowish externally; hind tarsus blackish in apical 1/2. Wings with slight yellowish tint, pterostigma yellow. Males unknown T a x o n o m i c a l r e m a r k : The species belongs to group E 1 sensu HILPERT 1525 (1992) and is characterized by the color pattern of 2nd and 3rd tergites, strongly enlarged tarsomeres and the semi-amblypygous metasoma with moderately elongate hypopygium (1992) and is characterized by the color pattern of 2nd and 3rd tergites, strongly enlarged tarsomeres and the semi-amblypygous metasoma with moderately elongate hypopygium. Ichneumon paravafer nov.sp. (figs 12, 33, 56, 75) Ichneumon paravafer nov.sp. (figs 12, 33, 56, 75) T a x o n o m i c a l r e m a r k : This species belongs to the group G 2.1 sensu HILPERT (1992) and runs to I. vafer meridionalis HEINRICH in his key. It differs by the red hind femur with scattered punctures in ventral ½ and the blackish 3rd tergite. Ichneumon paravulnerator nov.sp. (figs 13, 34, 57, 76) M a t e r i a l e x a m i n e d : Holotype: ♀ "1-5 vii 2003, 2000 m, SW Tannu-Ola Mt reg., Sogly vill., SW Tuwa reg., S Siberia, leg. Vastchenko" (coll. Riedel) D e s c r i p t i o n : Body length 11 mm. Antenna moderately lanceolate, with 37 flagellomeres; 1st flagellomere 1.8x longer than wide, c.8th flagellomere square, widest Ichneumon paravulnerator nov.sp. (figs 13, 34, 57, 76) M a t e r i a l e x a m i n e d : Holotype: ♀ "1-5 vii 2003, 2000 m, SW Tannu-Ola Mt reg., Sogly vill., SW Tuwa reg., S Siberia, leg. Vastchenko" (coll. Riedel) D e s c r i p t i o n : Body length 11 mm. Antenna moderately lanceolate, with 37 flagellomeres; 1st flagellomere 1.8x longer than wide, c.8th flagellomere square, widest D e s c r i p t i o n : Body length 11 mm. Antenna moderately lanceolate, with 37 flagellomeres; 1st flagellomere 1.8x longer than wide, c.8th flagellomere square, wides 1526 flagellomeres c.0.75x as long as wide; preapical flagellomere 0.9x as long as wide. Temple rather long, slightly narrowed behind eye. OOD 1.5. Frons coarsely and trans- versely rugose-punctate. Face coarsely and densely punctate, with fine granulation. Clypeus with dense punctures, smooth between punctures. Gena 1.2x as wide as eye, with dense punctures and fine striation in ventral 1/2. Malar space 1.25x as long as width of mandibular base and 1.15x as long as 1st flagellomere. Genal carina complete, not interrupted ventrally. Hypostomal carina slightly elevated. Mesosoma covered with brownish hairs. Mesoscutum coarsely and densely punctate, with fine granulation but shining. Mesopleuron and metapleuron coarsely rugose- punctate; coxal carina present. Scutellum moderately elevated, about as long as wide, with rather dense punctures becoming finer apically. Area superomedia slightly hexago- nal, 1.2x wider than long; costula absent. Area petiolaris without lateral border. Hind coxa densely punctate, without scopa. Hind femur 3.4x longer than wide, densely punc- tate. Hind tibia with c.8. denticular spurs apico-externally. 3rd mid tarsomere c.1.5x longer than wide. Metasoma strongly oxypygous, hypopygium short. Postpetiolus strongly widened, medi- an field with fine and regular aciculation. 2nd tergite very wide, 0.63x as long as wide. Gastrocoelus impressed, with several ridges; thyridium large, transverse, c.1.5x wider than the interval. 2nd to 4th tergites with dense punctures, granulate and ± opaque. Ovipositor sheath slightly extending beyond metasomal apex. Color: Black. Scapus, pedicel and flagellomeres 1-8 red, flagellomeres 9-12 more yellowish-red. Mandible shortly red centrally. Collare, tegula and scutellum red. 2nd and 3rd tergites red, 3rd tergite with black band in apical third. 6th and 7th tergites with roundish ivory spots medially. Coxae and trochanters black; legs otherwise red. Wings with slightly yellowish tint, pterostigma yellow. Males unknown. Ichneumon paravulnerator nov.sp. (figs 13, 34, 57, 76) T a x o n o m i c a l r e m a r k : The species belongs to group C sensu HILPERT (1992) and runs to Ichneumon norvegicus HILPERT in his key. It differs by less widened median flagellomeres, reddish scapus, pedicel, scutellum and 2nd tergite and an ivory spot on 6th tergite. Ichneumon pseudemancipatus nov.sp. (figs 14, 35, 58, 77) M a t e r i a l e x a m i n e d : Holotype: ♀ "S Siberia, Hakassia region, W Sayan mts 2400 m, Males unknown. Males unknown. T a x o n o m i c a l r e m a r k : The species belongs to group C sensu HILPERT (1992) and is closely related to Ichneumon emancipatus WESMAEL and Ichneumon emancipatops HEINRICH. It can be differentiated from the former species by shorter 1st flagellomere, absence of ivory spot on 5th tergite and red hind tibia and tarsus (except brownish distal tarsomere). It differs from the latter taxon by the black scapus and pedicel, black mesosoma (except scutellum), absence of ivory spot on 5th tergite and strongly orthogonal thyridia. Due to the limited East Palaearctic material, the status of Ichneumon emancipatops and Ichneumon pseudemancipatus remains unclear. It is possible that both species might be variants or subspecies of Ichneumon emancipatus (see HILPERT 1992: 108). Ichneumon rufolateralis nov.sp. (figs 15, 36, 59, 78) M a t e r i a l e x a m i n e d : Holotype: ♀ "S Siberia, Hakassia reg., Sallyghen-Taiga mt. Sallyghem-Taiga region, 5 vii 2004, leg. Osipov" (coll. Riedel). D e s c r i p t i o n : Body length 10 mm. Antenna moderately lanceolate, with 37 flagellomeres; 1st flagellomere 1.8x longer than wide, c. 7th flagellomere square, widest flagellomeres c.0.7x as long as wide; preapical flagellomere 1.05x longer than wide. Temple moderately and roundly narrowed behind eye. OOD 1.6. Frons coarsely rugose- punctate. Face coarsely punctate, rugose centrally, with fine granulation. Clypeus with rather dense punctures, finely granulate. Gena 1.1x as wide as eye, with rather dense punctures and fine striation in ventral 1/2. Malar space 1.5x as long as width of mandibu- lar base and 1.2x as long as 1st flagellomere. Genal carina complete, not interrupted ventrally. Hypostomal carina narrow. Mesosoma covered with brownish hairs. Mesoscutum densely punctate, smooth and shining between punctures. Mesopleuron and metapleuron coarsely rugose-punctate; coxal carina present. Scutellum moderately elevated, about as long as wide, with rather dense punctures. Area superomedia rectangular, about as long as wide, apical carina absent; costula absent. Area petiolaris without lateral carina. Hind coxa densely punctate and granulate, with rather dense and long brownish hairs, but without scopa. Hind femur 3.9x longer than wide, densely but superficially punctate and granulate. Hind tibia with c.4 denticular spurs apico-externally. 3rd mid tarsomere c.1.6x longer than wide. Metasoma strongly oxypygous, hypopygium short. Postpetiolus strongly widened, median field c. 1.9x wider than the lateral one, with some indistinct aciculation basally and fine irregular rugae apically. 2nd tergite 0.66x as long as wide. Gastrocoelus strongly impressed, with strong ridges; thyridium large, transverse, c.1.3x wider than the interval. 2nd and 3rd tergites with dense fine punctures, granulate and ± opaque, not rugose. Ovipositor sheath slightly extending behind metasomal apex. Color: Black. Scapus and pedicel black. Flagellomeres 1-8 red, flagellomeres 9-12 more yellowish-red. Scutellum yellow, mesosoma otherwise black. Median field of postpetiolus, 2nd and 3rd tergites red, 3rd tergite with black band in apical 1/4. 6th and 7th tergite with roundish ivory spots medially. Coxae and trochanters black; legs otherwise red; mid femur black in basal 1/2; hind femur black, red in basal and apical 1/5. Hind tibia and tarsus red, distal hind tarsomere partly brownish. Wings with moderate brown- ish tint, pterostigma yellow. Ichneumon pilulicornis HEINRICH, 1978 M a t e r i a l e x a m i n e d : East Siberia, 10 km east of Irkutsk, 1♀ 12.iv.1999, leg. Berlov (coll. Riedel). M a t e r i a l e x a m i n e d : East Siberia, 10 km east of Irkutsk, 1♀ 12.iv.1999, leg. Berlov (coll. Riedel). T a x o n o m i c a l r e m a r k : The available ♀ is smaller than the holotype and differs slightly in its coloration: Body length 8.5 mm. Antenna with 29 flagellomeres. Area superomedia rectangular, 1.4x longer than wide. Hind femur 2.8x longer than wide; 3rd mid tarsomere 1.5x longer than wide. 2nd tergite 0.83x as long as wide. Clypeus and face centrally reddish. Hind femur black, basal 1/8 red; hind tibia slightly darkened in apical 1/10. 3rd tergite completely red. Structure and coloration otherwise as described for the holotype (HEINRICH 1978: 35-36, HILPERT 1992: 243). D i s t r i b u t i o n : Only known from Far East Russia. D i s t r i b u t i o n : Only known from Far East Russia. Ichneumon pseudemancipatus nov.sp. (figs 14, 35, 58, 77) 1527 Ichneumon rufolateralis nov.sp. (figs 15, 36, 59, 78) 1528 reg., 2400 m, 10 vii 2005, leg. Osipov" (coll. Riedel). reg., 2400 m, 10 vii 2005, leg. Osipov" (coll. Riedel). D e s c r i p t i o n : Body length 11 mm. Antenna almost filiform, with 43 flagello- meres; 1st flagellomere 1.5x longer than wide, c.7th flagellomere square, widest flagello- meres c.0.75x as long as wide; preapical flagellomere 0.67x as long as wide. Temple slightly widened behind eye and slightly narrowed apically. OOD 1.8. Frons densely rugose-punctate and granulate. Face coarsely punctate and finely granulate; clypeus rather densely punctate, smooth between punctures. Gena wide, 1.4x as wide as eye, with scattered punctures in ventral 1/2. Malar space 1.25x as long as width of mandibular base and 1.3x as long as 1st flagellomere. Genal carina complete, not interrupted ventrally. Hypostomal carina narrow. Mesosoma covered with brownish hairs. Mesoscutum densely punctate and finely granu- late medially, lateral field with rather dense punctures centrally, smooth between punc- tures. Mesopleuron densely punctate, partly rugose; metapleuron coarsely rugose- punctate; coxal carina present. Scutellum slightly elevated, slightly longer than wide, with rather dense punctures. Area superomedia almost square; costula absent. Area petiolaris without lateral border. Hind coxa densely punctate, with large reddish scopa (2/5 of coxal length). Hind femur 3.7x longer than wide, with very scattered punctures basally and in ventral 1/2. Hind tibia with c.3 denticular spurs apico-externally. Tarso- meres distinctly widened, 3rd mid tarsomere 1.3x longer than wide. Metasoma strongly oxypygous, hypopygium short. Postpetiolus strongly widened, lateral field 0.5x as wide as the median one, median field with fine irregular aciculation and some subapical punctures. 2nd tergite 0.62x as long as wide. Gastrocoelus impressed, with fine ridges; thyridium slightly oblique, about as wide as the interval. 2nd and 3rd tergites very densely punctate and finely granulate, not rugose medially. Ovipositor sheath slightly extending behind metasomal apex. Color: Black. Scapus apically and flagellomeres 1-8 red, flagellomeres 9-13 darker red, following flagellomeres black. Mandible except teeth, collare and hind edge of prono- tum, tegula and scutellum red. Metasoma black; postpetiolus laterally and apically and 2nd to 3rd tergites laterally red; 7th tergite with an indistinct reddish spot medially. Coxae and trochanters black; legs otherwise red. Wings with yellowish tint, pterostigma yellow. Males unknown. T a x o n o m i c a l r e m a r k : This species belongs to group E 2 sensu HILPERT (1992). Ichneumon rufolateralis nov.sp. (figs 15, 36, 59, 78) The new taxon is closely related to Ichneumon lariae taimyrensis HEINRICH, but differs by larger number of flagellomeres, black mesoscutum and distinct scopa on hind coxa. Ichneumon sayanicus nov.sp. (figs 16, 37, 60, 79) Ichneumon sayanicus nov.sp. (figs 16, 37, 60, 79) M a t e r i a l e x a m i n e d : Holotype: ♀ "S Siberia, Hakassia, W Sayan Mts, Arada mountain range, Buyba vill., 25.viii.1990, leg. V. Gromenko" (coll. Riedel). Males unknown. T a x o n o m i c a l r e m a r k : This species belongs to group F sensu HILPERT (1992). It runs to Ichneumon subalpinus HOLMGREN in his key but differs by the less strongly lanceolate flagellum and black head and mesosoma (except the ivory scutellum). D e s c r i p t i o n : D e s c r i p t i o n : D e s c r i p t i o n : Body length 14.5 mm. Antenna moderately lanceolate, with 42 flagellomeres, 1st flagel- lomere 1.5x longer than wide; 4th flagellomere square, widest flagellomeres 0.57x as long as wide, preapical flagellomere 0.8x as long as wide. Temple moderately and roundly narrowed behind eye. OOD 1.6. Frons transversely rugose-punctate, finely granulate. Face densely punctate, smooth between punctures; clypeus with rather dense 1529 1529 punctures, smooth between punctures. Gena 1.2x as wide as eye, with rather dense punc- tures in ventral 1/2. Malar space 1.15x as long as width of mandibular base and 1.25x as long as 1st flagellomere. Genal carina complete, not interrupted ventrally. Hypostomal carina narrow. punctures, smooth between punctures. Gena 1.2x as wide as eye, with rather dense punc- tures in ventral 1/2. Malar space 1.15x as long as width of mandibular base and 1.25x as long as 1st flagellomere. Genal carina complete, not interrupted ventrally. Hypostomal carina narrow. Mesosoma covered with brownish hairs. Mesoscutum and mesopleuron densely punc- tate, smooth between punctures. Metapleuron coarsely rugose-punctate; coxal carina present. Scutellum slightly elevated, slightly wider than long, with scattered fine punc- tures. Area superomedia trapezoid, widest frontally, 1.1x wider than long; costula weakly indicated. Area petiolaris with lateral border defined by some irregular rugae in the api- cal half. Hind coxa densely punctate and granulate, without scopa or longitudinal edge. Hind femur 3.4x longer than wide, with scattered punctures in ventral 1/3. Hind tibia with c.4 denticular spurs apico-externally. Tarsomeres slender, 3rd mid tarsomere 1.7x longer than wide. Metasoma strongly oxypygous, hypopygium short. Postpetiolus strongly widened, medi- an field 1.4x wider than the lateral one, with fine aciculation. 2nd tergite 0.70x as long as wide. Gastrocoelus impressed, with distinct ridges; thyridium slightly oblique, 0.7x as wide as the interval. 2nd and 3rd tergites densely punctate and finely granulate, but shin- ing. 2nd tergite with fine longitudinal rugosity medially. Ovipositor sheath slightly ex- tending behind metasomal apex. Color: Black. Ring of flagellomeres 8-13, scutellum and roundish, equally sized spots on 5th to 7th tergites ivory. Coxae and trochanters black; legs otherwise red; tips of hind tibia and hind tarsomeres slightly darkened. Wings almost hyaline, pterostigma reddish- yellow. Males unknown. Ichneumon suspiciosus WESMAEL, 1845 M a t e r i a l e x a m i n e d : South Siberia, Hakassia, West Sayan mountains, Aradan mountain range, Buyba village, 2♀♀ 25.viii.1990, leg. V. Gromenko (coll. Riedel). M a t e r i a l e x a m i n e d : South Siberia, Hakassia, West Sayan mountains, Aradan mountain range, Buyba village, 2♀♀ 25.viii.1990, leg. V. Gromenko (coll. Riedel). T a x o n o m i c a l r e m a r k : Body length 15 mm. Antenna with 38-40 flagello- meres; 1st flagellomere 1.5x longer than wide. Hind femur 3.3x longer than wide, with scattered punctures in ventral 1/2. One ♀ has a completely red hind tibia without a sub- basal yellow stripe, both specimens have completely red hind tarsi. D i s t r i b u t i o n : Palaearctic region, known from Eastern Palaearctic region (UCHIDA 1926, MEYER 1933). D i s t r i b u t i o n : Palaearctic region, known from Eastern Palaearctic region (UCHIDA 1926, MEYER 1933). Ichneumon sibiricus ROMAN, 1904 M a t e r i a l e x a m i n e d : East Siberia, Lake Baikal near Anghasolka, overwintering under bark, 7♀♀ 6.iv.2007, leg. Berlov (coll. Riedel); Far East Russia, Primorskii Kray, Lazovski Zapovednik, c. 170 km E Vladivostok, Korpad, N 43°15.52´ E 134°07.45´, 174 m, 1♀ 1- 14.v.2001, flood plain, Malaise trap 441, leg. M. Quest (NHM). D i s t r i b u t i o n : Only known from Siberia. D i s t r i b u t i o n : Only known from Siberia. 1530 petiolaris without lateral border. Mid coxa with short scopa apically; hind coxa densely punctate and with strong and large scopa (0.5x coxal length). Hind femur stout, c.3.0x longer than wide, with scattered punctures in ventral 1/2. Hind tibia with c.6 denticular spurs apico-externally. All tarsomeres distinctly widened, 3rd mid tarsomere c.1.25x longer than wide. petiolaris without lateral border. Mid coxa with short scopa apically; hind coxa densely punctate and with strong and large scopa (0.5x coxal length). Hind femur stout, c.3.0x longer than wide, with scattered punctures in ventral 1/2. Hind tibia with c.6 denticular spurs apico-externally. All tarsomeres distinctly widened, 3rd mid tarsomere c.1.25x longer than wide. Metasoma strongly oxypygous, hypopygium short. Postpetiolus strongly widened, medi- an field with fine aciculation and some fine punctures. 2nd tergite c.0.7x as long as wide. Gastrocoelus impressed, with fine ridges; thyridium almost transverse, c.0.9x as wide as the interval. 2nd and 3rd tergites with fine dense punctures and fine longitudinal rugosity medially. Ovipositor sheath slightly extending behind metasomal apex. Color: Black. Scapus partly and flagellomeres 1-15 red, following flagellomeres black. Narrow frontal orbit, collare, hind edge of pronotum and tegula red. Scutellum yellowish-red. 2nd and 3rd tergites mainly red, darkened medially; sides of 4th tergite reddish basally. 6th and 7th tergites with narrow ivory stripes. Coxae and trochanters black, legs otherwise completely reddish. Wings with yellowish tint, pterostigma reddish. T a x o n o m i c a l r e m a r k : This new species belongs to group E2 sensu HILPERT (1992) and is closely related to I. rufolateralis nov.sp. It differs mainly by its larger size, stouter hind femur and distinct scopae on mid and hind coxae. Ichneumon scopator nov.sp. (figs 17, 38, 61, 80) M a t e r i a l e x a m i n e d : Holotype: ♀ "Russia, S Siberia, Hakassia reg., Sallyghem- Taiga mt. reg, 2400 m, 10 VII 2005, leg. Osipov" (coll. Riedel). M a t e r i a l e x a m i n e d : Holotype: ♀ "Russia, S Siberia, Hakassia reg., Sallyghem- Taiga mt. reg, 2400 m, 10 VII 2005, leg. Osipov" (coll. Riedel). D e s c r i p t i o n : Body length 15 mm. Antenna rather stout and almost filiform, with 45 flagellomeres; 1st flagellomere c.1.65x longer than wide, c.6th flagellomere square, widest flagellomeres c.1.3x wider than long; preapical flagellomere 0.6x as long as wide. Temple slightly widened behind eye, and slightly narrowed apically. OOD 1.5. Frons with coarse dense punctures, granulate. Face densely punctate; clypeus with rather dense punctures; both areas smooth between punctures. Gena wide, c.1.4x wider than eye, with scattered punctures in ventral 1/2. Malar space c.1.35x longer than width of mandibular base and 1.4x longer than 1st flagellomere. Genal carina complete, hypostomal carina narrow. Mesosoma covered with brownish hairs. Mesoscutum densely punctate, smooth between punctures. Mesopleuron coarsely and densely punctate, partly rugose; metapleuron coarsely rugose-punctate; coxal carina present. Scutellum slightly elevated, with rather dense fine punctures. Area superomedia c.1.2x wider than long; costula absent. Area 1530 D i s t r i b u t i o n : Palaearctic region, known from Siberia (ROMAN 1927, MEYER 1933). Ichneumon thyridiator nov.sp. (figs 18, 62, 81) M a t e r i a l e x a m i n e d : Holotype: ♀ "Russia, E Siberia, lake Baikal near Anghasolka, 6 iv 2007, leg. Berlov" "under bark of dead Pinus silvestris" (coll. Riedel). Paratype: ♀, with similar labels (coll. Riedel). M a t e r i a l e x a m i n e d : Holotype: ♀ "Russia, E Siberia, lake Baikal near Anghasolka, 6 iv 2007, leg. Berlov" "under bark of dead Pinus silvestris" (coll. Riedel). Paratype: ♀, with similar labels (coll. Riedel). D e s c r i p t i o n : Body length 10 mm. Antenna strongly lanceolate, with 32 flagel- lomeres; 1st flagellomere 2.0x longer than wide, c.8th flagellomere square, widest flagel- lomeres c.0.6x as long as wide; preapical flagellomere about square. Temple strongly and roundly narrowed apically. OOD 1.6. Frons coarsely rugose-punctate. Face densely punctate and finely granulate. Clypeus with scattered punctures, smooth and shining between punctures. Gena c.0.9x as wide as eye, with dense coarse punctures in ventral 1/2, striate-punctate dorsally. Malar space 1.0x as long as width of mandibular base and 0.8x as long as 1st flagellomere. Genal carina complete, hypostomal carina slightly elevated. Mesosoma covered with pale brownish hairs. Mesoscutum densely punctate and granu- late, ± opaque. Mesopleuron and metapleuron coarsely rugose-punctate; coxal carina present. Scutellum slightly elevated, with dense punctures and lateral carina in basal 0.2. Area superomedia rectangular, c.1.2x wider than long; costula absent. Area petiolaris bordered laterally with irregular rugae in apical half. Hind coxa densely punctate and granulate, without scopa. Hind femur 3.6x longer than wide, with dense punctures. Hind tibia without denticular spurs apico-externally. Tarsomeres slender, 3rd mid tarsomere c.1.8x longer than wide. Metasoma strongly oxypygous, hypopygium short. Postpetiolus strongly widened, medi- an field with strong aciculation and some fine punctures. 2nd tergite 0.78x as long as wide. Gastrocoelus impressed, with several ridges; thyridium slightly oblique, very wide, c.3.0x wider than the interval. 2nd and 3rd tergites with fine dense punctures, finely granu- late, 2nd tergite with longitudinal rugosity medially. Ovipositor sheath extending behind metasomal apex. Color: Black. Flagellomeres 7-13 and large spots on 6th and 7th tergites ivory. Mesosoma black, tegula reddish-brown or black. 2nd tergite mainly red, gastrocoelus darkened; sides of 3rd tergite reddish basally. Ichneumon thomsoni HOLMGREN, 1864 M a t e r i a l e x a m i n e d : South Siberia, Hakassia region, West Sayan mountains 2400 m, Sallyghem-Taiga region, 1♀ 5.vii.2004, leg. Osipov (coll. Riedel). Sallyghem-Taiga region, 1♀ 5.vii.2004, leg. Osipov (coll. Riedel). T a x o n o m i c a l r e m a r k : Antenna with 40 flagellomeres. Hind coxa with indistinct scopa. Petiolus and postpetiolus red, 2nd and 3rd tergites black medially, red laterally. D i s t r i b u t i o n : Palaearctic region known from Siberia (ROMAN 1927 MEYER T a x o n o m i c a l r e m a r k : Antenna with 40 flagellomeres. Hind coxa with indistinct scopa. Petiolus and postpetiolus red, 2nd and 3rd tergites black medially, red laterally. D i s t r i b u t i o n : Palaearctic region, known from Siberia (ROMAN 1927, MEYER 1933). 1531 M a t e r i a l e x a m i n e d : Far East Russia, Primorskij Kray, Sikhote-Alin, Oblachnaya, N 43°45´ E 134°15´, 850 m, 1♀ 2.vi.1993, leg. Taeger (SDEI). D i s t r i b u t i o n : Known from Siberia (MEYER 1933). M a t e r i a l e x a m i n e d : Far East Russia, Primorskij Kray, Sikhote-Alin, Oblachnaya, N 43°45´ E 134°15´, 850 m, 1♀ 2.vi.1993, leg. Taeger (SDEI). D i s t r i b u t i o n : Known from Siberia (MEYER 1933). Ichneumon zoologicus HILPERT, 1992 M a t e r i a l e x a m i n e d : Far East Russia, Primorskij Kray, Vladivostok: Sedanka, N 32°05´ E 131°53´, 4♀♀ 20.vi.1993, leg. Taeger (SDEI); Far East Russia, Khabarevsky Kray, Bostsovo 20 km North of Bikin, Bolshoi Solutsepyok Hill, N 47°02´ E 134°21´, 300 m, 1♀ 26.v.1993, leg. A. Taeger (SDEI); Far East Russia, Primorskij Kray, Ryazanovska, 14 km Southwest of Slavyanka, N 42°48´ E 131°12´, 50 m, 2♀♀ 16.vi.1993, leg. Taeger (SDEI). T a x o n o m i c a l r e m a r k : Antenna with 35-36 flagellomeres, otherwise typical. D i s t r i b u t i o n : Only known from Siberia (HILPERT 1992). Ichneumon veressi (KISS, 1915) M a t e r i a l e x a m i n e d : Far East Russia, Primorskii Kray, Lazovski Zapovednik, c. 170 km East of Vladivostok, Korpad, N 43°15.52´ E 134°07.45´, 174 m, 1♀ and 1♂ 20-27.vi.2001, flood plain, Malaise trap 489, 4♂♂ 05-26.viii.2001, sandy flood plain, Malaise trap 469, leg. M. Quest (NHM). D i s t r i b u t i o n : Known from the Western Palaearctic region, new record for Far East Russia. Ichneumon thyridiator nov.sp. (figs 18, 62, 81) Legs black; hind trochantellus completely and hind femur narrowly reddish at base; fore and mid femora apically and fore and mid tibiae and tarsi ± reddish; hind tibia reddish, infuscate in apical 1/3; hind tarsomere reddish-brown, with darkened tips. Wings almost hyaline; pterostigma ochreous. T a x o n o m i c a l r e m a r k : This new species belongs to group I sensu HILPERT (1992) and runs to Ichneumon boreellus THOMSON in his key. It can be differentiated by the black pronotum, black postpetiolus and 3rd tergite and slenderer 1st flagellomere. Ichneumon validicornis HOLMGREN, 1864 1532 (adopted from HILPERT 1992): (group C).9 - Thyridium smaller than the interval; if wider, then thyridium oblique and gastrocoelus short and only slightly impressed medially ...................................................... (group D).15 9 6th and 7th tergites with ivory spots ................................................................................... .10 - 5th to 7th tergites with ivory spots. (At least 2nd tergite mainly red, 3rd tergite often ± black apically or (in I. cinxiae) mandibular teeth of almost equal length) ......................... 11 10 Legs except black coxae and trochanters red. Scapus, pedicel and basal flagellomeres red. Scutellum red. Hind femur stout, 3.4x longer than wide. Siberia ................................... ...................................................................................................... I. paravulnerator nov.sp. - Hind femur mainly black. Scapus and pedicel black. Scutellum yellow. Hind femur slenderer, 3.9x longer than wide. Siberia ................................. I. pseudemancipatus nov.sp. 11 Mandible not distinctly narrowed apically, mandibular teeth of almost equal length (as fig. 43). Antenna with 34-36 flagellomeres; 1st flagellomere c.2.6x longer than wide. Body length 11.5-11.9 mm. 2nd tergite completely red. Hind femur mainly black; hind tibia red, basally and in apical 0.25 black. Hind tarsus black. Europe, Buyatsk region (HORSTMANN 2006) ............................................................ I. cinxiae KRIECHBAUMER, 1890 - Mandible normally narrowed apically, teeth of different length. 3rd tergite red. 5th tergite with large ivory spot. Hind femur largely red OR antenna with at least 37 flagellomeres ..................................................................................................................... 12 12 Mesoscutum and basal flagellomeres red. Hind femur red. 1st flagellomere c.2.3x longer than wide. Mid and hind coxae densely pilose, but hind coxa without scopa. Siberia ............................................................................................... I. mesonotator nov.sp. - Mesoscutum black ............................................................................................................. 13 13 Hind tibia almost completely red, at most black in apical 1/20. Thyridium rather short, but very wide and deep. Hind femur black, basally and apico-dorsally red. Antenna with 35 flagellomeres; 1st flagellomere 1.9x longer than wide. 2nd and 3rd tergite completely red. Body length 10.1 mm. Far East Russia .... I. emancipatops HEINRICH, 1978 - Hind tibia at least blackish in apical 1/7. Hind femur mainly red (if in I. gracilicornis rarely black, then temples strongly and almost linearly narrowed behind eye). Antenna with 34-42 flagellomeres. Scutellum always yellow ......................................................... 14 14 1st flagellomere slender, 2.4-3.1x longer than wide, antenna slightly lanceolate (widest flagellomeres 0.8-0.9x as long as wide). Color variable: Basal flagellomeres, width of ivory metasomal spots and color of hind femur variable. Europe, Far East Russia (ROMAN 1927) ............................................................. I. gracilicornis GRAVENHORST, 1829 - Hind femur black, hind coxa with large dorsal yellow spot. Propodeum with blunt apophyses. (adopted from HILPERT 1992): 1 Metasoma with color bands: all tergites yellow apically OR 6th tergite with ivory band AND 7th tergite black (or at most with narrow basal ivory band). Scutellum yellow .......... 2 - Metasoma without regular bands on the tergites. If 6th tergite with ivory band or stripe, then also 7th tergite. Often some tergites completely reddish or yellowish .......................... 5 2 All tergite with yellow apical bands. Hind coxa with large dorsal yellow spot. Hind tibia and tarsus completely reddish-yellow. Antenna with 40-44 flagellomeres. Europe, Russia, Mongolia (KOLAROV & GHAHARI 2005) ................................................................... ..................................................................... I. sexcinctus GRAVENHORST, 1829 (group A1) - 6th tergite with apical ivory band and 7th tergite black. Antenna with 33-39 flagellomeres .................................................................................................... (group A2).3 3 Apical bands on 2nd and 3rd tergites yellow. Smaller, body length 9-10 mm. Antenna with 33 flagellomeres. Europe, Far East Russia ................................. I. veressi (KISS, 1915) - Apical bands on tergites ± reddish. Larger, body length 11-13 mm. Antenna with 36-39 flagellomeres ....................................................................................................................... 4 4 2nd tergite red; 3rd tergite with yellow apical band, black. Whole Palaearctic region ............ ............................................................................................ I. lautatorius DESVIGNES, 1856 - 2nd and 3rd tergites red (ssp. sarcitorius) or reddish-yellow (ssp. caucasicus MEYER); 3rd tergite black basally. Europe. Uzbekistan, Kirgistan (HEINRICH, 1978) ................................ .............................................................................................. I. sarcitorius LINNAEUS, 1758 5 Hind coxa with yellow spot dorsally OR propodeum with blunt apophyses or at least apical transverse carina of propodeum with characteristic edge. Large, body length 16- 22 mm. Antenna with 39-47 flagellomeres, bristle-shaped, preapical flagellomeres square or longer than wide. Hind coxa without scopa ........................................ (group B) 6 - Hind coxa black or propodeum without blunt apophyses .................................................... 7 1533 1533 6 Hind femur red. Propodeum with blunt apophyses or at least apical transverse carina of propodeum with characteristic lateral edge. Metasoma black, with apical spots on (4th- 5th)- 6th-7th tergites. Antenna with 39-45 flagellomeres. Hind tibia narrowly darkened (1/15) at apex. Hind coxa black. Europe, Kirgistan, Far East Russia (MEYER 1933) ............ ............................................................................................ I. quaesitorius LINNAEUS, 1761 - Hind femur black, hind coxa with large dorsal yellow spot. Propodeum with blunt apophyses. Very large, body length 22-23 mm. Antenna with 44-57 flagellomeres. 2nd and 3rd tergites red, 5th to 7th tergites with ivory spots. Hind tibia black-yellow-black. Hind tarsus black. Europe, Far East Russia (UCHIDA 1926) .................................................. ............................................................................................... I. primatorius FORSTER, 1771 7 Antenna bristle-shaped, e.g. preapical flagellomeres square or longer than wide, rarely slightly widened. (adopted from HILPERT 1992): Hind coxa densely punctate, always without distinct scopa or longitudinal edge. If thyridium wider than the interval, than strongly transversal (orthogonal to the length axis of tergite) ............................................................................ .8 - Antenna filiform, e.g. preapical flagellomeres wider than long (seen from ventral). Hind coxa often differentiated: with scopa, longitudinal edge or scattered punctures (then the preapical flagellomere might be square). If thyridium wider than the interval, thyridium ± oblique to the length axis of the tergite ......................................................... .24 8 Thyridium at least as wide as the interval, usually much wider (fig. 76) (if rarely smaller, then 5th to 7th tergites with ivory spots or mandible with equally long teeth or mandible not distinctly narrowed apically.) ....................................................... (group C).9 - Thyridium smaller than the interval; if wider, then thyridium oblique and gastrocoelus short and only slightly impressed medially ...................................................... (group D).15 9 6th and 7th tergites with ivory spots ................................................................................... .10 - 5th to 7th tergites with ivory spots. (At least 2nd tergite mainly red, 3rd tergite often ± black apically or (in I. cinxiae) mandibular teeth of almost equal length) ......................... 11 10 Legs except black coxae and trochanters red. Scapus, pedicel and basal flagellomeres red. Scutellum red. Hind femur stout, 3.4x longer than wide. Siberia ................................... ...................................................................................................... I. paravulnerator nov.sp. - Hind femur mainly black. Scapus and pedicel black. Scutellum yellow. Hind femur slenderer, 3.9x longer than wide. Siberia ................................. I. pseudemancipatus nov.sp. 11 Mandible not distinctly narrowed apically, mandibular teeth of almost equal length (as fig. 43). Antenna with 34-36 flagellomeres; 1st flagellomere c.2.6x longer than wide. Body length 11.5-11.9 mm. 2nd tergite completely red. Hind femur mainly black; hind tibia red, basally and in apical 0.25 black. Hind tarsus black. Europe, Buyatsk region (HORSTMANN 2006) ............................................................ I. cinxiae KRIECHBAUMER, 1890 - Mandible normally narrowed apically, teeth of different length. 3rd tergite red. 5th tergite with large ivory spot. Hind femur largely red OR antenna with at least 37 flagellomeres ..................................................................................................................... 12 12 Mesoscutum and basal flagellomeres red. Hind femur red. 1st flagellomere c.2.3x longer than wide. Mid and hind coxae densely pilose, but hind coxa without scopa. Siberia ............................................................................................... I. mesonotator nov.sp. - Mesoscutum black ............................................................................................................. 13 13 Hind tibia almost completely red, at most black in apical 1/20. Thyridium rather short, but very wide and deep. Hind femur black, basally and apico-dorsally red. Antenna with 35 flagellomeres; 1st flagellomere 1.9x longer than wide. (adopted from HILPERT 1992): 2nd and 3rd tergite completely red. Body length 10.1 mm. Far East Russia .... I. emancipatops HEINRICH, 1978 - Hind tibia at least blackish in apical 1/7. Hind femur mainly red (if in I. gracilicornis rarely black, then temples strongly and almost linearly narrowed behind eye). Antenna with 34-42 flagellomeres. Scutellum always yellow ......................................................... 14 14 1st flagellomere slender, 2.4-3.1x longer than wide, antenna slightly lanceolate (widest flagellomeres 0.8-0.9x as long as wide). Color variable: Basal flagellomeres, width of ivory metasomal spots and color of hind femur variable. Europe, Far East Russia (ROMAN 1927) ............................................................. I. gracilicornis GRAVENHORST, 1829 6 Hind femur red. Propodeum with blunt apophyses or at least apical transverse carina of propodeum with characteristic lateral edge. Metasoma black, with apical spots on (4th- 5th)- 6th-7th tergites. Antenna with 39-45 flagellomeres. Hind tibia narrowly darkened (1/15) at apex. Hind coxa black. Europe, Kirgistan, Far East Russia (MEYER 1933) ............ ............................................................................................ I. quaesitorius LINNAEUS, 1761 6 Hind femur red. Propodeum with blunt apophyses or at least apical transverse carina of propodeum with characteristic lateral edge. Metasoma black, with apical spots on (4th- 5th)- 6th-7th tergites. Antenna with 39-45 flagellomeres. Hind tibia narrowly darkened (1/15) at apex. Hind coxa black. Europe, Kirgistan, Far East Russia (MEYER 1933) ............ ............................................................................................ I. quaesitorius LINNAEUS, 1761 - Hind femur black, hind coxa with large dorsal yellow spot. Propodeum with blunt apophyses. Very large, body length 22-23 mm. Antenna with 44-57 flagellomeres. 2nd and 3rd tergites red, 5th to 7th tergites with ivory spots. Hind tibia black-yellow-black. Hind tarsus black. Europe, Far East Russia (UCHIDA 1926) .................................................. ............................................................................................... I. primatorius FORSTER, 1771 7 Antenna bristle-shaped, e.g. preapical flagellomeres square or longer than wide, rarely slightly widened. Hind coxa densely punctate, always without distinct scopa or longitudinal edge. If thyridium wider than the interval, than strongly transversal (orthogonal to the length axis of tergite) ............................................................................ .8 - Antenna filiform, e.g. preapical flagellomeres wider than long (seen from ventral). Hind coxa often differentiated: with scopa, longitudinal edge or scattered punctures (then the preapical flagellomere might be square). If thyridium wider than the interval, thyridium ± oblique to the length axis of the tergite ......................................................... .24 8 Thyridium at least as wide as the interval, usually much wider (fig. 76) (if rarely smaller, then 5th to 7th tergites with ivory spots or mandible with equally long teeth or mandible not distinctly narrowed apically.) ....................................................... (adopted from HILPERT 1992): Very large, body length 22-23 mm. Antenna with 44-57 flagellomeres. 2nd and 3rd tergites red, 5th to 7th tergites with ivory spots. Hind tibia black-yellow-black. Hind tarsus black. Europe, Far East Russia (UCHIDA 1926) .................................................. ............................................................................................... I. primatorius FORSTER, 1771 1534 - 1st flagellomere stouter, 2.1x longer than wide, antenna distinctly lanceolate (widest flagellomeres 0.65x as long as wide). Europe, Far East Russia (ROMAN 1927) .................... ........................................................................................... I. emancipatus WESMAEL, 1845 15 Antenna with 28-35 flagellomeres .................................................................................... 16 - Antenna with at least 37 flagellomeres .............................................................................. 21 16 3rd tergite mostly or completely black. Antenna with 32-35 flagellomeres; 1st flagellomere c.2.2x longer than wide. Area superomedia slightly wider than long. Europe, Far East Russia (UCHIDA 1926, MEYER 1930) ......................................................... ................................................................................ I. submarginatus GRAVENHORST, 1829 - 3rd tergite completely red ................................................................................................... 17 17 Scutellum completely yellow ............................................................................................ 18 - Scutellum at most partly yellow, usually black ................................................................. 20 18 Malar space at least as long as 1st flagellomere. Antenna with 30-35 flagellomeres. Petiolus at spiracles with two elevated carinae, area between them ± concave. Postpetiolus red, median field with regular strong aciculation. Antenna except ivory ring always black, sometimes ivory spot at orbit opposite to antenna. Europe, Siberia ........ ............................................................................................. I. minutorius DESVIGNES, 1856 - Malar space shorter than 1st flagellomere. Petiolus without elevated longitudinal carinae ............................................................................................................................... 19 19 Hind femur slenderer, 3.6-4.2x longer than wide. Antenna with 32-35 flagellomeres. If femur rather stout, than with more flagellomeres. Very variable species. Europe, Irkutsk (KOKUJEV 1904) .................................................... I. sculpturatus HOLMGREN, 1864 - Hind femur stouter, 3.0-3.6x longer than wide. Antenna with 28-33 flagellomeres. Postpetiolus usually reddish. If femur rather slender, than with less flagellomeres. Scapus, pedicel and basal flagellomeres completely red. Europe, Iran ................................. ....................................................................................... I. fulvicornis GRAVENHORST, 1829 20 Antenna with weak or without pale ring, with 31-32 flagellomeres; basal flagellomeres red. Scutellum black. Area superomedia hexagonal. Postpetiolus apically, 2nd and 3rd tergites completely red. Thyridium distinctly smaller than the interval. Kazachstan ............ ............................................................................................... I. transaralius HILPERT, 1992 - Antenna with distinct pale ring, with 28 flagellomeres. Postpetiolus mainly red. Scutellum partly reddish. Area superomedia trapezoid (widest apically). Thyridium very small. Far East Russia. (According to HILPERT 1992 probably synonym to I. leucopeltis THOMSON) ......................................................... I. inoblidendus HEINRICH, 1978 21 5th to 7th tergites with large yellow spots. Antenna with 39-45 flagellomeres. Scutellum yellow and antenna with yellow ring. Pterostigma yellow or reddish. (adopted from HILPERT 1992): 4th tergite at most with small ivory spot ......................................................................................................... 22 - At most 6th and 7th tergites with large ivory spots, 5th tergite often with a smaller spot. Antenna with 42-45 flagellomeres .................................................................................... 23 22 Hind femur completely red and tergites mainly or completely black. 1st flagellomere 2.6x longer than wide. Hind tibia infuscate in apical 1/10. Palaearctic region, Far East Russia (MEYER 1933) ................................................ I. quaesitorius LINNAEUS, 1761 (part) - At least 2nd and 3rd tergite completely red (often apical tergites more or less red) or hind femur black. 1st flagellomere 1.9x longer than wide. Hind tibia infuscate in apical 1/10 or completely red. Subtegular ridge thick, usually widely yellow. Lateral field of mesoscutum often reddish. Europe, Altai region (HEINRICH 1978) ....................................... ................................................................................................... I. bellipes WESMAEL, 1845 23 Hind femur completely red; hind tibia red-black. Hind tarsus black. Scutellum black (if yellow: var. jakovlevi). 1st flagellomere slender, 3.0.3.2x longer than wide. Tergites completely black. Central Europe, Irkutsk (KOKUJEV 1904 as jakovlevi) ............................. ..................................................................................................... I. cessator MÜLLER, 1776 p ) 21 5th to 7th tergites with large yellow spots. Antenna with 39-45 flagellomeres. Scutellum yellow and antenna with yellow ring. Pterostigma yellow or reddish. 4th tergite at most with small ivory spot ......................................................................................................... 2 - At most 6th and 7th tergites with large ivory spots, 5th tergite often with a smaller spot. Antenna with 42-45 flagellomeres .................................................................................... 2 1535 - Hind femur black. Mandible with wide, blunt upper tooth and very small lower one. Hypopygium moderatly widened (metasoma semi-amblypygous). Hind tibia red, subbasally ± yellowish, apically not or narrowly darkened. Hind tarsus red. 1st flagellomeres 1.5x longer than wide. 2nd and 3rd tergites red, 3rd tergite basally black. Central Asia ...........................................................................I. mordaxiops HEINRICH, 1978 24 Thyridium not wider than the interval ............................................................................... 25 - Thyridium distinctly wider than the interval, oblique and deeply impressed, usually close to basal margin of 2nd tergite. 6th and 7th tergites or only 7th tergite with ivory spots ...................................................................................................................(group I).97 25 Hind coxa with scopa, longitudinal edge or scattered punctures ventrally ........................ 26 - Hind coxa without scopa or longitudinal edge; densely punctate ventrally, but punctures often even denser at area of scopa ..................................................................... 53 26 Hind coxa with a small dense scopa resembling a narrow elevation. 6th and 7th tergites with yellow spots, 5th tergite with small spot OR these spots are strongly widened. Scutellum yellow. (adopted from HILPERT 1992): Antenna ± lanceolate, widest flagellomeres 1.5-2.2x wider than long, preapical flagellomeres weakly to strongly transverse. Thyridium about as wide as the interval. Hind tarsus at least in the apical 3/4 or completely black....... (group E1)27 - Hind coxa without elevated small scopa; if scopa present, then larger .............................. 32 27 Tarsomeres distinctly widened (fig. 42). Hypopygium rather long (fig. 82), about as long as the distance between its apical margin and metasomal apex (Metasoma semi- amblypygous). Basal flagellomeres reddish. 3rd tergite reddish, with black basal band. Hind femur red. Far East Russia ...................................................... I. orientopodius nov.sp. - Tarsomeres slender. Hypopygium much shorter than the distance between its apical margin and metasomal apex .............................................................................................. 28 28 Larger, body length 13.2-15.5 mm. Antenna with 41-44 flagellomeres. Often all tergites (except yellow spots) black................................................................................... 29 - Smaller, body length 11-14 mm. Antenna with 36-41 flagellomeres. Some tergites always completely red ....................................................................................................... 30 29 Usually genal carina ventrally obsolete, hypostomal carina often distinctly elevated. 1st flagellomere 2.1x longer than wide. Hind femur 3.2x longer than wide, with scattered punctures in ventral 1/3. Coloration of hind femur and tibiae and metasoma variable. Europe, Iran, South Russia .................................................. I. tuberculipes WESMAEL, 1848 - Genal carina complete, hypostomal carina narrow. 1st flagellomere 1.9x longer than wide. Hind femur 3.7x longer than wide, densely punctate. Siberia...................................... ........................................................................................................ I. breviscopatus nov.sp. 30 Antenna with 36-38 flagellomeres, preapical flagellomeres distinctly transverse, 0.67x as long as wide. 3rd tergite rarely black basally. Hind femur black, 3.1x longer than wide. Europe, Irktusk (KOKUJEV 1904), Altai Region (HEINRICH 1978) ............................... .............................................................................................. I. cerebrosus WESMAEL, 1859 - Antenna with 37-41 flagellomeres, preapical flagellomere 0.92x as long as wide. 3rd tergite often blackish basally. Hind femur slenderer, at least 3.3x longer than wide ......... 31 31 Ivory spots on (5th-) 6th to 7th tergites very wide. Hind femur 3.3x longer than wide, usually large red. 3rd tergite red, without yellowish tint. Europe, Kazachstan (RASNITSYN & SIYTAN 1981) .................................................... I. balteatus WESMAEL, 1845 - Ivory spots on 6th and 7th tergites not distinctly widened. Hind femur slender, 4.1x longer than wide, black. 3rd tergite red, basally black, apically with yellowish tint. Germany, Altai region ............................................................... I. altaicola HEINRICH, 1978 32 Hind coxa with ± distinct scopa ..................................................................... (group E2) 33 - Hind coxa without scopa, but with scattered punctures or ± distinct longitudinal edge ventrally (if longitudinal edge present, then mandible distinctly thickened). (adopted from HILPERT 1992): Coloration of hind femur and tibiae and metasoma variable. Europe, Iran, South Russia .................................................. I. tuberculipes WESMAEL, 1848 - Genal carina complete, hypostomal carina narrow. 1st flagellomere 1.9x longer than wide. Hind femur 3.7x longer than wide, densely punctate. Siberia...................................... ........................................................................................................ I. breviscopatus nov.sp. 30 Antenna with 36-38 flagellomeres, preapical flagellomeres distinctly transverse, 0.67x as long as wide. 3rd tergite rarely black basally. Hind femur black, 3.1x longer than wide. Europe, Irktusk (KOKUJEV 1904), Altai Region (HEINRICH 1978) ............................... .............................................................................................. I. cerebrosus WESMAEL, 1859 - Antenna with 37-41 flagellomeres, preapical flagellomere 0.92x as long as wide. 3rd tergite often blackish basally. Hind femur slenderer, at least 3.3x longer than wide ......... 31 31 Ivory spots on (5th-) 6th to 7th tergites very wide. Hind femur 3.3x longer than wide, usually large red. 3rd tergite red, without yellowish tint. Europe, Kazachstan (RASNITSYN & SIYTAN 1981) .................................................... I. balteatus WESMAEL, 1845 - Ivory spots on 6th and 7th tergites not distinctly widened. Hind femur slender, 4.1x longer than wide, black. 3rd tergite red, basally black, apically with yellowish tint. Germany, Altai region ............................................................... I. altaicola HEINRICH, 1978 32 Hind coxa with ± distinct scopa ..................................................................... (group E2) 33 - Hind coxa without scopa, but with scattered punctures or ± distinct longitudinal edge ventrally (if longitudinal edge present, then mandible distinctly thickened). Widest flagellomeres sometimes c.2.3x wider than long. Antenna with 32-43 flagellomeres ....... 47 33 Widest flagellomeres at most 2.2x wider than long. Antenna with 27-48 flagellomeres ... 34 - Antenna strongly lanceolate, widest flagellomere c.2.5x wider than long, with 34-43 flagellomeres. Hind femur with scattered punctures in ventral 1/2 ................................... 42 - Hind femur black. Mandible with wide, blunt upper tooth and very small lower one. Hypopygium moderatly widened (metasoma semi-amblypygous). Hind tibia red, subbasally ± yellowish, apically not or narrowly darkened. Hind tarsus red. 1st flagellomeres 1.5x longer than wide. 2nd and 3rd tergites red, 3rd tergite basally black. Central Asia ...........................................................................I. mordaxiops HEINRICH, 1978 - Tarsomeres slender. Hypopygium much shorter than the distance between its apical margin and metasomal apex .............................................................................................. 2 28 Larger, body length 13.2-15.5 mm. Antenna with 41-44 flagellomeres. Often all tergites (except yellow spots) black................................................................................... 2 - Smaller, body length 11-14 mm. Antenna with 36-41 flagellomeres. Some tergites always completely red ....................................................................................................... 3 29 Usually genal carina ventrally obsolete, hypostomal carina often distinctly elevated. 1st flagellomere 2.1x longer than wide. (adopted from HILPERT 1992): Widest flagellomeres sometimes c.2.3x wider than long. Antenna with 32-43 flagellomeres ....... 47 33 Widest flagellomeres at most 2.2x wider than long. Antenna with 27-48 flagellomeres ... 34 - Antenna strongly lanceolate, widest flagellomere c.2.5x wider than long, with 34-43 flagellomeres. Hind femur with scattered punctures in ventral 1/2 ................................... 42 1535 - Hind femur black. Mandible with wide, blunt upper tooth and very small lower one. Hypopygium moderatly widened (metasoma semi-amblypygous). Hind tibia red, subbasally ± yellowish, apically not or narrowly darkened. Hind tarsus red. 1st flagellomeres 1.5x longer than wide. 2nd and 3rd tergites red, 3rd tergite basally black. Central Asia ...........................................................................I. mordaxiops HEINRICH, 1978 24 Thyridium not wider than the interval ............................................................................... 25 - Thyridium distinctly wider than the interval, oblique and deeply impressed, usually close to basal margin of 2nd tergite. 6th and 7th tergites or only 7th tergite with ivory spots ...................................................................................................................(group I).97 25 Hind coxa with scopa, longitudinal edge or scattered punctures ventrally ........................ 26 - Hind coxa without scopa or longitudinal edge; densely punctate ventrally, but punctures often even denser at area of scopa ..................................................................... 53 26 Hind coxa with a small dense scopa resembling a narrow elevation. 6th and 7th tergites with yellow spots, 5th tergite with small spot OR these spots are strongly widened. Scutellum yellow. Antenna ± lanceolate, widest flagellomeres 1.5-2.2x wider than long, preapical flagellomeres weakly to strongly transverse. Thyridium about as wide as the interval. Hind tarsus at least in the apical 3/4 or completely black....... (group E1)27 - Hind coxa without elevated small scopa; if scopa present, then larger .............................. 32 27 Tarsomeres distinctly widened (fig. 42). Hypopygium rather long (fig. 82), about as long as the distance between its apical margin and metasomal apex (Metasoma semi- amblypygous). Basal flagellomeres reddish. 3rd tergite reddish, with black basal band. Hind femur red. Far East Russia ...................................................... I. orientopodius nov.sp. - Tarsomeres slender. Hypopygium much shorter than the distance between its apical margin and metasomal apex .............................................................................................. 28 28 Larger, body length 13.2-15.5 mm. Antenna with 41-44 flagellomeres. Often all tergites (except yellow spots) black................................................................................... 29 - Smaller, body length 11-14 mm. Antenna with 36-41 flagellomeres. Some tergites always completely red ....................................................................................................... 30 29 Usually genal carina ventrally obsolete, hypostomal carina often distinctly elevated. 1st flagellomere 2.1x longer than wide. Hind femur 3.2x longer than wide, with scattered punctures in ventral 1/3. (adopted from HILPERT 1992): Hind femur 3.2x longer than wide, with scattered punctures in ventral 1/3. Coloration of hind femur and tibiae and metasoma variable. Europe, Iran, South Russia .................................................. I. tuberculipes WESMAEL, 1848 1536 1536 34 Scutellum black. Antenna without ivory ring, with 42-45 flagellomeres. Metasoma except ivory apical spots black. Hind femur red, tarsi slightly enlarged. Europe, Far East Russia (MEYER 1930) ................................................ I. melanosomus WESMAEL, 1855 - Scutellum ivory, yellow or red .......................................................................................... 35 35 Antenna with 27-28 flagellomeres, petiolus red. (5th-) 6th and 7th tergites with ivory spots. Hind femur with scattered punctures in ventral 1/2. Hind tibia yellow centrally. Preapical flagellomere strongly transverse, 0.66x as long as wide. Scopa often ± obsolete. Europe, Far East Russia (MEYER 1933) .............. I. validicornis HOLMGREN, 1864 - Antenna with at least 30 flagellomeres OR petiolus black ................................................ 36 36 All tarsi distinctly widened. Body length 11-12 mm ......................................................... 37 - Tarsi slender OR slightly and/or partly widened ............................................................... 39 37 Antenna with 33-37 flagellomeres. Scutellum yellow. 2nd and 3rd tergites completely red. Hind femur mainly black; stout, 2.5x longer than wide. Europe, Far East Russia (ROMAN 1927) ........................................................................ I. ligatorius THUNBERG, 1822 - Antenna with at least 43 flagellomeres. Hind femur red; at least 3.0x longer than wide. 2nd and 3rd tergite reddish with ± black coloration ............................................................. 38 38 Body length 11 mm. Antenna with 43 flagellomeres. Scutellum red. Hind femur slenderer, 3.7x longer than wide. Mid coxa without distinct scopa. Siberia .......................... .......................................................................................................... I. rufolateralis nov.sp. - Body length 15 mm. Antenna with 45 flagellomeres. Scutellum yellowish-red. Hind femur stouter, 3.0x longer than wide. Mid coxa with small apico-ventral scopa. Siberia ..... ................................................................................................................ I. scopator nov.sp. 39 Hind tibia yellow centrally. 2nd and 3rd tergites red (in I. crassifemur sometimes darkened) .......................................................................................................................... .40 - Hind tibia not yellow centrally .......................................................................................... 45 40 Hind femur with scattered punctures in ventral half. Metasoma almost completely black (except ivory apical spots). Hind coxa densely punctate. Antenna with 30-34 flagellomeres. Hind tibia black-yellow-black. Central Europe, Far East Russia (UCHIDA 1926, ROMAN 1927) ............................................................. I. crassifemur THOMSON, 1886 - Hind femur at most in ventral 1/3 or basally with scattered punctures, usually denser punctate ............................................................................................................................. 41 41 Hind tibia black-yellow-black, black in apical 2/5. Hind femur with scattered punctures in basal 1/2 and ventrally. Antenna with 33-36 flagellomeres, preapical flagellomere less wide, 0.64x as long as wide. Europe, Far East Russia (UCHIDA 1926) ........................... ............................................................................................... (adopted from HILPERT 1992): I. molitorius LINNAEUS, 1761 - Hind tibia red-yellow-red-black; black in apical 1/4. Hind femur densely punctate. Antenna with 30-34 flagellomeres, preapical flagellomere strongly transverse, 0.5x as long as wide. Europe, West Siberia (WOLDSTEDT 1881) ....................................................... .......................................................................................... I. confusor GRAVENHORST, 1820 42 Hind femur densely punctate externally. Collare ivory. Wings slightly infuscate. Hind leg black. 2nd and 3rd tergites red, 3rd tergite black basally OR 2nd tergite only slightly reddish. 6th and 7th tergites with ivory spots. Antenna with 37-43 flagellomeres. South Europe, Irkutsk (KOKUJEV 1904) ........................... I. haemorrhoicus KRIECHBAUMER, 1887 - Hind femur with scattered punctures in ventral 1/3 to 1/2 ................................................. 43 43 2nd and 3rd tergites red. Area superomedia distinctly longer than wide. Scopa on hind coxa weak. Europe, Irkutsk (KOKUJEV 1904), Far East Russia (ROMAN 1927) ..................... .............................................................................................. I. extensorius LINNAEUS, 1758 - 2nd and 3rd tergites blackish ............................................................................................... 44 44 Antenna with 43-47 flagellomeres. France, Altai region (HEINRICH 1978) ........................... ....................................................................................................... I. nebulosae HINZ, 1975 - Antenna with 32-33 flagellomeres. Far East Russia ................ I. magistratus HILPERT, 1992 45 Face, gena and mesoscutum mainly red. Collare ivory, with reddish margins. Siberia ......... ................................................................................................. I. zoologicus HILPERT, 1992 34 Scutellum black. Antenna without ivory ring, with 42-45 flagellomeres. Metasoma except ivory apical spots black. Hind femur red, tarsi slightly enlarged. Europe, Far East Russia (MEYER 1930) ................................................ I. melanosomus WESMAEL, 1855 1537 - Face not completely red. Mesoscutum black ..................................................................... 46 46 2nd and 3rd tergites completely red, 6th and 7th tergites with ivory spots. Antenna with 34-37 flagellomeres, preapical flagellomeres 0.83x as long as wide. Body length 10.5 mm. Hind tibia not yellow. Hind tarsus mainly red. Scopa of hind coxa weak. Europe, Altai region (HEINRICH 1978) ............................................. I. grandicornis THOMSON, 1886 - 2nd and 3rd tergites at most partly red, 5th tergite with small ivory spot. Hind coxa with rather weak scopa. Hind tarsus mainly black. Antenna with 34-39 flagellomeres, preapical flagellomeres 0.72x as long as wide. Body length 13-14 mm. Europe, Far East Russia (MEYER 1933) ................................................. I. computatorius MÜLLER, 1776 47 Hind coxa with longitudinal edge ventrally. Mandible thick, with parallel sides and blunt teeth (fig. 43). Gena strongly widened ventrally, c.1.5x wider than eye (seen from lateral). Siberia ................................................................................ I. mandibulatus nov.sp. - Hind coxa with ± scattered punctures apico-ventrally, without longitudinal edge. Mandible not thickened, sides narrowed apically, teeth pointed. Gena less strongly widened .......................................................................................................... (adopted from HILPERT 1992): (group E3) 48 48 Hind coxa with almost smooth area apico-ventrally (fig. 39). Lateral field of mesoscutum centrally with very scattered punctures. Fore and mid tarsomeres widened. Antenna slightly lanceolate. Siberia .............................................. I. brevipunctatus nov.sp. - Hind coxa with scattered punctures or rather dense punctures, without almost smooth areas .................................................................................................................................. 49 49 3rd tergite black basally. Basal flagellomeres red, antenna strongly lanceolate. Hind coxa rather densely punctate ventrally, but strongly shining between punctures. Hind femur stout, 2.9-3.1x longer than wide .............................................................................. 50 - 2nd and 3rd tergites almost always completely red, rarely with yellowish tint, not black basally, but sometimes both tergites completely black ...................................................... 51 50 Area superomedia longer than wide. 5th tergite black. 2nd and 3rd tergites red, with yellowish and blackish color patterns. Antenna with 35 flagellomeres. Hind femur 2.9x longer than wide. Hind tibia black in apical 1/8. Body length 10.7 mm. Far East Russia. (according to HILPERT 1992 probably a subspecies of I. alius TISCHBEIN) ............................ ................................................................................................ I. confundor HEINRICH, 1978 - Area superomedia wider than long. 5th tergite with wide ivory spot. 2nd and 3rd tergites red, 3rd tergite black basally. Antenna with 39 flagellomeres. Hind femur 3.1x longer than wide. Hind femur red, yellowish centrally. Body length 11-16 mm. Far East Russia ............................................................................. I. pseudocaedator HEINRICH, 1978 51 Large, body length 16-17 mm. Antenna with 44-45 flagellomeres. Hind tibia distinctly differentiated: with fewer spurs externally and sharp border to the densely pilose internal side. Hind femur with scattered punctures in ventral 2/3. Hind coxa with ± scattered punctures externally and weak scopa. Europe, Irkutsk (HEINRICH, 1931), Far East Russia (ROMAN 1927) ........................................................ I. hypolius THOMSON, 1888 - Smaller species. Antenna with lower number of flagellomeres ......................................... 52 52 2nd and 3rd tergites black, at most with reddish tint. Gena, frontal orbit and basal flagellomeres ventrally red. Hind coxa with very scattered punctures ventrally. Hind femur stout, 3.0x longer than wide, with scattered punctures in ventral 1/4. Antenna with 35-37 flagellomeres, preapical flagellomere distinctly transverse. Far East Russia ...... ...................................................................................................... I. helenae HILPERT, 1992 - 2nd and 3rd tergites red. Antenna strongly lanceolate, with 34-38 flagellomeres; widest flagellomere c.0.42x as long as wide. Europe, Irkutsk (KOKUJEV 1904) ............................... ....................................................................................................... I. alius TISCHBEIN, 1875 53 Metasoma except ivory marks black ................................................................. (group F).54 - Metasoma with red or yellow tergites ............................................................................... 62 54 (4th-) 5th-7th tergites with ivory spots. Antenna with 37-42 flagellomeres ......................... (adopted from HILPERT 1992): 55 - At most 6th and 7th tergites with ivory spots. Hind femur black. Antenna with 37-40 or 43-47 flagellomeres. Hind tibia and hind tarsus ± black apically ...................................... 57 . 46 - Face not completely red. Mesoscutum black .. - Face not completely red. Mesoscutum black ......................................................... - Face not completely red. Mesoscutum black . 46 2nd and 3rd tergites completely red, 6th and 7th tergites with ivory spots. Antenna with 34-37 flagellomeres, preapical flagellomeres 0.83x as long as wide. Body length 10.5 mm. Hind tibia not yellow. Hind tarsus mainly red. Scopa of hind coxa weak. Europe, Altai region (HEINRICH 1978) ............................................. I. grandicornis THOMSON, 188 1538 55 Head and mesosoma except yellow scutellum black (fig. 16). Femora, tibiae and tarsi red, hind tibia and hind tarsus slightly darkened apically. Siberia ......... I. sayanicus nov.sp. - Frontal orbit and upper margin of pronotum ± yellow. Hind femur black. Hind tibia and tarsus widely black ............................................................................................................ 56 red, hind tibia and hind tarsus slightly darkened apically. Siberia ......... I. sayanicus nov.sp. - Frontal orbit and upper margin of pronotum ± yellow. Hind femur black. Hind tibia and tarsus widely black ............................................................................................................ 56 56 Antenna with 39-42 flagellomeres, preapical flagellomere c.0.88x as long as wide, widest flagellomeres c.0.61x as long as wide. Upper margin of pronotum not yellow OR with yellow stripe frontally, then pale stripe widely interrupted. Frontal orbit narrowly ivory. Area superomedia slightly transverse. Hind tibia distinctly red centrally. Europe, Western Siberia (ROMAN 1927) ................ I. haglundi HOLMGREN, 1864 - Antenna with 38 flagellomeres, preapical flagellomere c.0.63x as long as wide. Upper margin of pronotum narrowly interrupted. Frontal orbit widely ivory up to vertex. Far East Russia ................................................................................. I. ustzazae HEINRICH, 1978 57 Antenna with 43-47 flagellomeres. Large, body length 15.2 mm. Hind femur with scattered punctures in ventral 1/2. Collare partly yellow. Hind coxa often with ± distinct scopa. France, Altai region (HEINRICH 1978) .................... I. nebulosae HINZ, 1975 - Antenna with 37-40 flagellomeres .................................................................................... 58 58 Hind tibia completely black. Hind coxa with variable punctures or with weak scopa. Preapical flagellomere 0.54x as long as wide. Scutellum completely yellow. Ivory spots on apical tergites wide. Median field of postpetiolus more than 2x wider than lateral field. South Europe, Irkutsk (KOKUJEV 1904, MEYER 1933) ................................................. ..................................................................... I. haemorrhoicus KRIECHBAUMER, 1887 (part) - Hind tibia infuscate in apical 1/3 OR preapical flagellomeres slightly transverse. (adopted from HILPERT 1992): Hind coxa with even punctuation. Scutellum almost completely yellow or ivory. Body length 12-14 mm ......................................................................................................................... .59 59 Antenna with 27-29 flagellomeres, 1st flagellomere c.2.x longer than wide. Body slender. Temple slightly widened behind eye. Hind femur with dense punctures. European Alps, Siberia (HILPERT 1992) .............................. I. buryas HEINRICH, 1949 (part) - Antenna with 35-40 flagellomeres. 1st flagellomere at most 1.8x longer than wide. Temple ± narrowed behind eye. Hind femur with scattered punctures in ventral 1/3 to 1/5 ..................................................................................................................................... 60 60 Antenna with 40 flagellomeres. 1st flagellomere 1.8x longer than wide. Hind tibia and tarsus red. Hind femur with scattered punctures in ventral 1/5. Siberia ................................ ........................................................................................................... I. inquinatops nov.sp. - Antenna with 35-38 flagellomeres, 1st flagellomere at most 1.5x longer than wide. Hind tibia ± infuscate apically, hind tarsus mainly black. Hind femur with scattered punctures in ventral 1/3 .................................................................................................... .61 61 Area superomedia square or slightly longer than wide, hexagonal. Thyridium narrow. Upper mandibular tooth wide and blunt. Inner orbit widely reddish-yellow. Central Europe, Far East Russia (MEYER 1933, UCHIDA 1935) ......................................................... ............................................................................................... I. inquinatus WESMAEL, 1845 - Area superomedia c.1.3x longer than wide, rectangular. Thyridium almost as wide as the interval. Mandible normal. Inner orbit yellow, edges of clypeus reddish. Siberia ........... ...................................................................................................... I. sibiricus ROMAN, 1904 62 Scutellum yellow OR antenna with at least 30 flagellomeres. If mesoscutum red, then scutellum often red or black .............................................................................................. 63 - Scutellum black or red. 5th tergite without ivory spot. Antenna with 22-29 flagellomeres .................................................................................................... (group H) 92 63 Hind tibia with clear yellow color centrally ................................................... (group G1) 64 - Hind tibia without yellow color centrally (or only in small parts) ..................................... 67 64 5th tergite at most with small ivory spot. 2nd and 3rd tergites red. Antenna with 33-38 flagellomeres, preapical flagellomeres strongly transverse, 0.59x as long as wide, widest flagellomere 0.58x as long as wide. Hind coxa with variable punctures ventrally. Hind tibia narrowly black basally, yellow subbasally, then weakly red and black in apical 1/3. Europe, West Siberia (WOLDSTEDT 1881) .............................................. .............................................................................................. I. melanotis HOLMGREN, 1864 56 Antenna with 39-42 flagellomeres, preapical flagellomere c.0.88x as long as wide, widest flagellomeres c.0.61x as long as wide. Upper margin of pronotum not yellow OR with yellow stripe frontally, then pale stripe widely interrupted. Frontal orbit narrowly ivory. Area superomedia slightly transverse. Hind tibia distinctly red centrally. Europe, Western Siberia (ROMAN 1927) ................ I. (adopted from HILPERT 1992): 68 Ivory spots on apical tergites not distinctly wide. Body length 10 15 mm. ....... (group G2.1) 6 - Ivory spots on apical tergites very wide. Face usually with extended red coloration. Body length 8-11 mm. ................................................................................. (group G2.2) 7 69 Mandible strongly thickened. Hypostomal carina widened. Hind tibia red, black apically. Body length 13.6-14.1 mm. Usually 2nd and 3rd tergite completely red, in I. bucculentus teberdensis HEINRICH 3rd tergite only medially red. Europe, Kazachastan (RASNITSYN & SIYTAN 1981) ................................................ I. bucculentus WESMAEL, 1845 - Mandible not thickened. Hypostomal carina narrow. Hind tibia completely red or black apically. Smaller, body length 10.4-11.5 mm .................................................................... 7 p y y g 70 Antenna with 33-35 flagellomeres. Area superomedia c1.1x longer than wide. Hind femur stouter, 3.2-3.3x longer than wide. Europe, Kazachstan (RASNITSYN & SIYTAN 1981) ....................................................................... I. curtulus KRIECHBAUMER, 1882 (part) - Antenna with 37-40 flagellomeres. Hind femur slenderer, 3.5-3.6x longer than wide. Wings with brownish tint, pterostigma reddish ................................................................ .71 71 Hind femur with scattered punctures in ventral 1/2. Hind femur red. Postpetiolus and 2nd tergite red, 3rd tergite blackish (fig. 12). Siberia .................................... I. paravafer nov.sp. - Hind femur with dense punctures, mainly black. Usually postpetiolus, 2nd and 3rd tergites completely red. 4th tergite with a small ivory spot. Iran ............................................ ................................................................................... I. vafer meridionalis HEINRICH, 1929 72 Hind tibia black in apical 1/6. Body length 8 mm. 1st flagellomere 1.1-1.6x longer than wide. Hind femur completely black, stout, 2.6x longer than wide. 3rd tergite usual black basally. Ivory spot on 5th tergite often narrow, sometimes absent. Basal flagellomeres red or black. Europe, Mongolia (RASNITSYN & SIYTAN 1981) .............................................. .......................................................................................... I. caedator GRAVENHORST, 1829 - Hind tibia yellow or red, not black apically. Body length 10-11 mm. 1st flagellomere longer, c.1.8x longer than wide. Hind femur less stout, 3.3x longer than wide. Ivory spot on 5th tergite usually very wide. Basal flagellomeres red. 3rd tergite not black basally. Europe, Kazachstan (RASNITSYN & SIYTAN 1981), Iran ........................................... ......................................................................................... I. curtulus KRIECHBAUMER, 1882 73 Antenna with 27-35 flagellomeres ...................................................................................... 74 - Antenna with at least 36 flagellomeres .............................................................................. 80 74 Antenna strongly lanceolate, widest flagellomere c.2x wider than long; 1st flagellomere stout, c.1.1x longer than wide. Basal flagellomeres and scutellum red (fig. 7). Siberia ........ ............................................................................................................ I. lanceolator nov.sp. - Antenna less strongly lanceolate. 1st flagellomere slenderer ............................................. 75 70 Antenna with 33-35 flagellomeres. Area superomedia c1.1x longer than wide. (adopted from HILPERT 1992): haglundi HOLMGREN, 1864 - Antenna with 38 flagellomeres, preapical flagellomere c.0.63x as long as wide. Upper margin of pronotum narrowly interrupted. Frontal orbit widely ivory up to vertex. Far East Russia ................................................................................. I. ustzazae HEINRICH, 1978 57 Antenna with 43-47 flagellomeres. Large, body length 15.2 mm. Hind femur with scattered punctures in ventral 1/2. Collare partly yellow. Hind coxa often with ± distinct scopa. France, Altai region (HEINRICH 1978) .................... I. nebulosae HINZ, 1975 - Antenna with 37-40 flagellomeres .................................................................................... 5 1539 1539 - 5th to 7th tergites with large ivory spots OR collare clearly ivory. Antenna with 31-41 flagellomeres ..................................................................................................................... 6 - 5th to 7th tergites with large ivory spots OR collare clearly ivory. Antenna with 31-41 flagellomeres ..................................................................................................................... 65 65 Ivory spots on 5th to 7th tergite roundish, not transverse. Antenna usually with 34-37 flagellomeres (rarely 32-33). 2nd to 3rd tergites red. Area superomedia distinctly longer than wide. Hind femur with scattered punctures in ventral 1/3-1/2. Hind tibia red- yellow-red-black. Europa, Far East Russia (UCHIDA 1926) . I. suspiciosus WESMAEL, 1845 - Ivory spots on 5th to 7th tergite wide and transverse. Antenna with 30-34 flagellomeres. Area superomedia about as long as wide ........................................................................... 66 Area superomedia about as long as wide ........................................................................... 66 66 Hind femur 2.9x longer than wide. Hind tibia red-yellow-red-black, hind tarsus black in apical 1/4. 3rd tergite red, somtimes black baso-medially. Europa, Kazachstan, Mongolia (RASNITSYN & SIYTAN 1981) .................. I. caedator GRAVENHORST, 1829 (part) - Hind femur 3.7x longer than wide. Hind tibia and tarsus mainly yellow, with reddish tips. 3rd tergite yellow, with basal and apical black bands (fig. 19). Kirgistan ...................... .......................................................................................................... I. altaicurtulus nov.sp. 67 5th-7th tergites with large ivory spots or apical bands AND antenna with at least 32 flagellomeres .................................................................................................. (group G2) 68 - 5th tergite at most with smaller ivory spot OR antenna with 27-31 flagellomeres ................. ....................................................................................................................... (group G4) 73 68 Ivory spots on apical tergites not distinctly wide. Body length 10-15 mm. ....... (group G2.1) 69 - Ivory spots on apical tergites very wide. Face usually with extended red coloration. Body length 8-11 mm. ................................................................................. (group G2.2) 72 69 Mandible strongly thickened. Hypostomal carina widened. Hind tibia red, black apically. Body length 13.6-14.1 mm. Usually 2nd and 3rd tergite completely red, in I. bucculentus teberdensis HEINRICH 3rd tergite only medially red. Europe, Kazachastan (RASNITSYN & SIYTAN 1981) ................................................ I. (adopted from HILPERT 1992): (group G4) 73 5 tergite at most with smaller ivory spot OR antenna with 27 31 flagellomeres ................. ....................................................................................................................... (group G4) 73 68 Ivory spots on apical tergites not distinctly wide. Body length 10-15 mm. ....... (group G2.1) 69 - Ivory spots on apical tergites very wide. Face usually with extended red coloration. Body length 8-11 mm. ................................................................................. (group G2.2) 72 69 Mandible strongly thickened. Hypostomal carina widened. Hind tibia red, black apically. Body length 13.6-14.1 mm. Usually 2nd and 3rd tergite completely red, in I. bucculentus teberdensis HEINRICH 3rd tergite only medially red. Europe, Kazachastan (RASNITSYN & SIYTAN 1981) ................................................ I. bucculentus WESMAEL, 1845 - Mandible not thickened. Hypostomal carina narrow. Hind tibia completely red or black apically. Smaller, body length 10.4-11.5 mm .................................................................... 70 70 Antenna with 33-35 flagellomeres. Area superomedia c1.1x longer than wide. Hind femur stouter, 3.2-3.3x longer than wide. Europe, Kazachstan (RASNITSYN & SIYTAN 1981) ....................................................................... I. curtulus KRIECHBAUMER, 1882 (part) - Antenna with 37-40 flagellomeres. Hind femur slenderer, 3.5-3.6x longer than wide. Wings with brownish tint, pterostigma reddish ................................................................ .71 71 Hind femur with scattered punctures in ventral 1/2. Hind femur red. Postpetiolus and 2nd tergite red, 3rd tergite blackish (fig. 12). Siberia .................................... I. paravafer nov.sp. - Hind femur with dense punctures, mainly black. Usually postpetiolus, 2nd and 3rd tergites completely red. 4th tergite with a small ivory spot. Iran ............................................ ................................................................................... I. vafer meridionalis HEINRICH, 1929 72 Hind tibia black in apical 1/6. Body length 8 mm. 1st flagellomere 1.1-1.6x longer than wide. Hind femur completely black, stout, 2.6x longer than wide. 3rd tergite usual black basally. Ivory spot on 5th tergite often narrow, sometimes absent. Basal flagellomeres red or black. Europe, Mongolia (RASNITSYN & SIYTAN 1981) .............................................. .......................................................................................... I. caedator GRAVENHORST, 1829 - Hind tibia yellow or red, not black apically. Body length 10-11 mm. 1st flagellomere longer, c.1.8x longer than wide. Hind femur less stout, 3.3x longer than wide. Ivory spot on 5th tergite usually very wide. Basal flagellomeres red. 3rd tergite not black basally. Europe, Kazachstan (RASNITSYN & SIYTAN 1981), Iran ........................................... ......................................................................................... I. curtulus KRIECHBAUMER, 1882 68 Ivory spots on apical tergites not distinctly wide. Body length 10-15 mm. ....... (group G2.1) 69 68 Ivory spots on apical tergites not distinctly wide. Body length 10-15 mm. ....... (group G2.1) 69 - Ivory spots on apical tergites very wide. Face usually with extended red coloration. (adopted from HILPERT 1992): bucculentus WESMAEL, 1845 - Mandible not thickened. Hypostomal carina narrow. Hind tibia completely red or black apically. Smaller, body length 10.4-11.5 mm .................................................................... 70 70 Antenna with 33-35 flagellomeres. Area superomedia c1.1x longer than wide. Hind femur stouter, 3.2-3.3x longer than wide. Europe, Kazachstan (RASNITSYN & SIYTAN 1981) ....................................................................... I. curtulus KRIECHBAUMER, 1882 (part) - Antenna with 37-40 flagellomeres. Hind femur slenderer, 3.5-3.6x longer than wide. Wings with brownish tint, pterostigma reddish ................................................................ .71 71 Hind femur with scattered punctures in ventral 1/2. Hind femur red. Postpetiolus and 2nd tergite red, 3rd tergite blackish (fig. 12). Siberia .................................... I. paravafer nov.sp. - Hind femur with dense punctures, mainly black. Usually postpetiolus, 2nd and 3rd tergites completely red. 4th tergite with a small ivory spot. Iran ............................................ ................................................................................... I. vafer meridionalis HEINRICH, 1929 72 Hind tibia black in apical 1/6. Body length 8 mm. 1st flagellomere 1.1-1.6x longer than wide. Hind femur completely black, stout, 2.6x longer than wide. 3rd tergite usual black basally. Ivory spot on 5th tergite often narrow, sometimes absent. Basal flagellomeres red or black. Europe, Mongolia (RASNITSYN & SIYTAN 1981) .............................................. .......................................................................................... I. caedator GRAVENHORST, 1829 - Hind tibia yellow or red, not black apically. Body length 10-11 mm. 1st flagellomere longer, c.1.8x longer than wide. Hind femur less stout, 3.3x longer than wide. Ivory spot on 5th tergite usually very wide. Basal flagellomeres red. 3rd tergite not black basally. Europe, Kazachstan (RASNITSYN & SIYTAN 1981), Iran ........................................... ......................................................................................... I. curtulus KRIECHBAUMER, 1882 73 Antenna with 27-35 flagellomeres ...................................................................................... 74 - Antenna with at least 36 flagellomeres .............................................................................. 80 74 Antenna strongly lanceolate, widest flagellomere c.2x wider than long; 1st flagellomere stout, c.1.1x longer than wide. Basal flagellomeres and scutellum red (fig. 7). Siberia ........ I l l t 66 Hind femur 2.9x longer than wide. Hind tibia red-yellow-red-black, hind tarsus black in apical 1/4. 3rd tergite red, somtimes black baso-medially. Europa, Kazachstan, Mongolia (RASNITSYN & SIYTAN 1981) .................. I. caedator GRAVENHORST, 1829 (part) - Hind femur 3.7x longer than wide. Hind tibia and tarsus mainly yellow, with reddish tips. 3rd tergite yellow, with basal and apical black bands (fig. 19). Kirgistan ...................... .......................................................................................................... I. altaicurtulus nov.sp. 67 5th-7th tergites with large ivory spots or apical bands AND antenna with at least 32 flagellomeres .................................................................................................. (group G2) 68 - 5th tergite at most with smaller ivory spot OR antenna with 27-31 flagellomeres ................. ....................................................................................................................... (adopted from HILPERT 1992): Hind femur stouter, 3.2-3.3x longer than wide. Europe, Kazachstan (RASNITSYN & SIYTAN 1981) ....................................................................... I. curtulus KRIECHBAUMER, 1882 (part) - Antenna with 37-40 flagellomeres. Hind femur slenderer, 3.5-3.6x longer than wide. Wings with brownish tint, pterostigma reddish ................................................................ .7 71 Hind femur with scattered punctures in ventral 1/2. Hind femur red. Postpetiolus and 2nd tergite red, 3rd tergite blackish (fig. 12). Siberia .................................... I. paravafer nov.sp 1540 75 Temple parallel or slightly widened behind eye. Antenna with 27-29 flagellomeres. Ventral part of central flagellomeres very narrow. Only 2nd tergite basally red. Area superomedia c.1.5x longer than wide. European Alps, Siberia (HILPERT 1992) .................... ..................................................................................................... I. buryas HEINRICH, 1949 - Temple usually ± narrowed behind eye. Antenna with 29-35 flagellomeres. Mid flagellomeres not as narrow, c.0.7x as long as wide. 2nd and 3rd tergites completely red ......... 76 76 Petiolus red and scutellum ivory. Smaller, body length 8.7 mm. Hind femur completely red. Thyridium smaller than the interval and only slightly impressed. Europe, Far East Russia (UCHIDA 1926) .................................................................. I. gratus WESMAEL, 1855 - Petiolus OR scutellum black ............................................................................................. 77 77 Tarsi moderately widened. Hind femur red-black. Body length 11 mm. Antenna with 28-30 flagellomeres, preapical flagellomeres 0.76x as long as wide. Area superomedia about square. Hind tarsus mainly black. Europe, Iran ............... I. eumerus WESMAEL, 1857 - Tarsi not widened. Hind femur usually completely black ................................................. 78 78 Ivory spots on apical tergites rather wide, seen from dorsal almost covering the whole width of tergites. Mandible rather thickened. 2nd tergite as long as wide. Antenna with 30-34 flagellomeres. Area superomedia about square. Europe, Mongolia (RASNITSYN & SIYTAN 1981) .......................................................... I. caedator GRAVENHORST, 1829 (part) - Ivory spots on apical tergites not that wide. Mandible not thickened ................................ 79 79 Basal flagellomeres red. Antenna with 32 flagellomeres. Area superomedia almost square. Hind tibia and hind tarsus except distal tarsomere completely red. 3rd tergite blackish basally (here also runs I. mordax). Siberia .............. I. pilulicornis HEINRICH, 1978 - Basal flagellomeres black. Antenna with 27-32 flagellomeres. Area superomedia c.1.25x longer than wide. Hind tibia black in apical 1/8. Hind tarsus completely red. 3rd tergite completely red. Europea, Altai region, Irkutsk (MEYER 1933) ................................... .............................................................................................. I. gracilentus WESMAEL, 1845 80 Antenna with 36-43 flagellomeres .................................................................................... 81 - Antenna with 44-47 flagellomeres .................................................................................... 91 81 Head and mesosoma punctate and distinctly granulate, ± opaque. Basal flagellomeres red. Hind femur red with black dorsal stripe centrally. (adopted from HILPERT 1992): Siberia ............ I. granulatus nov.sp. - Head and mesosoma punctate, smooth or with fine granulation between punctures. If hind femur mainly red, then mesoscutum ± red ................................................................ 82 82 Hind femur mainly red. Mesoscutum ± red ....................................................................... 83 - Hind femur mainly black ................................................................................................... 86 83 6th and 7th tergites with distinct ivory spots ....................................................................... 84 - Ivory spots on apical tergites absent OR at most 7th tergite with ± distinct spot. Mesoscutum, scutellum and legs except coxae and trochanters red. 1st and 2nd tergites red, 2nd tergite black apico-medially, 3rd tergite black with red basolateral stripes. Wings slightly infuscate. Hind tarsus black in apical 1/4 .................................................. 85 84 Scutellum red, sometimes reddish-yellow. Fore and mid tarsomeres moderately widened. Hind coxa sometimes with weak scopa. Hind femur stout, 3.2x longer than wide. Antenna with 39 flagellomeres, 1st flagellomere 1.2x longer than wide. Scandinavia, Siberia (ROMAN 1927) ...................................... I. thomsoni HOLMGREN, 1864 - Scutellum yellow to ivory. Tarsomeres slender. Hind coxa without scopa. Hind femur slenderer, 3.7x longer than wide. Antenna with 37-38 flagellomeres, 1st flagellomere 1.8x longer than wide. Far East Russia ................................... I. zherichini HEINRICH, 1978 85 Apical tergites without pale spots. Hind femur with scattered punctures in ventral 0.6. Mid tarsomeres slightly widened. Wings and pterostigma slightly infuscate. Hind coxa with unequal punctures ventrally. Smaller, body length 10.7 mm. Thyridium slightly smaller than the interval. Siberia ................................. I. lariae taimyrensis HEINRICH, 1978 - 7th tergite with apical spot, 6th tergite with narrowly ivory apical margin. Hind femur with dense punctures. Mid tarsomeres slender. Wings strongly infuscate, pterostigma blackish. Larger, body length 14.9 mm. Thyridium much smaller than the interval. Hind coxa with equal punctuation. Siberia................................ I. chernovi HEINRICH, 1978 1541 86 Genal carina widely interupted ventrally. Malar space longer, c.1.5x longer than 1st flagellomere (fig. 44). Antenna with 36 flagellomere, 1st flagellomeres 1.6x longer than wide. Hind tibia infuscate in apical 1/4. Hind femur stout, 2.9-3.0x longer than wide; hind tibia with multiple denticular spines externally (fig. 41). Siberia ............ I. genator nov.sp. wide. Hind tibia infuscate in apical 1/4. Hind femur stout, 2.9-3.0x longer than wide; hind tibia with multiple denticular spines externally (fig. 41). Siberia ............ I. genator nov.sp. - Genal carina not interupted ventrally. Malar space shorter ............................................... 87 87 Clypeus densely punctate and with coarse longitudinal rugae in apical half. Hind femur very slender, 4.1x longer than wide. (adopted from HILPERT 1992): 2nd tergite red; 3rd tergite black, with reddish sides. 6th and 7th tergites without ivory spots, diffusely reddish apically (fig. 10). Siberia ............. ................................................................................................... I. nigrostigmaticus nov.sp. - Clypeus with finer sculpture, usually ± punctate. Hind femur stouter, at most 3.6x longer than wide. 6th and 7th tergites with distinct ivory spots ........................................... 88 88 Hind tibia red, very narrowly black in apical 1/20. Hind femur black, narrowly at base and in apical 1/5 red. Hind tarsus completely red. Ivory spot on scutellum often obsolete. Area superomedia slightly wider than long. Lower mandibular tooth very small. Antenna stout, with 36-38 flagellomeres, red basally, with ivory ring centrally, 1st flagellomere 1.2x longer than wide; preapical flagellomere almost square, 0.91x as long as wide. Hind femur stout, 3.1x longer than wide. Postpetiolus partly red, 2nd tergite black in basal 2/5, then shortly red, apically yellowish, 3rd tergite reddish- yellow, black in basal 1/3. Hypopygium moderately elongate, metasoma semi- amblypygous. Siberia ......................................................... I. amblypygops HEINRICH, 1978 - Hind tibia at least in apical 1/4 black. Hind tarsus at least in distal 1/3 black ................... 89 89 Antenna completely black. Hind tibia narrowly black at base and in apical 1/5. 2nd tergite red, black in basal 0.5; 3rd tergite red, black in basal 1/3. Only 7th tergite with distinct ivory spot. Hind femur with scattered punctures in ventral 1/3. Wings slightly infuscate. Hind coxa with irregular punctuation. Hind tarsus completely black. Tadzikistan ......................................................................... I. kazdikistanus HEINRICH, 1980 - Ring of antenna and scutellum ivory ................................................................................. 90 90 Basal flagellomeres red, yellow ring not distinct. Inner orbit widely yellow-red. Scutellum yellow. 2nd and 3rd tergites red, 2nd tergite sometimes black basally, 3rd tergite basally, sometimes also apically black. Hind coxa and hind femur with regular punctuation. Hind tibia pale red, black in apical 1/3. Hind tarsus black in apical 1/2. Buryat .......................................................................................... I. obnixus HEINRICH, 1978 - Antenna except ivory ring black. 2nd tergite red, 3rd tergite black, with red lateral margins. Hind femur red, narrowly black apically. Hind tibia yellow-red, black in apical 1/8. Hind tarsus ± black. Siberia (MEYER 1933) ......................................................... ..................................................................................... I. insidiosus malaisei ROMAN, 1927 91 Tarsi widened. Hind coxa with unequal punctuation ventrally. Hind femur with scattered punctures in ventral 2/3. Hind tibia red, black in apical 1/4. Large, body length 17 mm. Europe, Far East Russia (ROMAN 1927) ........................................................ ......................................................................................... I. hypolius THOMSON, 1888 (part) - Tarsi not widened. (adopted from HILPERT 1992): Hind coxa with equal punctuation. Hind femur with scattered punctures in ventral 1/4. Mid and hind legs completely black. Body length 15 mm. Far East Russia. According to HILPERT (1992) probably conspecific with I. melanobatus GRAVENHORST ......................................................................... I. vitimensis HEINRICH, 1978 92 Apical tergites without ivory spots. Antenna with 22-23 flagellomeres. 1st tergite ± and hind coxa red. Very small, body length 6.4 mm. Area superomedia c.1.25x longer than wide. Europa, Irkutsk (WOLDSTEDT 1881) ...................... I. oblongus SCHRANK, 1802 (part) - At least 7th tergite with ivory spot. If 6th tergite without ivory spot, then coxae red OR antenna with more than 26 flagellomeres .......................................................................... 93 93 Coxae red (or at most 26 flagellomeres, body length less than 7.8 mm and area superomedia not strongly elongated). Hypopygium short ................................................. 94 - Coxae usually black; if red, then antenna at least with 25 flagellomeres .......................... 96 94 Very small, body length at most 6.2 mm. Antenna with 22-23 flagellomeres. Often coxae, pterostigma and 2nd and 3rd tergites dark. Hind femur black. Europe, Far East Russia (MEYER 1933) ..................................................... I. simulans TISCHBEIN, 1873 (part) - Genal carina not interupted ventrally. Malar space shorter ............................................... 8 87 Clypeus densely punctate and with coarse longitudinal rugae in apical half. Hind femur very slender, 4.1x longer than wide. 2nd tergite red; 3rd tergite black, with reddish sides. 6th and 7th tergites without ivory spots, diffusely reddish apically (fig. 10). Siberia ............. ................................................................................................... I. nigrostigmaticus nov.sp. - Clypeus with finer sculpture, usually ± punctate. Hind femur stouter, at most 3.6x longer than wide. 6th and 7th tergites with distinct ivory spots ........................................... 88 88 Hind tibia red, very narrowly black in apical 1/20. Hind femur black, narrowly at base and in apical 1/5 red. Hind tarsus completely red. Ivory spot on scutellum often obsolete. Area superomedia slightly wider than long. Lower mandibular tooth very small. Antenna stout, with 36-38 flagellomeres, red basally, with ivory ring centrally, 1st flagellomere 1.2x longer than wide; preapical flagellomere almost square, 0.91x as long as wide. Hind femur stout, 3.1x longer than wide. Postpetiolus partly red, 2nd tergite black in basal 2/5, then shortly red, apically yellowish, 3rd tergite reddish- yellow, black in basal 1/3. Hypopygium moderately elongate, metasoma semi- amblypygous. Siberia ......................................................... I. amblypygops HEINRICH, 1978 89 Antenna completely black. Hind tibia narrowly black at base and in apical 1/5. (adopted from HILPERT 1992): 2nd tergite red, black in basal 0.5; 3rd tergite red, black in basal 1/3. Only 7th tergite with distinct ivory spot. Hind femur with scattered punctures in ventral 1/3. Wings slightly infuscate. Hind coxa with irregular punctuation. Hind tarsus completely black. Tadzikistan ......................................................................... I. kazdikistanus HEINRICH, 1980 89 Antenna completely black. Hind tibia narrowly black at base and in apical 1/5. 2nd tergite red, black in basal 0.5; 3rd tergite red, black in basal 1/3. Only 7th tergite with distinct ivory spot. Hind femur with scattered punctures in ventral 1/3. Wings slightly infuscate. Hind coxa with irregular punctuation. Hind tarsus completely black. Tadzikistan ......................................................................... I. kazdikistanus HEINRICH, 1980 infuscate. Hind coxa with irregular punctuation. Hind tarsus completely black. Tadzikistan ......................................................................... I. kazdikistanus HEINRICH, 1980 - Ring of antenna and scutellum ivory ................................................................................. 90 90 Basal flagellomeres red, yellow ring not distinct. Inner orbit widely yellow-red. Scutellum yellow. 2nd and 3rd tergites red, 2nd tergite sometimes black basally, 3rd tergite basally, sometimes also apically black. Hind coxa and hind femur with regular punctuation. Hind tibia pale red, black in apical 1/3. Hind tarsus black in apical 1/2. Buryat .......................................................................................... I. obnixus HEINRICH, 1978 - Antenna except ivory ring black. 2nd tergite red, 3rd tergite black, with red lateral margins. Hind femur red, narrowly black apically. Hind tibia yellow-red, black in apical 1/8. Hind tarsus ± black. Siberia (MEYER 1933) ......................................................... ..................................................................................... I. insidiosus malaisei ROMAN, 1927 91 Tarsi widened. Hind coxa with unequal punctuation ventrally. Hind femur with scattered punctures in ventral 2/3. Hind tibia red, black in apical 1/4. Large, body length 17 mm. Europe, Far East Russia (ROMAN 1927) ........................................................ ......................................................................................... I. hypolius THOMSON, 1888 (part) - Tarsi not widened. Hind coxa with equal punctuation. Hind femur with scattered punctures in ventral 1/4. Mid and hind legs completely black. Body length 15 mm. Far East Russia. According to HILPERT (1992) probably conspecific with I. melanobatus GRAVENHORST ......................................................................... I. vitimensis HEINRICH, 1978 92 Apical tergites without ivory spots. Antenna with 22-23 flagellomeres. 1st tergite ± and hind coxa red. Very small, body length 6.4 mm. Area superomedia c.1.25x longer than wide. Europa, Irkutsk (WOLDSTEDT 1881) ...................... I. oblongus SCHRANK, 1802 (part) - At least 7th tergite with ivory spot. If 6th tergite without ivory spot, then coxae red OR antenna with more than 26 flagellomeres .......................................................................... (adopted from HILPERT 1992): 93 93 Coxae red (or at most 26 flagellomeres, body length less than 7.8 mm and area superomedia not strongly elongated). Hypopygium short ................................................. 94 - Coxae usually black; if red, then antenna at least with 25 flagellomeres .......................... 96 94 Very small, body length at most 6.2 mm. Antenna with 22-23 flagellomeres. Often coxae, pterostigma and 2nd and 3rd tergites dark. Hind femur black. Europe, Far East Russia (MEYER 1933) ..................................................... I. simulans TISCHBEIN, 1873 (part) 90 Basal flagellomeres red, yellow ring not distinct. Inner orbit widely yellow-red. Scutellum yellow. 2nd and 3rd tergites red, 2nd tergite sometimes black basally, 3rd tergite basally, sometimes also apically black. Hind coxa and hind femur with regular punctuation. Hind tibia pale red, black in apical 1/3. Hind tarsus black in apical 1/2. Buryat .......................................................................................... I. obnixus HEINRICH, 1978 90 Basal flagellomeres red, yellow ring not distinct. Inner orbit widely yellow-red. Scutellum yellow. 2nd and 3rd tergites red, 2nd tergite sometimes black basally, 3rd tergite basally, sometimes also apically black. Hind coxa and hind femur with regular punctuation. Hind tibia pale red, black in apical 1/3. Hind tarsus black in apical 1/2. Buryat .......................................................................................... I. obnixus HEINRICH, 1978 92 Apical tergites without ivory spots. Antenna with 22-23 flagellomeres. 1st tergite ± and hind coxa red. Very small, body length 6.4 mm. Area superomedia c.1.25x longer than wide. Europa, Irkutsk (WOLDSTEDT 1881) ...................... I. oblongus SCHRANK, 1802 (part) At least 7th tergite with ivory spot. If 6th tergite without ivory spot, then coxae red OR antenna with more than 26 flagellomeres .......................................................................... 93 - At least 7th tergite with ivory spot. If 6th tergite without ivory spot, then coxae red OR antenna with more than 26 flagellomeres .......................................................................... 93 93 Coxae red (or at most 26 flagellomeres, body length less than 7.8 mm and area superomedia not strongly elongated). Hypopygium short ................................................. 94 3 Coxae red (or at most 26 flagellomeres, body length less than 7.8 mm and area superomedia not strongly elongated). Hypopygium short ................................................. 94 - Coxae usually black; if red, then antenna at least with 25 flagellomeres .......................... 96 1542 - Larger, body length more than 6.2 mm. Antenna with 23-26 flagellomeres. Coxae only rarely black. 2nd and 3rd tergites completely red. Hind femur usally ± red. Postpetiolus red. Hind tibia black in apical 1/5-1/4 ............................................................................... 95 95 1st-4th tergites red. (adopted from HILPERT 1992): Ivory spot on 6th tergite small, much smaller than spot on 7th tergite. Area superomedia more than 1.3x longer than wide, sides not edged. Petiolus often dark medially ................................................................... I. oblongus SCHRANK, 1802 (part) - Only 1st-3rd tergites red, 4th tergite sometimes red at base. Ivory spot on 6th tergite large. Area superomedia at most 1.3x longer than wide, ± hexagonal ............................................ ....................................................................................... I. simulans TISCHBEIN, 1873 (part) 96 Petiolus black. Mandible moderately thickened. Temple only slightly narrowed behind eye. Hypopygium slighly enlarged. Metasomal apex somewhat compressed. 2nd tergite red, 3rd tergite with some diffuse reddish tint. Antenna with 28 flagellomeres, pale ring indistinct (structure similar to I. intricator WESMAEL). Central Asia .................................... ...........................................................................................I. medioasiaticus HILPERT, 1992 - Petiolus red. Mandible not thickend. Temple distinctly and roundly narrowed behind eye. Hypopygium narrow. Postpetiolus ± and 2nd and 3rd tergites red. Antenna with 28- 30 flagellomeres, with distinct ivory ring. Europe, Siberia (UCHIDA 1926, MEYER 1933) ........................................................................... I. memorator WESMAEL, 1845 (part) 97 Scutellum yellow. Antenna with 29-36 flagellomeres. Apical tergites without ivory spots. 2nd and 3rd tergites red; if black, then margins of 2nd tergite and apical margins of following tergites red. Area superomedia rectangular, large, wider than long. Hind femur red or black. Hind tibia completely red. Hind tarsus red, apical narrowly black. Thyridium often only slightly wider than the interval. Siberia .............................................. ..................................................................................................... I. modestus ROMAN, 1927 - Scutellum red or black ....................................................................................................... 98 98 6th and 7th tergites with large ivory spots. Scutellum black or reddish .............................. 99 - Apical tergite without ivory spots or only 7th tergite with narrow yellow stripe. Scutellum red, at least apically ........................................................................................ 100 99 Lateral field of mesoscutum and scutellum red (fig. 5). Femora, tibiae and tarsi except brownish distal tarsomeres red. Antenna with 34 flagellomeres; 1st flagellomere c.1.6x longer than wide. Siberia .................................................................... I. hakassiacus nov.sp. - Mesoscutum black, scutellum black or red. Hind femur mainly black. Antenna with at most 32 flagellomeres; 1st flagellomere usually longer ................................................... 102 100 Antenna with 27-30 flagellomeres; 1st flagellomeres 1.7x longer than wide. Body length 8.8 mm. Hind femur 3.3x longer than wide. 3rd tergite red. Hind coxa with slightly elevated ventral edge. Thyridium slightly wider than the interval. Europe, Siberia ......................................................................... I. memorator WESMAEL, 1845 (part) - Antenna with 32 flagellomeres; 1st flagellomere at least 2.0x longer than wide. Body length 9.5-10 mm. Hind femur at least 3.6x longer than wide. (adopted from HILPERT 1992): Thyridium at least 2x wider than the interval ..................................................................................................... 101 101 Widest flagellomeres about square. Thyridium c.2x wider than the interval (fig. 64). 1st flagellomere slender, 2.4x longer than wide. Scutellum reddish. 3rd tergite reddish- brown. Hind tibia completely yellowish. Area superomedia hexagonal, c.1.35x wider than long. Siberia ....................................................................................... I. berlovi nov.sp. - Widest flagellomeres c.1.6x wider than long. Thyridium c.3x wider than the interval (fig. 81). 1st flagellomere 2.0x longer than wide. Scutellum black. 3rd tergite mainly black. Hind tibia infuscate in apcial 1/3. Area superomedia rectangular, c.1.2x wider than long. Siberia ................................................................................. I. thyridiator nov.sp. 102 Antenna with 31-33 flagellomeres and distinct ivory ring, basal flagellomeres reddish. Apical tergites without ivory spots OR 7th tergite with narrow yellow stripe. 4th tergite partly red. Body length 10.2 mm. Hind coxa with long hairs indicating ± distinct scopa. Hind femur 3.6x longer than wide. Hind tibia usually black in apical 1/8. In Eastern Palaearctic material mesoscutum reddish, scutellum with dark reddish spot, frontal orbit widely yellow-red, hind femur black and distal hind tarsomere blackish. Europe, Far East Russia (ROMAN 1927 as kamtschaticus) .......... I. stigmatorius ZETTERSTEDT, 1838 1542 - Larger, body length more than 6.2 mm. Antenna with 23-26 flagellomeres. Coxae only rarely black. 2nd and 3rd tergites completely red. Hind femur usally ± red. Postpetiolus red. Hind tibia black in apical 1/5-1/4 ............................................................................... 95 95 1st-4th tergites red. Ivory spot on 6th tergite small, much smaller than spot on 7th tergite. Area superomedia more than 1.3x longer than wide, sides not edged. Petiolus often dark medially ................................................................... I. oblongus SCHRANK, 1802 (part 101 Widest flagellomeres about square. Thyridium c.2x wider than the interval (fig. 64). 1st flagellomere slender, 2.4x longer than wide. Scutellum reddish. 3rd tergite reddish- brown. Hind tibia completely yellowish. Area superomedia hexagonal, c.1.35x wider than long. Siberia ....................................................................................... I. berlovi nov.sp. - Widest flagellomeres c.1.6x wider than long. Thyridium c.3x wider than the interval (fig. 81). 1st flagellomere 2.0x longer than wide. Scutellum black. 3rd tergite mainly black. Hind tibia infuscate in apcial 1/3. Area superomedia rectangular, c.1.2x wider than long. Siberia ................................................................................. I. thyridiator nov.sp. 102 Antenna with 31-33 flagellomeres and distinct ivory ring, basal flagellomeres reddish. Apical tergites without ivory spots OR 7th tergite with narrow yellow stripe. 4th tergite partly red. Body length 10.2 mm. Hind coxa with long hairs indicating ± distinct scopa. Hind femur 3.6x longer than wide. 102 Antenna with 31-33 flagellomeres and distinct ivory ring, basal flagellomeres reddish. Apical tergites without ivory spots OR 7th tergite with narrow yellow stripe. 4th tergite partly red. Body length 10.2 mm. Hind coxa with long hairs indicating ± distinct scopa. Hind femur 3.6x longer than wide. Hind tibia usually black in apical 1/8. In Eastern Palaearctic material mesoscutum reddish, scutellum with dark reddish spot, frontal orbit widely yellow-red, hind femur black and distal hind tarsomere blackish. Europe, Far East Russia (ROMAN 1927 as kamtschaticus) .......... I. stigmatorius ZETTERSTEDT, 1838 Zusammenfassung In der vorliegenden Arbeit werden faunistische und taxonomische Angaben zu 42 Arten der Gattung Ichneumon LINNAEUS aus Sibirien gemacht. Sechs Taxa werden das erste Mal in der ostpaläarktischen Region nachgewiesen: Ichneumon acuticornis THOMSON, 1896, Ichneumon exilicornis WESMAEL, 1857, Ichneumon fulvicornis GRAVENHORST, 1829, Ichneumon karpathica HEINRICH, 1951, Ichneumon minutorius DESVIGNES, 1856 and Ichneumon veressi (KISS, 1915). In der vorliegenden Arbeit werden faunistische und taxonomische Angaben zu 42 Arten der Gattung Ichneumon LINNAEUS aus Sibirien gemacht. Sechs Taxa werden das erste Mal in der ostpaläarktischen Region nachgewiesen: Ichneumon acuticornis THOMSON, 1896, Ichneumon exilicornis WESMAEL, 1857, Ichneumon fulvicornis GRAVENHORST, 1829, Ichneumon karpathica HEINRICH, 1951, Ichneumon minutorius DESVIGNES, 1856 and Ichneumon veressi (KISS, 1915). Von 20 neuen Arten werden die Weibchen beschrieben und illustriert: Ichneumon altaicurtulus nov.sp., Ichneumon berlovi nov.sp., Ichneumon brevipunctatus nov.sp., Ichneumon breviscopatus nsp., Ichneumon genator nov.sp., Ichneumon granulatus nov.sp., Ichneumon hakassiacus nov.sp., Ichneumon inquinatops nov.sp., Ichneumon lanceolator nsp., Ichneumon mandibulatus nov.sp., Ichneumon mesonotator nov.sp., Ichneumon nigrostigmaticus nov.sp., Ichneumon orientopodius nov.sp., Ichneumon paravafer nov.sp., Ichneumon paravulnerator nov.sp., Ichneumon pseudemancipatus nov.sp., Ichneumon rufolateralis nov.sp., Ichneumon sayanicus nov.sp., Ichneumon scopator nov.sp. und Ichneumon thyridiator nov.sp. Ichneumon jakovlevi KOKUJEV, 1904 wird als neues Synynym zu Ichneumon cessator MÜLLER 1776 gestellt. Außerdem wird ein neuer Bestimmungsschlüssel für die Weibchen der bisher aus Sibirien bekann ten Ichneumon-Arten vorgelegt. 1543 - Antenna with 29-31 flagellomeres, black, at most with rudimental ring. Mesoscutum and scutellum red. Small, body length 7.1-8.4 mm. Apical tergites black, without pale spots. Hind tibia black in apical 1/3. Petiolus usally completely red. Hind femur slender, 3.8x longer than wide. Arctic Siberia .............................. I. asiaticus ROMAN, 1914 - Antenna with 29-31 flagellomeres, black, at most with rudimental ring. Mesoscutum and scutellum red. Small, body length 7.1-8.4 mm. Apical tergites black, without pale spots. Hind tibia black in apical 1/3. Petiolus usally completely red. Hind femur slender, 3.8x longer than wide. Arctic Siberia .............................. I. asiaticus ROMAN, 1914 Acknowledgement I thank S. Schmidt from the ZSM/Munich for his help and allowance to study the Heinrich collec- tion. I also want to thank G. Broad (NHM/London) and A. Taeger (SDEI/ Müncheberg) for the kind loan of their interesting Ichneumon material. (adopted from HILPERT 1992): Hind tibia usually black in apical 1/8. In Eastern Palaearctic material mesoscutum reddish, scutellum with dark reddish spot, frontal orbit widely yellow-red, hind femur black and distal hind tarsomere blackish. Europe, Far East Russia (ROMAN 1927 as kamtschaticus) .......... I. stigmatorius ZETTERSTEDT, 1838 1543 References HEINRICH G.H. (1931): Beitrage zur Systematik der Ichneumoninae Stenopneusticae (Hym. IV. — Mitteilungen der Deutschen Entomologischen Gesellschaft 2: 27-32. HEINRICH G.H. (1967-1968): Synopsis and Reclassification of the Ichneumonina Stenopneusticae of Africa South of the Sahara. — Vol 1-5, 1-1258. Farmington. HEINRICH G.H. (1978): [Eastern Palearctic Hymenopterous insects of the subfamily Ichneumoninae.] (in Russian). — Leningrad. 81 pp. HEINRICH G.H. (1980): Neue Ichneumoninae Stenopneusticae aus der paläarktischen Region (Hymenoptera, Ichneumonidae). — Mitteilungen der Münchner Entomologischen Gesellschaft 69: 9-27. HILPERT H. (1992): Zur Systematik der Gattung Ichneumon LINNAEUS, 1758 in der Westpalaearktis (Hymenoptera, Ichneumonidae, Ichneumoninae). — Entomofauna Suppl. 6: 1-389. HORSTMANN K. (2006): Revisionen von Schlupfwespen-Arten X. (Hymenoptera, Ichneumonidae, Braconidae). — Mitteilungen der Münchner Entomologischen Gesell- schaft 96: 5-16. 1544 KOKUJEV N.R. (1904): [Contributions a la faune des Hymenopteres de la prov. d'Irkoutsk. Ichneumonidae I.] (in Russian with Latin descriptions for new species). — Revue Russe d'Entomologie 4 (2-3): 80-84. KOKUJEV N.R. (1913): Contribution a la faune des Hymenopteres de la Russie III. — Revue Russe d'Entomologie 13: 161-170. KOKUJEV N.R. (1927): [Hymenoptera recueillies par V. Sovinskij sur les bords du lac Bajkal en 1902.] (in Russian with Latin descriptions). — Travaux de la Commission pour l'etude du lac Bajkal 2: 63-76. KOLAROV J. & H. GHAHARI (2005): A catalogue of Ichneumonidae (Hymenoptera) from Iran. — Linzer Biologische Beiträge 37 (1): 503-532. MEYER N.F. (1930): [Scientific results of the entomological expedition of the Zoological Museum in Ussur territory: I. Hymenoptera, Ichneumonidae]. — Ezhegodnik Zoologicheskago Muzeya. Akademii Nauk SSSR 31: 165-180. MEYER N.F. (1933): [Keys to parasitic Hymenoptera (family Ichneumonidae) of the USSR and adjacent countries. Vol. 1. Introduction and Ichneumoninae] (in Russian). — Zoological Institute of the Academy of Sciences of the USSR 9 (1): 1-458. RASNITSYN A.P. (1984): [Types of the Ichneumoninae (Hymenoptera, Ichneumonidae) preserved in the Zoological Institute, Academy of Sciences of the USSR. I. Taxa described from the USSR.] (in Russian). — Entomologicheskoye Obozreniye 63: 790- 801. RASNITSYN A.P. & U.V. SIYTAN: [Subfamily Ichneumoninae]. — In: KASPARYAN D.R. (ed.), [A guide to the insects of the European part of the USSR. Hymenoptera, Ichneumonidae] (In Russian). Opredeliteli po Faune SSSR. Leningrad: Nauka. 1981. Vol. 3. Pt.3.: 506– 636. ROMAN A. (1914): Die Ichneumoniden des arktischen Sibiriens nach der Sammlung der Russischen Polar-Expedition 1900-1903. — Mémoires de l'Académie Imperiale des Sciences de St.Petersbourg. Classe 8 Physico-Mathematique 29 (7): 1-14. ROMAN A. (1927): Entomologische Ergebnisse der schwedischen Kamtschatka-Expedition 1920-1922. 10. Ichneumonidae, Subfam. Dr. Matthias RIEDEL Zoologische Staatssammlung München Münchhausenstr. 21 D-81247 München, Germany E-mail: [email protected] References Ichneumoninae. — Arkiv för Zoologi 19A (7): 1-19. UCHIDA T. (1926): Erster Beitrag zur Ichneumoniden-Fauna Japans. — Journal of the Faculty of Agriculture, Hokkaido Imperial University 18: 43-173. UCHIDA T. (1935): Beiträge zur Kenntnis der Ichneumonidenfauna der Kurilen. — Insect Matsumurana 9: 108-122. WOLDSTEDT F.W. (1881): Fundorte russischer Ichneumoniden. — Horae Societatis Entomologicae Rossicae 16: 58-64. YU D.S.K., ACHTERBERG VAN C. & K. HORSTMANN. [electronic source]: World Taxapad 2016, Ichneumonoidea 2015. Taxonomy, Biology, Morphology and Distribution. 2016. — On USB flash-drive. www.taxapad.com, Nepean, Ontario, Canada. Dr. Matthias RIEDEL Zoologische Staatssammlung München Münchhausenstr. 21 D-81247 München, Germany E-mail: [email protected] Dr. Matthias RIEDEL Zoologische Staatssammlung München Münchhausenstr. 21 D-81247 München, Germany E-mail: [email protected] Address of the author: ©Biologiezentrum Linz, Austria; download unter www.zobodat.at 1545 Figs 1-4: Habitus of: (1) Ichneumon berlovi nov.sp.; (2) I. brevipunctatus nov.sp.; (3) I. genato nov.sp.; (4) I. granulatus nov.sp. Figs 1-4: Habitus of: (1) Ichneumon berlovi nov.sp.; (2) I. brevipunctatus nov.sp.; (3) I. genator nov.sp.; (4) I. granulatus nov.sp. Figs 1-4: Habitus of: (1) Ichneumon berlovi nov.sp.; (2) I. brevipunctatus nov.sp.; (3) I. genato nov.sp.; (4) I. granulatus nov.sp. 1546 Habitus of: (5). Ichneumon hakassiacus nov.sp.; (6) I. inquinatops nov.s or nov.sp.; (4) I. mandibulatus nov.sp. ©Biologiezentrum Linz, Austria; download unter www.zobodat.at ©Biologiezentrum Linz, Austria; download unter www.zobodat.at 1546 Figs 5-8: Habitus of: (5). Ichneumon hakassiacus nov.sp.; (6) I. inquinatops nov.sp.; (7) I. lanceolator nov.sp.; (4) I. mandibulatus nov.sp. Figs 5-8: Habitus of: (5). Ichneumon hakassiacus nov.sp.; (6) I. inquinatops nov.sp.; (7) I lanceolator nov.sp.; (4) I. mandibulatus nov.sp. ©Biologiezentrum Linz, Austria; download unter www.zobodat.at 1547 s 9-12: Habitus of: (9) Ichneumon mesonotator nov.sp.; (10) I. nigrostigmaticus nov.sp.; (1 ntopodius nov.sp.; (12) I. paravafer nov.sp. Figs 9-12: Habitus of: (9) Ichneumon mesonotator nov.sp.; (10) I. nigrostigmaticus nov.sp.; (11) I. orientopodius nov.sp.; (12) I. paravafer nov.sp. Figs 9-12: Habitus of: (9) Ichneumon mesonotator nov.sp.; (10) I. nigrostigmaticus nov.sp.; (11) I orientopodius nov.sp.; (12) I. paravafer nov.sp. 1548 3-16: Habitus of: (13) Ichneumon paravulnerator nov.sp.; (14) I. pseudemancipatus rufolateralis nov.sp.; (16) I. sayanicus nov.sp. ©Biologiezentrum Linz, Austria; download unter www.zobodat.at ©Biologiezentrum Linz, Austria; download unter www.zobodat.at 1548 Figs 13-16: Habitus of: (13) Ichneumon paravulnerator nov.sp.; (14) I. pseudemancipatus nov.sp.; (15) I. rufolateralis nov.sp.; (16) I. sayanicus nov.sp. Figs 13-16: Habitus of: (13) Ichneumon paravulnerator nov.sp.; (14) I. pseudemancipatus nov.sp (15) I. rufolateralis nov.sp.; (16) I. sayanicus nov.sp. References ©Biologiezentrum Linz, Austria; download unter www.zobodat.at 1549 -18: Habitus of: (17) Ichneumon scopator nov.sp.; (18) I. thyridiator nov.sp. -20: Metasoma of: (19) Ichneumon altaicurtulus nov.sp.; (20) I. brevipunctatus no Figs 17-18: Habitus of: (17) Ichneumon scopator nov.sp.; (18) I. thyridiator nov.sp. Figs 19-20: Metasoma of: (19) Ichneumon altaicurtulus nov.sp.; (20) I. brevipunctatus nov.sp. Figs 17-18: Habitus of: (17) Ichneumon scopator nov.sp.; (18) I. thyridiator nov.sp. Figs 19-20: Metasoma of: (19) Ichneumon altaicurtulus nov.sp.; (20) I. brevipunctatus nov.sp. 1550 Figs 21-26: Head from dorsal of: (21) Ichneumon berlovi nov.sp.; (22) I. brevipunctatus nov.sp.; (23) I. breviscopulatus nov.sp.; (24) I. genator nov.sp.; (25) I. granulatus nov.sp.; (26) I. hakassiacus nov.sp. ©Biologiezentrum Linz, Austria; download unter www.zobodat.at ©Biologiezentrum Linz, Austria; download unter www.zobodat.at 1550 1550 Figs 21-26: Head from dorsal of: (21) Ichneumon berlovi nov.sp.; (22) I. brevipunctatus nov.sp (23) I. breviscopulatus nov.sp.; (24) I. genator nov.sp.; (25) I. granulatus nov.sp.; (26) I hakassiacus nov.sp. ©Biologiezentrum Linz, Austria; download unter www.zobodat.at 1551 Figs 27-32: Head from dorsal of: (27) Ichneumon inquinatops nov.sp.; (28) I. lanceolator nov.sp.; (29) I. mandibulatus nov.sp.; (30) I. mesonotator nov.sp.; (31) I. nigrostigmaticus nov.sp.; (32) I. orientopodius nov.sp. 1551 1551 Figs 27-32: Head from dorsal of: (27) Ichneumon inquinatops nov.sp.; (28) I. lanceolator nov.sp (29) I. mandibulatus nov.sp.; (30) I. mesonotator nov.sp.; (31) I. nigrostigmaticus nov.sp.; (32) I orientopodius nov.sp. ©Biologiezentrum Linz, Austria; download unter www.zobodat.at 1552 Figs 33-38: Head from dorsal of: (33) Ichneumon paravafer nov.sp.; (34) I. paravulnerator nov.sp.; (35) I. pseudemancipatus nov.sp.; (36) I. rufolateralis nov.sp.; (37) I. sayanicus nov.sp.; (38) I. scopator nov.sp. ©Biologiezentrum Linz, Austria; download unter www.zobodat.at 1552 Figs 33-38: Head from dorsal of: (33) Ichneumon paravafer nov.sp.; (34) I. paravulnerator nov.sp.; (35) I. pseudemancipatus nov.sp.; (36) I. rufolateralis nov.sp.; (37) I. sayanicus nov.sp.; (38) I. scopator nov.sp. Figs 33-38: Head from dorsal of: (33) Ichneumon paravafer nov.sp.; (34) I. paravulnerator nov.sp (35) I. pseudemancipatus nov.sp.; (36) I. rufolateralis nov.sp.; (37) I. sayanicus nov.sp.; (38) I scopator nov.sp. ©Biologiezentrum Linz, Austria; download unter www.zobodat.at 1553 ©Biologiezentrum Linz, Austria; download unter www.zobodat.at 1553 Figs 39-40: Hind coxa from lateral of: (39) Ichneumon brevipunctatus nov.sp.; (40) I. breviscopatus nov.sp. Figs 41-42: (41) Hind tibia of Ichneumon genator nov.sp. with multiple denticular spurs apico-externally; (42) widened mid tarsus of Ichneumon orientopodius nov.sp. Figs 43-44: Head from frontal: (43) Ichneumon mandibulatus nov.sp.; (44) I. genator nov.sp. References ©Biologiezentrum Linz, Austria; download unter www.zobodat.at 1553 1553 Figs 39-40: Hind coxa from lateral of: (39) Ichneumon brevipunctatus nov.sp.; (40) I. breviscopatus nov.sp. Figs 41-42: (41) Hind tibia of Ichneumon genator nov.sp. with multiple denticular spurs apico-externally; (42) widened mid tarsus of Ichneumon orientopodius nov.sp. Figs 43-44: Head from frontal: (43) Ichneumon mandibulatus nov.sp.; (44) I. genator nov.sp. 1554 Figs 45-48: Scutellum and propodeum of: (45) Ichneumon berlovi nov.sp.; (46) I. brevipunctatus nov.sp.; (47) I. genator nov.sp.; (48) I. granulatus nov.sp. ©Biologiezentrum Linz, Austria; download unter www.zobodat.at ©Biologiezentrum Linz, Austria; download unter www.zobodat.at 1554 Figs 45-48: Scutellum and propodeum of: (45) Ichneumon berlovi nov.sp.; (46) I. brevipunctatus nov.sp.; (47) I. genator nov.sp.; (48) I. granulatus nov.sp. Figs 45-48: Scutellum and propodeum of: (45) Ichneumon berlovi nov.sp.; (46) I. brevipunctatu nov.sp.; (47) I. genator nov.sp.; (48) I. granulatus nov.sp. ©Biologiezentrum Linz, Austria; download unter www.zobodat.at 1555 Figs 49-52: Scutellum and propodeum of: (49) Ichneumon hakassiacus nov.sp.; (50) I. inquinatops nov.sp.; (51) I. lanceolator nov.sp.; (52) I. mandibulatus nov.sp. ©Biologiezentrum Linz, Austria; download unter www.zobodat.at 1555 Figs 49-52: Scutellum and propodeum of: (49) Ichneumon hakassiacus nov.sp.; (50) I. inquinatops nov.sp.; (51) I. lanceolator nov.sp.; (52) I. mandibulatus nov.sp. Figs 49-52: Scutellum and propodeum of: (49) Ichneumon hakassiacus nov.sp.; (50) I. inquinatops nov.sp.; (51) I. lanceolator nov.sp.; (52) I. mandibulatus nov.sp. Figs 49-52: Scutellum and propodeum of: (49) Ichneumon hakassiacus nov.sp.; (50) I. inquinatop nov.sp.; (51) I. lanceolator nov.sp.; (52) I. mandibulatus nov.sp. ©Biologiezentrum Linz, Austria; download unter www.zobodat.at 1556 Figs 53-56: Scutellum and propodeum of: (53) Ichneumon mesonotator nov.sp.; (54) I. nigrostigmaticus nov.sp.; (55) I. orientopodius nov.sp.; (56) I. paravafer nov.sp. ©Biologiezentrum Linz, Austria; download unter www.zobodat.at 1556 Figs 53-56: Scutellum and propodeum of: (53) Ichneumon mesonotator nov.sp.; (54) I. i ti ti no sp ; (55) I i t di no sp ; (56) I f no sp Figs 53-56: Scutellum and propodeum of: (53) Ichneumon mesonotator nov.sp.; (54) I nigrostigmaticus nov.sp.; (55) I. orientopodius nov.sp.; (56) I. paravafer nov.sp. ©Biologiezentrum Linz, Austria; download unter www.zobodat.at 1557 Figs 57-60: Scutellum and propodeum of: (57) Ichneumon paravulnerator nov.sp.; (58) I. pseudemancipatus nov.sp.; (59) I. rufolateralis nov.sp.; (60) I. sayanicus nov.sp. Figs 57-60: Scutellum and propodeum of: (57) Ichneumon paravulnerator nov.sp.; (58) I pseudemancipatus nov.sp.; (59) I. rufolateralis nov.sp.; (60) I. sayanicus nov.sp. ©Biologiezentrum Linz, Austria; download unter www.zobodat.at 1558 Figs 61-62: Scutellum and propodeum of: (61) Ichneumon scopator nov.sp.; (62) I. References thyridiator nov.sp. Figs 63-64: Postpetiolus and 2nd tergite of: (63) Ichneumon altaicurtulus nov.sp.; (64) I. berlovi nov.sp. Figs 61-62: Scutellum and propodeum of: (61) Ichneumon scopator nov.sp.; (62) I. thyridiator nov.sp. Figs 63-64: Postpetiolus and 2nd tergite of: (63) Ichneumon altaicurtulus nov.sp.; (64) I. berlovi nov.sp. Figs 61-62: Scutellum and propodeum of: (61) Ichneumon scopator nov.sp.; (62) I. thyridiator nov.sp. Figs 63-64: Postpetiolus and 2nd tergite of: (63) Ichneumon altaicurtulus nov.sp.; (64) I. berlovi nov.sp. 1559 Figs 65-68: Postpetiolus and 2nd tergite of: (65) Ichneumon breviscopatus nov.sp.; (66) I. genator nov.sp.; (67) I. granulatus nov.sp.; (68) I. hakassiacus nov.sp. ©Biologiezentrum Linz, Austria; download unter www.zobodat.at ©Biologiezentrum Linz, Austria; download unter www.zobodat.at 1559 Figs 65-68: Postpetiolus and 2nd tergite of: (65) Ichneumon breviscopatus nov.sp.; (66) I. genator nov.sp.; (67) I. granulatus nov.sp.; (68) I. hakassiacus nov.sp. Figs 65-68: Postpetiolus and 2nd tergite of: (65) Ichneumon breviscopatus nov.sp.; (66) I. genator Figs 65-68: Postpetiolus and 2nd tergite of: (65) Ichneumon breviscopatus nov.sp.; (66) I. genator nov.sp.; (67) I. granulatus nov.sp.; (68) I. hakassiacus nov.sp. Figs 65-68: Postpetiolus and 2nd tergite of: (65) Ichneumon breviscopatus nov.sp.; (66) I. genato nov.sp.; (67) I. granulatus nov.sp.; (68) I. hakassiacus nov.sp. 1560 Figs 69-72: Postpetiolus and 2nd tergite of: (69) Ichneumon inquinatops nov.sp.; (70) I. lanceolator nov.sp.; (71) I. mandibulatus nov.sp.; (72) I. mesonotator nov.sp. ©Biologiezentrum Linz, Austria; download unter www.zobodat.at ©Biologiezentrum Linz, Austria; download unter www.zobodat.at 1560 Figs 69-72: Postpetiolus and 2nd tergite of: (69) Ichneumon inquinatops nov.sp.; (70) I. lanceolator nov.sp.; (71) I. mandibulatus nov.sp.; (72) I. mesonotator nov.sp. Figs 69-72: Postpetiolus and 2nd tergite of: (69) Ichneumon inquinatops nov.sp.; (70) I. lanceolato nov.sp.; (71) I. mandibulatus nov.sp.; (72) I. mesonotator nov.sp. 1561 Figs 73-76: Postpetiolus and 2nd tergite of: (73) Ichneumon nigrostigmaticus nov.sp.; (74) I. orientopodius nov.sp.; (75) I. paravafer nov.sp.; (76) I. paravulnerator nov.sp. ©Biologiezentrum Linz, Austria; download unter www.zobodat.at ©Biologiezentrum Linz, Austria; download unter www.zobodat.at 1561 1561 Figs 73-76: Postpetiolus and 2nd tergite of: (73) Ichneumon nigrostigmaticus nov.sp.; (74) I orientopodius nov.sp.; (75) I. paravafer nov.sp.; (76) I. paravulnerator nov.sp. ©Biologiezentrum Linz, Austria; download unter www.zobodat.at 1562 Figs 77-80: Postpetiolus and 2nd tergite of: (77) Ichneumon pseudemancipatus nov.sp.; (78) I. rufolateralis nov.sp.; (79) I. sayanicus nov.sp.; (80) I. scopator nov.sp. ©Biologiezentrum Linz, Austria; download unter www.zobodat.at 1562 Figs 77-80: Postpetiolus and 2nd tergite of: (77) Ichneumon pseudemancipatus nov.sp.; (78) I. rufolateralis nov.sp.; (79) I. sayanicus nov.sp.; (80) I. scopator nov.sp. References Figs 81-82: (81) Ichneumon thyridiator nov.sp.: Postpetiolus and 2nd tergite; (82) Ichneumon orientopodius nov.sp.: Moderately elongated hypopygium. Figs 81-82: (81) Ichneumon thyridiator nov.sp.: Postpetiolus and 2nd tergite; (82) Ichneumon orientopodius nov.sp.: Moderately elongated hypopygium. Figs 81-82: (81) Ichneumon thyridiator nov.sp.: Postpetiolus and 2nd tergite; (82) Ichneumo orientopodius nov.sp.: Moderately elongated hypopygium.

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The Microbiome of Temporal Arteries

Pathogens & immunity

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Pathogens and Immunity - Vol 4, No 1 Pathogens and Immunity - Vol 4, No 1 21 Research Article Published February 12, 2019 Research Article Published February 12, 2019 Correction: This is a corrected version of this article, published on March 21, 2019. The original version of this article, published on February 12, 2019, contained an incorrect caption and legend for Figure 2. AUTHORSf Gary S. Hoffman1*, Ted M. Getz2*, Roshan Padmanabhan2, Alexandra Villa-Forte1, Alison H. Clifford1,5, Pauline Funchain2,3, Madhav Sankunny2, Julian D. Perry4, Alexander Blandford4, Gregory Kosmorsky4, Lisa Lystad4, Leonard H. Calabrese1, Charis Eng2,3,6,7 AFFILIATED INSTITUTIONS www.PaiJournal.com 1Center for Vasculitis Care and Research; Department of Rheumatic and Immunologic Diseases; Cleveland Clinic; Cleveland, Ohio 2Genomic Medicine Institute; Lerner Research Institute; Cleveland Clinic; Cleveland, Ohio 3Taussig Cancer Institute; Cleveland Clinic; Cleveland, Ohio 4Cole Eye Institute; Cleveland Clinic; Cleveland, Ohio 5Division of Rheumatology, University of Alberta, Canada 6Department of Genetics and Genome Sciences; Case Western Reserve University School of Med- icine; Cleveland, Ohio 7Germline High Risk Focus Group; CASE Comprehensive Cancer Center; Case Western Reserve University School of Medicine; Cleveland, Ohio *Joint first authors CORRESPONDING AUTHORS Gary S. Hoffman Cleveland Clinic Department of Rheumatic and Immunologic Diseases 9500 Euclid Avenue, A50 Cleveland, OH 44195 216-445-6996 [email protected] Charis Eng Cleveland Clinic Genomic Medicine Institute 9500 Euclid Avenue, NE50 Cleveland, OH 44195 216-444-3440 [email protected] SUGGESTED CITATION Hoffman GS, Getz TM, Padmanabhan R, Villa-Forte A, Clifford AH, Funchain P, Sankunny M, Perry JD, Blandford A, Kosmorsky G, Lystad L, Calabrese LH, Eng C. The Microbiome of Tempo- ral Arteries. Pathogens and Immunity. 2019;4(1):21-38. doi: 10.20411/pai.v4i1.270 ABSTRACT ABSTRACT Objective: A role for microorganisms in giant cell arteritis (GCA) has long been suspected. We describe the microbiomes of temporal arteries from patients with GCA and controls. Methods: Temporal artery biopsies from patients suspected to have GCA were collected under aseptic conditions and snap-frozen. Fluorescence in situ hybridization (FISH) and long-read 16S rRNA-gene sequencing was used to examine microbiomes of temporal arteries. Taxonomic clas- sification of bacterial sequences was performed to the genus level and relative abundances were calculated. Microbiome differential abundances were analyzed by principal coordinate analysis (PCoA) with comparative Unifrac distances and predicted functional profiling using PICRUSt. Results: Forty-seven patients, including 9 with biopsy-positive GCA, 15 with biopsy-negative GCA and 23 controls without GCA, were enrolled. FISH for bacterial DNA revealed signal in the arterial media. Beta, but not alpha, diversity differed between GCA and control temporal arteries (P = 0.042). Importantly, there were no significant differences between biopsy-positive and biop- sy-negative GCA (P > 0.99). The largest differential abundances seen between GCA and non-GCA temporal arteries included Proteobacteria (P), Bifidobacterium (g), Parasutterella (g), and Granu- licatella (g) [Log 2-fold change > 4]. Conclusion: Temporal arteries are not sterile, but rather are inhabited by a community of bacte- ria. We have demonstrated that there are microbiomic differences between GCA and non-GCA temporal arteries, but not between biopsy-positive and biopsy-negative GCA. Keywords: vasculitis; giant cell arteritis; microbiome CORRESPONDING AUTHORS www.PaiJournal.com Pathogens and Immunity - Vol 4, No 1 22 INTRODUCTION Giant cell arteritis (GCA) is the most common large vessel vasculitis in adults, with an estimated incidence of 15-25 cases/100,000 among persons > 50 years old [1]. The most frequently affected vessels are the extracranial branches of the carotid arteries, resulting in headache, scalp tender- ness, jaw claudication, and visual aberration or blindness. Temporal artery biopsies represent the most readily accessible source of tissue for confirmation of diagnosis; however, they may fail to reveal inflammatory infiltrates in up to 50% of cases, due to the presence of skip lesions or the predominance of large vessel disease [2]. Post-mortem and imaging studies have also revealed that involvement of the aorta and its primary branch vessels is common, if not universal, in GCA [3-5]. Indeed, in a post-mortem study of the aorta and primary branch vessels in 13 consecutive patients with GCA, all had features of vasculitis in spite of treatment with corticosteroids in 9 of 13 patients for several months to up to 9 years duration [3]. Although patients with GCA usual- ly respond quickly to treatment with high dose glucocorticoids, relapses occur frequently when doses are tapered, suggesting that the underlying driver of inflammation has not been addressed [6, 7]. Cellular infiltrates in GCA include activated vascular dendritic cells, which attract Th1 and Th17 lymphocytes and activated macrophages to the arterial wall. The antigen(s) responsible for initial activation of dendritic cells has not yet been identified [1]. Interferon gamma is the hallmark cy- tokine of the Th1 response and is a cytokine triggered in response to intracellular pathogens [8]. It is therefore plausible that infectious agents may be providing antigenic stimulation in GCA. www.PaiJournal.com 23 Pathogens and Immunity - Vol 4, No 1 A role for infection in GCA is supported by the observation of cyclical peaks in disease incidence [9]. Multiple viral [10-13] and bacterial [14-16] agents have previously been implicated; however, attempts at pathogen detection by epidemiologic, focused genetic, and immunohistochemical approaches have failed to provide consistent results. Recent reports of varicella zoster virus in temporal artery biopsies in GCA are intriguing, but require additional confirmation [17-20]. Sequencing of the bacterial-specific 16S ribosomal RNA gene from human tissue is a sensitive and culture-independent method for both pathogen and commensal detection, allowing for a more comprehensive and unbiased description of the temporal artery microbiome in GCA. Sample Accrual and Collection We prospectively enrolled consecutive consenting patients undergoing temporal artery biopsy for evaluation of possible GCA, under a study protocol conducted in compliance with the Helsinki Declaration and approved by the Institutional Review Board at the Cleveland Clinic. All partic- ipants provided written informed consent. The authors GSH and CE together have full access to the data and take responsibility for its integrity and data analysis. The temporal artery biopsies were collected under strictly aseptic conditions by a team of ophthalmologists. Biopsies did not include skin. Biopsies were split, with one-half sent for routine histopathological review and one- half snap-frozen under sterile technique for microbiome analysis. Patients were classified accord- ing to clinical phenotype, including corticosteroid use, clinical symptoms, co-morbidities and histopathology results as either biopsy-positive GCA (histopathology confirming inflammatory infiltrates and compatible clinical presentation), biopsy-negative GCA (histopathology without inflammatory infiltrates, but meeting the American College of Rheumatology 1990 Classification criteria for GCA [21] and a persistent clinical diagnosis of GCA at least 3 months post-biopsy), or as controls (patients in whom the diagnosis of GCA and other forms of vasculitis were sub- sequently ruled out). Following >3 years of collection, all samples were processed at the same time. Laboratory-based microbiome investigators were kept unaware of the patients’ clinical and pathological diagnoses. MATERIALS AND METHODS Sample Accrual and Collection 16S Ribosomal RNA-Gene Sequencing of Temporal Artery Biopsies 16S Ribosomal RNA Gene Sequencing of Temporal Artery Biopsies Total deoxyribonucleic acid (DNA) was isolated using the AllPrep DNA/RNA Isolation Kit according to the manufacturer’s protocol (Qiagen, Valencia, CA) with minor modifications [23]. Briefly, all beads, tubes, and non-enzymatic reagents were treated with UV light for 30 minutes prior to use; samples were digested with 20 µL of 20 ng/µL Proteinase K (Roche Diagnostics Corp., Indianapolis, IN) at 65oC for 1 hour, then transferred to 0.1 mm glass beaded tubes. After this the samples were homogenized using the TissueLyser LT (Qiagen). The quality and purity of the isolated total DNA were confirmed spectrophotometrically using a NanoDrop 2000 device (Fisher Scientific SAS, Illkirch, France). DNA concentration was quantified using the Qubit 2.0 instrument applying the Qubit dsDNA HS Assay (Life Technologies). Extracted DNA samples were stored at -20°C. Bacterial 16S rRNA-gene amplification and library construction were performed according to the 16S Metagenomic Sequencing Library Preparation guide from Illumina (Forest City, CA). In brief, 2 µL total DNA was amplified using primers targeting the 16S V3 and V4 region (Illumina) at 95oC for 5 minutes, followed by 35 cycles at 95oC for 30 seconds, 56°C for 30 seconds, and 72oC for 30 seconds with a final extension at 72oC for 10 minutes. The 16S rDNA amplicons were run out on a 1% agarose gel, size-selected at 450-500 bp, and gel-purified using QIAquick Gel Purification kit (Qiagen). A second round of PCR was performed to add Nextera XT indices (Illumina) to purified amplicons. Indexed PCR products were cleaned with Ampure XP beads (Beckman Coulter, Inc., Brea, CA) and resulting libraries quantified with the QuantiFluor dsDNA system according to the manufacturer’s protocol (Promega, Madison, WI). Samples were then normalized to 10nM and pooled into sequencing libraries. Pooled V3-V4 amplicon libraries were sequenced using the Illumina MiSeq platform with V3 reagent kit. The 300-bp paired end reads for each sample were demultiplexed and quality checked using FastQC 0.11.3. Temporal artery specimens were stored in sterile containers. Sterilized water was aliquoted into the containers and then removed and 16S rRNA gene sequencing performed, revealing no con- tamination. Fluorescence In Situ Hybridization (FISH) Two samples (TA6 Control and TA13 GCA) were examined by FISH to identify the possible pres- ence of bacteria within the vessel wall. Tissue had been snap-frozen for 16S rRNA gene analysis (described below) and also formalin-fixed and paraffin-embedded per routine protocols. FISH for bacterial 16S rRNA was performed using a previously published protocol [22]. Sections of 2 µm were cut and mounted on coated microscope slides. Sections were deparaffinized and then sub- jected to cell wall and protein degradation with incubations in lysozyme followed by proteinase K. Sections were hybridized, for 3.5 hours, with 100µM EUB 338, a previously published oligonucle- otide complementary to a universally conserved region of the bacterial 16S rRNA gene. Following this step, sections were counterstained with 0.025% (weight per volume) concanavalin A-Alexa Fluor 594 (Integrated DNA Technologies, Coralville, IA) for 20 minutes. Finally, sections were mounted with Vectashield-DAPI (Vector Laboratories, Burlingame, CA) and dried overnight. Concanavalin A is used as a counterstain for glycoproteins, while DAPI is used for identification of human nuclei. Images were acquired using a Leica TCS-SP2 spectral laser scanning confocal www.PaiJournal.com Pathogens and Immunity - Vol 4, No 1 24 microscope operated with Leica Confocal Software (Leica Microsystems, GmbH, Wetzlar, Ger- many). Fluorescence intensities from signals of FISH-positive bacteria were measured using Image J (NIH). The mean intensity was calculated by subtracting the background intensity for the green channel from the measured intensity within the region of interest (within the media, ie, not exter- nal to the adventitia). Microbiome Analysis A hybrid post-sequencing analysis methodology using QIIME and MICCA was adopted, and pre- processing was performed in QIIME and open-reference operational taxonomic unit (OTU) pick- ing was performed with MICCA and Phyloseq [24, 25]. After the biom files were created, down- stream analysis was performed with QIIME. The 250-bp Illumina paired-end reads were merged with FLASH [26], and low-quality reads (Phred < 20) were filtered out using the split_libraries.py command in QIIME (version 1.9) [27]. MICCA vsearch (version 1.9.5) [28] was utilized for clus- tering the sequences with a threshold of 97% similarity, and representative sequences were classi- www.PaiJournal.com Pathogens and Immunity - Vol 4, No 1 25 fied using RDP classifier (version 2.11) [29]. Multiple sequence alignments were performed using MUSCLE (version 3.8.31) [30, 31] against the Greengenes database (version 13.8) [32], filtered at 97% similarity, and FastTree (version 2.1.8) was used for phylogenetic tree construction [33]. fied using RDP classifier (version 2.11) [29]. Multiple sequence alignments were performed using MUSCLE (version 3.8.31) [30, 31] against the Greengenes database (version 13.8) [32], filtered at 97% similarity, and FastTree (version 2.1.8) was used for phylogenetic tree construction [33]. Data clean-up was done by removing the singletons and discarding taxa represented in fewer than 5% of total samples. The rarefaction value was set to 8,467 reads per sample to reduce sampling heterogeneity, and computation of alpha (Shannon diversity index) and beta diversity measures (unweighted UniFrac distances) were performed with PhyloSeq in R. Alpha diversity measures species richness (number of taxa) within a single microbial ecosystem. Beta diversity can be represented by UniFrac distances which describe similarities and dissimilarities between bacterial communities using phylogenetic information, taking into account the number of taxa and relative abundances within each taxon. F-tests based on sequential sums of squares derived from 1,000 permutations on UniFrac distance matrices were performed with the null hypothesis that there is no difference in community structure between groups. Note that PCoA and the calculation of P values are measurements of clustering strength. This is not based on linear correlations (because this is not linear). Differences (and the P value) are derived from measuring differences between UniFrac distances. To find which taxa are most likely to explain the differences between our clinical groupings, taxa summaries and differential abundances were analyzed with DESeq2. Microbiome Analysis This algorithm estimates variance-mean dependence in count data and tests for differential expression based on a model using the negative binomial distribution. Differentially abundant taxa that were statistically significant using an alpha of 0.05 and exceeded a Log2-fold change of ±2 were visually represented on box plots. A heatmap was generated from the top differentially dominant OTUs using the Bray-Curtis distance methods and where the plot was created using pheatmap in R. We analyzed functional composition of microbiomes using the PICRUSt 1.0.0-dev bioinformat- ics package [34]. We filtered out all de novo OTUs and used this OTU table as our input into the PICRUSt algorithm, which calculates contributions of various OTUs to known biological path- ways based on evolutionary modeling. The OTU picking was performed against the Greengenes (gg_13_8) database using a 97% similarity threshold. Welch’s t test was used to calculate P values, and corresponding Storey q-values were used to control for the false discovery rates associated with multiple testing. These values were calculated using DESeq2 and LEfSe and visualized as a cladogram or bar plot using Phyloseq or LEfSe [35]. Patients Forty-seven patients were enrolled in the study, including 9 patients with a final diagnosis of biopsy-positive GCA, 15 patients with biopsy-negative but clinically confirmed GCA, and 23 additional patients in whom the diagnosis of GCA was ruled out by histology and clinical course (Table 1). Median age at the time of biopsy was 71 years for those with GCA and 73 for non-GCA controls (P = 0.5). Two-thirds of the patients were female (P = 0.065 between GCA and non-GCA patients) [Table 1]. Thirty-eight of 49 patients (78%) had been receiving daily prednisone (mean dose > 50mg/day) prior to temporal artery biopsies (89% in the GCA biopsy-positive group, 86% in the GCA biopsy-negative group, and in 71% of controls). There was no difference between groups in prednisone use (P > 0.35), dose (P > 0.5) or duration of treatment (P = 0.56) [Table 1]. www.PaiJournal.com Pathogens and Immunity - Vol 4, No 1 26 Table 1. Patient baseline demographics at the time of biopsy    Controls (23) Bx- GCA* (15) Bx+ GCA (9) Total (47) Age (years) 72.9 +/-9.4 71.2+/- 2.4 75.5+/- 3.2 71.9+/-8.9 Female, no. (%) 15 (65%) 9 (60%) 4 (44%) 28 (60%)  Race, no. White (%) 21 (91%) 12 (80%)   8 (89%) 41 (87%)  Vascular Symptoms, no. (%) 16 (70%)  12 (80%)  8 (89%)   36 (77%) Systemic Symptoms, no. (%) 2 (9%) 6 (40%)  1 (11%)    9 (19%) ESR (mm/hr) (mean+/-SD, range)  39+/-6  48+/-7  32+/-9  40+/-25 CRP (mg/dl) (mean+/-SD, range) 1.8+/-1.1 4.5+/-1.5   5.6+/-1.6 3.5+/-4.5  Prednisone use (%) 15 (65%)   12 (80%) 8 (89%)  35 (74%)  Prednisone use >50mg/d (mean prior to biopsy) 12 (52%)   6 (40%) 5 (56%)  23 (49%)  Duration of prednisone (days) [mean+/-SD]  23+/-35 14+/-6  32+/-50 22+/-35  Other immunosuppression  2 (9%)**  0 (0%)   0 (0%)  2 (4%) *Biopsy-negative, but clinically confirmed positive GCA. **One patient each was receiving low dose methotrexate for rheumatoid arthritis and another for granulo- Table 1. Patient baseline demographics at the time of biopsy Biopsy negative, but clinically confirmed positive GCA. **One patient each was receiving low dose methotrexate for rheumatoid arthritis and another for granulo- matosis with polyangiitis. *Biopsy-negative, but clinically confirmed positive GCA. www.PaiJournal.com www.PaiJournal.com Pathogens and Immunity - Vol 4, No 1 27 Histopathology Temporal artery biopsies of the 9 patients with a final diagnosis of biopsy-proven GCA revealed arteritis, with mononuclear cell inflammatory infiltrates localized to the adventitia and media, fragmentation of the internal elastic lamina, and varying degrees of intimal proliferation and fibrinoid necrosis. All biopsies from patients who were biopsy-negative with clinically positive GCA and 20 of 23 TA from controls without GCA revealed arteriosclerosis, with intimal thicken- ing and rare, focal calcification; 3 TA from controls without GCA were normal. Bacterial DNA Detection in Temporal Arteries by FISH y Sterile, fresh frozen temporal artery biopsies from a control patient (TA6C) and a patient with GCA (TA13G) were fixed and paraffin-embedded. FISH using an oligonucleotide probe specif- ic for bacterial 16S rRNA revealed the presence of multiple single bacteria in the media of both control and GCA-involved arteries (Figure 1). No FISH signal corresponding to the presence of bacteria was apparent in the intimal layer, arterial lumen, or external border of the specimen. Corroborating the FISH microscopy, the mean intensity for the FISH-positive bacteria (Figure 1, bar graph) was higher in GCA-involved arteries compared to that of the control temporal artery (Figure 1B vs 1A), and no signal was ascertained at the external edge of a GCA-involved temporal artery (Figure 1C). www.PaiJournal.com In a temporal artery with histopathological evidence of GCA (B), bacterial DNA is scattered throughout the media and at a higher mean intensity than control (bar graph). Arterial layers are more disorganized and less distinct compared to a control temporal artery, as evidenced by weak autofluorescence and less distinct internal elastic lamina. There is an absence of bacterial DNA at the external edge of a GCA-involved temporal artery specimen (C, bar graph). Figure 1. Distribution of bacterial DNA in temporal arteries. Tissue sections were probed with fluores- cently labeled oligonucleotide probes against bacterial DNA (green). Sections were counterstained with DAPI (blue) and Concanavalin A (red) to delineate nuclei and glycoproteins, respectively. Sections were scanned by confocal microscopy. In a control temporal artery (A), bacterial DNA is scattered throughout the media, with select examples highlighted by green arrows. Notably, no/negligible bacterial DNA stain- ing is apparent in the lumen or intima (A, bar graph). The green channel emitted from the internal elastic lamina is a result of autofluorescence. In a temporal artery with histopathological evidence of GCA (B), bacterial DNA is scattered throughout the media and at a higher mean intensity than control (bar graph). Arterial layers are more disorganized and less distinct compared to a control temporal artery, as evidenced by weak autofluorescence and less distinct internal elastic lamina. There is an absence of bacterial DNA at the external edge of a GCA-involved temporal artery specimen (C, bar graph). www.PaiJournal.com Pathogens and Immunity - Vol 4, No 1 28 Figure 1. Distribution of bacterial DNA in temporal arteries. Tissue sections were probed with fluores- cently labeled oligonucleotide probes against bacterial DNA (green). Sections were counterstained with DAPI (blue) and Concanavalin A (red) to delineate nuclei and glycoproteins, respectively. Sections were scanned by confocal microscopy. In a control temporal artery (A), bacterial DNA is scattered throughout the media, with select examples highlighted by green arrows. Notably, no/negligible bacterial DNA stain- ing is apparent in the lumen or intima (A, bar graph). The green channel emitted from the internal elastic lamina is a result of autofluorescence. In a temporal artery with histopathological evidence of GCA (B), bacterial DNA is scattered throughout the media and at a higher mean intensity than control (bar graph). Arterial layers are more disorganized and less distinct compared to a control temporal artery, as evidenced by weak autofluorescence and less distinct internal elastic lamina. There is an absence of bacterial DNA at the external edge of a GCA-involved temporal artery specimen (C, bar graph). Figure 1. Distribution of bacterial DNA in temporal arteries. Tissue sections were probed with fluores- cently labeled oligonucleotide probes against bacterial DNA (green). Sections were counterstained with DAPI (blue) and Concanavalin A (red) to delineate nuclei and glycoproteins, respectively. Sections were scanned by confocal microscopy. In a control temporal artery (A), bacterial DNA is scattered throughout the media, with select examples highlighted by green arrows. Notably, no/negligible bacterial DNA stain- ing is apparent in the lumen or intima (A, bar graph). The green channel emitted from the internal elastic lamina is a result of autofluorescence. In a temporal artery with histopathological evidence of GCA (B), bacterial DNA is scattered throughout the media and at a higher mean intensity than control (bar graph) Figure 1. Distribution of bacterial DNA in temporal arteries. Tissue sections were probed with fluores- cently labeled oligonucleotide probes against bacterial DNA (green). Sections were counterstained with DAPI (blue) and Concanavalin A (red) to delineate nuclei and glycoproteins, respectively. Sections were scanned by confocal microscopy. In a control temporal artery (A), bacterial DNA is scattered throughout the media, with select examples highlighted by green arrows. Notably, no/negligible bacterial DNA stain- ing is apparent in the lumen or intima (A, bar graph). The green channel emitted from the internal elastic lamina is a result of autofluorescence. Microbiome in Temporal Arteries from Patients with and without Giant Cell Arteritis Culture-independent, long-read genomic sequencing was used to characterize the entire microbi- al communities of temporal arteries from the 47 research participants. After sequencing and qual- ity control, 4 samples were excluded from further analyses because of low read counts, leaving 43 samples comprising 20 with GCA (7 biopsy-positive, 13 biopsy-negative) and 23 without GCA. There were no alpha diversity differences between GCA and non-GCA temporal artery microbio- ta. In contrast, beta-diversity, as measured by unweighted UniFrac distances, differed between the GCA and non-GCA groups (P = 0.042, Figure 2A). Of note, there were no statistically significant differences between temporal arteries from those with biopsy-positive GCA and those with biop- sy-negative GCA (P = 1.0, Figure 2B). www.PaiJournal.com Pathogens and Immunity - Vol 4, No 1 29 Figure 2. Microbiomes from TAs with biopsy-positive and biopsy-negative GCA cluster together but differently from those from control patients. Principal component analysis (PCoA) of TA microbiomes. (A) β-diversity (not α, data not shown), differs between GCA and control groups (P = 0.042). (B) There were no statistically significant differences between TA microbiomes in those with biopsy-positive GCA vs those with biopsy-negative/clinically positive GCA (P > 0.99). Figure 2. Microbiomes from TAs with biopsy-positive and biopsy-negative GCA cluster together but differently from those from control patients. Principal component analysis (PCoA) of TA microbiomes. (A) β-diversity (not α, data not shown), differs between GCA and control groups (P = 0.042). (B) There were no statistically significant differences between TA microbiomes in those with biopsy-positive GCA vs those with biopsy-negative/clinically positive GCA (P > 0.99). In order to gain insight into the microbial inhabitants of temporal arteries, we compared the rela- tive abundances of bacterial OTUs within temporal artery biopsies with and without GCA (Fig- ure 3). At the phylum level, there were at least 2 classes of Firmicutes relatively over-represented (> + 4) in GCA temporal arteries compared to those without GCA, although there were 2 other classes of Firmicutes relatively under-represented in temporal arteries with GCA versus temporal arteries without GCA (< -4, Figure 3A). Proteobacteria and Actinobacteria were relatively un- der-represented in temporal artery samples from patients with GCA compared to those without GCA (< -4; Figure 3A). Microbiome in Temporal Arteries from Patients with and without Giant Cell Arteritis At the genus level, Granulicatella and Streptococcus, both belonging to phylum Firmicutes, were relatively over-represented whereas Parasutteralla, belonging to phylum Proteobacteria, and Bifidobacterium, belonging to phylum Actinobacteria, were relatively un- der-represented in temporal artery samples from patients with GCA compared to those without GCA (Figure 3B). www.PaiJournal.com (A) Bar blot representation from DESeq2 showing the most over-represented (+) and under-represented (-) phyla in TAs from patients with GCA compared to TAs from controls. (B) Bar blot representation from DESeq2 showing the most over-represented (+) and under-represented (-) genera in TAs from patients with GCA compared to TAs from controls. (C) Heat map of bacterial communities in TA with GCA (“inflammatory” blue bar) compared to those without GCA (“noninflammatory” pink bar) based on the top dominant OTUs. Columns and rows represent samples and dominant OTUs, respectively. Row names on the right of the heat map include Green Genes ID followed by family and genus. www.PaiJournal.com www.PaiJournal.com Pathogens and Immunity - Vol 4, No 1 30 Figure 3. Most differentially abundant taxa in temporal artery biopsies from patients with GCA and from control patients. (A) Bar blot representation from DESeq2 showing the most over-represented (+) and under-represented (-) phyla in TAs from patients with GCA compared to TAs from controls. (B) Bar blot representation from DESeq2 showing the most over-represented (+) and under-represented (-) genera in TAs from patients with GCA compared to TAs from controls. (C) Heat map of bacterial communities in TA with GCA (“inflammatory” blue bar) compared to those without GCA (“noninflammatory” pink bar) based on the top dominant OTUs. Columns and rows represent samples and dominant OTUs, respectively. Row names on the right of the heat map include Green Genes ID followed by family and genus. Figure 3. Most differentially abundant taxa in temporal artery biopsies from patients with GCA and from control patients. (A) Bar blot representation from DESeq2 showing the most over-represented (+) and under-represented (-) phyla in TAs from patients with GCA compared to TAs from controls. (B) Bar blot representation from DESeq2 showing the most over-represented (+) and under-represented (-) genera Figure 3. Most differentially abundant taxa in temporal artery biopsies from pa Figure 3. Most differentially abundant taxa in temporal artery biopsies from patients with GCA and f t l ti t (A) B bl t t ti f DES 2 h i th t t d ( ) Figure 3. Most differentially abundant taxa in temporal artery biopsies from patients with GCA and from control patients. (A) Bar blot representation from DESeq2 showing the most over-represented (+) and under-represented (-) phyla in TAs from patients with GCA compared to TAs from controls. (B) Bar blot representation from DESeq2 showing the most over-represented (+) and under-represented (-) genera in TAs from patients with GCA compared to TAs from controls. (C) Heat map of bacterial communities in TA with GCA (“inflammatory” blue bar) compared to those without GCA (“noninflammatory” pink bar) based on the top dominant OTUs. Columns and rows represent samples and dominant OTUs, respectively. Row names on the right of the heat map include Green Genes ID followed by family and genus. Figure 3. Most differentially abundant taxa in temporal artery biopsies from patients with GCA and from control patients. Predicting Functional Consequences of Differing Microbial Composition in GCA- and Non-GCA- Associ- ated Temporal Arteries We analyzed microbiomes by predicting their functional contributions to their host environments using the PICRUSt algorithm. There was relative downregulation of ion-coupled transporters and steroid biosynthesis pathways in the group with GCA (combining biopsy-positive and bi- opsy-negative) compared to temporal arteries from non-GCA controls (all P < 0.05, Figure 4A). When only the biopsy-positive GCA temporal arteries were compared to non-GCA controls, the steroid biosynthesis pathway was relatively downregulated while the cardiac muscle contraction pathway was relatively upregulated (Figure 4B). While the steroid biosynthesis pathway remained relatively downregulated in biopsy-negative GCA compared to controls, there were multiple other pathways that were relatively upregulated and downregulated (Figure 4C). Interestingly, predicted downregulation of metabolism pathways appears to be found between both biopsy-positive GCA compared to control and biopsy-negative GCA compared to control. www.PaiJournal.com Pathogens and Immunity - Vol 4, No 1 31 Figure 4. Predicted functional pathways differentially represented in GCA TA compared to con- trol (non-GCA) TA. Representation of PICRUSt DESeq2 analysis yielding relatively under-represented functional pathways in (A) GCA TA compared to control TA, (B) biopsy-positive GCA TA compared to control TA and (C) biopsy-negative GCA TA versus control TA. Figure 4. Predicted functional pathways differentially represented in GCA TA compared to con- trol (non-GCA) TA. Representation of PICRUSt DESeq2 analysis yielding relatively under-represented functional pathways in (A) GCA TA compared to control TA, (B) biopsy-positive GCA TA compared to control TA and (C) biopsy-negative GCA TA versus control TA. DISCUSSION This is corroborated by our FISH studies which demonstrate bacterial nucleic acid within the arterial walls, without any bacteria external to the adventitia nor in the arterial lumen. We were concerned about the effects of treatment on our analyses. It is generally recommend- ed that once the diagnosis of GCA is considered, treatment with corticosteroids should not be delayed while awaiting temporal artery biopsy [5]. Consequently, it was expected that most of our patients (74%) had been treated prior to biopsy. However, in comparing corticosteroid treated and untreated cases, treatment did not appear to influence the microbiome. Bhatt et al also note that indications for temporal artery biopsies in control patients are another factor that could impact GCA studies [36]. All biopsied individuals usually have had symptoms or findings suggestive of GCA, leading their physician to arrange for this procedure. Thus, inves- tigators should be concerned about whether controls are truly GCA biopsy-negative but clinically positive. We tried to minimize this risk by following patients for at least 3 months post-biopsy to ensure that while untreated, features of GCA did not emerge and initial symptoms had resolved or could be attributed to alternative diagnoses. We therefore have a high degree of confidence that our controls did not have undiagnosed GCA. Our results suggest that there does not appear to be a single bacterial pathogen characteristic of GCA. Species previously implicated in GCA pathogenesis such as Mycoplasma pneumonia [14], Chlamydia pneumonia [16], and Burkholderia pseudomallei-like organisms [15] were not found in our dataset. Given the high person-to-person species variability in our study, the inconsistency of named bacterial species in prior publications is not surprising. Additionally, many prior studies used formalin-fixed tissue, which is known to cause DNA cross-linking, nucleic acid shearing, reduction in yield, and sequencing artifacts [37]. We believe our fresh-frozen temporal artery samples, collected aseptically and specifically for microbiomic studies, most closely represent the actual bacterial constituents in temporal arteries. Our findings included differences in microbiome content and density for the phylum Firmic- utes, which was relatively over-represented, and Proteobacteria and Actinobacteria, which were relatively under-represented in temporal arteries from patients with GCA compared to controls. While differences were found in functional pathways between GCA and non-GCA cases, the sig- nificance of those changes is uncertain. The notion of a vascular microbiome is neither new nor novel [38]. DISCUSSION Our most reliable and important observation is that temporal arteries are not sterile, as previously assumed, but rather are inhabited by communities of bacteria in both the control and diseased state. Interestingly, the microbiomes of biopsy-negative, clinically confirmed GCA temporal arteries were similar to those of biopsy-positive GCA temporal arteries, suggesting a potential underlying similarity of temporal arteries from patients with GCA not reflected in histopatholog- ic review. Together, microbial communities in biopsy-positive and biopsy-negative GCA temporal arteries were also distinct from those in control GCA-negative samples. This observation raises an important question regarding why there are histopathologic differences between biopsy-positive and biopsy-negative individuals with GCA since the microbiome between these subsets is similar. The answer is unknown. One might speculate that if microbiomes played a role in pathogenesis of GCA, that role may be permissive, and at a later uncertain time interval, be followed by a histo- logically apparent inflammatory response. If such were the case, “skip lesions” in GCA biopsies, which are well known, may result from a GCA step-wise inflammatory response. Our study did not address this possibility. We believe that our GCA microbiome study is unique because of collecting and maintaining tissues using aseptic techniques, avoiding formalin fixation, paraffin embedding, and contamina- tion with known skin and external environmental organisms and not disclosing patient diagno- ses to our lab-based colleagues. Environmental contamination had been a concern of Bhatt and colleagues, who had identified Propionibacterium acnes and Escherichia coli as the most abundant microorganisms found in both formalin-fixed paraffin-embedded temporal arteries from patients with GCA and in controls [36]. Both quantitative and qualitative microbiome data were similar in their GCA cases and controls. Our study also differs in regard to clear qualitative differenc- es noted between GCA and control samples. The differences in microbiome between GCA and non-GCA temporal artery specimens also supports the conclusion that contamination was not www.PaiJournal.com Pathogens and Immunity - Vol 4, No 1 32 likely to have affected these specimens which were processed in identical fashion and analyzed at the same time. This is corroborated by our FISH studies which demonstrate bacterial nucleic acid within the arterial walls, without any bacteria external to the adventitia nor in the arterial lumen. likely to have affected these specimens which were processed in identical fashion and analyzed at the same time. DISCUSSION Many studies have demon- strated the presence of both bacteria and viruses within the walls of large and medium-sized blood vessels. Most compelling are the findings of bacteria in the lipid-rich core of plaques and within smooth muscle cells of atherosclerotic aortas and coronary arteries. Recent reports have also raised questions about the potential role of microbes in non-atherosclerotic arteries and apparently normal vessels. The most compelling of these studies are those that are metage- nome-DNA-based. One study included 56 fresh, sterile aortic aneurysm samples from patients with atherosclerosis and non-atherosclerotic disease [39, 40]. Using PCR with universal 16S rRNA primers, bacterial DNA was isolated from about 90% of samples. Ten samples were selected for speciation from patients with Marfan’s syndrome, idiopathic aortitis, aortic coarctation, and mechanical/degenerative aneurysms. All but 1 specimen revealed the presence of multiple bac- terial species [39]. While metagenomic techniques are unbiased and more sensitive, they cannot www.PaiJournal.com Pathogens and Immunity - Vol 4, No 1 33 determine whether the detected DNA is from living organisms or are remnants of microbes or contaminants. These unresolved questions can be applied to our findings in temporal artery biop- sies as well and represent universal limitations of such studies. determine whether the detected DNA is from living organisms or are remnants of microbes or contaminants. These unresolved questions can be applied to our findings in temporal artery biop- sies as well and represent universal limitations of such studies. Our study does have important strengths. It is the first to be performed on surgically sterile tem- poral arteries that were maintained under strict aseptic conditions throughout processing. Micro- biome studies were performed by some of the authors without knowledge of clinical or pathologi- cal diagnoses. Arguing against contamination are first, the FISH studies that revealed the absence of bacterial DNA on the adventitial or luminal surfaces of specimens, and second, differences in microbial communities in patients with GCA versus controls (beta diversity). In addition, spec- imens from clinically diagnosed but biopsy-negative patients with GCA were not eligible for the study until at least 3 months of follow-up during which other diagnoses did not become apparent and satisfactory responses to corticosteroid therapy were well documented. Conversely, controls were not classified as such until similar follow-up revealed alternative diagnoses. ACKNOWLEDGMENTS We thank the Nurses and Coordinators from the Cleveland Clinic Clinical Research Unit for help with study coordination and data collection, and especially the patients who volunteered their samples to this study. FUNDINGh This work was supported, in part, by the Fasenmyer Clinical Immunology Center (to GSH, LC and CE), the National Center for Advancing Translational Sciences (NCATS) of the NIH (UL- 1TR000439, to GSH), and a post-graduate fellowship training grant from the Union Chimique Belge-Canadian Rheumatology Association-The Arthritis Society (to AHC). PF was an Ambrose Monell Foundation Cancer Genomic Medicine Clinical Fellow and MS is an Ambrose Monell Foundation Cancer Genomic Medicine Fellow (PhD clinical research track) [to CE]. CE is the Sondra J. and Stephen R. Hardis Endowed Chair in Cancer Genomic Medicine at the Cleveland Clinic and an American Cancer Society Clinical Research Professor. DISCUSSION Our observations here are most important in emphasizing that temporal arteries are not sterile and that quantitative and qualitative microbiome and metabolic differences exist between those vessels in patients with GCA and controls. 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This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International License. h 4.0 International License. www.PaiJournal.com www.PaiJournal.com

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The non-muscle ADF/cofilin-1 controls sarcomeric actin filament integrity and force production in striated muscle laminopathies

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To cite this version: Nicolas Vignier, Maria Chatzifrangkeskou, Luca Pinton, Hugo Wioland, Thibaut Marais, et al.. The non-muscle ADF/cofilin-1 controls sarcomeric actin filament integrity and force production in striated muscle laminopathies. Cell Reports, 2021, 36 (8), pp.109601. ￿10.1016/j.celrep.2021.109601￿. ￿hal- 03350074￿ In brief Extracellular signal-regulated kinase (ERK) 1/2 has been shown to be important for the development of muscular dystrophy. Vignier et al. find that active ERK1/2 catalyzes phosphorylation of cofilin-1 on Thr25 and protects it from proteasomal degradation, which physically disrupts sarcomeric organization and causes loss of muscle force generation. Authors Graphical abstract Nicolas Vignier, Maria Chatzifrangkeskou, Luca Pinton, ..., Francesco Saverio Tedesco, Antoine Je´ gou, Antoine Muchir Nicolas Vignier, Maria Chatzifrangkeskou, Luca Pinton, ..., Francesco Saverio Tedesco, Antoine Je´ gou, Antoine Muchir Correspondence [email protected] Correspondence [email protected] HAL Id: hal-03350074 https://hal.sorbonne-universite.fr/hal-03350074v1 Submitted on 21 Sep 2021 L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. Article The non-muscle ADF/cofilin-1 controls sarcomeric actin filament integrity and force production in striated muscle laminopathies Highlights d Phosphorylation of cofilin-1 on Thr25 by p-ERK1/2 protects it from degradation by the UPS d Phospho(T25)-cofilin-1 localizes on I-bands in sarcomeres d Phosphorylation of cofilin-1 on Thr25 alters sarcomeric organization d Phospho(T25)-cofilin-1 alter skeletal muscle force and lead to muscular dystrophy ll ll ll OPEN ACCESS SUMMARY Cofilins are important for the regulation of the actin cytoskeleton, sarcomere organization, and force produc- tion. The role of cofilin-1, the non-muscle-specific isoform, in muscle function remains unclear. Mutations in LMNA encoding A-type lamins, intermediate filament proteins of the nuclear envelope, cause autosomal Em- ery-Dreifuss muscular dystrophy (EDMD). Here, we report increased cofilin-1 expression in LMNA mutant muscle cells caused by the inability of proteasome degradation, suggesting a protective role by ERK1/2. It is known that phosphorylated ERK1/2 directly binds to and catalyzes phosphorylation of the actin-depolyme- rizing factor cofilin-1 on Thr25. In vivo ectopic expression of cofilin-1, as well as its phosphorylated form on Thr25, impairs sarcomere structure and force generation. These findings present a mechanism that provides insight into the molecular pathogenesis of muscular dystrophies caused by LMNA mutations. Article The non-muscle ADF/cofilin-1 controls sarcomeric actin filament integrity and force production in striated muscle laminopathies Nicolas Vignier,1,11 Maria Chatzifrangkeskou,1,11,12 Luca Pinton,2,3 Hugo Wioland,4 Thibaut Marais,1 Me´ gane Lemaitre,5 Caroline Le Dour,1 Ce´ cile Peccate,1 De´ borah Cardoso,1 Alain Schmitt,6 Wei Wu,7,8 Maria-Grazia Biferi,1 Naı¨ra Naouar,1 Coline Macquart,1 Maud Beuvin,1 Vale´ rie Decostre,1 Gise` le Bonne,1 Guillaume Romet-Lemonne,4 Howard J. Worman,7,8 Francesco Saverio Tedesco,2,9,10 Antoine Je´ gou,4 and Antoine Muchir1,13,* 1Sorbonne Universite´ , INSERM, Institut de Myologie, Centre de Recherche en Myologie, 75013 Paris, France 2Department of Cell and Developmental Biology, University College London, London, UK 3Randall Centre for Cell and Molecular Biophysics, King’s College London, London, UK 4Universite´ de Paris, CNRS, Institut Jacques Monod, 75013 Paris, France 5Sorbonne Universite´ , UMS28, Phe´ notypage du Petit Animal, Paris, France 6Universite´ de Paris, INSERM, CNRS, Institut Cochin, 75005 Paris, France 7Department of Medicine, Vagelos College of Physicians and Surgeons, Columbia University, New York, NY, USA 10The Francis Crick Institute, London, UK 11These authors contributed equally 12Present address: Department of Oncology, University of Oxford, Oxford, UK 13Lead contact 12Present address: Department of Oncology, University of Oxford, Oxford, UK 13Lead contact *Correspondence: [email protected] https://doi.org/10.1016/j.celrep.2021.109601 Cell Reports 36, 109601, August 24, 2021 ª 2021 The Author(s). 1 This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). Cell Reports 36, 109601, August 24, 2021 ª 2021 The Author(s). 1 der the CC BY license (http://creativecommons.org/licenses/by/4.0/). 2 Cell Reports 36, 109601, August 24, 2021 INTRODUCTION Each skeletal muscle cell is composed of a repeated array of sarcomeres, the fundamental contractile units. Skeletal muscle cells are highly organized cells and necessary for voluntary move- ment induced by the somatic nervous system and to maintain posture. The coordinated contraction of all sarcomeres shortens the entire muscle cell and produces mechanical force. Despite the recent advances in deciphering the clinical description of muscular dystrophy caused by LMNA mutations (Madej-Pilarc- zyk, 2018), the molecular mechanisms leading to skeletal muscle damage remain to be determined. Loss of structural function and altered activation of tissue-specific signaling pathways have been proposed to partially explain the striated muscle dysfunc- tion in these diseases (Brull et al., 2018). We have previously shown that the extracellular signal-regulated kinase 1/2 (ERK1/ 2) activity was altered in the affected skeletal muscles expressing disease-causing A-type lamins variants, thus participating in LMNA encodes lamin A and lamin C, two components of the nu- clear lamina that are essential for nuclear architecture and regu- lation of chromatin organization (Aebi et al., 1986; Dechat et al., 2008; Lin and Worman, 1993). LMNA mutations are responsible for autosomal forms of Emery-Dreifuss muscular dystrophy (EDMD) (Bonne et al., 1999), a disorder characterized by pro- gressive muscle weakness and wasting associated with early contractures and dilated cardiomyopathy (Emery, 1987). LMNA mutations also cause limb girdle muscular dystrophy (Muchir et al., 2000), congenital muscular dystrophy (Quijano-Roy et al., 2008), or isolated cardiomyopathy without skeletal muscle involvement (Fatkin et al., 1999), expanding the phenotypic spectrum of striated muscle diseases linked to mutant A-type nuclear lamins. Article ll OPEN ACCESS Article ll OPEN ACCESS A A B C (legend on next page) Reports 36, 109601, August 24, 2021 B B B C (legend on next page) C C (legend on next page) 2 Cell Reports 36, 109601, August 24, 2021 Article ll OPEN ACCESS fast twitch extensor digitorum longus (EDL) muscle from old Lmnap.H222P/H222P mice (Figures S1A and S1B). pathogenesis (Muchir et al., 2013). However, insights into the mechanisms bridging abnormal ERK1/2 activation and defective skeletal muscle function are lacking. Figure 1. Abnormal soleus muscle structure and function in Lmnap.H222P/H222P mice (A) Histochemical analysis of soleus from young and old wild-type (WT) and Lmnap.H222P/H222P (H222P) mice. Sections of soleus muscles were stained with hematoxylin and eosin, modified Gomori’s trichrome, and Sirius red, showing an increase of fibrosis (arrows) in the old Lmna p.H222P/H222P (H222P) mice. Scale bar, 50 mm. (B) Expression of fibrosis-related genes (Col1a2, Col3a1, and Ctgf) in the soleus from young and old WT (n = 4) and Lmnap.H222P/H222P (H222P) (n = 4) mice. *p % 0.01. Data are represented as mean ± SD. (C) Top: representative curves of tetanic forces of soleus from young and old, WT and Lmnap.H222P/H222P (H222P) mice. Bottom: box-and-whisker plots showing median of tetanic forces of soleus from young, WT (n = 12) and Lmnap.H222P/H222P (H222P) (n = 12) mice and old, WT (n = 14) and Lmnap.H222P/H222P (H222P) (n = 9) mice. *p % 0 01 ***p % 0 0001 between old WT and Lmnap.H222P/H222P (H222P) Data are represented as mean ± SD Altered skeletal muscle actin dynamics in EDMD To identify the molecular mechanisms that underlie skeletal mus- cle alterations, we performed transcriptomic analysis on soleus muscle along the course of the muscular dystrophy in Lmnap.H222P/H222P mice. A principal-component analysis (Fig- We and others recently reported defective actin dynamics in cardiac muscle of mouse models of EDMD (Antoku et al., 2019; Chatzifrangkeskou et al., 2018; Ho et al., 2013). Therefore, we analyzed the expression of cofilin-1, cofilin-2, Neural Wiskott- Aldrich syndrome protein (N-WASP), actin-related protein 2 (ARP2), and profilin-1, proteins involved in the regulation of actin dynamics in soleus muscle (Figure 2A). We found increased cofi- lin-1 as well as decreased profilin-1 expression in the soleus mus- cleofold Lmnap.H222P/H222P mice(Figures 2B and 2C).The expres- sionoftheseproteinswasnotsignificantlyaffected atyoungerage (Figures S3A and S3B). Importantly, we also observed an activa- tion of cofilin-1 expression in skeletal muscle from a patient with EDMD carrying the p.E358K LMNA mutation (Figure 2D). When examined by immunoblotting, the ratio of F-actin to G-actin was significantly lower in soleus muscle from old Lmnap.H222P/H222P mice compared with WT mice (Figure 2E). These data indicate an actin filament disassembly in autosomal EDMD. Skeletal muscle pathology in soleus from Lmnap.H222P/H222P mice Severe cytoarchitectural abnormalities of skeletal muscles, particularly in soleus muscle, associated with increased connec- tive tissue have been previously observed in Lmnap.H222P/H222P mice, a mouse model of muscular dystrophy caused by LMNA mutations (Arimura et al., 2005). We confirmed these findings by histological analyses in soleus muscles from young and old Lmnap.H222P/H222P mice (Figure 1A). We also showed that the transcript levels of Col1a2 and Col3a1 encoding type I and III col- lagens, and Ctgf encoding connective tissue growth factor, were all correlated with the presence of interstitial fibrosis in the soleus muscle from old Lmnap.H222P/H222P mice (Figure 1B). We next determined whether these changes in structural organization of soleus muscle could impede force generation. In conjunction with the dystrophic pattern, maximal force production was reduced in soleus muscle from old Lmnap.H222P/H222P mice compared with wild-type (WT) mice (Table S1; Figure 1C). These structural and functional abnormalities were not observed in the Altered skeletal muscle actin dynamics in EDMD The actin cytoskeleton contributes to the functional and struc- tural organization of cells. Actin filaments are also a main compo- nent of sarcomeres, which play an active role in the contractile force of muscle. The ADF/cofilin family comprises small actin- binding proteins with key roles in tissue homeostasis and dis- ease. In mammals, three isoforms of the cofilin family have been described: ADF, cofilin-1, and cofilin-2 (Maciver and Hus- sey, 2002). The functions of each ADF/cofilin are not clearly defined, mostly because many of them may overlap. Cofilin-1 is a protein known to enhance actin filament turnover by severing and promoting dissociation of filamentous actin (F-actin) poly- mers into globular actin (G-actin) monomers (Bamburg and Bern- stein, 2008). However, cofilin-2 was the only isoform reported to control the actin filaments turnover in mature muscle sarcomeres (Kremneva et al., 2014; Vartiainen et al., 2002). Here, we have uncovered a function of ERK1/2 signaling that catalyzes the phosphorylation of the cofilin-1 on Thr25, to protect it from degra- dation by the ubiquitination-proteasome pathway. This tight regulation of phospho(T25)-cofilin-1 protein levels by ERK1/2 signaling is important for the maintenance of sarcomere structure and force generation, which participate in the development of muscular dystrophy caused by LMNA mutations. To identify the molecular mechanisms that underlie skeletal mus- cle alterations, we performed transcriptomic analysis on soleus muscle along the course of the muscular dystrophy in Lmnap.H222P/H222P mice. A principal-component analysis (Fig- ure S2A) and a heatmap of unsupervised hierarchical cluster analysis (Figure S2B) performed on all the probes sets showed clear separation between young and old Lmnap.H222P/H222P mice and WT mice. We next used a supervised learning method to distinguish probe sets representing genes with significant dif- ferences in expression. This analysis identified up and downre- gulated genes between young and old Lmnap.H222P/H222P mice and WT mice and also along the progression of the disease for Lmnap.H222P/H222P mice (Figures S2C and S2D; Table S2). These results were validated by reverse transcription quantitative poly- merase chain reaction (RT-qPCR) (Figure S2E). We then analyzed functional class scoring, which improves sensitivity by statistically evaluating genes in biologically meaningful groups. Alteration of transcripts encoding genes relating to sar- comeric structure and structural organization of the muscle fiber was strongly correlated with the progression of the muscular dystrophy in Lmnap.H222P/H222P mice (Figure S2F). Activated ERK1/2 signaling affects the stability of cofilin-1 These results raised the question whether the abnormal skeletal muscle activation of ERK1/2 signaling in muscular dystrophy Cell Reports 36, 109601, August 24, 2021 3 Article ll OPEN ACCESS A C D E B Figure 2. Increased cofilin-1 expression alters actin dynamics in Emery-Dreifuss muscular dystrophy (EDMD) (A) Schematic representation of actin dynamics mechanisms. (B) Immunoblots showing pERK1/2, ERK1/2, cofilin-1, profilin-1, ARP2, and N-WASP protein level in soleus from old WT (n = 5) and Lmnap.H222P/H222P (H222P) (n = 5) mice. GAPDH is shown as loading control. (C) Quantification of pERK1/2, ERK1/2, cofilin-1, profilin-1, ARP2, and N-WASP protein expression level in soleus from old WT (n = 5) and Lmnap.H222P/H222P (H222P) (n = 5) mice. **p % 0.001 between old WT and Lmnap.H222P/H222P (H222P). Data are represented as mean ± SD. (D) Immunoblots showing cofilin-1protein level in skeletal muscle from EDMD patient carrying LMNA mutation. GAPDH is shown as loading control. Data are represented as mean ± SD. (E) Immunoblot showing G-actin and F-actin protein levels in soleus from old WT (n = 3) and Lmnap.H222P/H222P (H222P) (n = 3) mice. B A B A D C D D C E E Figure 2. Increased cofilin-1 expression alters actin dynamics in Emery-Dreifuss muscular dystrophy (EDMD) Figure 2. Increased cofilin-1 expression alters actin dynamics in Emery-Dreifuss muscular dystrophy (EDMD) Figure 2. Increased cofilin-1 expression alters actin dynamics in Emery-Dreifuss muscular dystrophy (EDMD) (A) Schematic representation of actin dynamics mechanisms. (B) Immunoblots showing pERK1/2, ERK1/2, cofilin-1, profilin-1, ARP2, and N-WASP protein level in soleus from old WT (n = 5) and Lmnap.H222P/H222P (H222P) (n = 5) mice. GAPDH is shown as loading control. (C) Quantification of pERK1/2, ERK1/2, cofilin-1, profilin-1, ARP2, and N-WASP protein expression level in soleus from old WT (n = 5) and Lmnap.H222P/H222P (H222P) (n = 5) mice. **p % 0.001 between old WT and Lmnap.H222P/H222P (H222P). Data are represented as mean ± SD. (D) Immunoblots showing cofilin-1protein level in skeletal muscle from EDMD patient carrying LMNA mutation. GAPDH is shown as loading control. Data are represented as mean ± SD. (E) Immunoblot showing G-actin and F-actin protein levels in soleus from old WT (n = 3) and Lmnap.H222P/H222P (H222P) (n = 3) mice. causedbyLMNAmutationswasresponsiblefortheincreased co- filin-1 expression. 4 Cell Reports 36, 109601, August 24, 2021 Activated ERK1/2 signaling affects the stability of cofilin-1 We used stably transfected C2C12 mouse myoblasts expressing either WT (C2-WT) or the p.H222P lamin A variant (C2-H222P) (Choi et al., 2012), a simple model system where activation of ERK1/2 signaling has been well characterized (Chatzifrangkeskou et al., 2018; Choi et al., 2012; Muchir et al., 2013). We first investigated the levels of proteins involved in actin dynamics. We showed that cofilin-1 expression was increased in C2-H222P myoblasts compared with C2-WT cells (Figure 3A). Pharmacological inhibition of the ERK1/2 cascade with selumeti- nib, a selective MEK1/2 inhibitor, decreased cofilin-1 expression and increased N-WASP, ARP2, and profilin-1 levels (Figure 3A). Selumetinib washout experiments on C2-H222P cells suggested that regulation of cofilin-1, N-WASP, ARP2, and profilin-1 expres- sion was related to ERK1/2 activity (Figure 3B). To further validate whether ERK1/2 regulated directly the protein levels of cofilin-1, we transiently transfected C2-WT cells with WT ERK2 or MEK1 constructs.Thisledtoincreasedcofilin-1proteinlevelscompared with non-transfected cells (Figure 3C). Conversely, the inhibition of endogenous ERK2 in C2-H222P cells, upon transfection of ki- nase-dead (ERK2-K52R) or dominant-negative ERK2 (ERK2- T183A/Y185F) mutants constructs, led to decreased cofilin-1 expression compared with non-transfected C2-H222P cells (Figure 3C). Next, we analyzed the effect of ERK1/2 activity on 4 Cell Reports 36, 109601, August 24, 2021 ll OPEN ACCESS Article A C E G F H D B Figure 3. Increased cofilin-1 expression is under the control of ERK1/2 signaling (A) Representative immunoblots and quantification of cofilin-1, N-WASP, ARP2, and profilin-1 protein expression in C2C12 cells stably expressing WT (C (n = 3) or p.H222P (C2-H222P) (n = 3) lamin A. GAPDH is shown as loading control. C2-H222P cells were either untreated or treated with selumetinib. **p % between C2-WT and C2-H222P ± selumetinib. Data are represented as mean ± SD. (B) Representative immunoblot and quantification of effects of washout of selumetinib on cofilin-1, N-WASP, ARP2 and profilin-1 protein expression leve H222P cells. Data are represented as mean ± SD. (C) Representative immunoblot showing effects of transfection with ERK2 and MEK1 constructs on cofilin-1 protein expression in C2-WT and C2-H222P GAPDH is shown as loading control. (legend continued on next Article A A B A B C D D C E E F F F G H G H Figure 3. Activated ERK1/2 signaling affects the stability of cofilin-1 These data suggest that phosphorylation of ERK1/2 modulates cofilin- 1 protein level. To identify whether other LMNA mutations have the same effect on cofilin-1 expression through pERK1/2 activa- tion, we transiently transfected C2C12 cells with plasmids encod- ing lamin A variants found in EDMD (i.e., E358K, L271P, and N456I),whichcaused pERK1/2activation (Figure 3F).Theexpres- sion of these lamin A variants led to an elevated cofilin-1 protein level. These results suggest that LMNA mutations lead to increased cofilin-1 as a result of ERK1/2 hyperactivation. We next showed that depleting cofilin-1 by small interfering RNA (siRNA) in C2-H222P cells (Figure 3G) rescued the actin dy- namics, as evidenced by normalization of the F/G-actin ratio compared with control (Figure 3H). This result demonstrates that the increased of cofilin-1 protein level in cells expressing disease-causing lamin A variants is responsible for actin depolymerization. Activated ERK1/2 signaling affects the stability of cofilin-1 Cofilin-1 levels decreased at 6 h after the cyclohexi- mide-mediated inhibition of protein synthesis in C2-H222P cells, indicating that cofilin-1 becomes unstable. We further showed that cofilin-1 protein levels decreased faster after the cyclohexi- mide-mediated inhibition of protein synthesis in C2-H222P cells when the cells were treated with selumetinib. These results sug- gest that ERK1/2 signaling enhances cofilin-1 protein stability. We then assessed the role of ERK1/2 signaling on cofilin-1 expression in vivo using Lmnap.H222P/H222P mice lacking Erk1 (Wu et al., 2014). We showed that the soleus muscles from these mice have a reduction of interstitial fibrosis compared with Lmnap.H222P/H222P mice (Figure S4A and S4B). The inhibition of ERK1/2 cascade in Lmnap.H222P/H222P mice lacking Erk1 led to a decreased cofilin-1 expression (Figures S4C and S4D). These data suggest that phosphorylation of ERK1/2 modulates cofilin- 1 protein level. To identify whether other LMNA mutations have the same effect on cofilin-1 expression through pERK1/2 activa- tion, we transiently transfected C2C12 cells with plasmids encod- ing lamin A variants found in EDMD (i.e., E358K, L271P, and N456I),whichcaused pERK1/2activation (Figure 3F).Theexpres- sion of these lamin A variants led to an elevated cofilin-1 protein level. These results suggest that LMNA mutations lead to increased cofilin-1 as a result of ERK1/2 hyperactivation. We next showed that depleting cofilin-1 by small interfering RNA (siRNA) in C2-H222P cells (Figure 3G) rescued the actin dy- namics, as evidenced by normalization of the F/G-actin ratio compared with control (Figure 3H). This result demonstrates that the increased of cofilin-1 protein level in cells expressing disease-causing lamin A variants is responsible for actin depolymerization. cofilin-1 protein stability in a cycloheximide chase assay (Figures 3D and 3E). Cofilin-1 levels decreased at 6 h after the cyclohexi- mide-mediated inhibition of protein synthesis in C2-H222P cells, indicating that cofilin-1 becomes unstable. We further showed that cofilin-1 protein levels decreased faster after the cyclohexi- mide-mediated inhibition of protein synthesis in C2-H222P cells when the cells were treated with selumetinib. These results sug- gest that ERK1/2 signaling enhances cofilin-1 protein stability. We then assessed the role of ERK1/2 signaling on cofilin-1 expression in vivo using Lmnap.H222P/H222P mice lacking Erk1 (Wu et al., 2014). We showed that the soleus muscles from these mice have a reduction of interstitial fibrosis compared with Lmnap.H222P/H222P mice (Figure S4A and S4B). The inhibition of ERK1/2 cascade in Lmnap.H222P/H222P mice lacking Erk1 led to a decreased cofilin-1 expression (Figures S4C and S4D). Cofilin-1 controls sarcomere integrity Given that members of the ADF/cofilin family have been reported to be essential regulators of actin dynamics in sarcomeres (Krem- neva et al., 2014), we next assessed the sarcomere structure in skeletal muscles from EDMD. The structure of soleus muscle from old Lmnap.H222P/H222P mice exhibited sarcomere disorgani- zation compared with age-matched WT mice (Figures 5A and 5B). Sarcomere disorganization was not observed in soleus mus- cle from young Lmnap.H222P/H222P mice (Figures S5A and S5B). Notably, similar sarcomere abnormalities were present in muscle biopsy specimens from EDMD patients carrying LMNA mutations (Figure 5C). This sarcomere disorganization was not observed in the fast-twitch EDL muscle from old Lmnap.H222P/H222P mice (Fig- ures S5C and S5D). We showed that these structural abnormal- ities were reduced in soleus muscle from Lmnap.H222P/H222P mice lacking Erk1 (Figure S5E). To further validate this finding, we tested whether sarcomere abnormalities were also present in LMNA mutant human myotubes from striated muscle lamino- pathies. To this aim, we differentiated three human induced pluripotent stemcell(hiPSC)lines frompatientswithskeletalmus- cle laminopathies carrying LMNA p.K32del, p.L35P, and p.R249W mutations (Steele-Stallard et al., 2018) into skeletal Activated ERK1/2 signaling affects the stability of cofilin-1 Increased cofilin-1 expression is under the control of ERK1/2 signaling (A) Representative immunoblots and quantification of cofilin-1, N-WASP, ARP2, and profilin-1 protein expression in C2C12 cells stably expressing WT (C2-WT) (n = 3) or p.H222P (C2-H222P) (n = 3) lamin A. GAPDH is shown as loading control. C2-H222P cells were either untreated or treated with selumetinib. **p % 0.001 between C2-WT and C2-H222P ± selumetinib. Data are represented as mean ± SD. (B) Representative immunoblot and quantification of effects of washout of selumetinib on cofilin-1, N-WASP, ARP2 and profilin-1 protein expression level in C2- H222P cells. Data are represented as mean ± SD. (C) Representative immunoblot showing effects of transfection with ERK2 and MEK1 constructs on cofilin-1 protein expression in C2-WT and C2-H222P cells. GAPDH is shown as loading control. Cell Reports 36, 109601, August 24, 2021 5 Article ll OPEN ACCESS A ti l tinib treatment (Figure 4C). To rule out the possibility that this ef- fect might arise from an impaired function of the proteasome in C2-H222P cells, we examined the proteasome activity in these cells. Protein extracts from C2-WT and C2-H222P cells showed no difference in proteasome activity and treatment of C2-H222P cells with selumetinib had no effect (Figure 4D). These data sug- gest that ERK1/2 signalingincreases cofilin-1 stability by prevent- ing its degradation via the proteasome-ubiquitin pathway. We recently showed that active phosphorylated ERK1/2 catalyzes the phosphorylation of the actin depolymerizing factor cofilin-1 on Thr25 (Chatzifrangkeskou et al., 2018). To test the stability of this phospho(T25)-cofilin-1, we ectopically expressed mCherry- tagged cofilin-1 as well as mCherry-tagged cofilin-1(T25A), a non-phosphorylatable variant, and cofilin-1(T25D), a phospho- mimetic variant, in C2-WT cells. While both cofilin-1 WT and cofi- lin-1(T25D) protein levels were decreased in cells treated with selumetinib compared with untreated cells, the expression of the mutant cofilin-1(T25A) remained unchanged (Figures 4E and 4F). Adding MG132 to the selumetinib treating cells rescued the expression of cofilin-1 variants (Figures 4E and 4F). These results strongly suggest that ERK1/2-mediated (T25)phophorylation pro- tects cofilin-1 from proteosomal degradation. Given that other types of ERK1/2-dependent phosphorylation of cofilin-1 have been recently identified (Chatzifrangkeskou et al., 2018), it is possible that ERK1/2 signaling could regulate the activity of cofi- lin-1 through phosphorylation on other residues. cofilin-1 protein stability in a cycloheximide chase assay (Figures 3D and 3E). (D) Cycloheximide chase experiment using C2C12 cells stably expressing WT (C2-WT) or p.H222P (C2-H222P) lamin A, treated or not with selumetinib. Cells were treated with 50 mM cycloheximide and lysed at the indicated times for western blot analysis using anti-cofilin-1 antibody. GAPDH was used as a loading control. (E) Quantification of cofilin-1 signal intensity normalized to GAPDH content and expressed as the percent change from time zero, which was set at 100%. Data are represented as mean ± SD. (D) Cycloheximide chase experiment using C2C12 cells stably expressing WT (C2-WT) or p.H222P (C2-H222P) lamin A, treated or not with selumetinib. Cells were treated with 50 mM cycloheximide and lysed at the indicated times for western blot analysis using anti-cofilin-1 antibody. GAPDH was used as a loading control. (E) Quantification of cofilin-1 signal intensity normalized to GAPDH content and expressed as the percent change from time zero, which was set at 100%. Data are represented as mean ± SD. (F) Representative immunoblots showing effects of transfection with different mutated lamin A constructs on cofilin-1 expression in C2C12 cells. (G) Representative immunoblot showing the effect of cofilin-1 siRNA on cofilin-1 expression. GAPDH is shown as a loading control. (H) Representative immunoblot showing the effect of cofilin-1 siRNA on G-actin and F-actin expression in C2-H222P cells. Cytochalasin D (cytoD) induces actin depolymerization. (H) Representative immunoblot showing the effect of cofilin-1 siRNA on G-actin and F-actin expression in C2-H222P cells. Cytochalasin D (cytoD) induces actin depolymerization. owing the effect of cofilin-1 siRNA on G-actin and F-actin expression in C2-H222P cells. Cytochalasin D (cytoD) induces actin degradation through the proteasome We next sought to identify the underlying mechanism that regu- lates cofilin-1 expression in cells expressing pathogenic lamin A variants. The ubiquitin proteasome pathway is responsible for the targeted degradation of proteins, such as cofilin-1 (Goldberg, 2003; Yoo et al., 2010). We thus speculated that pERK1/2 might protect cofilin-1 from proteasomal degradation. To test our hy- pothesis,we firstexamined the influenceof theproteasomeinhib- itor MG132 on cofilin-1 expression. MG132 led to increased expression of endogenous cofilin-1 in C2-H222P cells compared with cells treated with selumetinib (Figure 4A). Similarly, treating C2-WT cells with MG132 increased endogenous cofilin-1 expres- sion (Figure 4B). To further validate our hypothesis, we examined potential ubiquitination of cofilin-1 by immunoprecipitation. We showed a decreased ubiquitination of cofilin-1 in C2-H222P cells compared with C2-WT cells, which was prevented upon selume- (D) Cycloheximide chase experiment using C2C12 cells stably expressing WT (C2-WT) or p.H222P (C2-H222P) lamin A, treated or not with selumetinib. Cells were treated with 50 mM cycloheximide and lysed at the indicated times for western blot analysis using anti-cofilin-1 antibody. GAPDH was used as a loading control. (E) Quantification of cofilin-1 signal intensity normalized to GAPDH content and expressed as the percent change from time zero, which was set at 100%. Data are represented as mean ± SD. e immunoblots showing effects of transfection with different mutated lamin A constructs on cofilin-1 expression in C2C12 cells ve immunoblot showing the effect of cofilin 1 siRNA on cofilin 1 expression GAPDH is shown as a loading control (F) Representative immunoblots showing effects of transfection with different mutated lamin A constructs on cofilin-1 expression in C2C12 cells. (G) Representative immunoblot showing the effect of cofilin-1 siRNA on cofilin-1 expression. GAPDH is shown as a loading control. (H) Representative immunoblot showing the effect of cofilin-1 siRNA on G-actin and F-actin expression in C2-H222P cells. Cytochalasin D (cytoD) induces actin depolymerization. 6 Cell Reports 36, 109601, August 24, 2021 ll OPEN ACCESS A C E F B D gure 4. pERK1/2 protects cofilin-1 from degradation by the ubiquitin-proteasome pathway ) Immunoblot showing effect of treatment with proteasome inhibitor MG132 on cofilin-1 expression in C2-H222P cells untreated or treated with selum APDH is shown as loading control. ) Immunoblot showing effect of treatment with proteasome inhibitor MG132 treatment on cofilin-1 expression in C2-WT cells. GAPDH is shown as l ntrol. ofilin-1 is involved in muscle force generation i Cofilin 1 is involved in muscle force generation in vivo Force in striated muscles is produced by actin thin filaments sliding past the myosin thick filaments, resulting in sarcomere contraction (Huxley and Hanson, 1954; Huxley and Niedergerke, 1958). Given that elevated expression of cofilin-1 phosphory- lated on Thr25 severely impacts the sarcomere structure, we hy- pothesized that it may affect the force production in skeletal muscles. We injected AAV expressing cofilin-1 into the soleus of 3-month-old WT mice (Figure 6A). Three months after injec- tion, the expression of cofilin-1, but not cofilin-2, was increased in the injected soleus (Figure 6B), muscle actin dynamics was altered (Figure 6C), and myofilaments were disorganized (Fig- ure S9). Tetanic force was significantly decreased in soleus mus- cles from cofilin-1-injected WT mice compared with non-treated WT mice (Figure 6D). Similar observations were made when so- leus muscles were injected with AAVs expressing cofilin- 1(T25D), but not AAVs expressing cofilin-1(T25A) (Figures 6B– 6D). Fibrosis in WT soleus transduced with AAVs expressing co- filin-1(T25A) and cofilin-1(T25D) was unchanged compared with non-injected soleus (Figures 6E and 6F), demonstrating that fibrosis is not the reason for the reduced force production. In conclusion, we propose that cofilin-1 phosphorylated on Thr25 can impair skeletal muscle contractile properties by modulating sarcomeric actin dynamics. We then investigated whether the activation of cofilin-1 expres- sion could contribute to myofilament disorganization. Transduc- tion of WT differentiated primary mouse myofibers (Figure S7A) withanadeno-associatedvirus(AAV)encodingcofilin-1increased the G-actin pool (Figure S7B) and led to altered myofibrillar orga- nization compared with the untransfected condition (Figure S7C). We next hypothesized that phosphorylation of Thr25 on cofilin-1 has detrimental effects on striated muscle cells. Transduction with an adenovirus encoding cofilin-1(T25D) in WT differentiated primary mouse myofibers induced depolymerization of actin (Fig- ure S7B) and altered myofibrillar organization (Figures S7C and S7D). However, transduction with an adeno-associated virus encoding cofilin-1(T25A) had no effect on both cellular actin dy- namics (Figure S7B) and myofibrillar organization (Figures S7C and S7D). To study the role of cofilin-1 as well as its Thr25 phos- phorylated form in muscle sarcomeres, we examined its localiza- tioninsoleusmuscle(FiguresS8Aand S8B).Immunofluorescence microscopy revealed regular striated pattern of cofilin-1 as well as its Thr25 phosphorylated form in WT mice (Figures S8A and S8B). ofilin-1 is involved in muscle force generation i In old Lmnap.H222P/H222P mice, we observed a punctuated pattern alongside regular striated pattern (Figures S8A and S8B, arrows), reminiscentofsarcomeredisorganization (Figures5Aand5B).The co-localization with titin (antibody against PVEK domain) demon- strated that cofilin-1 as well as its Thr25 phosphorylated form lo- calizes at I-bands in sarcomeres. These findings suggest that sarcomere disorganization arises from expression of phos- pho(T25)-cofilin-1, which participates in the development of muscular dystrophy caused by LMNA mutations. DISCUSSION We have unraveled a protective role of phosphorylated ERK1/2 for cofilin-1 by blunting its degradation through the ubiquitination- proteasome pathway. This participates in the development of muscular dystrophy (Figure 7). Non-muscle cofilin-1 is a small actin-binding protein that accelerates actin turnover by disassem- bling actin filaments. In striated muscle cells, actin and several scaffolding and regulatory proteins are arranged into contractile myofilament (Ono, 2010). Actin-binding proteins are important for actin dynamics, which contribute to controlling myofilament structure and organization. This mechanism is regulated by me- chanical forces (Fukuda et al., 2019; Skwarek-Maruszewska et al., 2009). The two ADF/cofilin isoforms, cofilin-1 and cofilin-2, are expressed in striated muscles, cofilin-2 being the dominant isoform(Ono etal.,1994;Vartiainenetal.,2002).Wedemonstrated that the non-muscle cofilin-1, as well as its Thr25 phosphorylated form, localizes on I-bands in sarcomeres and is an essential regu- lator of proper actin dynamics and sarcomeric organization. Abnormalities of actin dynamics hamper myofilament organiza- tion, alter skeletal muscle force, and ultimately lead to muscular To validate that cofilin-1 phosphorylated on Thr25 alters the sarcomeric organization, we assessed its molecular activity in vitro (Wioland et al., 2017), by exposing actin filaments to the phosphomimetic eGFP-cofilin1(T25D). We observed that co- filin-1(T25D) disassembles filaments, creating cofilin domains that grow similarly to WT (Figure 5D) although filaments severing at cofilin domain boundaries was slightly less efficient than WT cofilin-1 (Figure 5E). Overall, our in vitro results show that cofi- lin-1(T25D) is active and able to disassemble actin filament, as opposed to phosphomimetic cofilin-1(S3D) (Elam et al., 2017; Wioland et al., 2017). We thus conclude that phospho(T25)-cofi- lin-1 can alter actin dynamics and sarcomeric organization in striated muscle cells. (A) Left: immunofluorescence micrographs of sarcomeric a-actinin (green) and sarcomeric a-actin (red) labeled soleus muscle from old, WT and Lmnap.H222P/H222P (H222P) mice. Scale bar, 5 mm. Right: immunofluorescence micrographs of titin (green) and sarcomeric a-actinin (red) labeled soleus muscle from old, WT and Lmnap.H222P/H222P (H222P) mice. Scale bar, 8 mm. degradation through the proteasome Article ll OPEN ACC A B B C C D D E F E Figure 4. pERK1/2 protects cofilin-1 from degradation by the ubiquitin-proteasome pathway (A) Immunoblot showing effect of treatment with proteasome inhibitor MG132 on cofilin-1 expression in C2-H222P cells untreated or treated with selumetinib. GAPDH is shown as loading control. (B) Immunoblot showing effect of treatment with proteasome inhibitor MG132 treatment on cofilin-1 expression in C2-WT cells. GAPDH is shown as loading control. (C) Immunoprecipitation of cofilin-1 showing ubiquitination levels in C2-WT and C2-H222P cells untreated or treated with selumetinib. Input is shown as loading control. (D) Proteasome activity in C2-WT and C2-H222P cells untreated or treated with selumetinib. Data are represented as mean ± SD. (E) Immunoblot showing effect of selumetinib and MG132 on ectopically expressed mCherry-tagged cofilin-1, cofilin-1(T25A), and cofilin-1(T25D) in C2-WT cells. GAPDH is shown as loading control. (F) Quantification of mCherry signal intensity normalized to GAPDH content in C2-WT cells treated with the different conditions (n = 3). Data are represented as mean ± SD. Figure 4. pERK1/2 protects cofilin-1 from degradation by the ubiquitin-proteasome pathway ( ) G C Cell Reports 36, 109601, August 24, 2021 7 Article ll OPEN ACCESS A B C D E (legend on next Artic OPEN ACCESS A A B A A B B B B C C C C C D E (legend on next page) D D E E (legend on next page) (legend on next page) 8 Cell Reports 36, 109601, August 24, 2021 8 Cell Reports 36, 109601, August 24, 2021 Article ll OPEN ACCESS myogenic cells (Maffioletti et al., 2015). Immunofluorescence an- alyses of sarcomeric proteins suggested that some abnormalities were detectable in the newly formed myotubes (Figure S6), albeit less evident than in the aforementioned EDMD muscle biopsy specimens (Figure 5C). Figure 5. Alteration of sarcomere organization in EDMD (A) Left: immunofluorescence micrographs of sarcomeric a-actinin (green) and sarcomeric a-actin (red) labeled soleus muscle from old, WT and Lmnap.H222P/H222P (H222P) mice. Scale bar, 5 mm. Right: immunofluorescence micrographs of titin (green) and sarcomeric a-actinin (red) labeled soleus muscle from old, WT and Lmnap.H222P/H222P (H222P) mice. Scale bar, 8 mm. (B) Electron microscopy showing sarcomeric disorganization in soleus muscles from old WT and Lmnap.H222P/H222P (H222P) mice. Scale bar, 2 mm. (C) Left: electron microscopy showing sarcomeric disorganization in striated muscles from EDMD patients carrying LMNA mutations. Right: striated muscle from human control. Scale bar, 10 mm (D) Growth rate of single cofilin domains for eGFP-cofilin-1 WT and T25D, observed on individual actin filaments in vitro, in the presence of 200 nM cofilin-1 (n = 10 filaments for each condition). The distributions are plotted as violin plots, with the white dot representing its median. The statistical test was a t test for the means of two independent samples with unequal variance. (E) Single eGFP-cofilin-1 WT or T25D domain severing rate. Time t = 0 is defined for every domain as the frame on which they nucleate. n = 152 and 202 cofilin domains for eGFP-cofilin-1 WT and T25D, respectively. Figure 5. Alteration of sarcomere organization in EDMD (A) Left: immunofluorescence micrographs of sarcomeric a-actinin (green) and sarcomeric a-actin (red) labeled soleus muscle from old, WT and Lmnap.H222P/H222P (H222P) mice. Scale bar, 5 mm. Right: immunofluorescence micrographs of titin (green) and sarcomeric a-actinin (red) labeled soleus muscle from old, WT and Lmnap.H222P/H222P (H222P) mice. Scale bar, 8 mm. H222P/H222P (D) Growth rate of single cofilin domains for eGFP-cofilin-1 WT and T25D, observed on individual actin filaments in vitro, in the presence of 200 nM cofilin-1 (n = 10 filaments for each condition). The distributions are plotted as violin plots, with the white dot representing its median. The statistical test was a t test for the means of two independent samples with unequal variance. (D) Growth rate of single cofilin domains for eGFP-cofilin-1 WT and T25D, observed on individual actin filaments in vitro, in the presence of 200 nM cofilin-1 (n = 10 filaments for each condition). The distributions are plotted as violin plots, with the white dot representing its median. The statistical test was a t test for the means of two independent samples with unequal variance. (E) Single eGFP-cofilin-1 WT or T25D domain severing rate. Time t = 0 is defined for every domain as the frame on which they nucleate. n = 152 and 202 cofilin domains for eGFP-cofilin-1 WT and T25D, respectively. (E) Single eGFP-cofilin-1 WT or T25D domain severing rate. Time t = 0 is defined for every domain as the frame on which they nucleate. n = 152 and 202 cofilin domains for eGFP-cofilin-1 WT and T25D, respectively. Cell Reports 36, 109601, August 24, 2021 9 Article ll OPEN ACCESS A C E F B D igure 6. Cofilin-1 is involved in the muscle force generation in vivo A) Schematic representation of the experimental procedure followed for transduction with AAV vectors expressing cofilin-1 constructs of soleus muscl oung WT mice A B A B D C D C E E F re 6 Cofilin-1 is involved in the muscle force generation in vivo F Figure 6. Cofilin-1 is involved in the muscle force generation in vivo (A) Schematic representation of the experimental procedure followed for transduction with AAV vectors expressing cofilin-1 constructs of soleus muscles in young WT mice. (A) Schematic representation of the experimental procedure followed for transduction with AAV vectors expressing cofilin-1 constructs of soleus muscles in young WT mice. Figure 5. Alteration of sarcomere organization in EDMD cofilin-1 protein levels in soleus from WT mice non-injected or injected with either PBS or AAV vector expressing cofilin-1 oading control. (C) Representative immunoblot showing the effect of AAV expressing cofilin-1 construct on G-actin and F-actin expression in the soleus from WT mice non- injected or injected with either PBS or AAV vector expressing cofilin-1 constructs. (F) Expression of fibrosis-related genes (Col1a2, Col3a1, and Ctgf) in the soleus from WT mice non-injected or injected with either PBS or AAV vector expressing cofilin-1 constructs. Quantification of soleus muscle from Lmnap.H222P/H222P (H222P) is shown as control. *p % 0.01 between WT and Lmnap.H222P/H222P (H222P). Data are represented as mean ± SD. (F) Expression of fibrosis-related genes (Col1a2, Col3a1, and Ctgf) in the soleus from WT mice non-injected or injected with either PBS or AAV vector expressing cofilin-1 constructs. Quantification of soleus muscle from Lmnap.H222P/H222P (H222P) is shown as control. *p % 0.01 between WT and Lmnap.H222P/H222P (H222P). Data are represented as mean ± SD. 10 Cell Reports 36, 109601, August 24, 2021 10 p-ERK1/2 Cof-1 Ub Ub Ub Ub p-ERK1/2 Cof-1 proteasome control EDMD muscular dystrophy Cof-1 Cof-1 Cof-1 Cof-1 Article Article ll OPEN ACCESS Figure 7. Schematic representation of the mechanism of pERK1/2-mediated protec- tion of cofilin-1 from proteasome degrada- tion and its consequences on sarcomeric actin depolymerization in striated muscle diseases caused by LMNA mutations Figure 7. Schematic representation of the mechanism of pERK1/2-mediated protec- tion of cofilin-1 from proteasome degrada- tion and its consequences on sarcomeric actin depolymerization in striated muscle diseases caused by LMNA mutations EDMD muscular dystrophy be important both to identify and charac- terizemechanismsthatregulatetheprotea- some-ubiquitination system and to find ways to prevent dysfunction of cell death mechanisms where abnormal protein ag- gregation has occurred. Altogether, these findings provide clues of a balance between protein stabilization and degradation, which are causing myopathies. dystrophy. In support of these findings, missense mutations in UNC-60B, the C. elegans homolog of ADF/cofilin, lead to defects in actin organization in the muscles (Ono et al., 1999). Our results indicate that cofilin-1-mediated alteration of actin dynamics is a cellular consequence of ERK1/2 activation in skel- etal muscle cells. Specifically, we have shown that activation of ERK1/2 signaling protects cofilin-1 from proteasome-dependent degradation. Figure 5. Alteration of sarcomere organization in EDMD We further demonstrated that muscular dystrophy is associated with muscle cofilin-1 activation, which generated sarcomere disruption and alteration of force production, suggest- ing that cofilin-1 overexpression is a pathological event in muscular dystrophy caused by LMNA mutations. Enhanced acti- vation of cofilin-1 in skeletal muscles from a mouse model of EDMD and from patients with this disease supports our conclu- sion that this mechanism contributes to the pathology. Similar pathogenic mechanism of cofilin-1-mediated modulation of sar- comeric actin dynamics may play a role in other muscular dystro- phies, in whichthere appears to be abnormalactivationof ERK1/2 signaling (Barton, 2006; Griffin et al., 2005; Kumar et al., 2004, 2011; Lang et al., 2004; Muchir et al., 2007; Smythe and Forwood, 2012). These findings suggest that therapeutic approaches that could correct impaired actin dynamics may ameliorate muscular dystrophy caused by LMNA mutations. Our work brings insight into a role played by actin regulators in muscular dystrophies. LMOD3, the gene encoding leimodin-3, a sarcomeric actin nucleator (Chereau et al., 2008), was identified as a cause of nemaline myopathies (Yuen et al., 2014). Similarly, mutated cofilin-2 has been previously shown to cause nemaline myopathy (Agrawal et al., 2012). Histopathological analysis of the skeletal muscle from cofilin-2 null mice revealed extensive myofilament disruptions (Agrawal et al., 2007). To date, cofilin-2 was the only known member of the ADF/cofilin family involved in the disassembly of actin skeletal muscle sarcomeres (Agrawal et al., 2012). Cofilin-1 and cofilin-2, both expressed in skeletal muscle cells, contribute to the dynamic turnover of F-actin (Krem- nevaetal.,2014).Werecentlyshowedthatonlycofilin-1wasphos- phorylated on Thr25 by pERK1/2 and leads to disruption of myofil- aments in the presence of LMNA mutations (Chatzifrangkeskou et al., 2018). Zebrafish cofilin-1 mutants also exhibit myofilament disruption associated with alteration of muscle contraction (Fu- kuda et al., 2019). Together, our results show that the ERK1/2-co- filin-1 axis regulates myofilament organization in skeletal muscles. A higher level of ERK1/2 activity in skeletal muscle prevents the proteasome-dependent degradation of cofilin-1 protein, increasing its accumulation. Our results suggest that this escape from the proteasome machinery may be the consequence of increased phosphorylation of the cofilin-1by ERK1/2 (Chatzifrang- keskou et al., 2018). Similarly, the stability of the FRA-1 is depen- dent on its phosphorylation status, which is regulated by ERK1/2 signaling (Casalino et al., 2003). In addition the proteasome- dependent degradation of c-FOS can be inhibited by ERK1/2 (Musti et al., 1997). Figure 5. Alteration of sarcomere organization in EDMD Furthermore, loss of KLHL40 results in a severe lethal form of nemaline myopathy associated with destabilization of actin (Garg et al., 2014). KLHL40 binds to and stabilizes leimo- din-3 by blocking its ubiquitination. In addition, loss of KLHL40 wasassociatedwithabsenceofleimodin-3proteininskeletalmus- cle.Itisnowgenerallyacceptedthatmisregulationofthetwomajor proteolytic systems, ubiquitin-proteasome and autophagy, can lead to abnormal protein accumulation that might be involved in the pathophysiology of several disorders, including muscular dys- trophies (Sandri et al., 2013). In line with this, a previous report has suggested the involvement of autophagy in cardiomyopathy in autosomal EDMD (Choi et al., 2012). To develop therapeutic stra- tegies for muscular dystrophy caused by LMNA mutations, it will STAR+METHODS Detailed methods are provided in the online version of this paper and include the following: REFERENCES Emery, A.E. (1987). X-linked muscular dystrophy with early contractures and cardiomyopathy (Emery-Dreifuss type). Clin. Genet. 32, 360–367. Aebi, U., Cohn, J., Buhle, L., and Gerace, L. (1986). The nuclear lamina is a meshwork of intermediate-type filaments. Nature 323, 560–564. Aebi, U., Cohn, J., Buhle, L., and Gerace, L. (1986). The nuclear lamina is a meshwork of intermediate-type filaments. Nature 323, 560–564. Fatkin, D., MacRae, C., Sasaki, T., Wolff, M.R., Porcu, M., Frenneaux, M., Atherton, J., Vidaillet, H.J., Jr., Spudich, S., De Girolami, U., et al. (1999). 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Normal myofibrillar development followed by progressive sarcomeric disruption with actin accumulations in a mouse Cfl2 knockout demonstrates requirement of cofilin-2 for muscle maintenance. Hum. Mol. Genet. 21, 2341–2356. Goldberg, A.L. (2003). Article B Cofilin-1 binding and severing activity B Protein extraction and immunoblotting B F/G actin ratio measurements B Proteasome activity assay B Immunoprecipitation B Histological analyses B Immunofluorescence microscopy B Electron microscopy B Contractile properties of isolated muscle in vitro QUANTIFICATION AND STATISTICAL ANALYSIS B Cofilin-1 binding and severing activity B Cofilin-1 binding and severing activity Arimura, T., Helbling-Leclerc, A., Massart, C., Varnous, S., Niel, F., Lace` ne, E., Fromes, Y., Toussaint, M., Mura, A.-M., Keller, D.I., et al. (2005). 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Cofilin-1 phosphorylation catalyzed by ERK1/2 alters cardiac actin dynamics in dilated cardiomyopathy caused by lamin A/C gene mutation. Hum. Mol. Genet. 27, 3060–3078. Conceptualization, A.M.; investigation, N.V., M.C., C.L.D., C.P., D.C., W.W., H.J.W., C.M., and A.M.; transcriptomic analysis, N.V., N.N., and A.M.; AAV production and injection, T.M. and M.-G.B.; force measurement, M.L. and V.D.; electron microscopy, A.S. and M.B.; iPSC experiments: L.P. and F.S.T.; single-filament experiment: H.W., G.R.-L., and A.J.; writing – original draft, A.M.; writing – review & editing, all authors; funding acquisition, A.M. and F.S.T.; supervision, A.M. Chereau, D., Boczkowska, M., Skwarek-Maruszewska, A., Fujiwara, I., Hayes, D.B., Rebowski, G., Lappalainen, P., Pollard, T.D., and Dominguez, R. (2008). Leiomodin is an actin filament nucleator in muscle cells. Science 320, 239–243. Choi, J.C., Muchir, A., Wu, W., Iwata, S., Homma, S., Morrow, J.P., and Wor- man, H.J. (2012). Temsirolimus activates autophagy and ameliorates cardio- myopathy caused by lamin A/C gene mutation. Sci. Transl. Med. 4, 144ra102. SUPPLEMENTAL INFORMATION Supplemental information can be found online at https://doi.org/10.1016/j. celrep.2021.109601. Bonne, G., Di Barletta, M.R., Varnous, S., Be´ cane, H.-M., Hammouda, E.-H., Merlini, L., Muntoni, F., Greenberg, C.R., Gary, F., Urtizberea, J.-A., et al. (1999). Mutations in the gene encoding lamin A/C cause autosomal dominant Emery-Dreifuss muscular dystrophy. Nat. Genet. 21, 285–288. DECLARATION OF INTERESTS myopathy caused by lamin A/C gene mutation. Sci. Transl. Med. 4, 144ra102. H.J.W. is on the scientific advisory board and owns equity in AlloMek Thera- peutics. The remaining authors declare no competing interests. Dechat, T., Pfleghaar, K., Sengupta, K., Shimi, T., Shumaker, D.K., Solimando, L., and Goldman, R.D. (2008). Nuclear lamins: major factors in the structural organization and function of the nucleus and chromatin. Genes Dev. 22, 832–853. Received: March 23, 2020 Revised: June 9, 2021 Accepted: August 4, 2021 Published: August 24, 2021 Received: March 23, 2020 Revised: June 9, 2021 Accepted: August 4, 2021 Published: August 24, 2021 Received: March 23, 2020 Revised: June 9, 2021 Accepted: August 4, 2021 Published: August 24, 2021 Elam, W.A., Cao, W., Kang, H., Huehn, A., Hocky, G.M., Prochniewicz, E., Schramm, A.C., Negro´ n, K., Garcia, J., Bonello, T.T., et al. (2017). Phosphomi- metic S3D cofilin binds but only weakly severs actin filaments. J. Biol. Chem. 292, 19565–19579. Accepted: August 4, 2021 Published: August 24, 2021 metic S3D cofilin binds but only weakly severs actin filaments. J. Biol. Chem. 292, 19565–19579. ACKNOWLEDGMENTS Brull, A., Morales Rodriguez, B., Bonne, G., Muchir, A., and Bertrand, A.T. (2018). The pathogenesis and therapies of striated muscle laminopathies. Front. Physiol. 9, 1533. The authors would like to thank P. Zammit for helpful discussions. This work was supported by the Association Franc¸ aise contre les Myopathies, Cure CMD, and Fundacion Andres Marcio (A.M.). This work was also supported by the European Research Council, BBSRC, MDUK, and the National Institute for Health Research (F.S.T.). Casalino, L., De Cesare, D., and Verde, P. (2003). Accumulation of Fra-1 in ras- transformed cells depends on both transcriptional autoregulation and MEK- dependent posttranslational stabilization. Mol. Cell. 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Cell Reports 36, 109601, August 24, 2021 13 Article ll OPEN ACCESS ll OPEN ACCESS STAR+METHODS KEY RESOURCES TABLE STAR+METHODS KEY RESOURCES TABLE REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Rabbit polyclonal to ERK1 + ERK2 abcam Cat# 17942; RRID:AB_2297336 Mouse monoclonal Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (E10) Cell Signaling Cat# 9106; RRID:AB_331768 Rabbit monoclonal Cofilin (D3F9) XP Cell Signaling Cat #5175; RRID:AB_10622000 Rabbit polyclonal Cofilin2 Invitrogen Cat# PA5-22301; RRID:AB_11156612 Rabbit polyclonal Profilin1 Cell Signaling Cat# 3237; RRID:AB_2236990 Rabbit polyclonal N-WASP (30D10) Cell Signaling Cat #4848; RRID:AB_10694415 Rabbit polyclonal ARP2 abcam Cat# 47654; RRID:AB_1139848 Rabbit polyclonal Ubiquitin Cell signaling Cat# 3933; RRID:AB_2180538 Mouse monoclonal Actin (a-Sarcomeric) Merck Cat# A2172; RRID:AB_476695 Rabbit polyclonal Sarcomeric Alpha Actinin abcam Cat# 137346 Rabbit polyclonal pan actin Cytoskelton Cat# AAN01; RRID:AB_10708070 Mouse monoclonal Titin DSHB Cat# 9D10; RRID:AB_528491 Mouse monoclonal Lamin A/C (E-1) Santa Cruz Cat# sc-376248; RRID:AB_10991536 Mouse monoclonal Lamin A/C Leica Biosystems Cat# NCL-LAM-A/C; RRID:AB_563846 Mouse monoclonal GAPDH (6C5) Abcam Cat# 8245; RRID:AB_2107448 Goat Anti-Mouse IgG StarBright Blue 520 BioRad Cat# 12005867 Goat Anti-Mouse IgG StarBright Blue 700 BioRad Cat# 12004159; RRID:AB_2884948 Goat Anti-Rabbit IgG StarBright Blue 520 BioRad Cat# 12005870; RRID:AB_2884949 Goat Anti-Rabbit IgG StarBright Blue 700 BioRad Cat# 12004161; RRID:AB_2721073 Goat anti-Rabbit IgG (H+L) Cross- Adsorbed Secondary Antibody, Alexa Fluor 488 Invitrogen Cat# A-11008; RRID:AB_143165 Goat anti-Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 546 Invitrogen Cat# A-11010; RRID:AB_2534077 Goat anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 405 Invitrogen Cat# A48254; RRID:AB_2890548 Goat anti-Mouse IgG (H+L) Cross- Adsorbed Secondary Antibody, Alexa Fluor 488 Invitrogen Cat# A-11001; RRID:AB_2534069 Goat anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 546 Invitrogen Cat# A-11030; RRID:AB_144695 Goat anti-Mouse IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 405 Invitrogen Cat# A-31553; RRID:AB_221604 Goat anti-Mouse IgM (Heavy chain) Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 Invitrogen Cat# A-21042; RRID:AB_141357 Goat anti-Mouse IgM (Heavy chain) Cross-Adsorbed Secondary Antibody, Alexa Fluor 546 Invitrogen Cat# A-21045; RRID:AB_2535714 Bacterial and virus strains DH5a Competent Cells ThermoFisher Scientific EC0112 (Continued on next page) Cat# A-21042; RRID:AB_141357 Cat# A-21045; RRID:AB_2535714 e1 Cell Reports 36, 109601, August 24, 2021 ll OPEN ACCESS Continued REAGENT or RESOURCE SOURCE IDENTIFIER Biological samples Human skeletal muscle tissue Kindly provided by Myobank-AFM, Paris, France. 23-year-old patient carrying the LMNA p.R249Q mutation Human skeletal muscle tissue Kindly provided by Myobank-AFM, Paris, France. 14-year-old patient carrying the LMNA p.E358K mutation Human skeletal muscle tissue Kindly provided by Myobank-AFM, Paris, France. STAR+METHODS KEY RESOURCES TABLE Age match controls 129S2/svPasCrl mouse primary myoblastes This paper N/A 129S2/svPasCrl Lmnap.H222P/H222P mouse primary myoblastes This paper N/A Rabbit muscle acetone powder Pel-Freeze 41995
1 Human erythrocytes Etablissement Francais du Sang N/A Chemicals, peptides, and recombinant proteins Mouse eGFP-cofilin-1 Kremneva et al., 2014 Uniprot: P18760 Mouse eGFP-(T25D)-cofilin-1 This paper N/A Alexa488- succimidyl ester Life Technologies Cat#A20000 Critical commercial assays Proteasome activity assay kit Abcam ab107921 G-actin/F-actin in vivo assay kit Cytoskeleton BK037 RNeasy Mini Kit QIAGEN 74104 Agilent RNA 6000 Nano Kit Agient 5067-1511 SuperScript III First-Strand Synthesis System for RT-PCR Invitrogen 18080-051 LightCycler 480 SYBR Green I Master Roche 04887352001 GeneChip Mouse Gene 2.0 ST Array ThermoFisher Scinetific 902119 GeneChip WT Pico Kit ThermoFisher Scinetific 902622 QuikChange II Site-Directed Mutagenesis Kit Agilent 200523 Lipofectamine 2000 Transfection Reagent ThermoFisher Scinetific 11668019 Deposited data Transcriptome analysis Affimetrix GeneChip Mouse Gene 2.0 ST Array GEO number: GSE146112 Experimental models: Cell lines C2C12 immortalized mouse myoblast cell lines that constitutively expressed wild type Lmna gene Kindly provided by Dr. Howard J. Worman, Department of Medicine, College of Physicians and Surgeons, Columbia University, New York, New York 10032, USA N/A C2C12 immortalized mouse myoblast cell lines that constitutively expressed mutated c.665A > C Lmna gene Kindly provided by Dr. Howard J. Worman, Department of Medicine, College of Physicians and Surgeons, Columbia University, New York, New York 10032, USA N/A (hiPSC) lines from patients carrying LMNA p.K32del Cellular Dynamics International Inc. and Cure Congenital Muscular Dystrophy (CureCMD; https://www.curecmd.org) https://fujifilmcdi.com https://www.curecmd.org (hiPSC) lines from patients carrying LMNA p.L35P Cellular Dynamics International Inc. and Cure Congenital https://fujifilmcdi.com https://www.curecmd.org (hiPSC) lines from patients carrying LMNA p.R249W Cellular Dynamics International Inc. and Cure Congenital https://fujifilmcdi.com https://www.curecmd.org (Continued on next page) Article ll OPEN ACCESS https://fujifilmcdi.com https://www.curecmd.org https://fujifilmcdi.com https://www.curecmd.org https://fujifilmcdi.com https://www.curecmd.org (Continued on next page) (Continued on next page) Cell Reports 36, 109601, August 24, 2021 e2 Continued REAGENT or RESOURCE SOURCE IDENTIFIER C2C12 myoblast cell line ATCC CRL-1772 Human iPSCs: Healthy Control UCLi007-A Tedesco laboratory (https://hpscreg.eu/ cell-line/UCLi007-A) UCLi007-A or STM2012CTRL03(401) Experimental models: Organisms/strains Mouse 129S2/svPasCrl wild-type Janvier Labs https://www.janvier-labs.com Mouse 129S2/svPasCrl Lmnap.H222P/H222P Kindly provided by Dr. Gise` le Bonne, INSERM UMR-S 974, Paris, France N/A Mouse C57BL/6JRj wild type Janvier Labs https://www.janvier-labs.com Mouse C57BL/6JRj Lmnap.H222P/H222P,ERK1KO/KO Kindly provided by Dr. Howard J. STAR+METHODS KEY RESOURCES TABLE Worman, Department of Medicine, College of Physicians and Surgeons, Columbia University, New York, New York 10032, USA N/A Oligonucleotides List of primers This paper Table S3 SignalSilence Cofilin siRNA I Cell Signaling Cat #6267 Recombinant DNA virus: AAV-rh10-Cfl1 Kindly provided by Dr. Maria-Grazia Bieferi, INSERM UMR-S 974, Paris, France N/A virus: AAV-rh10-Cfl1(p.T25A) Kindly provided by Dr. Maria-Grazia Bieferi, INSERM UMR-S 974, Paris, France N/A virus: AAV-rh10-Cfl1(p.T25D) Kindly provided by Dr. Maria-Grazia Bieferi, INSERM UMR-S 974, Paris, France N/A plasmid: GFP-ERK2 Kindly provided by P. Stork, Oregon Health and Science University N/A plasmid: RFP-MEK1 Kindly provided by P. Stork, Oregon Health and Science University N/A plasmid: GFP-ERK2K52R Kindly provided by P. Stork, Oregon Health and Science University N/A plasmid: GFP-ERK2T183A/Y185F Kindly provided by P. Stork, Oregon Health and Science University N/A plasmid: GFP-lamin A Ostlund et al., 2001 N/A plasmid: GFP-lamin AE358K Ostlund et al., 2001 N/A plasmid: GFP-Lamin AL271P Ostlund et al., 2001 N/A plasmid: GFP-Lamin AN456I Ostlund et al., 2001 N/A plasmid: pmCherryC1-Cofilin1 Addgene Cat #27687 plasmid: pmCherryC1-Cofilin1T25A This paper N/A plasmid: pmCherryC1-Cofilin1T25D This paper N/A plasmid: Mouse eGFP-cofilin-1 Kremneva et al., 2014 Uniprot: P18760 plasmid: Mouse eGFP-(T25D)cofilin-1 This paper Uniprot: P18760 Software and algorithms ImageJ Schneider et al., 2012 https://imagej.net/ Prism 8 GraphPad Software, LLC https://www.graphpad.com GeneChip Command Console Software ThermoFisher Scientific https://www.thermofisher.com/us/en/ home.html Transcriptome Analysis Console (TAC) Software ThermoFisher Scientific https://www.thermofisher.com/us/en/ home.html Linear Models for Microarray and RNA-Seq Data Ritchie et al., 2015 https://bioconductor.org/packages/ release/bioc/html/limma.html Article ll OPEN ACCESS ll OPEN ACCESS (Continued on next page) e3 Cell Reports 36, 109601, August 24, 2021 Article ll OPEN ACCESS Continued REAGENT or RESOURCE SOURCE IDENTIFIER ErmineJ Lee et al., 2005 https://erminej.msl.ubc.ca GenEx software multid https://multid.se PowerLab System 4SP AD Instruments https://www.adinstruments.com Labchart 4 v8 AD Instruments https://www.adinstruments.com Jupyter for numpy/python analysis Jupyter https://jupyter.org GeneTraffic 3.0 Stratagene https://www.stratagene.com Article ll OPEN ACCESS Data and code availability d Affymetrix array transcriptome data are available at the GEO database: GSE146112 (https://www.ncbi.nlm.nih.gov/geo/query/ acc.cgi?acc=GSE146112) Thi d t t i i l d d Affymetrix array transcriptome data are available at the GEO database: GSE146112 (https://www.ncbi.nlm.nih.gov/geo/query/ acc.cgi?acc=GSE146112) d This paper does not report original code d Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request. d Any additional information required to reanalyze the data reported in this paper is available from the lea Animal skeletal muscles Animal skeletal muscles, soleus and/or EDL were harvested from dead, young and old, wild-type, Lmnap.H222P/H222P mice and Lmnap.H222P/H222P mice lacking ERK1. Animals were sacrifice by cervical dissociation according to the guidelines from Directive 2010/63/EU of the European Parliament on the protection of animals used for scientific purposes. Lead contact Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact Antoine Muchir ([email protected]) Animals All in vivo experiments were approved by the French Ministry of Agriculture (approval number #6455 and #20161). The animal exper- iments were performed according to the guidelines from Directive 2010/63/EU of the European Parliament on the protection of an- imals used for scientific purposes. Wild-type and Lmnap.H222P/H222P mice from the 129S2/SvPasCrl genetic background (Vignier et al., 2019) were housed in a disease-free barrier facility with 12 h/12 h light/dark cycles and received chow diet and water ad libitum. Young mice correspond to 2-4 months old animals, and old mice to 6-8 months old animal. Only males were chosen since the onset of the disease was earlier than in females (Arimura et al., 2005). Materials availability y In this work, the newly generated material is listed in the Key resources table and can be shared upon request. y this work, the newly generated material is listed in the Key resources table and can be shared upon req In this work, the newly generated material is listed in the Key resources table and can be shared upon request. Human skeletal muscles Human skeletal muscles were obtained from the Myobank-AFM, Paris, France (https://www.institut-myologie.org/en/recherche-2/ myobank-afm/). Patients and age-match controls were informed and gave consent. For biochemistry, we studied skeletal muscle from a 14-year-old man subject carrying the LMNA p.E358K mutation. For electron microscopy, we studied skeletal muscle from a 12-year-old man subject carrying the LMNA p.H222P mutation and a 23-year-old man carrying the LMNA p.R249Q mutation. Tis- sue samples without patient identifiers received from consent donors were not obtained specifically for this study. RESOURCE AVAILABILITY Lead contact Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact Antoine Muchir ([email protected]) Lead contact Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact Antoine Article USA. Cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum in 5% CO2 and 20% O2 at 37C. Cells were treated with 50 mM selumetinib (8 hours), 10 mM MG132 (5 hours), 10 mg/ml cycloheximide, 100 nM cytochalasin D, 10 mg/ml CHX for the indicated time points. USA. Cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum in 5% CO2 and 20% O2 at 37C. Cells were treated with 50 mM selumetinib (8 hours), 10 mM MG132 (5 hours), 10 mg/ml cycloheximide, 100 nM cytochalasin D, 10 mg/ml CHX for the indicated time points. LMNA mutant human iPSCs were kindly provided by Cellular Dynamics International Inc. (CDI; https://cellulardynamics.com) and Cure Congenital Muscular Dystrophy (CureCMD; https://www.curecmd.org). Human iPSC-derived myogenic cells were cultured and differentiated according to previously reported protocol (Maffioletti et al., 2015). In brief, cells were plated onto Matrigel-coated dishes and upon achievement of 90%–100% confluence, differentiation was induced by pulsing cells twice with 1mM 4-OH tamox- ifen: once in proliferation medium and the second time after 24 hours in differentiation medium (i.e., DMEM with 2% horse serum). Differentiation was maintained for 4 days changing the medium every other day. Work with human cells in the Tedesco laboratory was performed under approval of the NHS Health Research Authority Research Ethics Committee reference no. 13/LO/1826; IRAS proj- ect ID no. 141100. Mouse primary cells Mouse primary myoblast cells were obtained from 129S2/svPasCrl wild-type mouse. Primary mouse myoblasts were cultured on collagen-coated dishes in Ham’s F10 medium (Life Technologies) supplemented with 20% FBS (Eurobio), 2ng/ml basic fibroblast growth factor (FGF) (R&D Systems) and 1% penicillin/streptomycin (Pen/Strep) (Life Technologies). Myoblasts were plated at a den- sity of 1 3 104 cells per cm2. After 48 h, myoblasts were shifted to 5% horse serum and left to differentiate. RNA isolation and reverse-transcription qPCR Total RNA was extracted using the RNeasy isolation kit (QIAGEN) according to the manufacturer’s instructions. Adequacy and integ- rity of extracted RNA were determined with the 2100 Bioanalyzer system (Agilent) according the manufacturer’s instructions. cDNA was synthesized using the SuperScript III first-strand synthesis system according to the manufacturer’s instructions (Invitrogen). Real-time qPCR reactions were performed with SYBR Green I Master mix (Roche) using the LightCycler 480 (Roche). Relative levels of mRNA expression calculated using the DDCT method were normalized to housekeeping mRNA (Pfaffl, 2001). Microarray processing Transcriptome analysis was performed with GeneChip Mouse Gene 2.0 ST Array (Affymetrix), which contains 698,000 probes that covered 35,240 transcripts from RefSeq database. Complementary DNA synthesis, cRNA synthesis, and labeling were performed with GeneChip WT Pico Reagent Kit (Applied Biosystems) according to the manufacture’s instructions. Hybridization, washing, stain- ing andscanning of arrays wereperformedat theGeneChip CoreFacility of theCochin Hospital (GENOM’IC). Imagefileswere obtained through Affymetrix GeneChip software and analyzed by robust multichip analysis using Affymetrix microarray ‘‘.cel’’ image files and GeneTraffic 3.0 software (Stratagene). Gene expression analysis was realized using the Affymetrix Transcriptome Analysis Console (TAC). Genes were processed and normalized using the Robust Multichip Analysis (RMA), which consists in three steps: background correction, normalization, and probe set summarization (Irizarry et al., 2003). Paired comparisons between groups were realized using Limma (Ritchie et al., 2015) and the generated p values were corrected with Benjamini and Hochberg procedure (Reiner et al., 2003). Genes were finally identified as being differentially expressed (DEG) if they met a false discovery rate threshold of p < 0.05 and showed at least a 2-fold difference in expression independent of absolute signal intensity. Gene expression changes related to functional groups were analyzed using the class score method in the bioinformatics tools DAVID (https://david.abcc.ncifcrf.gov/) and ErmineJ (http://www.chibi.ubc.ca/ermineJ/) to provide a statistical confidence to groupings. These bioinformatics tools take as input the q- values of differentially expressed genes andidentifystatistically significantfunctional groupings(GOterms) using modified Fisher exact test in DAVID and Wilcoxon rank-sum test in ErmineJ. Significant GO terms were identified using a false discovery rate of p < 0.05. Adeno-associated virus (AAV) delivery AAV delivery was performed by intramuscular injection using a 29-G needle, at the dose of 1.1x1011 viral particles per 5 mg of tissue in a final volume of 5 ml. Phosphate-buffered saline; PBS 1X (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4) was use as placebo. Mice were anesthetized with intraperitoneal injection of xylazin (10 mg/kg)/ketamine (100 mg/kg) cocktail and place on a heating pad at 28C during the intervention. Wild-type males were injected with AAV-rh10-Cfl1, AAV-rh10-Cfl1(p.T25A) and AAV- rh10-Cfl1(p.T25D) at 90 days of age. AAV vectors of serotype rh10 (AAV-rh10-Cfl1, AAV-rh10-Cfl1(p.T25A), AAV-rh10-Cfl1(p.T25D)), carrying sequence of the wild-type cofilin-1 gene (accession NM_007687), cofilin-1 c.73A>G or cofilin-1 c.73ACA>GAT under the control of the cytomegalovirus imme- diate/early promoter was prepared by the triple transfection method in HEK293T cells as previously described (Biferi et al., 2017; Chatzifrangkeskou et al., 2018). Cell lines C2C12 mouse myoblast cell line was purchased at ATCC (RRID:CVCL_0188). Cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum in 5% CO2 and 20% O2 at 37C. C2C12 mouse myoblast cell lines that constitutively expressed wild-type Lmna gene (C2-WT) and mutated c.665A > C Lmna gene (C2-H222P) has been described previously (Choi et al., 2012). C2-WT and mutated C2-H222P were kindly provided by Dr. Dr. Ho- ward J. Worman, Department of Medicine, College of Physicians and Surgeons, Columbia University, New York, New York 10032, Cell Reports 36, 109601, August 24, 2021 e4 Article ll OPEN ACCESS Plasmids as ds Plasmids encoding GFP-ERK2, RFP-MEK1, GFP-ERK2 K52R and GFP-ERK2 T183A/Y185F were kindly provided by P. Stork (Oregon Health and Science University). Plasmids encoding GFP-lamin A, GFP-lamin A E358K, GFP-Lamin A L271P and GFP-LaminA N456I Plasmids encoding GFP-ERK2, RFP-MEK1, GFP-ERK2 K52R and GFP-ERK2 T183A/Y185F were kindly provided by P. Stork (Oregon Health and Science University). Plasmids encoding GFP-lamin A, GFP-lamin A E358K, GFP-Lamin A L271P and GFP-LaminA N456I e5 Cell Reports 36, 109601, August 24, 2021 Article ll OPEN ACCESS have been previously described (Ostlund et al., 2001). Cofilin-pmCherryC1 was purchased from Addgene (#27687). Mutagenesis was carried out using QuikChange II Site-Directed Mutagenesis Kit (Agilent Technologies) as previously described (Chatzifrangkeskou et al., 2018). Transient transfection experiments were performed using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instruc- tions. Briefly, cells were seeded at 3 3 105 cells per well in 6-well plates or at 1 3 106 cells per 10 cm Petri dish were transfected with 3 mg or 15 mg plasmid DNA respectively for 24 h. Immunoprecipitation Cells were treated with 10 mM MG132 and lysed in TBS 1X-1mM EDTA-1% NP-40 completed with proteinase inhibitor cocktail (Roche). Cell lysates were incubated with 20 mL protein A Dynabeads (Invitrogen) and 2 mg of the indicated antibodies for 2 h at 4C. Pelleted beads were collected in sample buffer NuPAGE LDS (Thermo Fisher Scientific) with 200 mM DTT and subjected to SDS-PAGE and immunoblotting. F/G actin ratio measurements The ratio of F-actin to G-actin was determined using the G-actin/ F-actin in vivo assay kit (Cytoskeleton) according to the manufac- turer’s instructions. Briefly, 2 mg of protein from cells or frozen soleus tissues were homogenized in Lysis and F-actin Stabilization Buffer and centrifuged at 2,000 rpm for 5 min to remove unbroken cells. F-actin was separated from G-actin by centrifugation at 100,000 g for 60 min at 37,C. The F-actin- containing pellet was resuspended in F-actin Depolymerizing Buffer at a volume equiv- alent to the G-actin-containing supernatant volume. The resuspended F-actin pellet was kept on ice for 60 min and was gently mixed every 15 min to dissociate F-actin. Proteins in equivalent volumes (10 ml) of supernatant and pellet were separated by SDS-PAGE and subjected to immunoblot analysis using an anti-pan actin antibody supplied in the kit. F/G actin ratio was quantified using ImageJ software. Histological analyses g y The skeletal muscles were briefly washed in PBS and snap frozen at
70C in isopentan. Frozen tissues were cut into 8-mm-thick sections. Haematoxylin and eosin, Gomori’s trichrome and Sirius Red stainings were performed according to standard procedures. I fl i The skeletal muscles were briefly washed in PBS and snap frozen at
70C in isopentan. Frozen tissues were cut into 8-mm-thick sections. Haematoxylin and eosin, Gomori’s trichrome and Sirius Red stainings were performed according to standard procedures. Cofilin-1 binding and severing activity The procedure is described in detail in Wioland et al. (2017). Briefly, single, fluorescently labeled, rabbit alpha-skeletal actin filaments are aged for 15 minutes and exposed to eGFP-cofilin-1 in a microfluidics chamber, in F-buffer (5 mM Tris HCl pH 7.4, 50 mM KCl, 1 mM MgCl2, 0.2 mM EGTA, 0.2 mM ATP, 10 mM DTT and 1 mM DABCO), at room temperature (RT). Acquisition is performed on a Nikon TiE inverted microscope, controlled by micromanager, using epifluorescence with a 120W Xcite lamp (Lumen dynamics) and images were acquired by an sCMOS Orca-Flash4.0 camera (Hamamatsu). All experiments were performed at least twice and at least one representative movie was analyzed as described in (Wioland et al. (2017). The Welch’s unequal variances t test was used to test for significant differences in the domain growth rates, using the ‘scipy’ python package. Protein extraction and immunoblotting Total proteins from mouse soleus tissue or cultured cells were extracted in Cell lysis buffer (Cell Signaling) completed with protease inhibitors (25 mg/ml aprotinin; 10 mg/ml leupeptin; 1 mM 4-[2-aminoethyl]-benzene sulfonylfluoride hydrochloride; 2 mM Na3VO4). The lysates were sonicated (3 pulses of 10 s at 30% amplitude) and protein concentration was measured with the Bicinchoninic Acid Assay protein assay (Pierce). Protein extracts were separated by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto nitrocellulose membranes (Invitrogen). Blocking and antibody incubations were performed in 5% bovine serum albumin. Membranes were incubated with fluorescent-conjugated anti-mouse or anti-rabbit secondary antibodies (BioRad) 1h at RT. Antibody detection was imaged using the ChemiDoc Touch Imaging System (BioRad). Quantification was done using ImageJ software. Proteasome activity assay y y Protein were extracted from C2-WT and C2-H222P cells in PBS 1X-0.5% NP-40. Proteasome activity was measured by proteasome activity assay kit (Abcam ab107921) according to the manufacturer’s instructions. Fluorescence was measured with a plate reader (BMG Labtech) in the presence/absence of MG132 at Ex/Em = 350/440 nm at 37C. Contractile properties of isolated muscle in vitro The isometric contractile properties of soleus and EDL muscles were studied in vitro according to previously published protocols (Agbulut et al., 2009; Mouisel et al., 2014). Muscles were immersed in an oxygenated (95% O2 and 5% CO2) Tyrode solution (58.5 mM NaCl, 24 mM NaHCO3, 5.4 mM KCl, 1.2 mM KH2PO4, 1.8 mM CaCl2, 1 mM MgSO4, and 10 mM glucose (pH 7.4)) at 22C. Muscles were connected at one end to an electromagnetic puller and at the other end to a lever arm of a servomotor system (Isometric Force Transducer FT50, Harvard Apparatus). The skeletal muscles were placed between two electrodes parallel to the muscle. Once the system was equilibrated (30 min), an electrical field was applied to the muscle. Twitch and tetanic contractions were performed and data were recorded and analyzed using the PowerLab System (4SP; AD Instruments) and software (Labchart 4 v8; AD Instruments). Absolute maximal isometric tetanic force (P0) was measured during tetanic contractions (frequency of 120 Hz, train of stimulation of 500 ms). The muscle length was adjusted to the optimum length (L0) that produced P0. Specific maximal iso- metric force (sP0) was calculated by dividing the force by the estimated cross-sectional area (CSA) of the muscle. Assuming muscles have a cylindrical shape and a density of 1.06 mg/mm3, the muscle cross-sectional area corresponds to the wet weight of the muscle divided by its fiber length (Lf). The fiber length to L0 ratio of 0.70 was used to calculate Lf (Mendias et al., 2006). Isometric twitch contractions were recorded at L0. The following parameters of the twitch contraction were measured: maximum twitch force (Pt), time to peak tension (TTP ms) and half relaxation time (HRT ms). Specific Pt (sPt mM/mm2) was calculated by dividing the force by the CSA of the muscle. Electron microscopy Freshly harvested skeletal muscles were cut into small pieces and immediately fixed (PBS 1X, 2.5% glutaraldehyde) 1 h at RT. After washing in PBS 1X, samples were post-fixed (PBS 1X, 1% OsO4), dehydrated in a graded series of acetone and embedded in an epoxy resin. Ultrathin sections were cut at 90 nm and stained with uranyl acetate and lead citrate, examined using a transmission electron microscope (JEOL 1011) and photographed with a digital Erlangshen 1000 camera (GATAN), using Digital Micrograph software. Article A ti l X-100, 1% BSA) 45 min at RT and blocked (PBS 1X, 10% goat serum, 0.2% Triton X-100, 1% BSA) 30 min at RT. Samples were incubated overnight at 4C with primary antibodies and washed three times (PBS 1X, 0.2% Triton X-100). Cells were incubated one hour at RT with secondary antibodies goat anti-mouse IgG, goat anti-mouse IgM, goat anti-rabbit IgG conjugated with Alexa Fluor 405, 488 and 546, respectively. Cells were washed with PBS 1X and slides were mounted in Vectashield mounting medium with DAPI (Vector Laboratories). Imaging of iPSC-derived myotubes was carried out using a Leica SPE inverted confocal microscope using a 63X lens and selecting random fields. Scoring of myotubes carrying sarcomere abnormalities and a-actinin or titin aggregates was performed blindly. The myofibers were considered disorganized when they showed no sarcomere organization/pattern across most of the myotube, lack of myofibrils-like structures and abundance of aggregates. X-100, 1% BSA) 45 min at RT and blocked (PBS 1X, 10% goat serum, 0.2% Triton X-100, 1% BSA) 30 min at RT. Samples were incubated overnight at 4C with primary antibodies and washed three times (PBS 1X, 0.2% Triton X-100). Cells were incubated one hour at RT with secondary antibodies goat anti-mouse IgG, goat anti-mouse IgM, goat anti-rabbit IgG conjugated with Alexa Fluor 405, 488 and 546, respectively. Cells were washed with PBS 1X and slides were mounted in Vectashield mounting medium with DAPI (Vector Laboratories). Imaging of iPSC-derived myotubes was carried out using a Leica SPE inverted confocal microscope using a 63X lens and selecting random fields. Scoring of myotubes carrying sarcomere abnormalities and a-actinin or titin aggregates was performed blindly. The myofibers were considered disorganized when they showed no sarcomere organization/pattern across most of the myotube, lack of myofibrils-like structures and abundance of aggregates. e7 Cell Reports 36, 109601, August 24, 2021 Immunofluorescence microscopy Frozen tissues were cut into 8-mm-thick sections. Cryosections were fixed (PBS 1X, 4% PFA) 15 min at RT, permeabilized (PBS 1X, 0.5% Triton X-100) 10 min at RT and blocked (PBS 1X, 0.3% Triton X-100, 5% BSA) one hour at RT. Sections were incubated over- night at 4oC with primary antibodies (PBS 1X, 0.1% Triton X-100, 1% BSA) and washed in PBS 1X. Sections were then incubated for 1 h with secondary anti-rabbit or anti-mouse IgG conjugated with Alexa Fluor 488 or Alexa Fluor 546 antibodies. Sections were washed with PBS 1X and slides were mounted in Vectashield mounting medium with DAPI (Vector Laboratories). For human iPSC-derived myotubes, cells were fixed using 4% PFA at RT for 10 min. Samples were permeabilized (PBS 1X, 0.2% Triton Cell Reports 36, 109601, August 24, 2021 e6 Article ll OPEN ACCESS QUANTIFICATION AND STATISTICAL ANALYSIS Immunoblots and gels were analyzed and quantified with Fiji software. Statistical analyses were performed using GraphPad Prism software. Statistical significance between groups of mice was analyzed with a corrected non-parametric test, Mann-Whitney test when compared two sets of data, or Kruskal Wallis test with Dunn’s test post-test when compared multiple sets data. The statistical details of experiments are presented in the relevant figure legends. A p value of < 0.05 was considered significant. e7 Cell Reports 36, 109601, August 24, 2021

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4I4U Concept citizen engagement of engineering students and high school students Elisa Sayrol Clols, Thierry Monteil, Benoît Desjeux, María del Mar Pérez Cambra, Nathalie Bruyère, Inés Aquilué Junyent, Eduardo José Alarcón Cot, Mélanie Julliot To cite this version: Elisa Sayrol Clols, Thierry Monteil, Benoît Desjeux, María del Mar Pérez Cambra, Nathalie Bruyère, et al.. 4I4U Concept citizen engagement of engineering students and high school students. 50th Annual conference of The European Society for Engineering Education (SEFI 2022), European Society For Engineering Education (SEFI), Sep 2022, Barcelona, Spain. pp.1564-1572, ￿10.5821/conference- 9788412322262.1419￿. ￿hal-04279699￿ Distributed under a Creative Commons Attribution 4.0 International License HAL Id: hal-04279699 https://hal.science/hal-04279699v1 Submitted on 10 Nov 2023 L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. 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Distributed under a Creative Commons Attribution 4.0 International License 1 Corresponding Author: [email protected] 2 Co-Funded by EIT Urban Mobility Conference Key Areas: Student Engagement, Regional Involvement and Innovation Keywords: Innovation, Entrepreneurship, International Education, Citizen Engagement, Urban Mobility Conference Key Areas: Student Engagement, Regional Involvement and Innovation Keywords: Innovation, Entrepreneurship, International Education, Citizen Engagement, Urban Mobility 4I4U CONCEPT CITIZEN ENGAGEMENT OF ENGINEERING STUDENTS AND HIGH SCHOOL STUDENTS Sayrol, Elisa1 Universitat Politècnica de Catalunya Barcelona, Spain ORCID: 0000-0002- 0526-97 Pérez, Mar CARNET, Technology Center UPC, Barcelona, Spain Monteil, Thierry IRIT, University of Toulouse, INSA Toulouse, France ORCID: 0000- 0001-6031-5555 Desjeux, Benoît Lycée Joseph Gallieni & Campus des métiers et des qualifications d'excellence Mobilité et Transport Intelligent, Toulouse, France Julliot, Mélanie Toulouse Métropole, France Bruyère, Nathalie ISDAT, Institut Supérieure des Arts et du Design de Toulouse, France Aquilué, Inés CARNET, Technology Center UPC, Barcelona, Spain ORCID: 0000- 0002-2813-6191 Aquilué, Inés CARNET, Technology Center UPC, Barcelona, Spain ORCID: 0000- 0002-2813-6191 Alarcon, Eduard Universitat Politècnica de Catalunya Barcelona, Spain ORCID: 0000-0001- 7663-7153 Alarcon, Eduard Universitat Politècnica de Catalunya Barcelona, Spain ORCID: 0000-0001- 7663-7153 Alarcon, Eduard Universitat Politècnica de Catalunya Barcelona, Spain ORCID: 0000-0001- 7663-7153 1.1 Citizen Engagement of young people The aim of citizen engagement is to ensure that users are part of the ideation process by achieving social and gender inclusion and stakeholder engagement, creating an opportunity to experiment and share information on urban mobility. 4I4U focuses on students aged 15 - 23 as they are rarely involved in the ideation process. Moreover, one of the added values compare to previous experiences for example in India-ICT standardization European project [1] on the smart city part is that they do not just experiment new products, system of services and try to create a new one by themselves as done in cities of Delhi, Kanpur, Bangalore, Chandigarh, Pune, Goa. In 4I4U, they not only try to develop an idea, they also have a phase of improving skills and exchange ideas in urban mobility with a strong connection with researchers, city services, and mobility companies. This allows them to advance in urban mobility and feel included. 1.2 Context Partners of the project in both cities were willing to involve young students in the area of urban mobility, including university students, not necessarily from the civil engineering field, and high-school students. The common goals are to rise awareness and empower them as citiziens to act and be part of the solution for future mobility in our cities. Barcelona and Toulouse have some historical ties in both sides of the Pyrenées, also a common focus in teaching students who could work in the automotive or urban mobility sector with a sustainable mindset. ABSTRACT The 4I4U project2, which runs through 2022, aims to engage the community of citizens made up of students between the ages of 15 to 23 in the urban mobility of the future while empowering them to become actors of change who can participate in the development and evolution of their city. Led by a European Consortium composed of two cities, Barcelona in Spain and Toulouse in France, as well as education entities from high school to universities and clusters of companies in the field of urban mobility, and co-funded by the EIT Urban Mobility, the main objectives of 4I4U focus on raising awareness among young citizens in the context of urban mobility, developing their capabilities to become actors, highlighting their needs and finally creating a methodology and an environment that help students from ideation to action for the mobility of the future. The objectives of 4I4U are achieved by planning a set of seminars, round tables and brainstorming sessions to raise awareness or understanding of different aspects of urban mobility. To move from ideation to action, small groups of students design and produce a first ideation to understand and be able to project themselves into the proposed solution. This activity will be carried out under the mentoring of cities and industrialists, using the means of training institutions. 1.3 Goals and Scope of the 4I4U Project The community of citizens made up of students aged 15 to 23 has and will have a growing impact on urban mobility. Making students aware of this issue as early as possible will have an important effect for the future, both in everyday life but also in the following generations. We believe that young citizens are not involved in this issue because they think they cannot have an impact on the city. They do not master the process that goes from the awareness of a need, the formalization of an idea, the integration of the right actors and contacts to finally have impactful results. However, it is necessary to adapt the tools that are put in place to interest them and put them in a position to be protagonists of the necessary changes and innovations. y g More specifically, the objectives of 4I4U are: • OB1 Raise awareness of young citizens on the context of urban mobility with the help and expertise of teachers and trainers, city services and industrialists in this field. • OB2 Develop the capacities of students to become actors of future urban mobility with the creation of networking between students, city and industry on this topic. • OB3 Be able to highlight the needs and hope of this class of citizens. • OB4 Create a methodology and an environment to help students from ideation to action in this field. The scope of the activities focus on The scope of the activities focus on • Propose, implement and evaluate a methodology based on a set of tools (seminars, round tables, workshop) addressed to the student population between 15 and 23 years old in a co-creation process tutored by city services, industrialists and academics. The use of these tools and, above all, the fact that they are part of the ideation process, will allow students, not only to be informed about science and innovation with different classes, but to be empowered and feel actors of urban mobility projects. • The proposed methodology and activities are developed in two cities with specific and common events. 1.3 Goals and Scope of the 4I4U Project The development of skills through access and exchange with experts, the identification of levers through the presence of city services and industrialists in this field, the networking to bring together all the players around the same approach should enable this population of citizens to understand and change their thinking and behavior in terms of urban mobility. 1.4 Towards a new future in engineering education The 4I4U project includes some elements that can contribute to the future of engineering as it tries to engage engineering students in city challenges by involving them in the ideation of solutions and at the same encouraging younger students, who are currently in high school, in this design thinking approach. The focus is on citizen engagement and urban mobility and bringing together two cities close enough but from two different countries. The methodology of the process is explained below. Task 3. Assessment of the ideation and engagement process During the beginning of the project, the program has been refined in terms of events and communication [2]. At the end of the project a summary of the process implemented and the results obtained will be made. CARNET experience in other projects [3] will help develop and assess the methodology for evaluating the ideation and the engagement of students in urban mobility. Partners will collaborate in the drafting of the good practices guide. This guide will produced and distributed on the Erasmus platforms, the EIT Urban Mobility community, and in the multiplier events organised by each partner for dissemination purposes. Task 1. Networking debate and problem finding This task concerns the sharing of information and the networking of the various participants at the beginning of the project. Its objective is to share information on the existing situation and the solutions envisaged around the world, but also to report the problems perceived by people in each partner city. The goal is to have well-informed and motivated students. It takes the form of events with focused seminars, discussion forums that can be shared or specific to each partner city. The events require of setting up the necessary structures, organizing the recruitment of external speakers, managing students and creating a space of physical and virtual conviviality conducive to exchanges. We rely in particular on a digital tool called DecidiUM and social networks to fuel and maintain exchanges. Seminars for example on the evolution of cities, the challenges of mobility, the city and the citizen, the industrial perspectives on mobility. Task 2. Ideation phase and selection of proposals This task is dedicated to the ideation phase by each group of students to propose a solution to an open problem that has been debated in the previous phase with citizens and stakeholders. The problem solved could be specific for Toulouse or Barcelona or for both/any cities. Educational establishments provide their equipment and fablab. The objective is to obtain a prototype demonstrating the philosophy of the proposed solution without having a level of operational maturity. During a final workshop at the end of the year, each group will demonstrate their solutions and obtain various feedback to improve their proposal. They will present their idea and prototype in front of a jury bringing together different actors: users, cities, manufacturers, incubators, etc. The jury selects the best solutions. Each city together with the stakeholders decides about the future of the proposals. 2.1 Implementation of the Project The project is divided in three tasks. The first task aims to understand and increase students' competencies in the field of urban mobility. It creates transdisciplinarity groups of students. This task specifically addresses objective OB1. The second task aims to create, produce ideas and think about their implementation. It addresses objective OB2 of the project. Finally, the third task creates a synthesis of the methodology implemented and the results in terms of ideas and proposed actions. This task integrates the dissemination of the events and the results of the activities. It addresses objectives OB3 and OB4 of the project. Environmental: The methodology of working will raise awareness of young generations of students to the need that future urban mobility should be sustainable and active. The approach is to imagine that the project ideations that the students are going to be involved in are sustainable. We estimate that at least 3 of the ideas selected by the jury will have an impact on the environment. The change of scale in production will allow for the development of local employment but above all will avoid the transport of semi- industrialized parts around the world. Social: We expect more than 200 students involved in 4I4U in different degrees of implication. So far around 21 posters and videos have been created by ISDAT students, as explained in section 3.5, and 10 telecom engineering students have develop an app, as explained in section 3.4. The hackathon organized in Barcelona in December will gather around 150 students from Toulouse and Barcelona. Activities with high-schools in Barcelona are planned for the fall semester involving engineering students as “ambassador” of the project. We have to add to this numbers other students that have attended the seminars so far.They will become well-informed and increase their capacity to become actors of the future urban mobility and more generally actors of the city. Moreover, connection will be made between the different visions of the city from: learners, citizens, city management, companies and teaching entities. Furthermore, common work for high school students, undergraduate and postgraduate students can encourage high school students to project their future as citizens engaged in sustainable activities in their cies. Finally, teachers will also increase their knowledge on this topic and be able to integrate this experience in their courses. Economic: Economic: The expected outcomes and impacts are the possible creation of products on the domain of the future mobility, an opportunity given to ideate in the city an innovative solution and help to argue for finding founds, the production of forms with easily shared techniques, the perception of which is easily understood by all thanks to a work of popularisation of information and maintenance and finally sharing new ideas with industry. We expect at least 2 ideas that have a real impact and economic potential for commercialisation. 3 All seminars could be found on the following link: https://engage.eiturbanmobility.eu/processes/Project4I4U/f/41/meetings?filter%5Bsearch_text%5D=&filter% 5Bdate%5D%5B%5D=&filter%5Btype%5D%5B%5D=&filter%5Bactivity%5D=&filter%5Bactivity%5D=all 3 All seminars could be found on the following link: https://engage.eiturbanmobility.eu/processes/Project4I4U/f/41/meetings?filter%5Bsearch_text%5D=& 5Bdate%5D%5B%5D=&filter%5Btype%5D%5B%5D=&filter%5Bactivity%5D=&filter%5Bactivity%5D=all 3.1 Seminars shared between the two cities the First Semestrer Twelve hybrid events are held simultaneously in Toulouse and Barcelona. These seminars, round-tables and brainstorming sessions have taken place in order to understand different aspects of urban mobility and raise awareness on the subject as it can be seen in the topics of the events that follow.3 y t can be seen in the topics of the events that follow.3 p 1. “Draw me the Automobile of the Future for Urban Mobility” by Jean Luc Maté on February 15th 2022 4PM. 2. “Project Design from the perspective of city services for citizens” by Ángel López on March, 3rd 2022 5PM. 3. “Non-Financial Report of ICT projects” by Eva Vidal, on March, 8th 2022 at 2PM. 4. “Autofiction, a biography on the automobile object” by Olivier Peyricot on March 9th 2022 at a6PM 5. “The role of Technology Innovation in Urban Mobility” by Jordi Ortuño on March 17th 2022 at 5PM 6. “Challenges for sustainable urban logistics” by François de Bertier on March 24th 2022 at 4:30 PM. 7. “Smart mobility. Urban Mobility challenges” by Ángel López on March 31st 2022 at 5PM. 8. “When smart mobility means smart infraestructures” by Pierre Emmanuel Maire on April14th 2022 at 5PM. 9. “Female mobility patterns” by Imma Ribas on April 28th 2022 at 5PM 10. “5G technology and smart mobility” by Bruno Pouget, Frederic Gardes, Stéphane Vialle and André Bottaro on May 12th 2022 at 4PM. 11. “Mobility 4.0. and cognitive ergonomics: thinking about the human in an autonomous and connected environment” by Céline Lemercier on May 16th 2022 at 4PM. 11. “Mobility 4.0. and cognitive ergonomics: thinking about the human in an autonomous and connected environment” by Céline Lemercier on May 16th 2022 at 4PM. 12. “Urban mobility and Pollution Citizen Maps. Young Population study in Barcelona” by Universitat Politècnica de Catalunya on May 19th 2022 at 5PM. 12. “Urban mobility and Pollution Citizen Maps. Young Population study in Barcelona” by Universitat Politècnica de Catalunya on May 19th 2022 at 5PM. p g results of their own work are presented to the university community at the last eminar in Barcelona. 3.2 Barcelona Activities (current and to be done) A core group of 10 final year undergraduate students in the area of Telecommunication Engineering, not related to urban mobility, automotive or environmental studies, are taking a 12 ECTS challenge-based course during the first semester of 2022. The proposed challenge is to develop a product/service with the following requirements: to estimate the exposure to pollutants while moving around the city in their daily life, especially when they go to study; to raise awareness among the young population between 15 and 23 years old about the carbon footprint caused by the means of transport they use; finally, to use the knowledge in their own studies as a way to see that urban mobility is a transversal field where any professional can contribute. As a product they decided to create an app that shows three alternative routes to go from one place to another in the cities of Barcelona and Terrassa, that is, the fastest route and two alternatives with less pollutant exposure. The app also shows the carbon footprint generated according to the chosen route and depending on the mode of transport. The app contains projections of carbon footprint estimates for 1, 5 and 10 years. Finally, a ranking, as a game between friends from the same high school to see who is the "champion" generating less emissions in their daily mobility around the city. This project has required them, first of all, to become aware themselves of the environmental, social and economic impact of pollution through their own research study and by attending parallel seminars organized by 4I4U. Thanks to this awareness of the magnitude of the consequences of pollution, they are now ambassadors and together with 4I4U partners they visit high schools to show students younger than them their own experience in acquiring knowledge and on the other hand in their contribution by developing an application that can help this awareness and at the same time have fun. The university students also want to get feedback on the product they are developing. Some results of their own work are presented to the university community at the last 4I4U seminar in Barcelona. Common Activities for the Second Semester Beyond the sharing that takes place in the first semester, during the seminars, we wanted the students to also benefit from the richness of their different cultures between Toulouse and Barcelona. For this, a 2-day event will allow them on the one hand to attend together at a conference and exhibition on urban mobility in Barcelona and on the other hand to share the knowledge they have acquired during the project 4I4U on a one-day and night hackathon on short projects on urban mobility involving technologies of connected objects, communication and design (see for example A humanitarian technology hackathon [4] and some good practices in [5]). 3.3 Toulouse Activities (current and to be done) Following the networking phase, a first set of solution proposals in the form of posters and videos was produced by ISDAT. This material will be exhibited and shown to other students from other establishments to give them a first ideas of solution (idea0) and allow them to co-construct either from this first batch of ideas or from new ideas, their will propose their final proposal of idea (idea1) for the end of June. We will then begin the mentored implementation phase of the various ideas (idea1) by industrialists, city services and teachers. In December, groups will finalise their first prototypes (proto1). A jury will evaluate these prototypes and select those that can have a real impact and a possibility of deployment in the city. Students will then be able to gather around these few selected prototypes to improve them and move on to a prototype version (proto2) that can be deployed and used by a subset of citizens in real-life situations in the city. Assessment The 4I4U assessment of the ideation and engagement process includes the quantification of three main aspects: 1) Social impact 2) Environmental impact and 3) Feasibility . 1) Regarding the social impact the assessment will focus on how university students from 18 up to 23 years old understand and increase the transversal fields related to urban mobility. Considering the students of secondary school and University the assessment is focused on building the awareness of sustainable mobility for the youngest and increasing it for the oldest taking into account the effects of a non sustainability mobility. 2) Regarding the environmental assessment, for instance, in one of the projects it is based on pollutant parameter measurements of the students’ mobility patterns. This is achieved by an app created by a group of students. 3) Feasibility assessment is based on how this project can be repeated in time or how the environment and the social impact can be reproduced through the outputs of the project and thanks to citizen associations which are aligned with 4I4U values. 4 CONCLUSIONS AND PERSPECTIVES The first results of the project show that the choice to have developed an offer of face- to-face seminars, streaming and recording makes it possible to adapt to this audience of 15-25 year olds who consume information at their own rythm. The final conclusions will have to wait until the end of the project with the implementation phase done and the final feedback from the partners involved and the students on the whole of what they have learned and achieved. Nevertheless, the first presentations of the concept to college, and undergraduate school outside the project have aroused an interest in this type of approach with a desire to participate in future editions. REFERENCES [1] India-EU ICT Standarisation, April 2022, https://www.indiaeu-ictstandards.in [2] 4I4U Project, 2022, EIT Urban Mobility, April 2022, https://engage.eiturbanmobility.eu/processes/Project4I4U [3] Aquilué, I.; Caicedo, A.; Moreno, J.; Estrada, M.; Pagès, L. A Methodology for Assessing the Impact of Living Labs on Urban Design: The Case of the Furnish Project. Sustainability 2021, 13, 4562. https://doi.org/10.3390/su13084562 [4] Linnell N, Figueira S, Chintala N, Falzarano L, Ciancio V (2014), Hack for the homeless: A humanitarian technology hackathon, IEEE Global Humanitarian Technology Conference (GHTC 2014) [5] Brennan, J. (2020), Civic Hackathons for Youth, National Civic Review, Vol. 108, No. 4, (Winter 2020) pp. 55-64. https://www.jstor.org/stable/10.32543/naticivirevi.108.4.0055 [1] India-EU ICT Standarisation, April 2022, https://www.indiaeu-ictstandards [2] 4I4U Project, 2022, EIT Urban Mobility, April 2022, https://engage.eiturbanmobility.eu/processes/Project4I4U [3] Aquilué, I.; Caicedo, A.; Moreno, J.; Estrada, M.; Pagès, L. A Methodology for Assessing the Impact of Living Labs on Urban Design: The Case of the Furnish Project. Sustainability 2021, 13, 4562. https://doi org/10 3390/su13084562 [3] Aquilué, I.; Caicedo, A.; Moreno, J.; Estrada, M.; Pagès, L. A Methodology for Assessing the Impact of Living Labs on Urban Design: The Case of the Furnish Project. Sustainability 2021, 13, 4562. https://doi.org/10.3390/su13084562 [4] Linnell N, Figueira S, Chintala N, Falzarano L, Ciancio V (2014), Hack for the homeless: A humanitarian technology hackathon, IEEE Global Humanitarian Technology Conference (GHTC 2014) [4] Linnell N, Figueira S, Chintala N, Falzarano L, Ciancio V (2014), Hack for the homeless: A humanitarian technology hackathon, IEEE Global Humanitarian Technology Conference (GHTC 2014) [5] gy ( ) [5] Brennan, J. (2020), Civic Hackathons for Youth, National Civic Review, Vol. 108, No. 4, (Winter 2020) pp. 55-64. https://www.jstor.org/stable/10.32543/naticivirevi.108.4.0055 [5] Brennan, J. (2020), Civic Hackathons for Youth, National Civic Review, Vol. 108, No. 4, (Winter 2020) pp. 55-64. https://www.jstor.org/stable/10.32543/naticivirevi.108.4.0055

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https://aacr.figshare.com/articles/journal_contribution/Supplementary_Table_1_from_CAMKK2_Promotes_Prostate_Cancer_Independently_of_AMPK_via_Increased_Lipogenesis/22420535/1/files/39866711.pdf

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Supplementary Table 1 from CAMKK2 Promotes Prostate Cancer Independently of AMPK via Increased Lipogenesis

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Supplementary Table 1. List of primers used for quantitative PCR. Gene Forward Primer Reverse Primer 36B4 (RPLP0) GGACATGTTGCTGGCCAATAA GGGCCCGAGACCAGTGTT CAMKK2 TCCAGACCAGCCCGACATAG CAGGGGTGCAGCTTGATTTC FKBP5 ATTATCCGGAGAACCAAACG CAAACATCCTCCCACCACAG PSA AGGAGTTCTTGACCCCAAAGAAAC TTGCGCACACACGTCATTG TMPRSS2 GCGGCTGTCACGATCC GCGGCTGTCACGATCC Acaca (Acc1) ATGGGCGGAATGGTCTCTTTC TGGGGACCTTGTCTTCATCAT Camkk2 GGAGGACGAGAACTGCACAC TTCGCTGCCTTGCTTCGTGA Fasn TGCTCCCAGCTGCAGGC GCCCGGTAGCTCTGGGTGTA Hmbs CTGGGCTCCTCTTGGAATG GATGGGCAACTGTACCTGACTG Ppia CAAATGCTGGACCAAACACAAACG GTTCATGCCTTCTTTCACCTTCCC Rps14 GACCAAGACCCCTGGACCT CCCCTTTTCTTCGAGTGCTA Human gene names are in capitals, mouse genes in lowercase. Common aliases are shown in parentheses. Supplementary Table 1. List of primers used for quantitative PCR. Gene Forward Primer Reverse Primer 36B4 (RPLP0) GGACATGTTGCTGGCCAATAA GGGCCCGAGACCAGTGTT CAMKK2 TCCAGACCAGCCCGACATAG CAGGGGTGCAGCTTGATTTC FKBP5 ATTATCCGGAGAACCAAACG CAAACATCCTCCCACCACAG PSA AGGAGTTCTTGACCCCAAAGAAAC TTGCGCACACACGTCATTG TMPRSS2 GCGGCTGTCACGATCC GCGGCTGTCACGATCC Acaca (Acc1) ATGGGCGGAATGGTCTCTTTC TGGGGACCTTGTCTTCATCAT Camkk2 GGAGGACGAGAACTGCACAC TTCGCTGCCTTGCTTCGTGA Fasn TGCTCCCAGCTGCAGGC GCCCGGTAGCTCTGGGTGTA Hmbs CTGGGCTCCTCTTGGAATG GATGGGCAACTGTACCTGACTG Ppia CAAATGCTGGACCAAACACAAACG GTTCATGCCTTCTTTCACCTTCCC Rps14 GACCAAGACCCCTGGACCT CCCCTTTTCTTCGAGTGCTA Human gene names are in capitals, mouse genes in lowercase. Common aliases are shown in parentheses. Supplementary Table 1. List of primers used for quantitative PCR.

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Tegaderm transparent dressing (3M) with antibiotic ointment for treatment of infectious corneal ulcer: Antibiotic Wet Therapy

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Tegaderm transparent dressing (3M) with antibiotic ointment for treatment of infectious corneal ulcer: Antibiotic Wet Therapy Shingo Yasuda  (  [email protected] ) Wakayama Medical University https://orcid.org/0000-0003-1019-2459 Takayoshi Sumioka  Wakayama Medical University Yukihisa Takada  Wakayama Medical University Yuka Okada  Wakayama Medical University Shizuya Saika  Wakayama Medical University Abstract To retrospectively review cases of infectious corneal ulcer treated with Tegaderm transparent dressing (3M) with an antibiotic ointment. Results Nineteen eyes in which the causative bacteria were not identified and five eyes in which the causative bacteria (Pseudomonas aeruginosa, methicillin-susceptible Staphylococcus aureus (MSSA), Streptococcus, Corynebacterium.) were identified were successfully treated using AWT with ofloxacin ointment. methicillin-resistant Staphylococcus aureus (MRSA) was detected in 3 of 5 eyes in which AWT with ofloxacin was not successful, and filamentous fungi were detected in one eye. Two of the three eyes MRSA detected were healed by changing to vancomycin hydrochloride eye ointment, one of which was severely affected and was later enucleated. One eye with filamentous fungi was treated after changing to pimaricin eye ointment. A causative pathogen was not identified in the other eye, but because it was suspected to be fungal the ointment was changed to a pimaricin eye ointment, which led to healing. Subjects and methods: A series of 29 eyes of 29 patients with possible infectious corneal keratitis were treated in Wakayama Medical University Hospital between January 1st, 2016 and December 31st, 2018. The age of the patients ranged from 26 to 92 years (mean 68.0 ± 17.8). At the first visit, the corneal infection lesion was scraped from the epithelium to the substantially superficial layer, and the scraped matter was submitted to Gram staining and culture examination. Cases suspected of fungi and Acanthamoeba were examined directly with potassium hydroxide (KOH) treatment. For several days until drug sensitivity results were obtained, initial treatment was Antibiotic Wet Therapy (AWT) with ophthalmic eye ointment and transparent Tegaderm (3M) dressing. We retrospectively reviewed the culture results of corneal scrapings (detected bacteria and drug resistance) and the therapeutic outcome of AWT. Conclusion AWT may be an option for initial treatment of corneal infections when the causative organism is unknown. Original research Keywords: cornea, infectious corneal ulcer, antibiotic ointment Posted Date: March 11th, 2021 DOI: https://doi.org/10.21203/rs.3.rs-276979/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Posted Date: March 11th, 2021 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Page 1/10 Page 1/10 Introduction Page 2/10 It is important that the injured or infected cornea remains transparent after treatment [1–3]. Scarring following wound healing in the corneal stroma results in opacification and irregular astigmatism, both of Page 2/10 which impair visual function [4, 5]. Therefore, initial treatment of corneal infection is critical for a final outcome of healing with minimal scarring. In a clinical setting, application of a broad-spectrum antibiotic eye drop is the first choice of treatment until bacteriology examination identifies the specific pathogen [6, 7]. For over ten years, we have treated infectious corneal ulcers using Antibiotic Wet Therapy (AWT, an antibiotic ophthalmic ointment with Tegaderm (3M) transparent dressing) as the initial treatment. In this study, we retrospectively reviewed the culture results of corneal scrapings (detected bacteria and drug resistance) and the therapeutic outcomes of AWT in cases from January 1st, 2016 to December 31st, 2018. AWT was successful in most cases, even without identification of a causative pathogen. Subjects This retrospective study was approved by the Ethical Review Board of Wakayama Medical University Hospital (#2620). The study included a series of 29 eyes of 29 patients with suspected infectious corneal disorder treated in Wakayama Medical University Hospital during the between January 1st, 2016 and December 31st, 2018. Patients were aged 26 to 92 years (mean 68.0±17.8). At the first visit, the corneal infection lesion was scraped from the epithelium to the substantially superficial layer, and the scraped matter was submitted to Gram staining and culture examination. Cases suspected of fungi and Acanthamoeba were examined directly with potassium hydroxide (KOH) treatment [8, 9]. Pimaricin eye ointment was used to treat confirmed fungal infection. Ophthalmic eye ointment was used for suspected pathogens sensitive to new quinolone, such as Pseudomonas aeruginosa, as the initial treatment for unknown pathogens. When methicillin-resistant Staphylococcus aureus (MRSA) or fungi were detected with culture examination, treatment was changed from ophthalmic ointment to vancomycin hydrochloride or pimaricin ointment. The AWT method involves applying about 0.5g eye ointment in the lower conjunctival sac and affixing Tegaderm transparent dressing (3M) (Figure 1) to discourage eye opening and to develop a tear-filled environment inside the film. Our hospital policy is to only remove the protective film for treatment, such as for corneal abrasion at examination. This usually occurs once per day, but if the wet state cannot be maintained, additional exchange is performed. We aim to ensure the eye is always moist, but film removal did not exceed 3 times per day if possible to avoid damaging the skin around the eyelid. Methods In this study, we retrospectively reviewed the culture results of corneal scrapings (including bacteria and drug resistance) and the therapeutic outcomes of AWT. The eyes were grouped two [Therapeutic response (+) group and Therapeutic response (-) group], and causative pathogens were assayed in each group. Page 3/10 Page 3/10 Representative case was also shown here. In this patient we did not detect causative specific pathogens were detected, but AWT was successful. Representative case was also shown here. In this patient we did not detect causative specific pathogens were detected, but AWT was successful. Result AWT was successful in the treatment of 19 eyes in which the causative bacteria were not identified and five eyes in which causative bacteria were identified as Pseudomonas aeruginosa, methicillin-sensitive Staphylococcus aureus (MSSA), Streptococcus and Corynebacterium. (Table 1) Table 1 Number and types of causative pathogens with positive or negative response to AWT. Therapeutic response +   24   Pseudomonas aeruginosa 3   MSSA 1   Streptococcus 1   Corynebacterium 1     (Partially duplicated)   No detection 19 Therapeutic response −   5   MRSA 3   fungus 1   No detection 1 Of a further five eyes in which AWT had no therapeutic effect, three eyes had MRSA infection, one eye had causative fungus infection and one eye had an unknown cause of infection. Of a further five eyes in which AWT had no therapeutic effect, three eyes had MRSA infection, one eye had causative fungus infection and one eye had an unknown cause of infection. For the three eyes with MRSA infection, ofloxacin ointment was changed to vancomycin ointment and inflammation was improved in two out of three eyes, but one eye with severe MRSA infection required enucleation. The eye with a fungal cause of infection was improved by changing ofloxacin to pimaricin ointment, and in the eye with unknown cause of infection, but suspected to be fungal, the ointment was changed to a pimaricin eye ointment, which led to healing. In this retrospective study, there was no recurrence of corneal infection. Representative Case Report Representative Case Report Page 4/10 A 67-year-old male patient complained of left eye pain and decreased visual acuity and was diagnosed with infectious keratitis. Two days later, a corneal ulcer appeared, and he visited our hospital. The findings at the first visit were visual acuity: Right = 0.7 (1.0) Left = 0.05 (no improvement with refractive correction). Congestion of the bulbar conjunctiva was prominent, and a central corneal ring-shaped ulcer and inflammatory cells in the anterior chamber were found. (Fig. 2) As initial treatment, corneal lesion scrubbing and Gram staining was performed, but no causative bacteria were identified. Infectious corneal ulcer was suspected on the basis of clinical findings, and AWT was started. One week later, the genus Streptococcus was detected by culture examination, and sensitivity to levofloxacin was confirmed. The visual acuity on day 18 after the start of AWT was Right = 0.7 (1.0) and Left = 0.5 (no improvement with refractive correction). Bulbar conjunctival hyperaemia was reduced and inflammatory cells in the anterior chamber had disappeared. Although corneal opacity remained, ( ) As initial treatment, corneal lesion scrubbing and Gram staining was performed, but no causative bacteria were identified. Infectious corneal ulcer was suspected on the basis of clinical findings, and AWT was started. One week later, the genus Streptococcus was detected by culture examination, and sensitivity to levofloxacin was confirmed. The visual acuity on day 18 after the start of AWT was Right = 0.7 (1.0) and Left = 0.5 (no improvement with refractive correction). Bulbar conjunctival hyperaemia was reduced and inflammatory cells in the anterior chamber had disappeared. Although corneal opacity remained, epithelial surface was observed and the ulcer had healed. (Fig. 3) The treatment was terminated at this point, but it was noted that the patient had already lapsed treatment without relapse of corneal ulcer or keratitis. epithelial surface was observed and the ulcer had healed. (Fig. 3) The treatment was terminated at this point, but it was noted that the patient had already lapsed treatment without relapse of corneal ulcer or keratitis. Discussion In the current study, 24 of 29 cases were successfully treated using AWT. The efficacy of the treatment strategy might depend on water retention and long-standing exposure of antibiotics to the lesion. The common use of dressings in treatment of cutaneous burn or decubitus [10, 11] led us to hypothesize that maintenance of a moist environment may be favourable in the treatment of corneal injury or infection. A wound-healing effect of tear retention might depend on activation of cell behaviours and provision of wound healing-related bioactive components, such as growth factors and extracellular matrix components like fibronectin [12–14]. Physiological tear clearance is calculated as the sum of tear secretion, including tears lost from the conjunctiva, tear evaporation, and tear discharge to the nasolacrimal duct, and occurs at a stable rate. Human tear turnover rate is said to be around 10%-20% per minute [15, 16]. With frequent eye drops, the quantity of tears temporarily increases, but it is thought that this quantity increases over time and as the tears flow from the ocular surface. By applying Tegaderm to form an enclosed environment, the tears that would have evaporated were retained and accumulated in the space inside the Tegaderm transparent dressing. Antibiotic ointments mixed with tears were also stored inside this environment over a sustained period. Therefore, Tegaderm transparent dressing may have been useful in keeping antibiotics, growth factors and extracellular matrix components in contact with the eye for a long time. Ethics approval and consent to participate This retrospective study was approved by the Ethical Review Board of Wakayama Medical University Hospital (#2620). Funding Not applicable. Conclusion In the cases reported here we applied ofloxacin ophthalmic ointment prior to obtaining results of bacteriology culture testing. In 19 cases the lesion was successfully treated with AWT treatment even though the bacteriology testing did not identify a specific pathogen. In five cases the bacteria were Page 5/10 sensitive to ofloxacin with favourable outcome. In five cases with MRSA or fungi, AWT did not yield satisfactory outcomes. However, the drug suitable to these pathogens showed effectiveness. We consider it is not suitable to continue AWT if effectiveness is not seen following several days of treatment. Another treatment strategy for bacterial keratitis is frequent instillation of an antibiotic eye drop. However, AWT treatment involves reduced drug instillation, which may be beneficial to both patient and medical team. sensitive to ofloxacin with favourable outcome. In five cases with MRSA or fungi, AWT did not yield satisfactory outcomes. However, the drug suitable to these pathogens showed effectiveness. We consider it is not suitable to continue AWT if effectiveness is not seen following several days of treatment. Another treatment strategy for bacterial keratitis is frequent instillation of an antibiotic eye drop. However, AWT treatment involves reduced drug instillation, which may be beneficial to both patient and medical team. Consent for publication Written informed consent for publication was obtained. Written informed consent for publication was obtained. AWT Antibiotic Wet Therapy (AWT) MSSA methicillin-susceptible Staphylococcus aureus MRSA Availability of data and material The datasets used during the current study are available from the corresponding author on reasonable request. Competing interests There is no financial disclosure. Authors' contributions All authors contributed to project conception and critical review of manuscript. The author(s) read and approved the final manuscript. Acknowledgements Not applicable. Authors' information (optional) Not applicable. Page 6/10 Page 6/10 References 1. Brayden B, Sarah E, Amy W et al (2019) Corneal injury: Clinical and molecular aspects. Exp Eye Res 186:107709 2. Andre A, Abirami S, Jiahuli W et al (2016) The corneal fibrosis response to epithelial-stromal injury. Exp Eye Res 142:110–118 3. Alexander V, Mehrnoosh S (2015) Progress in corneal wound healing. Prog Retin Eye Res 49:17–45 3. Alexander V, Mehrnoosh S (2015) Progress in corneal wound healing. Prog Retin Eye Res 49:17–45 4. Yureeda Q, Gilbert W, Bryan M et al. Corneal transparency: genesis, maintenance and dysfunction. 2010;81:198–210 4. Yureeda Q, Gilbert W, Bryan M et al. Corneal transparency: genesis, maintenance and dysfunction. 2010;81:198–210 5. Arsen A, Dilek I, Sibel P et al (2007) Does blunt ocular trauma induce corneal astigmatism? Cornea 26:539–542 6. Alexandra J, Brian K, Heuy-Ching W et al (2020) Multidrug-Resistant Organisms from Ophthalmic Cultures: Antibiotic Resistance and Visual Acuity. Mil Med 185:e1002–e1007 7. McLeod S, LaBree L, Tayyanipour R et al (1995) The importance of initial management in the treatment of severe infectious corneal ulcers. Ophthalmology 102:1943–1948 8. Anita R, Shaffie B, Prabhu V et al (2018) Diagnostic Evaluation of Co-Occurrence of Acanthamoeba and Fungi in Keratitis: A Preliminary Report. Cornea 37:227–234 8. Anita R, Shaffie B, Prabhu V et al (2018) Diagnostic Evaluation of Co-Occurrence of Acanthamoeba and Fungi in Keratitis: A Preliminary Report. Cornea 37:227–234 9. Noopur G, Radhika T (2008) Investigative modalities in infectious Keratitis. Indian J Ophthalmol 56:209–213 9. Noopur G, Radhika T (2008) Investigative modalities in infectious Keratitis. Indian J Ophthalmol 56:209–213 10. Jason W, Heather C, Fiona C et al (2013) Dressings for superficial and partial thickness burns. Cochrane Database Syst Rev 2013:CD002106 10. Jason W, Heather C, Fiona C et al (2013) Dressings for superficial and partial thickness burns. Cochrane Database Syst Rev 2013:CD002106 11. Daniel B, Ashkan J (2008) Pressure ulcers: prevention, evaluation, and management. Am Fam Physician 78:1186–1194 11. Daniel B, Ashkan J (2008) Pressure ulcers: prevention, evaluation, and management. Am Fam Physician 78:1186–1194 12. Mingyue Z, Chenglei T, Tingjun F et al (2019) Fibronectin regulates the self-renewal of rabbit limbal epithelial stem cells by stimulating the Wnt11/Fzd7/ROCK non-canonical Wnt pathway. Exp Eye Res 185:107681 12. Mingyue Z, Chenglei T, Tingjun F et al (2019) Fibronectin regulates the self-renewal of rabbit limbal epithelial stem cells by stimulating the Wnt11/Fzd7/ROCK non-canonical Wnt pathway. Exp Eye Res 185:107681 13. Invest Ophthalmol Vis Sci 36:2120–2126 15. Mochizuki H, Yamada M, Hatou S et al (2009) Turnover rate of tear-film lipid layer determined by fluorophotometry. Br J Ophthalmol 93:1535–1538 16. Luigina S, Trefford S, Stephanie V et al (2004) Tear turnover rate is reduced in patients with symptomatic dry eye. Cont Lens Anterior Eye 27:15–20 16. Luigina S, Trefford S, Stephanie V et al (2004) Tear turnover rate is reduced in patients with symptomatic dry eye. Cont Lens Anterior Eye 27:15–20 16. Luigina S, Trefford S, Stephanie V et al (2004) Tear turnover rate is reduced in patients with symptomatic dry eye. Cont Lens Anterior Eye 27:15–20 References Zhao M, Dick A, Forrester J et al (1999) Electric field-directed cell motility involves up-regulated expression and asymmetric redistribution of the epidermal growth factor receptors and is enhanced by fibronectin and laminin. Mol Biol Cell 10:1259–1276 13. Zhao M, Dick A, Forrester J et al (1999) Electric field-directed cell motility involves up-regulated expression and asymmetric redistribution of the epidermal growth factor receptors and is enhanced by fibronectin and laminin. Mol Biol Cell 10:1259–1276 14. Maldonado B, Furcht L (1995) Epidermal growth factor stimulates integrin-mediated cell migration of cultured human corneal epithelial cells on fibronectin and arginine-glycine-aspartic acid peptide. 14. Maldonado B, Furcht L (1995) Epidermal growth factor stimulates integrin-mediated cell migration of cultured human corneal epithelial cells on fibronectin and arginine-glycine-aspartic acid peptide. Page 7/10 Page 7/10 Invest Ophthalmol Vis Sci 36:2120–2126 15. Mochizuki H, Yamada M, Hatou S et al (2009) Turnover rate of tear-film lipid layer determined by fluorophotometry. Br J Ophthalmol 93:1535–1538 16. Luigina S, Trefford S, Stephanie V et al (2004) Tear turnover rate is reduced in patients with symptomatic dry eye. Cont Lens Anterior Eye 27:15–20 Figures Page 8/10 Figure 1 A: Approximately 0.5g appropriate eye ointment is applied to the lower eyelid. B: T dressing (3M) is affixed over and around the eye. C: The eye is closed under Tega dressing (3M). Figure 1 A: Approximately 0.5g appropriate eye ointment is applied to the lower eyelid. B: Tegaderm transparent dressing (3M) is affixed over and around the eye. C: The eye is closed under Tegaderm transparent dressing (3M). Figure 1 A: Approximately 0.5g appropriate eye ointment is applied to the lower eyelid. B: Tegaderm transparent dressing (3M) is affixed over and around the eye. C: The eye is closed under Tegaderm transparent dressing (3M). A: Approximately 0.5g appropriate eye ointment is applied to the lower eyelid. B: Tegaderm transparent dressing (3M) is affixed over and around the eye. C: The eye is closed under Tegaderm transparent dressing (3M). Page 9/10 Figure 2 Central corneal ring ulcer with opacity. A: Anterior segment photograph. B: Photograph showing fluorescein staining. Central corneal ring ulcer with opacity. A: Anterior segment photograph. B: Photograph showing fluorescein staining. Central corneal ring ulcer with opacity. A: Anterior segment photograph. B: Photograph showing fluorescein staining. Central corneal ring ulcer with opacity. A: Anterior segment photograph. B: Photograph showing fluorescein staining. Figure 3 The ulcer and edema disappeared, although opacity remained in the central cornea. This resolved without recurrence even after discontinuation of AWT. A: Anterior segment photograph. B: Photograph showing fluorescein staining. Figure 3 The ulcer and edema disappeared, although opacity remained in the central cornea. This resolved without recurrence even after discontinuation of AWT. A: Anterior segment photograph. B: Photograph showing fluorescein staining. The ulcer and edema disappeared, although opacity remained in the central cornea. This resolved without recurrence even after discontinuation of AWT. A: Anterior segment photograph. B: Photograph showing fluorescein staining. Page 10/10 Page 10/10

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New vessels detected on wide-field imaging compared to two-field and seven-field imaging: implications for diabetic retinopathy screening image analysis

British journal of ophthalmology

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Stephen James Talks,1 Vina Manjunath,1 David H W Steel,2 Tunde Peto,3 Roy Taylor4 Method A consecutive series of treatment naïve patients with DR, referred from DRSS with pre- proliferative or proliferative DR or diabetic maculopathy, were imaged with Optomap colour images, within 3 months of DRSS referral. The incidence and distribution of NVs were recorded in relation to two-field and seven-field areas. ABSTRACT Introduction Wide-field retinal imaging (Optomap), used for detecting diabetic retinopathy (DR), has been shown to compare well with seven-field early treatment diabetic retinopathy study (ETDRS) photographs. An Optomap 200° image covers 80% of the retinal surface, compared with the standard seven-field, 30° images, covering 30% of the retinal surface. In England, DR screening is performed by grading two, 45° images per eye, by the DR screening service (DRSS). eye, by the DR screening service (DRSS). Purpose To assess how often retinal new vessels (NVs) are observed on Optomap imaging, outside the DRSS two fields and standard seven-field photography, in a cohort of patients referred by the DRSS. Method A consecutive series of treatment naïve patients with DR, referred from DRSS with pre- proliferative or proliferative DR or diabetic maculopathy, were imaged with Optomap colour images, within 3 months of DRSS referral. The incidence and distribution of NVs were recorded in relation to two-field and seven-field areas. Purpose To assess how often retinal new vessels (NVs) are observed on Optomap imaging, outside the DRSS two fields and standard seven-field photography, in a cohort of patients referred by the DRSS. Correspondence to Stephen James Talks, Newcastle Eye Centre, Royal Victoria Infirmary, Queen Victoria Road, Newcastle upon Tyne NE1 4LP, UK; [email protected] Correspondence to Stephen James Talks, Newcastle Eye Centre, Royal Victoria Infirmary, Queen Victoria Road, Newcastle upon Tyne NE1 4LP, UK; [email protected] Method A consecutive series of treatment naïve patients with DR, referred from DRSS with pre- proliferative or proliferative DR or diabetic maculopathy, were imaged with Optomap colour images, within 3 months of DRSS referral. The incidence and distribution of NVs were recorded in relation to two-field and seven-field areas. METHOD A Received 1 February 2015 Revised 23 March 2015 Accepted 21 May 2015 Published Online First 13 August 2015 A consecutive series of treatment naïve patients, referred from two DRSS in England, were imaged with Optomap colour images, within 3 months of referral. Referral from DRSS occurs if ‘referable’ DR is detected on analysis of two standard 45° photographs. At DRSS images are graded for the level of DR, and diabetic maculopathy (DMac): no DR is denoted as R0; mild DR as R1; pre- proliferative DR as R2; proliferative DR as R3. Potentially clinically significant DMac is repre- sented by the M1 grade. R2, R3 and M1 are then subsequently referred to hospital eye services. Results NVs were found in 102 of 1562 treatment naïve eyes (6.5%) of 781 patients. Of these, 72 were referred from DRSS as having NVs, but an additional 30 eyes (29% of NVs detected) from 25 patients were referred with a lesser degree of DR. In 25 of the 30 eyes without NVs reported on referral, NVs were located outside the standard two fields taken at DRSS, and in 12, NVs were outside the area covered on seven-field imaging (11.7% of eyes with NVs). Conclusions Wide-field imaging with Optomap detected approximately 30% more NVs than standard two-field imaging in patients referred from a UK DRSS. At the hospital eye clinic, certified medical photo- graphers took three wide-field Optomap images per eye after mydriasis, using the Optomap P2000 scan- ning laser ophthalmoscope; straight-ahead and up and down, with eye steering, which involves the patient following a fixation target (figure 1). Open Access Scan to access more free content Clinical science Clinical science on October 23, 2024 by guest. Prote http://bjo.bmj.com/ Br J Ophthalmol: first published as 10.1136/bjophthalmol-2015-306719 on 13 August 2015. Downloaded from Received 1 February 2015 Revised 23 March 2015 Accepted 21 May 2015 Published Online First 13 August 2015 Stephen James Talks,1 Vina Manjunath,1 David H W Steel,2 Tunde Peto,3 Roy Taylor4 on October 23, 2024 by guest. Protected by copyright. http://bjo.bmj.com/ hed as 10.1136/bjophthalmol-2015-306719 on 13 August 2015. Downloaded from 1Newcastle Eye Centre, Royal Victoria Infirmary, Newcastle upon Tyne, UK 2Sunderland Eye Infirmary, Sunderland and Institute of Genetic Medicine, Newcastle upon Tyne, UK 3NIHR Biomedical Research Centre at Moorfields Eye Hospital NHS Foundation Trust and UCL Institute of Ophthalmology, London, UK 4Magnetic renounce centre, Campus for ageing and vitality, Newcastle University, Newcastle upon Tyne, UK 1Newcastle Eye Centre, Royal Victoria Infirmary, Newcastle upon Tyne, UK 2Sunderland Eye Infirmary, Sunderland and Institute of Genetic Medicine, Newcastle upon Tyne, UK 3NIHR Biomedical Research Centre at Moorfields Eye Hospital NHS Foundation Trust and UCL Institute of Ophthalmology, London, UK 4Magnetic renounce centre, Campus for ageing and vitality, Newcastle University, Newcastle upon Tyne, UK in a population referred from the community could not be assessed. Grading is based on the ETDRS studies that related retinal findings to the likelihood of progression of the retinopathy and is based on seven-field colour imaging. It is still unknown how often more severe DR changes are found outside the standard seven-field, in particular new vessel (NV) formation. In the English DR screening service (DRSS) two images with nominal 45° fields are taken per eye, one centred on the fovea and the other on the disc. This is said to have a sensitivity of 80.2% and specificity of 92.9% for detecting refer- able DR compared to slit lamp biomicroscopy.5 In this study, we aimed to assess how often NVs were seen with wide-field Optomap imaging when com- pared to the areas covered by DRSS’s two-field and standard seven-field photography, in a cohort of patients referred from a DRSS. ABSTRACT Introduction Wide-field retinal imaging (Optomap), used for detecting diabetic retinopathy (DR), has been shown to compare well with seven-field early treatment diabetic retinopathy study (ETDRS) photographs. An Optomap 200° image covers 80% of the retinal surface, compared with the standard seven-field, 30° images, covering 30% of the retinal surface. In England, DR screening is performed by grading two, 45° images per eye, by the DR screening service (DRSS). Purpose To assess how often retinal new vessels (NVs) are observed on Optomap imaging, outside the DRSS two fields and standard seven-field photography, in a cohort of patients referred by the DRSS. New vessels detected on wide-field imaging compared to two-field and seven-field imaging: implications for diabetic retinopathy screening image analysis Stephen James Talks,1 Vina Manjunath,1 David H W Steel,2 Tunde Peto,3 Roy Taylor 1Newcastle Eye Centre, Royal Victoria Infirmary, Newcastle upon Tyne, UK 2Sunderland Eye Infirmary, Sunderland and Institute of Genetic Medicine, Newcastle upon Tyne, UK 3NIHR Biomedical Research Centre at Moorfields Eye Hospital NHS Foundation Trust and UCL Institute of Ophthalmology, London, UK 4Magnetic renounce centre, Campus for ageing and vitality, Newcastle University, Newcastle upon Tyne, UK INTRODUCTION Wide-field retinal imaging (Optomap), used for detecting diabetic retinopathy (DR), has been shown to compare well with seven-field early treat- ment diabetic retinopathy study (ETDRS) photo- graphs.1–3 As it provides a wider field of view, it would not be surprising that more DR is seen; however, it is thought that most potentially sight- threatening pathology occurs between the posterior pole and retinal mid-periphery. Silva et al4 reported that 10% of a cohort of 206 eyes were given a higher DR grade on Optomap images compared to seven-field images, predominantly due to the finding of more haemorrhages per quadrant. In his study, the patients were chosen from a tertiary eye clinic to represent a range of DR severity, and so, incidence rates of previously not recorded findings The images were then graded by an independent reading centre and the number of eyes with NVs, R3, recorded. The R3 images were then further assessed to map the distribution of NVs in relation to two-field and seven-field standard images using a standard field map (figure 2). If there was more than one area of NVs and any were located inside either the two-field or seven-field areas, then they were counted as being detected by that method. In a few cases, where the distinction between haemor- rhage, intra-retinal microvascular abnormalities (IRMA) and small NVs was uncertain, a fundus fluorescein angiogram (FFA) was performed, at the examiners discretion, on a second visit. 23, 2024 by guest. Protected by copyright. Open Access Scan to access more free content Talks SJ, et al. Br J Ophthalmol 2015;99:1606–1609. doi:10.1136/bjophthalmol-2015-306719 1606 on October 23, 2024 by guest. Protected by copyrig http://bjo.bmj.com/ Br J Ophthalmol: first published as 10.1136/bjophthalmol-2015-306719 on 13 August 2015. Downloaded from Clinical science Red free Optomap images of a left eye of a diabetic referred with maculopathy in the other eye, (R1, M1), the left eye being referred as, A) Up-steered, showing new vessels, (B) with zoom, (C) straight ahead showing new vessels outside two fields and on the edge of the ven-field images, (D) down steered, showing new vessels and (E) better seen with on zoom. http://bjo.b Br J Ophthalmol: first published as 10.1136/bjophthalmol-2015-306719 on 13 August 2015. Downloaded from on October 23, 2024 by guest. Protec http://bjo.bmj.com/ n 13 August 2015. RESULTS d d Independent assessment of the wide-field imaging found NVs in 102 of 1562 treatment naïve eyes (6.5%) of 781 patients referred from DRSS. Of these, 72 were referred from DRSS as having NVs, but 30 eyes (29% of NVs detected) from 25 patients were referred with a lesser degree of DR: 14 were referred as R2 and 16 as R1. In these eyes, 18 had been referred with Dmac, (M1), the other 12 were graded as M0. The fellow eyes of these patients were graded as R3 (proliferative DR) in three cases, R2 in eight and R1 in nine. In nine cases both eyes were referred with R1, having been referred because of DMac. Figure 1 shows NVs outside two fields and the benefit of using up and down eye steering. Figure 2 shows NVs outside seven fields confirmed on FFA. INTRODUCTION Downloaded from Figure 1 Red free Optomap images of a left eye of a diabetic referred with maculopathy in the other eye, (R1, M1), the left eye being referred as, (R1, M0). (A) Up-steered, showing new vessels, (B) with zoom, (C) straight ahead showing new vessels outside two fields and on the edge of the standard seven-field images, (D) down steered, showing new vessels and (E) better seen with on zoom. The images were reviewed using the proprietary image review software (Optos V2 Vantage Dx Review V.2.5.0.135; Optos, Dunfermline, UK). Grading for each wide-field image involved viewing the colour composite, green-wavelength and red- wavelength images using all the available image enhancement tools, including localised optimisation and magnification. However, true NVs were recorded only if the NV was present on FFA where avail- able and also where the NV remained clearly visible on the original image once spotted on the manipulated image, where appropriate. FFAs were requested in 31 patients where the examiner had some uncertainty between R2 and R3. In 14 of these cases, the examiner had diagnosed NVs and this was confirmed in 10 cases but not found in 4 cases and in 17 cases the examiner thought they were seeing, R2 changes, this was confirmed in 9 cases, but NVs were found in 8 cases. In 25 of the 30 eyes, in which NVs were not reported on the two DRSS images, the NVs were found outside the standard two-field and in 12 of these outside seven-field (11.7% of eyes with NVs); three were just within the field of view of the two fields but had been referred as R2 with IRMA. Two had very small disc NVs but also had NVs beyond two fields. Images from 23 eyes of the referred 1562 (1.4%) were deemed ungrad- able on Optomap due to poor image quality. 2024 by guest. Protected by copyright. DISCUSSION Figure 2 Red free Optomap and fundus fluorescein angiogram of the left eye of a diabetic referred due to maculopathy, (R1, M1 right; R1, M1 left), showing new vessels outside standard two-field (A); and seven-field (B) in the left eye. One factor that may have led us to detect this rate of NVs was the use of three images per eye, using eye steering, as less pathology is likely to be missed due to defocus or masking from eyelashes.9 The Optos camera can take a 200° image, but the resolution is best in a central band between the two arcades. The focus for the top and bottom areas of the retina is better by taking the image with the patient looking up and down. Looking at three images per eye takes extra time compared to one. Montage software is being developed to merge three pic- tures, which will help with analysis, but is not commercially available yet. A study comparing wide-field photographs, taken with undi- lated and dilated pupils, found that this did not statistically change the agreement with seven-field imaging, but reduced the ungradable rate from 4.5% to 0%.4 We had an ungradable rate of 1.4% using dilation and three images per eye. Figure 2 Red free Optomap and fundus fluorescein angiogram of the left eye of a diabetic referred due to maculopathy, (R1, M1 right; R1, M1 left), showing new vessels outside standard two-field (A); and seven-field (B) in the left eye. Our patients with NVs not detected on DRSS images were not ‘missed’ cases, as they were correctly referred for further medical assessment. All registered patients with diabetes are offered annual DRSS photography in England and this has led to fewer patients being referred from DRSS with severe NVs, and so, our incidence figures of more peripheral pathology may be higher than in unscreened populations. It is possible that if patients had small NVs outside the two-field images they would have been eventually referred as more posterior pathology developed. patient and on the photographer to use this protocol in every- day clinical practice. In the Eurodiab paper justifying the use of 45° imaging criteria only 48 eyes were compared.6 The findings supported the use of two-field imaging as a practical method, with the agreement for correct DR between several examiners, ranging between 28 and 43 of the 48 eyes, mean of 37 eyes. DISCUSSION ETDRS seven-field colour imaging is still considered the gold standard for assessing DR; however, it is hard both on the Talks SJ, et al. Br J Ophthalmol 2015;99:1606–1609. doi:10.1136/bjophthalmol-2015-306719 1607 on October 23, 2024 by guest. Protected by copyrig http://bjo.bmj.com/ Br J Ophthalmol: first published as 10.1136/bjophthalmol-2015-306719 on 13 August 2015. Downloaded from Clinical science patient and on the photographer to use this protocol in every- day clinical practice. In the Eurodiab paper justifying the use of 45° imaging criteria only 48 eyes were compared.6 The findings supported the use of two-field imaging as a practical method, with the agreement for correct DR between several examiners, ranging between 28 and 43 of the 48 eyes mean of 37 eyes Figure 2 Red free Optomap and fundus fluorescein angiogram of the left eye of a diabetic referred due to maculopathy, (R1, M1 right; R1, M1 left), showing new vessels outside standard two-field (A); and seven-field (B) in the left eye. Our study shows that on two-field DRSS imaging there is only a small risk of missing NVs, 30/1562 (1.9%), but these repre- sented 29% of the total number of eyes graded as having NVs. The NVs were found outside even the seven-field area in 11.7%. This is a higher rate than previously reported. In a study of 206 eyes of 103 patients, 10% were given a more severe DR grade with wide-field imaging, using one image per eye.4 In relation to our findings, 46 had NVs, but only two of these were found outside the seven-field area (4% of NVs). Our study population was much larger and represents a consecutive series referred from DRSS, rather than a group from a highly specialised clinic. A study using wide-field FFA on 118 patients found a total of 22 eyes (10%) had pathology visible only outside a simulated seven-field boundary. Of those eyes, 13 had peripheral retinal non-perfusion (8%) and 9 of 54 cases (17% of NVs) had per- ipheral NV outside seven-field. While using a different technique for identification of cases with NVs, this study draws a similar conclusion to ours on the relative proportion of NVs found outside seven-field.8 In the cases where we did use FFA some changes were made in the grading and eight additional cases of NVs were found. Acknowledgements This article presents independent research funded by the National Institute for Health Research (NIHR) under its Research for Patient Benefit (RfPB) Programme (Grant Reference Number PB-PG-0609-19117). The views expressed are those of the authors and not necessarily those of the NHS, the NIHR or the Department of Health. We thank the photography departments at Royal Victoria Infirmary, Newcastle upon Tyne and Sunderland Eye Infirmary for their Provenance and peer review Not commissioned; externally peer reviewed. Open Access This is an Open Access article distributed in accordance with the terms of the Creative Commons Attribution (CC BY 4.0) license, which permits others to distribute, remix, adapt and build upon this work, for commercial use, provided the original work is properly cited. See: http://creativecommons.org/ licenses/by/4.0/ 6 Adlington SJ, Kohner EM, Meuer S, et al. Methodology for retinal photography and assessment of diabetic retinopathy: the EURODIAB IDDM complications study. Diabetologia 1995;38:437–44. g 7 Diabetic eye screening. www.diabeticeye.screening.nhs.uk 8 Wessel MM, Aaker GD, Parlitsis G, et al. Ultra-wide-field angiography improves the detection and classification of diabetic retinopathy. Retina 2012;32:785–91. 9 Rasmussen ML, Broe R, Frydkjaer U, et al. Comparison between Early Treatment Diabetic Retinopathy Study 7-field retinal photos and non-mydriatic, mydriatic and mydriatic steered widefield scanning laser ophthalmoscopy for assessment of diabetic retinopathy. J Diabetes Complications 2014;29:99–104. DISCUSSION The kappa for interobserver and intraobserver comparisons was good at 0.83 and 0.85, respectively. Two-field imaging, where approximately 80% of patients are imaged yearly, using this protocol meets the appropriate sensitivity and specificity required for a screening programme and was therefore rolled out with scale, as shown by the England DRSS.7 This study also does not clarify how much risk there is in missing peripheral NVs, as they were not detected as a result of a patient presenting with the complications of proliferative DR, rather as a result of imaging a cohort of patients. However, if NVs are missed on DRSS images, and the patient is referred because of Dmac, appropriate management depends on the clin- ician detecting these NVs, which may not occur in a busy streamlined macular service. We would therefore advocate the use of steered wide-field images in ophthalmology clinics, and in the future hope that automated software can be developed to allow for fast, reliable and valid identification of abnormal vessels. In one study, seven-field ETDRS stereo images were ungrad- able by strict grading criteria in 31.6% and in 15.3% with a more lenient approach.5 The same paper reported good agree- ment for detecting the difference between referable and non- referable retinopathy between slit lamp biomicroscopy, 2×45° field and 7×30° field photography. However, there was only agreement on finding proliferative DR in 51/88 (58%) patients when comparing seven-field ETDRS stereo images with slit lamp examination. It is not clear how many had already had laser which may have lead to confusion on definitions between active or inactive NVs. In two cases, the clinician found NVs outside seven-field. For the comparison of two-field to seven- field only correlations between detecting referable from non- referable DR were presented. 23, 2024 by guest. Protected by copyright. guest. Protected by copyright. Acknowledgements This article presents independent research funded by the National Institute for Health Research (NIHR) under its Research for Patient Benefit (RfPB) Programme (Grant Reference Number PB-PG-0609-19117). The views expressed are those of the authors and not necessarily those of the NHS, the NIHR or the Department of Health. We thank the photography departments at Royal Victoria Infirmary, Newcastle upon Tyne and Sunderland Eye Infirmary for their Talks SJ, et al. Br J Ophthalmol 2015;99:1606–1609. doi:10.1136/bjophthalmol-2015-306719 1608 Clinical science assistance during this study. Photographers Gill O’Brien and Jade Ward prepared the figures. DISCUSSION TP was supported by the NIHR Biomedical Research Centre at Moorfields Eye Hospital NHS Foundation Trust and UCL Institute of Ophthalmology. assistance during this study. Photographers Gill O’Brien and Jade Ward prepared the figures. TP was supported by the NIHR Biomedical Research Centre at Moorfields Eye Hospital NHS Foundation Trust and UCL Institute of Ophthalmology. examination for evaluation of diabetic retinopathy. Am J Ophthalmol 2012;154:549–59. examination for evaluation of diabetic retinopathy. Am J Ophthalmol 2012;154:549–59. 2 Kernt M, Haritoglou C, Hadi I, et al. Assessment of diabetic retinopathy using nonmydriatic ultra-widefield scanning laser ophthalmoscopy (Optomap) compared with ETDRS 7-field stereo photography. Diabetes Care 2012;35:2459–63. Contributors All the authors contributed to the design of the study. TP led the independent reading centre. SJT and VM carried out the study, recruiting and consenting the patients. All the authors contributed to writing the paper. 3 Soliman AZ, Silva PS, Aiello LP, et al. Ultra-wide field retinal imaging in detection, classification, and management of diabetic retinopathy. Semin Ophthalmol 2012;27:221–6. Funding National Institute for Health Research. Funding National Institute for Health Research. Competing interests None declared. Competing interests None declared. Competing interests None declared. 4 Silva PS, Cavallerano JD, Sun JK, et al. Peripheral lesions identified by mydriatic ultrawide field imaging: distribution and potential impact on diabetic retinopathy severity. Ophthalmology 2013;120:2587–95. Ethics approval Ethical approval was obtained from the Northern Regional Ethics Committee and the NHS Trust Research departments at the Royal Victoria Infirmary, Newcastle upon Tyne and the Sunderland Eye Infirmary. 5 Scanlon PH, Malhotra R, Greenwood RH, et al. Comparison of two reference standards in validating two field mydriatic digital photography as a method of screening for diabetic retinopathy. Br J Ophthalmol 2003;87:1258–63. Provenance and peer review Not commissioned; externally peer reviewed. REFERENCES 1 Silva PS, Cavallerano JD, Sun JK, et al. Nonmydriatic ultrawide field retinal imaging compared with dilated standard 7-field 35-mm photography and retinal specialist Talks SJ, et al. Br J Ophthalmol 2015;99:1606–1609. doi:10.1136/bjophthalmol-2015-306719 1609

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Transformation of the resource basis of specialized dermatovenerologic establishments during the upgrading of the healthcare system in Russian Federation

Vestnik dermatologii i venerologii

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Преобразования ресурсной базы специализированных дерматовенерологических учреждений в период модернизации системы здравоохранения Российской Федерации М.М. Бутарева, Л.Е. Мелехина, М.А. Каспирович М.М. Бутарева, Л.Е. Мелехина, М.А. Каспирович Tr р р р ф Ключевые слова: дневной стационар, круглосуточный стационар, стационарозамещающие технологии, койко- место, место, койка круглосуточного стационара, здравоохранение, кожно-венерологический диспансер, амбулаторно-поликлиническое учреждение, лечебно-профилактическое учреждение, полномочия в сфере здравоохранения, модернизация здравоохранения. № 3, 2012 14 № 3, 2012 14 Преобразования ресурсной базы специализированных дерматовенерологических учреждений в период модернизации системы здравоохранения Российской Федерации М.М. Бутарева, Л.Е. Мелехина, М.А. Каспирович П Организация здравоохранения и эпидемиология 15 специализированной службы по профилю «Дерма- товенерология» за период 2000—2010 гг. произошли следующие значимые структурные преобразования. Сокращение числа кожно-венерологических диспан- серов в целом по Российской Федерации более чем на 30% (2000 г. — 341; 2010 г. — 219) привело, соот- ветственно, к снижению обеспеченности населения диспансерами данного типа с 0,23 до 0,16 на 10 000 населения (рис. 1). специализированной службы по профилю «Дерма- товенерология» за период 2000—2010 гг. произошли следующие значимые структурные преобразования. на стационарное обслуживание [4]. В странах Евро- пейского союза разработан и действует «Протокол оценки обоснованности использования стационарной помощи», в котором отражены основные критерии, оценивающие необходимость использования стаци- онарных или поликлинических ресурсов при выборе терапии индивидуально для каждого пациента, что позволяет более рационально использовать финансо- вые ресурсы [5]. Сокращение числа кожно-венерологических диспан- серов в целом по Российской Федерации более чем на 30% (2000 г. — 341; 2010 г. — 219) привело, соот- ветственно, к снижению обеспеченности населения диспансерами данного типа с 0,23 до 0,16 на 10 000 населения (рис. 1). Значительное сокращение числа медицинских уч- реждений было отчасти обусловлено вступлением в силу Федерального закона 122-ФЗ от 22.08.2004 г. «О внесении изменений в законодательные акты Рос- сийской Федерации и признании утратившими силу некоторых законодательных актов Российской Федера- ции в связи с принятием Федеральных законов «О вне- сении изменений и дополнений в Федеральный закон «Об общих принципах организации законодательных (представительных) и исполнительных органов госу- дарственной власти субъектов Российской Федерации» и «Об общих принципах организации местного само- управления в Российской Федерации»», в соответствии с которым оказание специализированной медицинской помощи стало обязательством субъектов Российской Федерации и было возложено на территориальные ор- ганы его исполнительной власти [8]. Происходящее реформирование национального здравоохранения существенно не повлияло на пере- ориентацию деятельности медицинских организаций. Одним из основных направлений в совершенствова- нии оказания медицинской помощи по-прежнему оста- ется развитие первичной медико-санитарной помощи на базе муниципального здравоохранения и перерас- пределение части объектов медицинской помощи из стационарного звена в амбулаторный сектор — пере- мещение с госпитального звена контингента больных, медицинская помощь которым может быть оказана на амбулаторном уровне [6]. Согласно данным официальной государственной статистики, последние годы характеризуются высоким показателем заболеваемости хроническими дермато- зами среди трудоспособного населения, а также уве- личением частоты встречаемости тяжелых форм дер- матозов, резистентных к различным видам терапии. Хроническое рецидивирующее течение заболеваний требует поиска новых, экономически более целесоо- бразных форм организации лечебного процесса, тем самым обеспечивается внедрение принципов эконо- мически обоснованного менеджмента в процесс ока- зания медицинской помощи. g p p p p Кey words: day hospital, round the clock hospital, hospital replacement technologies, bed, place, round the clock hospital bed, healthcare, dermatovenerologic dispensary, dispensary health unit, prevention and treatment facility, authorities in the field of healthcare, healthcare upgrading. ции по сравнению с аналогичным средним показате- лем по Европейскому союзу указывает на высокую «затратность» бюджета отечественного здравоохра- нения на данный вид помощи. Вместе с тем в Рос- сийской Федерации отмечается избыточный уровень госпитализации, при которой каждый третий случай с медико-экономической точки зрения является не- обоснованным [2, 3]. ции по сравнению с аналогичным средним показате- лем по Европейскому союзу указывает на высокую «затратность» бюджета отечественного здравоохра- нения на данный вид помощи. Вместе с тем в Рос- сийской Федерации отмечается избыточный уровень госпитализации, при которой каждый третий случай с медико-экономической точки зрения является не- обоснованным [2, 3]. „ „ Амбулаторно-поликлиническая медицинская помощь в структуре национального здравоохранения в настоящее время занимает лидирующие позиции, ввиду того что более 70% населения, обращающегося за медицинской помощью, получают услуги в условиях амбулаторно-поликлинических учреждений. Несмотря на это в 2010 г. из бюджета Российской Федерации было выделено более 60% средств (от общего объема финансирования здравоохранения) на долю наиболее ресурсоемкой стационарной медицинской помощи [1]. Во многих зарубежных странах, таких как Канада, Италия, Великобритания, лечебная сеть функциони- рует в условиях жестких финансовых ограничений. Развитие сети отделений краткосрочного пребыва- ния и стационаров на дому сократило часть расходов Превосходящее в два раза значение интегрально- го показателя объемов госпитализации (число койко- дней в расчете на человека) в Российской Федера- © М.М. Бутарева и соавт., 2011 Vestn Dermatol Venerol 2012; 3: 14—21. Контактная информация: [email protected] Организация здравоохранения и эпидемиология Однако, несмотря на реорганизацию специализи- рованной дерматовенерологической службы, обеспе- ченность населения кадровым потенциалом медицин- ских работников не изменилась и осталась на уровне 0,7 на 10 000 населения [9]. Число физических лиц врачей дерматовенерологов на занятых должностях в конце 2000 г. составило 9973, а в конце 2010 г. — 10142 (рис. 2). Проведенный нами анализ данных форм офици- альной государственной статистики ресурсов здра- воохранения показал, что в ресурсном обеспечении Реструктуризацию коечного фонда медицинских организаций наглядно можно продемонстрировать на 341 Рис. 1. Динамика ресурсов дерматовенерологической службы Российской Федерации — число КВД (2000—2010 гг.) 400 350 300 250 200 150 2000 г. 2001 г. 2002 г. 2003 г. 2004 г. 2005 г. 2006 г. 2007 г. 2008 г. 2009 г. 2010 г. 338 331 330 318 308 263 240 229 226 219 Рис. 1. Динамика ресурсов дерматовенерологической службы Российской Федерации — число КВД (2000—2010 гг.) № 3, 2012 16 Рис. 2. Структурные изменения кадровых ресурсов дерматовенерологической службы Р й й Ф (2005 2010 ) 10000 8000 6000 4000 2000 12000 2000 г. 2001 г. 2002 г. 2003 г. 2004 г. 2005 г. 2006 г. 2007 г. 2008 г. 2009 г. 2010 г. Число врачей дерматовенерологов Обеспеченность врачами дерматовенерологами 9973 0,7 0,7 0,7 0,7 0,7 0,7 0,7 0,7 0,7 0,7 0,7 9987 9983 9959 10 512 10 486 10 439 10 402 10 397 10 291 10 142 Число врачей дерматовенерологов Обеспеченность врачами дерматовенерологами Рис. 2. Структурные изменения кадровых ресурсов дерматовенерологической службы Российской Федерации (2005—2010 гг.) Это обстоятельство было обусловлено реорганиза- цией части круглосуточных дерматовенерологических коек в койко-места дневных стационаров [9, 10]. Оценить мощность коечного фонда дневных стаци- онаров возможно лишь начиная с 2002 г., поскольку информация стала собираться только с этого года. В 2002 г. для больных дерматовенерологического про- филя было развернуто всего 2899 койко-мест в днев- ных стационарах при больничных учреждениях, в том Это обстоятельство было обусловлено реорганиза- цией части круглосуточных дерматовенерологических коек в койко-места дневных стационаров [9, 10]. изменениях, произошедших в учреждениях дермато- венерологического профиля. За последние восемь лет коечный фонд дермато- венерологических учреждений снизился более чем на 40% (с 23 594 в 2002 г. до 15 713 в 2010 г.) и, со- ответственно, обеспеченность населения круглосуточ- ными дерматовенерологическими койками в расчете на 10 000 населения также снизилась с 1,96 в 2000 г. до 1,1 в 2010 г. (рис. 3). Оценить мощность коечного фонда дневных стаци- онаров возможно лишь начиная с 2002 г., поскольку информация стала собираться только с этого года. Организация здравоохранения и эпидемиология 17 Организация здравоохранения и эпидемиология 17 Важно отметить, что из числа больных, получивших лечение в дневных стационарах при больничных уч- реждениях, среди взрослых и детей нуждались в про- должении дальнейшего лечения в условиях круглосу- точного стационара только 1 и 2% соответственно. числе 2800 койко-мест для взрослых, 99 — для детей. Количество койко-мест дневных стационаров дерма- товенерологического профиля при больничных уч- реждениях составило 3,6% от общего коечного фонда дневных стационаров, организованных при больнич- ных учреждениях в целом по Российской Федерации. числе 2800 койко-мест для взрослых, 99 — для детей. Количество койко-мест дневных стационаров дерма- товенерологического профиля при больничных уч- реждениях составило 3,6% от общего коечного фонда дневных стационаров, организованных при больнич- ных учреждениях в целом по Российской Федерации. Несколько иная ситуация прослеживается с коеч- ным фондом дневных стационаров дерматовенеро- логического профиля, развернутых при амбулатор- но-поликлинических учреждениях (при АПУ). Наблю- дается интенсивное наращивание коечного фонда дневных стационаров при АПУ для взрослых. Так, ес- ли в 2002 г. общее число среднегодовых мест состав- ляло 755, то к концу 2008 г. число мест увеличилось до 1945, соответственно увеличивается и число боль- ных, получивших в них лечение; в 2002 г. было проле- чено — 17 227, а в 2010 г. — 45 998 (рис. 6). у р р За период с 2002 по 2008 г. коечный фонд дневных стационаров при больничных учреждениях для взрос- лых практически не изменился и составил в 2010 г. 2833 среднегодовых койко-места против 2800 в 2002 г. При этом следует отметить, что коечный фонд дневных стационаров при больничных учреждениях для детей увеличился по сравнению с 2002 г. в 2,5 раза и составил 253 среднегодовых койко-места в 2010 г. против 99 среднегодовых койко-мест в 2002 г. (рис. 4). За период с 2002 по 2008 г. коечный фонд дневных стационаров при больничных учреждениях для взрос- лых практически не изменился и составил в 2010 г. 2833 среднегодовых койко-места против 2800 в 2002 г. При этом следует отметить, что коечный фонд дневных стационаров при больничных учреждениях для детей увеличился по сравнению с 2002 г. в 2,5 раза и составил 253 среднегодовых койко-места в 2010 г. против 99 среднегодовых койко-мест в 2002 г. (рис. 4). Несмотря на то что коечный фонд дневных стациона- ров при больничных учреждениях для взрослых не изме- нился, а для детей увеличился в 2,5 раза, интенсивность использования койко-мест увеличилась в дневных ста- ционарах как для взрослого населения, так и для детей. Число пролечившихся больных к 2010 г. Организация здравоохранения и эпидемиология В 2002 г. для больных дерматовенерологического про- филя было развернуто всего 2899 койко-мест в днев- ных стационарах при больничных учреждениях, в том 23 594 1,7 1,6 1,5 1,4 1,4 1,3 1,2 1,16 1,1 22 421 21 498 20 526 19 497 18 692 17 369 16 476 15 713 25 000 20 000 15 000 10 000 5 000 2002 г. 2003 г. 2004 г. 2005 г. 2006 г. 2007 г. 2008 г. 2009 г. 2010 г. Рис. 3. Динамика ресурсов дерматовенерологической службы РФ — коечный фонд КВД, обеспеченность населения койками (2000—2010 гг.) Число коек в круглосуточных стационарах Обеспеченность дерматовенерологическими койками на 10 000 населения 23 594 1,7 1,6 1,5 1,4 1,4 1,3 1,2 1,16 1,1 22 421 21 498 20 526 19 497 18 692 17 369 16 476 15 713 25 000 20 000 15 000 10 000 5 000 2002 г. 2003 г. 2004 г. 2005 г. 2006 г. 2007 г. 2008 г. 2009 г. 2010 г. Обеспеченность дерматовенерологическими койками на 10 000 населения Рис. 3. Динамика ресурсов дерматовенерологической службы РФ — коечный фонд КВД, обеспеченность населения койками (2000—2010 гг.) Вестник дерматологии и венерологии Организация здравоохранения и эпидемиология 17 увеличилось на 20% и в том, и в другом случае; в 2002 г. в дневных стационарах при больничных учреждениях лечение по- лучил 47 941 взрослый больной, а в 2010 г. — 57 814; в дневных стационарах для детей — в 2002 г. — 1430, в 2010 г. — 4796 детей (рис. 5). К 2008 г. число мест в дневных стационарах при АПУ для детей снизилось почти на 20% (81 место в 2002 г. против 66 в 2008 г.), однако число пролечен- ных больных за этот период увеличилось почти в 2,5 раза (с 487 в 2002 г. до 1324 в 2008 г.) (рис. 7). Если количество среднегодовых мест дневных ста- ционаров при АПУ для взрослых на протяжении изуча- емого периода ежегодно увеличивалось — в среднем на 300 мест, то число мест в дневных стационарах при АПУ для детей колебалось, то снижаясь, то увеличи- ваясь (рис. 8). 2500 2000 1500 1000 500 3000 3500 2002 г. 2003 г. 2004 г. 2005 г. 2006 г. 2007 г. 2008 г. 2009 г. 2010 г. Число среднегодовых мест (дети) Число среднегодовых мест (взрослые) 2800 99 157 165 222 214 236 229 265 253 2949 2888 2902 2819 2811 3017 2946 2833 Рис. 4. Динамика ресурсов дерматовенерологической службы РФ — коечный фонд дневных стационаров при больничных учреждениях (2002—2010 гг.) 2500 2000 1500 1000 500 3000 3500 2002 г. 2003 г. 2004 г. 2005 г. 2006 г. 2007 г. 2008 г. 2009 г. 2010 г. 2800 99 157 165 222 214 236 229 265 253 2949 2888 2902 2819 2811 3017 2946 2833 Число среднегодовых мест (взрослые) Число среднегодовых мест (взрослые) Рис. 4. Динамика ресурсов дерматовенерологической службы РФ — коечный фонд дневных стационаров при больничных учреждениях (2002—2010 гг.) № 3, 2012 18 № 3, 2012 18 Рис. 5. Число больных, пролеченных в дневных стационарах при больничных учреждениях дерматовенерологического профиля в РФ (2002—2010 гг.) 50 000 40 000 30 000 20 000 10 000 60 000 2002 г. 2003 г. 2004 г. 2005 г. 2006 г. 2007 г. 2008 г. 2009 г. 2010 г. Число пролеченных больных (взрослые) Число пролеченных больных (дети) 47 941,5 1430,5 2447,5 2893,5 4049 4106 4268,5 4304,5 4941,5 4896 51 942,5 58 283 55 083,5 57 367,5 56 141 58 263,5 59 451,5 57 814,5 Рис. 6. Организация здравоохранения и эпидемиология 17 Динамика ресурсов дерматовенерологической службы РФ — коечный фонд дневных стационаров при АПУ для взрослых, число пролеченных в них больных (2002—2008 гг.) 25 000 20 000 15 000 10 000 5000 30 000 45 000 40 000 35 000 50 000 2002 г. 2003 г. 2004 г. 2005 г. 2006 г. 2007 г. 2008 г. Число пролеченных больных (взрослые) Число среднегодовых мест 17 227 755 1036 1338 1394 1679 1909 1945 23 850,5 30 707 30 615 39 094 44 899 45 997,5 Рис. 5. Число больных, пролеченных в дневных стационарах при больничных учреждениях дерматовенерологического профиля в РФ (2002—2010 гг.) 50 000 40 000 30 000 20 000 10 000 60 000 2002 г. 2003 г. 2004 г. 2005 г. 2006 г. 2007 г. 2008 г. 2009 г. 2010 г. Число пролеченных больных (взрослые) Число пролеченных больных (дети) 47 941,5 1430,5 2447,5 2893,5 4049 4106 4268,5 4304,5 4941,5 4896 51 942,5 58 283 55 083,5 57 367,5 56 141 58 263,5 59 451,5 57 814,5 50 000 40 000 30 000 20 000 10 000 60 000 2002 г. 2003 г. 2004 г. 2005 г. 2006 г. 2007 г. 2008 г. 2009 г. 2010 г. 47 941,5 1430,5 2447,5 2893,5 4049 4106 4268,5 4304,5 4941,5 4896 51 942,5 58 283 55 083,5 57 367,5 56 141 58 263,5 59 451,5 57 814,5 Число пролеченных больных (взрослые) Число пролеченных больных (дети) Рис. 5. Число больных, пролеченных в дневных стационарах при больничных учреждениях дерматовенерологического профиля в РФ (2002—2010 гг.) Рис. 6. Динамика ресурсов дерматовенерологической службы РФ — коечный фонд дневных стационаров при АПУ для взрослых, число пролеченных в них больных (2002—2008 гг.) 25 000 20 000 15 000 10 000 5000 30 000 45 000 40 000 35 000 50 000 2002 г. 2003 г. 2004 г. 2005 г. 2006 г. 2007 г. 2008 г. Число пролеченных больных (взрослые) Число среднегодовых мест 17 227 755 1036 1338 1394 1679 1909 1945 23 850,5 30 707 30 615 39 094 44 899 45 997,5 25 000 20 000 15 000 10 000 5000 30 000 45 000 40 000 35 000 50 000 2002 г. 2003 г. 2004 г. 2005 г. 2006 г. 2007 г. 2008 г. 17 227 755 1036 1338 1394 1679 1909 1945 23 850,5 30 707 30 615 39 094 44 899 45 997,5 Число пролеченных больных (взрослые) Рис. 6. Организация здравоохранения и эпидемиология 17 Динамика ресурсов дерматовенерологической службы РФ — коечный фонд дневных стационаров при АПУ для взрослых, число пролеченных в них больных (2002—2008 гг.) Вестник дерматологии и венерологии Организация здравоохранения и эпидемиология 19 19 Рис. 7. Динамика ресурсов дерматовенерологической службы РФ — коечный фонд дневных стационаров при АПУ для детей, число пролеченных в них больных (2002—2008 гг.) 1000 800 600 400 200 1200 1400 2002 г. 2003 г. 2004 г. 2005 г. 2006 г. 2007 г. 2008 г. Число пролеченных больных (дети) Число среднегодовых мест 487 81 40 35 47 39 48 66 1180 1039,5 614,5 507 739,5 1323,5 Рис. 8. Динамика числа среднегодовых мест в дневных стационарах при АПУ для взрослых и детей (2002—2008 гг.) 1000 800 600 400 200 1200 1400 2000 1800 1600 2002 г. 2003 г. 2004 г. 2005 г. 2006 г. 2007 г. 2008 г. Число среднегодовых мест дс при АПУ (взрослые) Число среднегодовых мест дс при АПУ (дети) 755 81 40 35 47 39 48 66 1036 1394 1338 1679 1909 1945 1000 800 600 400 200 1200 1400 2002 г. 2003 г. 2004 г. 2005 г. 2006 г. 2007 г. 2008 г. Число пролеченных больных (дети) Число среднегодовых мест 487 81 40 35 47 39 48 66 1180 1039,5 614,5 507 739,5 1323,5 Рис. 7. Динамика ресурсов дерматовенерологической службы РФ — коечный фонд дневных стационаров при АПУ для детей, число пролеченных в них больных (2002—2008 гг.) 1000 800 600 400 200 1200 1400 2002 г. 2003 г. 2004 г. 2005 г. 2006 г. 2007 г. 2008 г. Число пролеченных больных (дети) Число среднегодовых мест 487 81 40 35 47 39 48 66 1180 1039,5 614,5 507 739,5 1323,5 Рис. 7. Динамика ресурсов дерматовенерологической службы РФ — коечный фонд дневных стационаров при АПУ для детей, число пролеченных в них больных (2002—2008 гг.) Рис. 8. Динамика числа среднегодовых мест в дневных стационарах при АПУ для взрослых и детей (2002—2008 гг.) 1000 800 600 400 200 1200 1400 2000 1800 1600 2002 г. 2003 г. 2004 г. 2005 г. 2006 г. 2007 г. 2008 г. Число среднегодовых мест дс при АПУ (взрослые) Число среднегодовых мест дс при АПУ (дети) 755 81 40 35 47 39 48 66 1036 1394 1338 1679 1909 1945 1000 800 600 400 200 1200 1400 2000 1800 1600 2002 г. 2003 г. 2004 г. 2005 г. 2006 г. 2007 г. 2008 г. Организация здравоохранения и эпидемиология 17 755 81 40 35 47 39 48 66 1036 1394 1338 1679 1909 1945 Число среднегодовых мест дс при АПУ (дети) Число среднегодовых мест дс при АПУ (взрослые) Рис. 8. Динамика числа среднегодовых мест в дневных стационарах при АПУ для взрослых и детей (2002—2008 гг.) Рис. 8. Динамика числа среднегодовых мест в дневных стационарах при АПУ для взрослых и детей (2002—2008 гг.) № 3, 2012 20 № 3, 2012 20 Анализ данных официальной статистической от- четности Российской Федерации за период 2002— 2010 гг. показал более эффективную работу кругло- суточной койки дерматовенерологического профиля и койко-места дневного стационара при больничном учреждении. Среднее число дней работы круглосуточной дер- матовенерологической койки в 2010 г. составило 310 дней против 300 дней в 2002 г. Несколько увеличилось среднее число дней работы койко-места дневного ста- ционара при больничном учреждении, достигнув 296 дней в 2010 г., против 290 — в 2002 г. Вместе с тем Вестник дерматологии и венерологии Рис. 9. Число дней работы койки (койко-места) в стационарах дерматовенерологического профиля (2002—2010 гг.) 300 290 307 201 301 280 282 300 305 290 289 293 301 325 307 293 292 300 300 296 310 295 295 297 294 294 290 1000 900 800 700 600 500 400 300 200 100 2002 г. Круглосуточный стационар Дневной стационар при больнице Дневной стационар при АПУ 2003 г. 2004 г. 2005 г. 2006 г. 2007 г. 2008 г. 2009 г. 2010 г. Рис. 10. Число дней пребывания больного в стационарах дерматовенерологического профиля (2002—2010 гг.) 18 16,9 13,4 12,6 13,1 12,7 12,6 14,9 13 13 12,9 16,6 15,8 15,4 15,1 14,7 15,1 16,4 14,7 14,6 16,4 17,7 16,7 17,3 17 16,9 16,6 2002 г. Круглосуточный стационар Дневной стационар при больнице Дневной стационар при АПУ 2003 г. 2004 г. 2005 г. 2006 г. 2007 г. 2008 г. 2009 г. 2010 г. и койко-места дневного стационара при больничном учреждении. ционара при больничном учреждении, достигнув 296 дней в 2010 г., против 290 — в 2002 г. Вместе с тем 300 290 307 201 301 280 282 300 305 290 289 293 301 325 307 293 292 300 300 296 310 295 295 297 294 294 290 1000 900 800 700 600 500 400 300 200 . 9. Число дней работы койки (койко-места) в стационарах дерматовенерологического профиля (2002—2010 гг.) Рис. 10. Организация здравоохранения и эпидемиология Организация здравоохранения и эпидемиология 21 Таким образом, реструктуризация ресурсного обе- спечения медицинских организаций данного профиля, а именно, сокращение количества кожно-венероло- гических диспансеров в целом по Российской Феде- рации и перераспределение коечного фонда между круглосуточными и дневными стационарами, не от- разилась на обеспеченности населения врачами дер- матовенерологами и объемах оказания медицинской помощи данного вида. среднее число работы места дневного стационара при АПУ снизилось с 307 дней в 2002г. до 289 дней в 2010 г. (рис. 9). Средняя длительность пребывания больного как на койке в круглосуточном стационаре дерматовене- рологического профиля, так и на койко-месте (месте) в дневных стационарах при больничном учреждении и при АПУ за изучаемый период снизилась. В кругло- суточном стационаре длительность пребывания боль- ного на койке снизилась на 3% — с 18,0 дней в 2001 г. до 16,4 дня лечения в 2010 г. Если в 2002 г. анало- гичный показатель в дневном стационаре при боль- ничном учреждении составлял 16,9 дня, то к 2010 г. — 14,6 дня лечения (на 2,5%). В дневном стационаре при АПУ изучаемый показатель снизился до 12,9 дня лечения в 2010 г. по сравнению с 13,4 дня в 2002 г. (на 1,7%) (рис. 10). Интенсификация и рациональное использование коечного фонда привели к уменьшению объемов стационарной помощи при одновременном уве- личении объемов стационарозамещающих ресур- соемких технологий, что соответствует одному из основных условий успешной реализации стратегии совершенствования системы современного здраво- охранения [5]. Организация здравоохранения и эпидемиология 17 Число дней пребывания больного в стационарах дерматовенерологического профиля (2002—2010 гг.) 18 16,9 13,4 12,6 13,1 12,7 12,6 14,9 13 13 12,9 16,6 15,8 15,4 15,1 14,7 15,1 16,4 14,7 14,6 16,4 17,7 16,7 17,3 17 16,9 16,6 2002 г. Круглосуточный стационар Дневной стационар при больнице Дневной стационар при АПУ 2003 г. 2004 г. 2005 г. 2006 г. 2007 г. 2008 г. 2009 г. 2010 г. Рис. 10. Число дней пребывания больного в стационарах дерматовенерологического профиля (2002—2010 гг.) Вестник дерматологии и венерологии Литература 9. Сборник «Ресурсы и деятельность учрежде- ний здравоохранения» Министерство здра- воохранения и социального развития Россий- ской Федерации, Департамент организации медицинской профилактики, медицинской помощи и развития здравоохранения. Цен- тральный научно-исследовательский инсти- тут организации и информатизации здраво- охранения за 2000—2010 гг. Москва. 1. Денисов И.Н. Актуальные аспекты формиро- вания первичной медико-санитарной помо- щи. Главврач. 2010; 7: 29—31. 5. Комаров Ю.М. Медицинское страхование: опыт зарубежного здравоохранения. Вестн. гос. соц. страх. 2009; (1): 65—75. р 2. Анопченко Т.Ю., Максимов Д.А. Организа- ция стационарной медицинской помощи населению крупного города в современных условиях. Экономические аспекты стратегии модернизации России. Сборник научных трудов / Под ред. проф. В.А. Алешина, проф. М.А. Чернышева, проф. Т.Ю. Анопченко. Ро- стов н/Д.: Изд-во «Ака-демЛит», 2011; 208. 6. Доклад о результатах экспертной работы по актуальным проблемам социально-экономи- ческой стратегии России на период до 2020 г. Стратегия-2020: Новая модель роста — но- вая социальная политика. 7. Бутарева М.М., Знаменская Л.Ф., Марты- нов А.А. Лечение больных псориазом в случае резистентности к инфликсимабу. Вестн. дер- мат. и венер. — 2011. [6]: 69—72. 10. Сводная отчетная форма № 14-ДС «Сведе- ния о деятельности дневных стационаров лечебно-профилактического учреждения» по Российской Федерации за 2002—2010 гг. 3. Шабунова А.А. Здоровье населения в России: состояние и динамика. Монография. Волог- да: ИСЭРТ РАН, 2010: 408. 8. Федеральный закон 122-ФЗ от 22.08.2004 г. «О внесении изменений в законодательные акты Российской Федерации и признании утратившими силу некоторых законодатель- ных актов Российской Федерации в связи с принятием Федеральных законов «О вне- сении изменений и дополнений в Федераль- ный закон «Об общих принципах органи- зации законодательных и исполнительных органов государственной власти субъектов Российской Федерации» и «Об общих прин- ципах организации местного самоуправления в Российской Федерации». 4. Мартынчик С.А., Тимчинский Д.Л. Совершен- ствование механизмов оплаты стационарной помощи в системе добровольного медицин- ского страхования. Здравоохранение 2008; (5): 67—74.

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A review and meta‐analysis of the impact of intestinal worms on child growth and nutrition

Maternal and child nutrition

public-domain

WestminsterResearch http://www.wmin.ac.uk/westminsterresearch A review and meta-analysis of the impact of intestinal worms on child growth and nutrition. Andrew Hall1 Gillian Hewitt1 Veronica Tuffrey1 Nilanthi de Silva2 1 Centre for Public Health Nutrition, School of Integrated Health, University of Westminster 2 Faculty of Medicine, University of Kelaniya WestminsterResearch http://www.wmin.ac.uk/westminsterresearch A review and meta-analysis of the impact of intestinal worms on child growth and nutrition. Andrew Hall1 Gillian Hewitt1 Veronica Tuffrey1 Nilanthi de Silva2 1 Centre for Public Health Nutrition, School of Integrated Health, University of Westminster 2 Faculty of Medicine, University of Kelaniya 1 Centre for Public Health Nutrition, School of Integrated Health, University of Westminster 2 Faculty of Medicine, University of Kelaniya This is an electronic version of an article published in Maternal & Child Nutrition, 4 (s1). pp. 118-236, April 2008. © Sage Publications. The definitive version is available online at: http://dx.doi.org/10.1111/j.1740-8709.2007.00127.x http://dx.doi.org/10.1111/j.1740-8709.2007.00127.x The WestminsterResearch online digital archive at the University of Westminster aims to make the research output of the University available to a wider audience. Copyright and Moral Rights remain with the authors and/or copyright owners. Users are permitted to download and/or print one copy for non-commercial private study or research. Further distribution and any use of material from within this archive for profit-making enterprises or for commercial gain is strictly forbidden. The WestminsterResearch online digital archive at the University of Westminster aims to make the research output of the University available to a wider audience. C i ht d M l Ri ht i ith th th d/ i ht The WestminsterResearch online digital archive at the University of Westminste aims to make the research output of the University available to a wider audience Copyright and Moral Rights remain with the authors and/or copyright owners. Users are permitted to download and/or print one copy for non-commercial private study or research. Further distribution and any use of material from within this archive for profit-making enterprises or for commercial gain is strictly forbidden. Whilst further distribution of specific materials from within this archive is forbidden, you may freely distribute the URL of the University of Westminster Eprints (http://www.wmin.ac.uk/westminsterresearch). In case of abuse or copyright appearing without permission e-mail [email protected]. Keywords Intestinal worms, anthelmintics, children, growth, nutrition A review and meta-analysis of the impact of intestinal worms on child growth and nutrition Andrew Hall1, Gillian Hewitt1, Veronica Tuffrey1 and Nilanthi de Silva2 1Centre for Public Health Nutrition, University of Westminster, 115 New Cavendish Street, London W1W 6UW, U.K. 1Centre for Public Health Nutrition, University of Westminster, 115 New Cavendish Street, London W1W 6UW, U.K. 2 Faculty of Medicine, University of Kelaniya, Ragama, Sri Lanka. Author for correspondence: Andrew Hall, Centre for Public Health Nutrition, University of Westminster, 115 New Cavendish Street, London W1W 6UW Tel: 0207 911 5000 E-mail: [email protected] Abstract More than a half of the world’s population are infected with one or more species of intestinal worms of which the nematodes Ascaris lumbricoides, Trichuris trichiura and the hookworms are the most common and important in terms of child health. This paper: a) introduces the main species of intestinal worms with particular attention to intestinal nematodes; b) examines how such worms may affect child growth and nutrition; c) reviews the biological and epidemiological factors that influence the effects that worms can have on the growth and nutrition of children; c) considers the many factors that can affect the impact of treatment with anthelmintic drugs; d) presents the results of a meta-analysis of studies of the effect of treating worm infections on child growth and nutrition; e) discusses the results in terms of what is reasonable to expect that deworming alone can achieve; f) describes some important characteristics of an ideal study of the effects of deworming; and g) comments on the implications for programmes of recommendations concerning mass deworming. Introduction 1. Parasitic worms are among the most common and widespread infections of humans in the world today. Using recent estimates of the prevalence of the four main species of intestinal nematode worm (de Silva et al., 2003) a simple calculation that assume the probability of infection with one worm is independent of infection with another, indicates that about 48% of the 5 billion or so people living in the developing world are infected with at least one species, while almost 10% are infected with at least two species. But, if some 2.3 billion people in the developing world are infected with intestinal nematode worms, why isn’t disease due to worms more common, and why don’t they seem to have a greater and more noticeable impact on the health of children? This review will attempt to explain why, it will review the studies that have been done to examine the impact of treating intestinal nematode worms on children’s nutritional status and growth, and it will examine the scientific and experimental problems with estimating the impact that worms in general have on human health. Key messages • The effects of intestinal worms depend on the species, the mixture of species, the duration of infection and on the number of worms. • The distribution of worms among hosts is highly skewed so that only a minority of infected individuals have moderate to heavy infections and are likely to be diseased. • The impact of infections will also depend on the size and nutritional status of the host. • Treating worms can lead to improvements in growth and nutritional status but deworming alone does not treat any underlying nutritional deficits that have been caused or made worse by worms, so extra energy, protein and micronutrients are required. 2 2 1.1 The gastro-intestinal ecosystem The human intestinal tract provides a protected habitat for several hundred species of viruses, bacteria, yeasts, protozoa and worms. All the organisms that live in the intestinal ecosystem are parasites, because they are dependent for their existence on their host, and the basis of this dependence is usually nutritional (Hall, 1985). The parasitic life-style is highly successful for worms in general mainly because, once established within a host, there are no predators and life is a sheltered steady state with a constant supply of nutrients that is sustained by the host’s homeostatic mechanisms. Because the gut is a cavity within the host, it is said to be immunologically privileged, as the organisms living there are not exposed to the full force of the human immune system. Nevertheless, intestinal worms do elicit an immune response, and hookworms and whipworms in particular come into contact with both the cellular and humoral immune systems to elicit a Th- 2 responses and cause a rise in the concentration of IgE (Else, 2005). But the fact that intestinal worms persist and are not expelled from the gut indicates that they are able to evade these immune responses, although the mechanisms by which they achieve this are unclear. The only important non-specific barrier to infection is hydrochloric acid, secreted as ions into the stomach lumen by the parietal cells in the gut wall. Although this acid can kill infectious stages of many potential pathogens, paradoxically it is also a necessary stimulus for the establishment of many parasites: exposure to acid is required for the excystment of Giardia duodenalis (al-Tukhi et al., 1991, Hautus et al., 1988) and may be a necessary stimulus for the eggs of Ascaris lumbricoides to hatch, along with warmth and exposure to bile salts. But once in the intestine, the site where the worms come to maturity, all parasites of the gut have a body surface that is resistant to the action of the host’s digestive enzymes, while some worms have developed specific anti-enzymes (Uglem and Just, 1983), presumably for self-protection. The role of anti-enzymes in causing malnutrition is putative rather than proven, and it seems most likely that they act locally to prevent damage to the worms’ surface by host enzymes, rather than being secreted to have a widespread effect in the gut and thus perhaps on human nutrition. 1.1 The gastro-intestinal ecosystem The infectious stages of parasites have an easy way to enter their host, usually through the mouth as a contaminant of food, water or fingers, while the next generation leaves the body in faeces through the anus in the form of spores, cysts, eggs or larvae. There are exceptions: a few parasites of the gut enter the body through the skin, notably the larvae of hookworms and Strongyloides stercoralis. The major problem for parasitic worms is to get from one host to another, a journey that is facilitated in several different ways: • by producing large numbers of infectious stages to increase the chances of infecting a new host; a fertilised female A.lumbricoides, for example, may produce up to 200,000 eggs a day (Sinniah, 1982), therefore millions in a life-time; • by producing resistant infectious stages that can withstand adverse conditions; the eggs of species of Ascaris can survive for several months or years in warm, humid and sheltered conditions (Gaasenbeek and Borgsteede 1998) and are resistant even to 10% (Gaasenbeek and Borgsteede, 1998), and are resistant even to 10% (Gaasenbeek and Borgsteede, 1998), and are resistant even to 10% 2 formalin (Sandars, 1951) though not to exposure to ultra-violet light or to desiccation (Crompton, 1989); by infecting an intermediate host in which the parasite both multiplies and is dispersed, a feature of the life-cycle of many trematodes, a group of flatworms whose species often reproduce in snails from which are released larval stages that are infectious to humans ; • by infecting or encysting on foods that are consumed by a new host (Fried et al., 2004); • by the behaviour of infected people that puts others at risk of infection, such as defecating in the open, so that infectious stages are spread in the environment (Kilama, 1989); • and the behaviours that put people at risk of infection, such as pica (Geissler et al., 1998); by using fresh human faeces (sometimes called ‘nightsoil’) as a fertiliser (Needham et al., 1998, Pan et al., 1954); and by poor personal hygiene. Because the gastro-intestinal ecosystem offers such a rich habitat it has been colonised by an enormous number of different species. The next section introduces the major species of worms that live in the human gastro- intestinal tract. 1.2 Groups of intestinal parasitic worms that infect humans p p The variety of general and specific names given to worms can be quite confusing to a novice (see Box 1). The terms ‘helminths’ and ‘worms’ are generic names for metazoan (multicellular) parasites that are classified by helminthologists into two main Phyla: • Nematoda: the nematodes or roundworms, such as Ascaris lumbricoides and Trichuris trichiura; • Platyhelminthes: the flatworms, which contain two important Classes of parasites of humans: o Trematoda: the flukes, such as Fasciolopsis buski and Metagonimus yokogawa; o Cestoidea, sub-class Eucestoda: the tapeworms, such as o Cestoidea, sub-class Eucestoda: the tapeworms, such as Taenia saginata and Diphyllobothrium latum. Taenia saginata and Diphyllobothrium latum. 3 3 4 4 Box 1 How worms are named Worms go under a variety of general and specific names derived from different languages, but mostly old English, Latin or Greek. ‘Worm’ is derived from the old English word wyrm, meaning a snake or a dragon. ‘Worm’ is also associated with the Latin vermis from which comes the English words vermicide and vermifuge, a drug for treating worms. The generic term ‘helminths’ is an English word derived from the Greek word for worms, helmins. From this root is derived the term anthelmintic (sometimes anthelminthic or antihelminthic), a drug to treat worms. The name of the phylum Platyhelminthes combines the Greek terms platys, meaning broad or flat, and the word for worm. The Platyhelminths include two groups: the tapeworms (Old English ‘tape’ meaning tape, combined with ‘worm’) or cestodes (derived from the Greek word kestos meaning a strap); and the flukes (Old English term derived from the name for a type of fish called a plaice or flounder, which the worms look like) also called trematodes (derived from the Greek word trema meaning orifice or hole and eidos, meaning ‘in form’). The Phylum Nematoda (derived from the Greek nema, a thread, and eidos, meaning ‘in form’) are classified as helminths (but not Platyhelminths) as they are roundworms (derived from old French rond meaning round), not flat worms. Each species has a name in Latin that is a noun (which takes an upper case first letter) followed by an adjective (which takes a lower case first letter). For example the Latin name Ascaris lumbricoides is derived from the Greek word Askaris, meaning intestinal worm, and the Latin word lumbricus, meaning worm-like. 1.2 Groups of intestinal parasitic worms that infect humans The worm was given its name in 1758 by Carolus Linnæus, the father of nomenclature, who apparently had not heard of tautology. 5 5 Over 340 species of helminths have been recorded in association with humans (Coombs and Crompton, 1991) but most are rare zoonoses – infections of animals that can be contracted by humans. Table 1 lists the names of the most common species of helminths that live in the human intestine. The following section describes the life cycles of the most common species of intestinal flukes, tapeworms and roundworms. 1.2a Flukes or trematodes The two main species of intestinal trematode that infect humans listed in Table 1 are not widespread, although Fasciolopsis buski occurs focally in south-east Asian countries such as Thailand and the Philippines (Waikagul, 1991) and in the Indian sub-continent (Gilman et al., 1982, Chandra, 1984). Fasciolopsis buski is a zoonosis, and usually infects dogs and pigs, two animals closely associated with humans (Mas-Coma et al., 2005). A study in China reported an association between malnutrition and infection with flukes including F.buski, but the prevalence of this species was relatively low and the main pathogenic species was judged to be Schistosoma japonicum (Zhou et al., 2005). Some 16 species of Echinostoma have been reported to infect humans (Carney, 1991, Huffman and Fried, 1990), which makes it the most common genus of intestinal fluke, but another seven species of gut flukes from a variety of Trematode families have been recorded including the Fasciolidae, Heterophyidae, Lecithodriidae, Microphallidae, Paramphistomatidae and Plagiorchiidae (Waikagul, 1991). They are all zoonoses: infections of humans occurs by eating freshwater fish or shellfish, and the normal hosts are fish- eating animals such as cats and birds. Infections in humans are mainly found in adults in Asia who eat undercooked intermediate hosts such as crabs, frogs or fish, or in children who swallow metacercariae that have encysted on vegetation, such as water caltrop. Infections with intestinal flukes are not common, even among adults, and are rarer still among children, so there is no known association with malnutrition. The members of the order Schistosoma are not included in this review because they are not parasites of the intestinal lumen: they live in the portal 6 blood vessels around the gut (S.mansoni and S.japonicum) or urinary bladder (S.haematobium). 1.2b Tapeworms or cestodes Of the most common tapeworms of humans, the three species of Taenia tend not to be found among children as they are transmitted by eating undercooked beef (T.saginata) or pork (T.solium = Taeniarhynchus solium and Taenia asiatica) (Macpherson, 2005, Eom and Rim, 1993). These foods are not commonly eaten by poor children, or are proscribed in some parts of the world. The adult worms live in the small intestine and release their eggs in packets called proglottides, a living section that breaks off the posterior end of a growing worm (Pawlowski and Schultz, 1972). 1.2a Flukes or trematodes The proglottides of T.saginata are motile and can crawl away from a human stool deposited on the ground (see Figure 1), whereas the proglottides of T.solium do not show this activity. This behaviour occurs because cattle do not eat human faeces, but pigs do. When the eggs of Taenia species are swallowed by a suid or bovid species, they hatch, penetrate tissues and develop in muscles or organs to become infective cysticercoids. Humans become infected by eating raw or undercooked beef or pork. Infections can be common among people who habitually eat undercooked meat, such as ethnic groups that live in the Rift Valley of East Africa (Hall et al., 1981). Because the eggs are passed in packets rather than loose in the faeces, infections can be missed during the microscopical examination of faecal samples (Hall et al., 1981). Insert Figure 1 here The main concern for disease in humans is the possibility that the eggs of T.solium may hatch in the human intestine and develop in tissues to cause cysticercosis. If this happens in the brain it can lead to epilepsy (Newell et al., 1997). When pigs infected with T.solium were given to appease guerrillas fighting the government in west New Guinea, there was an outbreak of cysticercosis. This came to attention when people with severe burns appeared at hospitals: they had experienced an epileptic fit when sleeping next to a fire for warmth at night and had fallen into the flames (Gajdusek, 1978). 7 The effect of tapeworms in the intestine is minimal, probably because their relative mass is small in comparison with their host. It is also thought that the presence of existing worms may perhaps inhibit the establishment of additional worms, though this is hard to prove without deliberately infecting people. There is no known association between infections with Taenia spp and malnutrition in children. Hymenolepis nana is a widespread parasite of children, but the reported prevalence rarely exceeds 20% and is usually less than 5% (Khalil et al., 1991, Mason and Patterson, 1994, Sirivichayakul et al., 2000). The worm can persist by means of autoinfection, a process in which eggs hatch and mature in the human gut to form adults, without passing into the environment in the normal way to infect an insect intermediate host. Hymenolepis diminuta is usually a parasite of rodents, but it is found in children in situations in which they come into contact with rat or human faeces containing the worms’ eggs. Both species of Hymenolepis are associated with malnutrition, in that they tend to occur amongst children living in poor and unhygienic communities, but there have been no studies looking at the impact of treatment to suggest that they cause malnutrition. Diphyllobothrium latum is a notable tapeworm because it selectively absorbs vitamin B12 from the diet of its host or may interfere with absorption, which occurs only in the last third of the ileum; this can lead to pernicious anaemia (Nyberg, 1963). Infections occur by eating raw freshwater fish containing a plerocercoid larva, and were once common in Scandinavian countries such as Finland (Raisanen and Puska, 1984). This species has been reported all over the world, but mostly as curious case reports. Insert Figure 1 here It is not a common cause of anaemia in young children mainly because fish is an expensive food, and in most communities it is not commonly eaten raw, especially by children. 1.2c Roundworms or nematodes 1.2c Roundworms or nematodes Of the six species of nematode worms listed in Table 1, Enterobius vermicularis is found worldwide but is rarely a cause of serious disease, and is more a cause of irritation. The female worms lay their eggs around the anus 8 8 at night. This causes itching and pruritis that may occasionally lead to peri- anal sepsis in young children (Mahomed et al., 2003), probably because they scratch themselves. Infections have been reported to cause enuresis (Otu- Bassey et al., 2005) and are very rarely associated with appendicitis (Arca et al., 2004). Enterobius vermicularis tends to be most common among very young children, especially in kindergartens or among children living in institutions, probably because their personal habits are not well developed and they are in close physical contact with other children (Remm, 2006, Song et al., 2003). It is a difficult infection to diagnose efficiently because the eggs are not often seen in faeces, so it is necessary to press sticky cellophane tape over the peri-anal skin of a child, usually after a night’s sleep when the worms have laid their eggs, and examine the tape under a microscope (Celiksoz et al., 2005). The itching may affect a child’s sleep, but is not known to be a cause of malnutrition or poor growth. Infections with Strongyloides stercoralis are also associated with poor hygiene, close contact between people, and a lack of sanitary facilities. Infections have been reported among children in nursery schools and among adults in psychiatric institutions (Braun et al., 1988, Gatti et al., 2000). The worm can persist by a process of auto-infection in which larvae hatch in the large intestine and burrow directly into the gut wall, so emulating a naturally acquired infection (Schad, 1989). Infections with S.stercoralis can be transmitted directly from person-to- person by exposure to fresh faeces in the immediate living environment. A study in Bangladesh found that infections with S.stercoralis in people living in an urban slum were associated with households that lacked a latrine and had an earthen floor which may help larvae to survive (Hall et al., 1994). But when these factors were controlled for, the aggregation of infections may have been due not only to shared risk factors, but to a genetic predisposition that could also have contributed to infection (Conway et al., 1995). a The Japanese Organization for International Cooperation in Family Planning (JOICFP) 1.2c Roundworms or nematodes Although hyperinfections with Strongyloides stercoralis can be dangerous in immunocompromised patients (Keiser and Nutman, 2004), such as those being treated with immunosupressants (Schaeffer et al., 2004) or in the elderly, little is known about how many children are infected in the world 9 today, so the worm’s status as a cause of malnutrition and poor growth is unknown. The four main nematode worms most commonly associated with malnutrition and disease in children are Ascaris lumbricoides, Trichuris trichiura and both species of hookworms, Ancylostoma duodenale and Necator americanus. These worms are sometimes called soil-transmitted helminths. As this term refers to their mode of transmission, the generic term intestinal nematodes will be used here, which infers direct consequences for human health, and is perhaps more informative. Ascaris lumbricoides is the largest intestinal nematode worm to infect humans. An adult female A.lumbricoides typically weighs between 4 and 7 g, but can weigh up to 9 g and grow as long as 40 cm. Male worms are smaller, and weigh 2 – 3 g. Adult worms usually inhabit the jejunum (Crompton, 1989) where they feed on intestinal contents, but worms may be found higher and lower in the gut when present in large numbers, perhaps because of competition for living space. Worms may sometimes migrate into unusual sites such as the bile or pancreatic ducts, which they can block and cause acute and life-threatening disease (Ferreyra and Cerri, 1998, Sandouk et al., 1997). Adult A.lumbricoides have a tendency to wander if irritated and worms have been extracted from the nose and Eustachian tube (Fagan and Prescott, 1993, Jain and Pahuja, 1988). Adult A.lumbricoides maintain their position in the intestine by swimming against the flow of food, and when they die, they are carried out of the body in the faeces. Ascaris lumbricoides is the only intestinal nematode worm that is easily seen and identified in faeces, and is the only species of nematode for which anthelmintic treatment is visibly successful. This was the basis of a long-running Japanese family planning programme:a because the expulsion of A.lumbricoides from the gut offered manifest evidence of the effectiveness of treatment it provided an entry point to households to encourage women to use family planning. 1.2c Roundworms or nematodes An adult female A.lumbricoides may produce up to 200,000 eggs a day (Sinniah, 1982) in a life-span of 12 – 18 months (Anderson and May, 1991), 10 but there is good evidence of both density dependent fecundity (Hall and Holland, 2000) as well as geographical variation in the number of eggs produced per female worm (Hall and Holland, 2000). This means that worms produce fewer eggs when there are many present in the gut, and that there is not a linear relationship between the number of worms in a host and the concentration of eggs in faeces. The consequence is that the concentration of eggs in a faecal sample from a Bangladeshi child, for example, is not necessarily equivalent in terms of worm burden to the same concentration of eggs in a sample from an Iranian child (Hall and Holland, 2000). As any given concentration of eggs in faeces may not reflect the same number of worms in different parts of the world, it means that the use of fixed ranges of egg counts to classify the intensity of infection with intestinal nematode worms is scientifically dubious. This is discussed in more detail in Section 2. Freshly excreted A.lumbricoides eggs are not immediately infectious and take 10 – 14 days to embryonate in the environment at 30 C, or about 50 days at 17 C (Pawlowski and Arfaa, 1984). This means that old faeces are a source of infection, not fresh faeces, and the soil on which they lie may no longer bear evidence of faecal contamination. A new infection occurs when mature eggs are swallowed as a contaminant of food or fingers. When an egg comes into contact with bile acids, the larva breaks out of the egg case and burrows through the intestinal wall. After a few days migration through the blood stream to the liver and then to the lungs, the developing larvae break into the alveoli and are coughed up and swallowed. Large numbers of larvae can cause a verminous pneumonia (Tomashefski et al., 1989, Valentine et al., 2001, Gelpi and Mustafa, 1968). The larvae pass through the stomach and into the small intestine for a second time, where they grow and mature to become adults. 1.2c Roundworms or nematodes Although this migration through tissues exposes the immune system to Ascaris antigens and stimulates an immune response, it does not seem to lead to protective immunity, at least not in all individuals, although some individuals may develop partial immunity (see Section 2.4). The adults of Trichuris trichiura live in the large intestine and caecum (Bundy and Cooper, 1989). These nematode worms insert their whip-like anterior end into the gut wall and secrete enzymes and a specific protein that causes a syncytium to form (Drake et al., 1994), which provides an easily 11 ingested liquid food. The penetration of the worm into tissues also causes inflammation and bleeding so that, when large numbers of worms are present, they can cause dysentery and even rectal prolapse (Bundy and Cooper, 1989). Each female worm produces 3,000 – 20,000 eggs a day (Bundy and Cooper, 1989). The eggs mature in the environment to form an infectious larva within the egg shell in about 10 – 14 days. When a mature egg is swallowed the larva hatches from the egg in the stomach and is propelled down the gut by peristalsis to the worm’s habitat in the large in intestine. The two main species of hookworm that infect humans, Ancylostoma duodenale and Necator americanus, are usually considered together, for two reasons. First, because they mostly now have an overlapping geographical distribution and occur worldwide, even if their origins were in the old and new world respectively. Second, the eggs of each species cannot be told apart when examined under a microscope, so only a diagnosis of hookworm can be made. It is necessary either to hatch the eggs and examine the larvae to tell which species is which, or to expel adult worms from the gut and recover them from the faeces. Both of these procedures are difficult and the first carries a risk of infection. The eggs of both species of hookworm are passed in the faeces. A female A.duodenale is estimated to lay 10,000 – 25,000 eggs a day and a female N.americanus 5,000 – 10,000 eggs a day (Pawlowski et al., 1991). Again, like A.lumbricoides, the average number of eggs produced per female worm declines as the number of worms increases, a mechanism believed to contribute to the stability of worm populations (Bundy, 1990) but which may also serve to help prevent massive infections occurring. 1.2c Roundworms or nematodes Hookworm larvae hatch out onto the soil within 48 - 96 hours of being passed in the faeces, although the speed of maturation depends on the temperature and humidity (Smith, 1990). The larvae do not feed, so have a finite life-span measured in a few weeks, again depending on the temperature, the degree of humidity to prevent desiccation, and whether the larvae are shaded from sunlight or not, such as by vegetation (Smith, 1990). Hookworm larvae are thought to survive longest on light, sandy soil rather than on heavy, clay soil, and in places where the relative humidity is high (Mabaso et al., 2003). 12 Infection with both species of hookworm occurs when the third stage larvae on the soil come into contact with bare skin. The infectious larvae burrows through the epidermis by a process of mechanical penetration facilitated by secreted protease enzymes (Salafsky et al., 1990). In large numbers this can cause an allergic reaction called “ground itch” (Gilles, 1990). Infections with A.duodenale can also occur if the larvae are swallowed (Schad, 1990). Once in the human body, hookworm larvae migrate through the blood system and heart to the pulmonary blood vessels, where they bore into the alveoli. The action of the cilia lining the bronchioles carries the larvae upwards, into the oesophagus, where they are swallowed, pass through the stomach, and reach the small intestine. The worms take about 4 - 5 weeks to mature and start producing eggs, called the pre-patent period. There is circumstantial evidence that larval worms may get into breast milk, because hookworm eggs have been seen in the faeces of infants too young to have been exposed to larvae (Schad, 1990). The buccal cavity of both species of hookworms that infect humans contains sharp plates or ‘teeth’ used to grasp and cut gut tissue to enable the worms to suck up blood and tissue fluids (Roche and Layrisse, 1966). Both species of hookworm secrete an anticoagulant to maintain the flow of blood (Roche and Layrisse, 1966, Hotez and Cerami, 1983). It has been estimated that a single A.duodenale causes blood loss of 0.2 ml per day (range 0.14 – 0.26 ml) compared with 0.04 ml per day (range 0.02 – 0.07 ml) by N.americanus (Roche and Layrisse, 1966). 1.2c Roundworms or nematodes When expressed in terms of the number of worms needed to lose 5 ml of blood each day, this corresponds to 25 A.duodenale and 110 N.americanus (Pawlowski et al., 1991). Some of the blood and iron ingested by hookworms is excreted into the host’s gut, and is available for absorption lower down the intestine. It has been estimated that as much as 40 – 60% of the iron lost into the gut may be reabsorbed by anaemic people (Roche and Layrisse, 1966). Whether or how quickly any given individual develops anaemia will depend on five factors: the number of worms; the duration of infection (see Section 2.3); the initial haemoglobin concentration; the size of the existing reserves of iron in the bone marrow; The buccal cavity of both species of hookworms that infect humans contains sharp plates or ‘teeth’ used to grasp and cut gut tissue to enable the worms to suck up blood and tissue fluids (Roche and Layrisse, 1966). Both species of hookworm secrete an anticoagulant to maintain the flow of blood (Roche and Layrisse, 1966, Hotez and Cerami, 1983). It has been estimated that a single A.duodenale causes blood loss of 0.2 ml per day (range 0.14 – 0.26 ml) compared with 0.04 ml per day (range 0.02 – 0.07 ml) by N.americanus (Roche and Layrisse, 1966). When expressed in terms of the number of worms needed to lose 5 ml of blood each day, this corresponds to 25 A.duodenale and 110 N.americanus (Pawlowski et al., 1991). Some of the blood and iron ingested by hookworms is excreted into the host’s gut, and is available for absorption lower down the intestine. It has been estimated that as much as 40 – 60% of the iron lost into the gut may be reabsorbed by anaemic people (Roche and Layrisse, 1966). Whether or how quickly any given individual develops anaemia will depend on five factors: the number of worms; the duration of infection (see Section 2.3); the initial haemoglobin concentration; the size of the existing reserves of iron in the bone marrow; 13 and, most importantly, the amount and bioavailability of iron in the diet (Crompton and Whitehead, 1993, Gilles, 1990). 1.2c Roundworms or nematodes 1.3 How worms may affect human nutrition and growth There are several mechanisms by which intestinal nematodes could affect the nutritional status of their host: • By feeding on the contents of the host’s gut, including the host’s secretions that make up the exoenteric circulation; • By feeding on host tissues, including blood and serum, that leads to a loss of iron and protein; • By causing maldigestion or malabsorption of nutrients; • By inflammatory responses that lead to the production of substances that may affect appetite and food intake, or substances that modify the metabolism and storage of key nutrients such as iron; • And through contingent responses to infection, such as fever, leading to an increased metabolic rate; by causing hypertrophy of muscles; and by immune responses to infection, all of which result in the diversion or use of nutrients and energy for purposes that would not have been necessary had worms not been present. All intestinal parasites obtain their nutrients either from the food and intestinal secretions of their host, or from their host’s tissues and body fluids. The nutritional needs of parasites are relatively small compared with a well nourished host, mainly because their relative biomass is small (see Box 2). This means that worms such as A.lumbricoides only take a relatively small proportion of the host’s food from the gut. A study of tapeworms in protein malnourished rats indicated that the amount of protein in worms was only about 1% of the total protein intake, even if there were enough worms to fill the small intestine (Hall, 1983). Although worms do not have an aerobic metabolism and are relatively wasteful of substrates to generate energy, it is also likely that the worm’s excretory products are absorbed, metabolised and excreted by the human host. 14 Box 2 The nutrition of worms There is a common belief that worms make children thin by consuming the food in their intestine or that they increase children’s weight by their presence.(SC/UK, 2004) This is a fallacy because the biomass of worm tissue is relatively small in comparison with the biomass of an infected child. For example a female Ascaris lumbricoides, which is the largest intestinal nematode worm that infects humans, has an average weight of some 3.2 g and a maximum of 9.0 g. Male worms are half the size. 1.2c Roundworms or nematodes A study of the worm burdens of 268 infected children aged 4 – 10 years old found an average of 23 A.lumbricoides per child which weighed an average of just under 50 g. This was 0.3% of the average weight of the children. If 70% of the weight of worm tissue is metabolically active (excluding the chitinous exoskeleton and the pseudocoelomic fluid), and if the metabolic rate and need for energy of the worm is the same as its homeothermic host (77 kcal/kg/day), then a biomass of 50 g would require about 2.7 kcal of energy a day. As helminths have an anaerobic metabolism which only generates about 5% of the energy as aerobic metabolism, this biomass of worms would require some 54 kcal, equivalent to 13 g of glucose, a day. However as the metabolic by-products of the worm’s metabolism are likely to be absorbed and metabolised by the host, in whom they could produce energy, the inefficiency of energy production by worms may be mitigated. Some unique metabolites of A.lumbricoides can be detected in human urine in proportion to the number of worms in the host (Hall and Romanova, 1990). These rough estimates indicate that the nutritional needs of most worm burdens are small in relation to a child host, though during a severe shortage of food, the loss of any nutrients to a moderate or large worm burden may exacerbate undernutrition. 15 The impact of worms’ nutritional requirements may be more significant to a host if the worms feed directly on host tissues, because the physical damage they do may have important consequences, in addition to the effects of nutrient losses due to feeding. For example when hookworms move from a site at which they have been feeding, it may continue to bleed into the gut as a result of the persistent effects of the anticoagulant secreted from the worm’s salivary glands (Hotez and Cerami, 1983). Moderate to heavy infections with hookworm are strongly associated with anaemia (Roche and Layrisse, 1966) which has consequences for growth (Stephenson et al., 1993a), physical fitness (Stephenson et al., 1993a, Latham et al., 1990b, Stephenson et al., 1990) and worker productivity (Gilgen et al., 2001, Selvaratnam et al., 2003, Hunt, 2002). The feeding of hookworms can also cause a loss of blood proteins and the development of hypo-albumenaemia (Gilles, 1990). 1.2c Roundworms or nematodes Maldigestion and malabsorption may occur as a result of physical damage to the gut surface. The presence of moderate burdens of Ascaris suum in experimentally infected pigs has been shown to cause flattening of villi as well as villous atrophy and fusion (Martin et al., 1984), all of which could lead to a loss of brush border enzymes and a reduced surface area for digestion and absorption. Damage to villi might be expected to lead to the loss of lactase. A study of African children infected with intestinal parasites, including A.lumbricoides, did not find evidence of lactose malabsorption (Gendrel et al., 1992) although this may be a result of a failure of the study to take into account the worm burden (see Section 2.3). Another study, of Panamanian children, did find differences between groups of infected and uninfected children in the results of hydrogen breath tests, an indicator of lactose malabsorption (Carrera et al., 1984). Another possible cause of malabsorption could be bacterial overgrowth of the small intestine due to the presence of worms, though this is more commonly associated with infections such as Giardia duodenalis (de Boissieu et al., 1996, Farthing, 1993, Müller and von Allmen, 2005, Tandon et al., 1977). A loss of appetite has been reported as a consequence of worm infections (Easton, 1999, Symons, 1985, Hadju et al., 1996) but it is hard to 16 study, because it would mean leaving some infected children untreated while others were given an anthelmintic. However several studies have measured improvements in the appetite of children after treating worms (Hadju et al., 1996, Latham et al., 1990b, Stephenson et al., 1993a), which has provided quite convincing evidence of an important mechanism by which worms can impair children’s nutrition and growth. The contingent responses to infection, which have been described in a previous review (Hall, 1985), lead to a waste – or at least an unnecessary diversion – of resources as a result of the physical and immunological responses to infection. These are hard to quantify in humans, so experimental animals are often used. For example, experimental infection of pigs with moderate numbers of A.suum, a species very similar to A.lumbricoides that can also infect humans, has been shown to lead to an increase by 50 – 100% in the wet weight of the small intestine compared with uninfected controls, mainly due to hypertrophy of the tunica muscularis (Stephenson, 1987). 1.2c Roundworms or nematodes This is likely to be in response to the need for increased muscularity to push food past worms in the small intestine by peristaltic contractions. Histological cross-sections of the mucosa also show changes in tissues in addition to the flattened villi described above: the lamina propria becomes infiltrated with mast cells and eosinophils as a result of immune reactions to the presence of worms in the intestine, while goblet cells show hyperplasia as a result of producing more mucus, perhaps to try to protect the villi from erosion (Stephenson, 1987, Stephenson et al., 1980a). These may be usefully adaptive and protective responses to infection, but they represent a diversion of nutrients that should not be necessary and could be better used for growth if they happen in an already undernourished child. 1.4 Design of studies to estimate the impact of worms 1.4 Design of studies to estimate the impact of worms The main problem with studying the impact that worms have on child growth and nutrition has been touched upon in the previous section: the need for untreated controls. If worms impair growth, and if treating worms leads to extra or catch-up growth, then it is necessary to measure the difference that treatment makes between treated and untreated subjects, not just the absolute amount of growth that occurs after treatment. This is because some 17 amount of growth and weight gain should occur naturally in all children, unless they are severely undernourished or have a hormonal disease. It could be argued that it is enough to express weight gain as a change in proportion to a reference value, such as a higher z-score of weight-for- height, or a greater percentage of the median value. But such improvements in anthropometric status could occur as a result of secular changes in the food supply, or as a result of better health because of seasonality in the transmission of other diseases, such as malaria and diarrhoea. Concurrent and untreated controls are essential to the validity of the conclusions of any study of the impact of treating intestinal worms on child growth and nutritional status and are an important criterion for including any study in a meta- analysis. 1.5 Aims 2. Factors affecting the impact of intestinal worms 2. Factors affecting the impact of intestinal worms In order to understand the impact that intestinal nematode worms have on the nutritional status and growth of children by any of the mechanisms proposed in Section 1, it is necessary to understand the factors that are likely to influence the degree or magnitude of their effects. 1.5 Aims The aim of this review is: The aim of this review is: • To describe the epidemiological factors that influence the impact that intestinal worms have on human nutritional status and growth; • To describe the factors that affect the impact of anthelmintic treatment; • To undertake a meta-analysis of the effects of intestinal worms on children’s nutritional status and growth. 2.1 Species of intestinal worm The most important species in terms of disease are Ascaris lumbricoides, Trichuris trichiura and the hookworms. These worms live in different parts of the intestine, differ in the route they take to reach their adult habitat, and feed in different ways. This has been described in Section 1.2. 18 Although A.lumbricoides is undoubtedly the most common species worldwide, it is very hard to distinguish from A.suum (Crompton, 1989). It is quite likely that both species occur together, especially in places where pigs are allowed to roam freely in their search for food in an environment inhabited by people (Maruyama et al., 1997, Kofie and Dipeolu, 1983). Although the two hookworm species are considered together because there is no easy way to tell them apart, there is evidence that A.duodenale is more pathogenic than N.americanus because it consumes more blood per worm (Roche and Layrisse, 1966). As well as measurements of blood loss using radioactive isotopes (Roche and Layrisse, 1966), there is epidemiological evidence from a study of schoolchildren in Pemba, a small Tanzanian island where both types of hookworms occur, that in schools where the prevalence of A.duodenale is high there may be more anaemia and iron deficiency than in schools where N.americanus is the predominant species (Albonico et al., 1998). Although the two hookworm species are considered together because there is no easy way to tell them apart, there is evidence that A.duodenale is more pathogenic than N.americanus because it consumes more blood per worm (Roche and Layrisse, 1966). As well as measurements of blood loss using radioactive isotopes (Roche and Layrisse, 1966), there is epidemiological evidence from a study of schoolchildren in Pemba, a small Tanzanian island where both types of hookworms occur, that in schools where the prevalence of A.duodenale is high there may be more anaemia and iron deficiency than in schools where N.americanus is the predominant species (Albonico et al., 1998). The conclusion is that different species will have different effects on the nutritional status and growth of children. Insert Table 3 here Insert Table 4 here Figure 2 shows the typical relationship between age and the prevalence of infection with A.lumbricoides derived from a study of people living in an urban slum in Bangladesh; a similar relationship is commonly observed for T.trichiura. Figure 2 indicates that infections are acquired in the first two years of life and that around 80% of all age classes are infected, a high but typical proportion. 2.2 Prevalence of infection The first important epidemiological parameter that describes the potential effect of worms on human health is the proportion infected, or prevalence. Infections are usually diagnosed by seeing the characteristic eggs of each species of worm in faeces examined under a microscope, which simply indicates that there is present in the gut at least one sexually mature female and one male worm. The exception is A.lumbricoides, because unfertilised female worms can produce infertile or “decorticated” eggs; they can be identified because they are longer and narrower than fertilised eggs (Crompton, 1989, WHO, 1994a). Infections with intestinal worms may therefore be missed if there are only female worms present, or only male worms, or only immature worms. Such infections are not clinically important, but they will lead to an underestimate of the prevalence 19 Infections may also be missed if an insensitive method of diagnosis is used, such as a direct faecal smear, and if the concentration of eggs in faeces is low. A study in Bangladesh found that some 8% of infections with A.lumbricoides were missed when infections were diagnosed using a moderately sensitive ether sedimentation method (Hall, 1981) and compared with a diagnosis made by expelling worms using an effective anthelmintic drug (Hall et al., 1999). Table 2 shows the range in numbers of eggs estimated to be produced daily by a female worm of each species. They suggest that the sensitivity of diagnosing a light infection of a few worm varies between species, probably in the rank order A.lumbricoides, A.duodenale, T.trichiura and lastly N.americanus. Insert Table 2 here Infections with the three main types of intestinal worms number among the most common infections of children in the world today. Table 3 presents some recent estimates of the numbers of people infected with A.lumbricoides, T.trichiura and the hookworms, by age range. Table 4 presents recent estimates of the prevalence of these infections in young school-age children, aged 5 – 9 years. This age group is particularly likely to have moderate to heavy infections and is vulnerable to their impact on nutritional status. Insert Figure 2 here Hookworms tend to show a different pattern in which the prevalence typically increases with age, reaching a peak in late adolescence and adulthood (Bundy et al., 1992a). The reason for this difference is not clear, especially as children are less likely to wear shoes than adults and so could be considered to be more to be exposed to hookworm larvae on the soil. But 20 the differences in prevalence may reflect where worms are transmitted in what have been called “domains of infection” (Cairncross et al., 1996). The transmission of both A.lumbricoides and T.trichiura is thought to occur within or around the household, in the domestic domain, while hookworms may be transmitted beyond the household, in the public domain which is frequented more by adults than children (Cairncross et al., 1996). Although the prevalence of infection in children may provide some indication of the importance of each worm in terms of health and nutrition, there is no clear threshold prevalence associated with disease, largely because the relationship between prevalence and the intensity of infection is strikingly non-linear. Figure 3 shows the relationship between the prevalence of infection with intestinal worms and the worm burden, derived from data collected in Bangladesh on A.lumbricoides (Hall et al., 1999). It can be seen that, below a prevalence of about 50%, the mean worm burden is relatively low, but rises almost exponentially above a prevalence of 60%. This means that even a prevalence of up to about 50% is associated with a low average worm burden, and that the prevalence of infection is a poor indicator of the probability of disease unless it is 70% or greater. This relationship helps to explain the use of a threshold prevalence of 50% for administering mass anthelmintic treatment for both soil-transmitted helminths and schistosomiasis, a threshold that was endorsed by a WHO Expert Committee in 2001 (WHO, 2002a). This threshold has subsequently been lowered by the WHO to 20% for mass treatment once a year in what they classify as “low risk” communities, and the WHO now apply the 50% threshold to define a “high risk” community where mass treatment twice a year is warranted (WHO, 2006). Insert Figure 3 here The relationship between prevalence and disease is further complicated by the mix of species: in many parts of the world it is common to find all three major types of worms together, so that some children have two or three infections. Although the prevalence of A.lumbricoides and T.trichiura is often correlated (Booth and Bundy, 1992), it seems that an infection with one species does not predispose to the presence of another. Figure 4 shows a diagrammatic representation of the percentage of individuals who have 21 multiple infections when the prevalence of A.lumbricoides is 60%, T.trichiura is 50% and the hookworms is 40%, all arbitrary but typical figures. If the probability of having one infection is independent of having another, then it can be estimated that 32% of individuals have at least two infections and 18% have all three infections (Figure 4). There is no method to assess the impact of multiple infections: they could be additive but might be multiplicative or even antagonistic if species occur in the same location in the gut, such as A.lumbricoides and the hookworms. As multiple infections may be as common or more common than single infections, it is hard to estimate the relative benefit of treating each different species, especially as the drugs used to treat intestinal nematode worms are effective against all species, if to differing degrees (see Section 3.2). In conclusion the WHO threshold of 50% infection provides a reasonable basis for applying mass treatment. Below this threshold few people have moderate to heavy worm burdens that cause disease and the majority are uninfected; above this threshold the likelihood of moderate to heavy infections increases exponentially. This is not to say that worms do not cause disease below a prevalence of 50%, but the effect on a very small minority may be lost in the group average. A prevalence of 50% was taken as a minimum for studies of deworming to be included in the meta-analysis reported below, and studies such as those of (Garg et al., 2002) in which the prevalence of infection with any worm before treatment with an anthelmintic was only 11%, were excluded. This threshold may seem somewhat arbitrary, but it is based on the relationship shown in Figure 3 and it would be unusual to include any uninfected individuals in a trial of a drug to treat a bacterial diseases. Number and distribution of worms 2.3 Each worm that becomes established in a host represents the successful hatching, migration, establishment and development of a single fertile egg. Nematode worms do not multiply within their host, except for Strongyloides stercoralis (see above) the eggs of which can hatch within the lower bowel to release infective filariform larvae that penetrate the gut wall in a process called auto-infection (Schad, 1989). This explains the persistence of S.stercoralis in British soldiers who were prisoners of the Japanese in Asia during the second world war and who have developed strongyloidiasis in their old age (Gill and Bell, 1979, Gill and Bell, 1987, Gill et al., 2004). Each worm that becomes established in a host represents the successful hatching, migration, establishment and development of a single fertile egg. Nematode worms do not multiply within their host, except for Strongyloides stercoralis (see above) the eggs of which can hatch within the lower bowel to release infective filariform larvae that penetrate the gut wall in a process called auto-infection (Schad, 1989). This explains the persistence of S.stercoralis in British soldiers who were prisoners of the Japanese in Asia during the second world war and who have developed strongyloidiasis in their old age (Gill and Bell, 1979, Gill and Bell, 1987, Gill et al., 2004). For each of the four major types of intestinal worms the probability of disease is related to the number of worms in the host, called the worm burden. Figure 5 shows the average number of A.lumbricoides recovered from males in 11 age classes in an urban slum in Bangladesh who were given a drug that paralysed their worms so that they were expelled by peristalsis, then recovered, washed and counted. It shows that the heaviest average infections were found in school-age children from 5 – 15 years old, a characteristic typical of many helminth infections. The notable exception is the hookworms, for which the prevalence and mean worm burden tend to increase with age so that adolescents and adults tend to be most heavily infected (Bradley et al., 1992, Bundy, 1990). Insert Figure 3 here A prevalence of 50 – 100% represents a typical range in many human communities in which mass anthelmintic treatment is given, and the effectiveness of treatment in such studies represent common epidemiological circumstances. 22 Number and distribution of worms Insert Figure 5 here There are two theories to explain the shape of the distribution in Figure 5. First, it could reflect differences in behaviour, because children are more exposed to worm eggs than adults, perhaps as a result of playing on faecally contaminated ground and poor personal hygiene. Second, it could reflect the development of a partially effective immune response to infection, or perhaps a more effective immune response in some individuals than others as a result of repeated exposure to worm larvae. The study in Bangladesh however found a statistically significant difference in the mean worm burden of A.lumbricoides between adult males (who leave their community to work during the day) and adult females (who stay at home in the crowded, unsanitary environment), which suggests that exposure more than immunity influences this distribution (Hall et al., 1999). 23 When the distribution of worms among hosts is examined it is typical to find that it is highly skewed, so that most individuals have light infections while a minority have moderate to heavy infections. Figure 6 illustrates this distribution using data on the number of A.lumbricoides recovered from 1,765 people who were treated with pyrantel pamoate to paralyse and expel their worms. The distribution shown in Figure 6 is best described by the negative binomial, which is purely an empirical fit, and implies nothing about the biological reason why worms should be distributed in this way. But this distribution, which is described as aggregated or over-dispersed, is typical of most helminth infections and has been observed even in pigs each of which have been infected experimentally with the same number of fertile eggs of A.suum (Boes et al., 1998, Stephenson et al., 1980a). This suggests that the distribution derives from differences between hosts in the establishment of worms rather than in differences in exposure to eggs. Insert Figure 6 here The equation that describes this distribution is defined by three parameters, the prevalence (P), the arithmetic mean worm burden (M), and by (k), a parameter that varies inversely with the degree of aggregation or clumping of worms in a few hosts so that: P = (1 – (1 + M/k)-k) * 100 P = (1 – (1 + M/k)-k) * 100 The parameter k captures the degree to which worms in a small proportion of hosts tend to be aggregated, clumped or over-dispersed (terms commonly used in this context). Small values of k, typically less than 1, indicate a high degree of aggregation of worms, irrespective of age, sex or any other factor. The study in Bangladesh indicated that k varied with the mean worm burden so that, as the mean burden increased, the degree of aggregation decreased (Hall et al., 1999). The implication is that, when the average worm burden is large, more people are likely to have moderate to heavy infections and may be diseased as worms are less aggregated. Figure 7 shows the cumulative percentage of all worms recovered from 1,765 people in urban Dhaka plotted against the cumulative percentage of hosts, for worm burdens between zero and 187 worms. It shows that a half of all subjects expelled only 10% of all worms, and that 80% of all subjects contained only 24 40% of all worms. The remaining 60% of worms were recovered from only 20% of individuals. Insert Figure 7 here If it is typical to find that a small proportion of people harbour a large proportion of worms, what happens when they are treated? Evidence from studies of reinfection after treatment have found that heavily infected individuals tended to become heavily reinfected again while lightly infected people become lightly reinfected, leading to the theory that some individuals are predisposed to infection and others are not (Bundy and Cooper, 1988, Chan et al., 1994, Forrester et al., 1990, Haswell-Elkins et al., 1987, Holland et al., 1989, Kightlinger et al., 1995). If this is so, could efforts be concentrated on the individuals who were predisposed to worms? This was examined in a study of infection and reinfection with A.lumbricoides in 880 individuals in Bangladesh (Hall et al., 1992). It was found that, although there was evidence of a predisposition to moderate to heavy infections, over three rounds of treatment and two periods of reinfection of 6 months, about two thirds of all subjects were moderately or heavily infected at least once (Hall et al., 1992). This suggested that there was no benefit in identifying moderately to heavily infected people on the assumption that they were predisposed individuals, and mass treatment should be given rather than any form of selective treatment. The biological implications of the aggregation of worms are that most individuals in a community at any one time have light infections, but a minority ranging from < 1 – 40% will have moderate to heavy infections and are most likely to be diseased. If treatment is given periodically and reinfection occurs (which will happen because eggs can persist in the environment for many months, if not years), then over a period of 2 – 3 years a majority of individuals will be moderately to heavily infected at some point. This has implications for measuring the impact of mass treatment, as a minority will benefit more than the majority in the short term, but over a longer period of periodic treatment, an increasingly larger proportion will benefit. A light infection probably has little effect on the nutritional status of a host, and the worm burden is the key indicator of the probability of disease. This requires that worms are expelled and counted, something that is difficult 25 to do, and it destroys the worm burden at the same time, so that infected subjects can no longer be followed. Insert Figure 7 here So in most surveys of worms the concentration of eggs in faeces is used as an indicator of the intensity of infection. However the perception of egg counts tends to be relative, so that what is seen to be a moderate egg count in one place could be considered to be high or even low in another. To provide some guidance on assessing egg counts a WHO Expert Committee in 1987 suggested threshold egg counts to classify light, moderate and heavy infections with A.lumbricoides and T.trichiura (WHO, 1987). In 2002 a new Expert Committee added hookworm to the list although the previous committee had stated that egg counts for this worm could not be given because the “critical worm load differs locally depending on age, sex, iron intake and species of hookworm” (WHO, 2002a). These thresholds are shown in Table 5. Insert Table 5 here Since the Expert Meeting in 1987 it has been clearly shown for A.lumbricoides at least, that the relationship between worm burden and egg count is both non-linear and differs between worms in different countries (Hall and Holland, 2000). For example 20 worms was associated with around 1,300 eggs/g of faeces or 2,300 egg/g in two studies in Bangladesh, with 17,300 eggs/g in Iran, with 22,000 eggs/g in Nigeria and Madagascar, with 27,000 eggs/g in Burma, and with 44,500 eggs/g in Mexico (Hall and Holland, 2000). This indicates that there is no consistent relationship between egg counts and worm burdens that can be applied universally, for A.lumbricoides at least. 2.4 Duration of infection One of the main factors besides the worm burden that is likely to contribute to the development of disease is the duration of infection, something that is not usually known. Table 6 shows the estimated life span of the major species of intestinal nematodes of humans. Insert Table 6 here But even if a worm such as A.lumbricoides can live for as long as two years and is then expelled from the gut when it dies, new worms are continually acquired so that people remain persistently infected with slowly 26 changing numbers of worms. Figure 2 indicates that just over 60% of children aged 1 – 2 years in the study in Bangladesh were infected with A.lumbricoides and Figure 5 indicates that they contained an average of about 7 worms. As both the prevalence and mean worm burden are higher in older age groups, it suggests that most people are infected throughout their life. 2.5 Rate of reinfection If the population of worms in a community of hosts is perturbed by giving mass treatment with an anthelmintic, reinfection can occur immediately and the number of worms will rebound to a similar number as before, a state called equilibrium. Without treatment there may be fluctuations in time in the numbers of worms in individuals as some die and others are gained, but the number of worms in the community seems to reach a relatively steady state, perhaps driven by factors such as sanitation, the contamination of the environment, behaviours that put people at risk, and environmental conditions that favour the survival of infectious stages. Figure 8 shows the prevalence of infection with A.lumbricoides in 880 people at three rounds of treatment, six months apart and Figure 9 shows the mean worm burden on the same occasions (Hall et al., 1992). Insert Figure 9 here The prevalence, shown in Figure 8, rebounded very quickly after treatments in the school-age children, perhaps because the force of infection was greater in this age class, but was lower at each round in the four adult age classes. The mean worm burden shown in Figure 9 was, however, lower at each round of treatment in all age classes except for the very youngest cohort, whose exposure to worm eggs probably increased as they became mobile and were increasingly exposed to worm eggs. Figures 8 and 9 show that periodic treatment may do little to perturb the prevalence of infection but, if given often enough, it can help to prevent reinfection with the same number of worms. In an environment such as an urban slum in Bangladesh, it may be necessary to give treatment for worms at least three times a year rather than twice annually, in order to sustain low worm burdens and reduce the probability of disease. 27 Figures 8 and 9 also illustrate that the prevalence is a poor indicator of the effectiveness of worm control by periodic deworming, and that an indicator of the intensity of infection is better (Bundy et al., 1992a). Ideally both should be measured during a programme of mass deworming. 2.6 Summary The probability that an infected child has disease or malnutrition caused by intestinal worms is related to: • The species of worm; • The mixture of species; • The worm burden of each species. • And the duration of infection before treatment. The number of worms is difficult to assess without expelling them from the gut. The concentration of eggs in faeces may only represent a worm burden in a specific locality, because fecundity varies with the number of worms and perhaps between strains of worms in different locations around the world. There are no threshold numbers of worms to classify burdens as light, moderate or heavy. There are no methods to combine the probability of disease due to different numbers of worms of different species in the same individual. 3. Factors affecting the impact of treatment Section 2 has described how the biology of each species of worm and the distribution of worms among hosts is likely to influence the effect that they have on the growth and nutritional status of children. As it is unethical to infect children with worms and measure prospectively their impact on growth, evidence of the effect of worms on humans comes either from cross-sectional surveys or from experimental studies in which changes in key indicators of health, growth and nutrition are measured after giving treatment. Cross-sectional studies are difficult to interpret. Although several have shown an association between infections with intestinal worms and undernutrition or stunted growth (Moore et al., 2001, Muniz et al., 2002, Al- 28 Mekhlafi et al., 2005, Gupta, 1990, Saldiva et al., 1999, Egger et al., 1990, Cerf et al., 1981), other studies have not (Pegelow et al., 1997) or have had mixed results (Mahendra Raj et al., 1997b). The best analysed studies have controlled for factors such as age, sex and socio-economic status, and have examined associations not just with the presence of infection, but with an estimate of the intensity of infection. The main problem with doing such analyses lies in the lack of data on the actual worm burden rather than on egg counts, which do not have a consistent relationship with worm burden, and with quantifying the combined effects of more than one species of worm. There is also considerable potential for publication bias in cross- sectional studies because only interesting and biologically plausible associations are likely to be written up and submitted for publication, while negative findings are either not submitted or are less likely to be accepted by a scientific journal. But the main scientific problem with cross-sectional studies is related to temporality: did malnutrition predispose children to infection with worms, or did worms cause the malnutrition? It is also unlikely that worms, and only worms, are responsible for all undernutrition or stunted growth. Worms are associated with poverty and a poor diet, so if worms have a harmful effect, it is likely to be in addition to underlying chronic malnutrition. Second, infection with worms reflects exposure to human faeces, a waste material that contains many other pathogens in addition to worm eggs, such as viruses, bacteria and protozoa. 3. Factors affecting the impact of treatment Repeated episodes of diarrhoea and other infectious illnesses associated with living in an impoverished and unhygienic environment can also affect nutritional status through their effects on appetite, absorption and metabolic rate, as the studies of Mata and colleagues in Guatemala vividly demonstrated (Mata, 1978). Infection with worms is an indicator of life in an environment contaminated with human faeces. These complexities mean that it is not adequate simply to compare the growth of a group of infected children after treatment with a control group who were not infected with worms (Mahendra Raj et al., 1997a, Mahendra Raj and Naing, 1998), nor is it adequate to explain the magnitude of weight gain after treatment in terms of prior egg counts (Stephenson et al., 1980b). 29 The need for untreated controls is reinforced when it comes to measuring the impact of treatment on weight gain or growth. Even when children are chronically undernourished, some growth can be achieved, so without a control group it is not possible to know whether a treatment has led to extra weight gain or extra growth over any given period. Making comparisons with a reference population to assess the degree of change in undernutrition will not provide an answer either. Even if an improvement in anthropometric indices is recorded, or a fall in the percentage of children who are in some way undernourished, such changes can occur as a result of secular improvements in food supply or a decrease in the transmission of other diseases such as malaria or diarrhoea over the period of a study. This section describes factors that influence the impact of treatment on children’s growth and nutritional status. 3.1 Study design: controls and randomisation In order to estimate reliably the magnitude and statistical significance of the effect of a treatment on growth or nutritional outcome it is necessary to have an untreated control group (Stephenson, 1987). To ensure that naturally occurring differences between subjects are evenly distributed between groups before treatment is given, individuals also need to be randomly assigned to each study group. As there can be large differences between individuals in the intensity of infection because of the aggregated distribution of worms, if the sample size is small then subjects may need to be stratified first by egg count, before random allocation. If the sample size is large and the prevalence is high, then random allocation is likely to be sufficient. If the treatments are allocated by clusters, such as villages or schools rather than to individuals, there must be a sufficient number of clusters to distribute any variation between clusters in the prevalence and intensity of infections evenly between study groups. Randomised cluster designs have the potential to provide the large sample sizes that are needed to be able to detect the effects of treatment on the minority of subjects who will benefit most from treatment. Ideally all subjects in a control group in a simple randomised trial should be given an identical placebo. This is less easy to do in cluster 30 randomised trials, especially if anthelmintics are given through the health system. Effectiveness trials, such as the one done as a part of Child Health Fairs in Uganda (Alderman et al., 2006), typically have untreated controls, but they usually receive nothing rather than a placebo. This opens the study to potential bias because of self-treatment with anthelmintics among the control group. 3.2 Anthelmintic drugs and other treatments The drugs used to treat infections with intestinal nematodes can be divided into two main types: • Drugs that act on the nervous system to paralyse worms so that they are expelled from the gut by normal peristalsis, such as piperazine, levamisole and pyrantel pamoate; • Drugs that inhibit metabolic processes, such as the benzimidazole derivatives albendazole, mebendazole and tiabendazole, which block the uptake of glucose by microtubules in the mitochondria of worms (Horton, 2002, Horton, 2000), and the relatively new treatment nitazoxanide, which acts in protozoa by inhibiting the enzyme pyruvate ferredoxin oxidoreductase (Esposito et al., 2005, Sisson et al., 2002, White, 2003). 3.1 Study design: controls and randomisation There are differences between these types of drug in the efficacy with which they treat each species of intestinal worm (de Silva et al., 1997). Efficacy is assessed in two ways: • As the cure rate, which is the percentage of infected subjects in whose faeces worm eggs are no longer found after treatment (although some immature female or male worms could still remain, thereby over- estimating the cure rate); • As the egg reduction rate, which is the percentage reduction in the arithmetic or geometric mean concentration of eggs in the faeces of infected individuals. The concentration of eggs reflects the number of female worms, and an even sex ratio is usually assumed. The cure rate and egg reduction rate are typically estimated by collecting and examining under a microscope a faecal sample collected a few 31 days before treatment and then again some 14 – 21 days afterwards. The gap after treatment is advisable because there is evidence that the drugs may temporarily inhibit egg production by worms that have survived treatment (Hall and Nahar, 1994). Because of their different modes of action the two types of anthelmintic drugs differ in their efficacy: drugs that paralyse worms tend to be less effective than those that inhibit metabolic processes, perhaps because the paralysis can wear off more quickly than the effects of a metabolic inhibitor, especially if worms are anchored to the gut wall. (If A.lumbricoides are expelled from the gut using a drug such as pyrantel or levamisole and placed in warm saline, they can recover their motility after a few hours, showing the transitory effect of the paralysis). Table 7 gives a rough guide to the range in efficacy of each type of drug against the main species of intestinal nematode worms at the usual dosage given. Data are generally lacking for the drugs that paralyse worms, because they are older and less well studied than the highly effective benzimidazole drugs, mebendazole and albendazole, which superseded levamisole and pyrantel in 1975 and 1980 respectively (Horton, 2003b). In general the benzimidazoles are less effective against T.trichiura than against A.lumbricoides and the hookworms, and are less effective against hookworm than against A.lumbricoides. Insert Table 7 here There also seem to be differences in the efficacy of the two benzimidazole anthelmintics, even though they are chemically similar. Table 8 shows data from a large study in Zanzibar of treating intestinal nematode infections with albendazole or mebendazole which revealed statistically significant differences in the efficacy of treating hookworm (Albonico et al., 1994). A reason for the lower efficacy of drugs to treat hookworm compared with A.lumbricoides may be because hookworms are usually physically attached to the gut and may be better able to resist the transient effects of anthelmintic drugs than A.lumbricoides which can only maintain their position in the gut by actively swimming against the intestinal flow. 32 The reason for the lower efficacy of T.trichiura compared with both A.lumbricoides and the hookworms may be because they live in the large intestine, where anthelmintic drugs may be more dilute than in the small intestine. Adult T.trichiura are also typically embedded in the gut wall by their whip-like anterior end, which may provide an anchor so that they, like hookworms, can withstand any transitory effects of anthelmintic drugs. A study in Bangladesh showed that, in order to achieve a cure rate for T.trichiura of 80%, 400 mg albendazole had to be given daily for three consecutive days (Hall and Nahar, 1994). There is also evidence that there may be a lower cure rate for T.trichiura in Asia than in Africa: an analysis of trials of albendazole showed a median cure rate of 33% in Asia compared with 61% in Africa (Bennett, 2000), There is obvious concern that repeated mass treatment could lead to the development of drug resistance, something that has been shown to develop as a result of repeated treatment (Albonico et al., 2003). There are substantial lessons to be learned from veterinary medicine in which the same anthelmintic drugs have been used for many years and far more intensively than in humans (Coles, 1995). Strategies to avoid or delay the development of drug resistance should be applied (WHO, 2002a), such as alternating drugs with different modes of action, and the efficacy of treatment should be assessed periodically at sentinel sites during mass treatment programmes, the interval to depend on the frequency of treatment (WHO, 2002a). The main limitation to alternating treatments has been the lack of effective alternatives to the albendazole and mebendazole, as single dose treatments. Insert Table 7 here One possibility is a combination of pyrantel and oxantel which has been shown to be quite effective in comparison with mebendazole (Albonico et al., 2002, Rim et al., 1975), but this mixture is not widely available. A combination of albendazole and ivermectin has been tested as a treatment for lymphatic filariasis (Beach et al., 1999b, Belizario et al., 2003, Horton et al., 2000) and has effects on the major species of soil-transmitted helminths as well. The advantage of giving two drugs in combination is that the chance of genes that confer resistance to both treatments at the same time is greatly reduced. 33 A relatively new drug called nitazoxanide has been shown to be an effective treatment for all major species of intestinal nematode worms as well as Hymenolepis spp (Juan et al., 2002, Romero Cabello et al., 1997). But the drug currently has to be given twice a day for three days whereas the others are single dose treatments, which ensures compliance in mass treatment campaigns. The major advantage of nitazoxanide is that it is an effective treatment for intestinal protozoa, including Giardia duodenalis and Cryptosporidium spp, as well as anaerobic bacteria such as Helicobacter pylori and Clostridium difficile (Bobak, 2006, Megraud et al., 1998). Such a drug could have a significant impact on growth and nutritional status, as studies of giving metronidazole, another broad-spectrum antibacterial and anti-protozoal drug have suggested (Gupta and Urrutia, 1982, Khin Maung et al., 1990). The differences in the efficacy of treatments for intestinal nematode worms will therefore depend on the drug, the dose of drug, the species of worm, and the strain of the worm perhaps, which will all have consequences for the impact of treatment on nutritional status and growth. In some studies included in this review a single treatment was given, in others, repeated treatment was given using the same drug. And in one study included in the review, two different drugs were given: pyrantel pamoate at the first treatment and then mebendazole for subsequent bi-monthly treatments (Northrop- Clewes et al., 2001). The situation is further complicated if anthelmintic drugs to treat other types of worms are given, such as praziquantel or metrifonate to treat infections with Schistosoma species. This is a genus of blood flukes found in Africa, the Middle East, Asia and South America, often concurrently with infections with intestinal nematode worms. Insert Table 7 here Schistosoma haematobium causes bleeding into the urinary bladder as a result of the passage of the sharp-spined eggs through tissues, while the passage of the eggs of Schistosoma mansoni and Schistosoma japonicum through the gut wall also causes internal bleeding. Studies of the impact of treatment in areas in which schistosomes occur tend to give a drug to treat these infections, as well as a drug to treat intestinal nematode worms (Beasley et al., 1999, Friis et al., 2003). As well as treating schistosomes, the drug metrifonate is also an 34 effective treatment for hookworm (Kurz et al., 1986), which complicates studies and measuring outcomes due to treatment. Finally, if any study is to assess the impact of treating intestinal worms alone, then additional treatments cannot be given unless a factorial design is used and an untreated control group is provided. One study of the effect of treating worms with mebendazole also gave all subjects metronidazole as well, a broad-spectrum anti-bacterial drug that also treats infections with intestinal protozoa (Marinho et al., 1991). This meant that the effect of mebendazole could not be separated from the effect of metronidazole. In other studies supplements of micronutrients or food were also provided which also may have had independent effects on nutritional status and growth so making it impossible to estimate the impact of deworming alone (Latham et al., 1990a, Lind et al., 2004, Majumdar et al., 2003). 3.3 Intervals between treatments In an infected human population the death rate of worms tends to a reach a balance with the rate at worms are acquired, so an equilibrium develops. The effect of mass treatment is to cure a proportion of people, which reduces the prevalence of infection, and treatment expels a proportion of the total worm population, which reduces the mean worm burden. But, as there is no fully protective immunity to worms, reinfection can occur immediately, especially as infective eggs can survive in the environment for months in suitable conditions. The rate of reinfection is strongly related to the prior prevalence and mean worm burden, as well as to local sanitation, which influences the number of infective stages in the environment. Insert Table 7 here Studies of reinfection after treatment and mathematical models that simulate treatment and reinfection have shown that the prevalence of infection can rebound within a few months after treatment, but that the worm burden takes considerably longer to reach the pre-existing number (Hall et al., 1992). This means that the prevalence may show only a small difference between rounds of treatment, but the mean worm burden may decline. Figure 10 shows data on the prevalence of infection with A.lumbricoides among 445 school-age children in Bangladesh at the first treatment and after two more rounds of treatment, each six months apart. 35 The dotted line shows the trend which, when extrapolated, suggests only a small decline over two future rounds of treatment. Figure 10 also shows the mean worm burden at each round of treatment, which declines by almost 60% between the first and third treatment. If the linear decline was extrapolated it would lead to a substantial reduction in mean worm burden after five rounds of treatment. The relatively slow decline in worm burden achieved in the study in Bangladesh suggests that treatment would actually be best given every 3 – 4 months or 3 – 4 times a year rather than twice a year. Insert Figure 10 here Figure 10 makes the point that the interval between treatments will influence the average intensity of reinfection that occurs, assuming a high efficacy, especially in the absence of any measures to prevent exposure to infectious stages in the environment or if only a proportion of the population is treated. Ideally the intervals between treatment should prevent the prevalence rising above 50%, the threshold at which the probability of moderate to heavy infections begins to increase exponentially (see Figure 3). The conclusion is that the frequency of treatment is therefore likely to affect the impact of treatment, and the aim should be to prevent moderate to large worm burdens from being reacquired. The consequence for this review is that it is difficult to compare studies of different numbers of treatments given over different periods. Nevertheless it can be useful to standardise gains in weight or any other parameter per unit of time, such as per year (Alderman et al., 2006). But, if reinfection is rapid, then two or even three treatments a year may have a greater impact than one, assuming that worms are the only constraint on growth. Insert Figure 11 here The duration of follow up is important because, as Section 2.4 has shown, a relatively small proportion of infected subjects will benefit from treatment so it is likely to take some time before the effects of repeated treatment are detectable in comparison with an untreated infected group. Some studies have attempted to measure a difference between groups in weight gain or growth over a very short period, such as 3 or 7 weeks after treatment (Hadju et al., 1996). Even though there was a statistically significant difference between study groups in such studies, it is too short a period to warrant inclusion in the review. Another study attempted to measure a change in haemoglobin and iron status over a period of 10 days after treatment (Karyadi et al., 1996), which is also a very short time in which to expect an improvement. 3.5 Outcomes measured and the need for controls The primary outcomes usually measured during studies of the effect of anthelmintic treatment are changes in body weight, height, mid upper arm circumference and skinfold thickness. A change in haemoglobin concentration may also be expected if hookworm or T.trichiura occur. 3.5 Outcomes measured and the need for controls The primary outcomes usually measured during studies of the effect of anthelmintic treatment are changes in body weight, height, mid upper arm circumference and skinfold thickness. A change in haemoglobin concentration may also be expected if hookworm or T.trichiura occur. Measuring vitamin A status is hard to do as the concentration of retinol in serum is usually sustained from liver stores, so some sort of dose response test is usually used. Secondary outcome measures are anthropometric indices such as height-for-age and weight-for-age, which both require age to be known, ideally to within a month, and weight-for-height or body mass index. Indices of weight-for-height can only be calculated for girls younger than 10 years and boys younger than 11.5 years if the National Centre for Health Statistics reference values are used (WHO, 1983) which may limit their application in studies of school-age children. Z-scores of anthropometric Measuring vitamin A status is hard to do as the concentration of retinol in serum is usually sustained from liver stores, so some sort of dose response test is usually used. 3.4 Duration of follow up It follows from the discussion in the previous section that, as well as the number of treatments given, the duration of follow up after treatment will affect the magnitude of any difference between a treated and an untreated control group, and therefore its statistical significance. This is illustrated in Figure 11 which shows hypothetical data on the weight of two cohorts of undernourished children, 300 treated and 300 untreated, over a period of three years during which the treated group were kept almost free of worms. The children were 36 five years old at the start of the study and were underweight, with an average body weight that was 2 standard deviations below the NCHS median. If the periodic treatment of worms led to an extra gain in weight of 0.5 kg a year, or 3.5% of the initial mean weight, then it can be seen from the confidence intervals around the means in Figure 11 that it is not until the third year that a statistically significant difference between groups is achieved Insert Figure 11 here Insert Figure 11 here Secondary outcome measures are anthropometric indices such as height-for-age and weight-for-age, which both require age to be known, ideally to within a month, and weight-for-height or body mass index. Indices of weight-for-height can only be calculated for girls younger than 10 years and boys younger than 11.5 years if the National Centre for Health Statistics reference values are used (WHO, 1983) which may limit their application in studies of school-age children. Z-scores of anthropometric 37 indices are useful when controlling for the initial nutritional status of subjects based on the assumption that, when there is a large initial deficit, the impact of treatment is likely to be greater than if there is a small initial growth deficit. A change in z-score can also help to give a perspective to the magnitude and nutritional significance of an improvement. It cannot be assumed that a gain in body weight is due totally to an increase in lean body mass, because some increase could result from the deposition of fat in adipose tissue. Although there are now available weighing scales that can estimate the percentage body fat by applying standard equations to measurements of electrical impedance, they cannot usually be applied to young children, and may not apply to undernourished children in a developing country either. Measurements of mid-upper arm circumference and triceps skinfold thickness tend to be under-used in studies of child growth after deworming. Their value lies in the ability to estimate the surface area of sub-cutaneous fat and muscle in the arm, assuming a standard area of bone (Gibson, 1990) which could indicate both growth in lean tissue and the deposition of fat reserves. A statistically significant weight gain can be achieved over a relatively short period, especially if food is available ad libitum, while a gain in height can take relatively longer. A period of about 12 weeks seems to be about the minimum period of follow up. A study of vitamin A and iron given to Tanzanian children after treatment with albendazole and praziquantel showed statistically significant extra gains in weight and height after 3 months of supplementation (Mwanri et al., 2000). Studies of the effects of treating worms that cause blood loss require controls because the haemoglobin concentration can fluctuate naturally. Insert Figure 11 here Studies of giving iron supplements to school-age children after deworming have shown statistical significance only because the mean value of the control group may go down in comparison with the treated children whose haemoglobin was sustained by the iron (Hall et al., 2002, Roschnik et al., 2004, Aguayo, 2000). Natural fluctuations in haemoglobin may occur as a result of changes in the diet and diseases such as malaria, both or which may be seasonal. 38 Initial nutritional status 3.6 The impact of treatment on any indicator of nutritional status is likely to be related to the prior degree of undernutrition or deficiency, thus the margin for potential improvement. This was not the case in a study in South Africa of the effect of anthelmintic treatment and micronutrient fortified biscuits (Jinabhai et al., 2001b). One of the outcome measures was anthropometric status, but at the start of the study only 7.3% of children were stunted and 0.8% were underweight (Jinabhai et al., 2001b), not a large margin for improvement. It may seem obvious, but it is also important that treatment and control groups should be similar before any treatment is given. A study of the impact of treatment with mebendazole given to young children as a part of an evaluation of the Integrated Management of Childhood Illness in western Kenya reported statistically significant differences in gains in weight, height and weight-for-age when infected and treated children were compared with infected and untreated controls (Garg et al., 2002). But in this study the treated children were significantly more undernourished than the control group: the z-score of weight-for-height of the group given the placebo was much greater than the treated children (Treatment -0.89 vs Control -0.19, P < 0.001) and there was a statistically significant difference in their initial weight- for-age as well (-1.43 vs -1.01, P < 0.001) (Garg et al., 2002). If the infected and treated children gained more weight it could easily have been because they started from a lower initial nutritional status and had a greater potential to achieve more catch-up growth. Both the treatment and control groups need to be similar if the impact of treatment on growth or most nutritional variables is to be compared reliably. 3.7 Age of subjects The age of subjects will influence the measurement of the impact of anthelmintic treatment. This is because growth is not linear during childhood and shows two spurts, the first during the first two years of life, and the second during adolescence. Although the prevalence and intensity of worm infections tends not to be as high during the first two years of life as during the school-age years, the potential impact of worms early in life is greater, mainly because of the relative size of worms to their hosts, and the relative 39 magnitude of any extra weight gain achieved after treatment. An extra weight gain of 500 g over a year by a 5 year old girl whose weight is 2 S.D. below the mean is 3.6% of initial weight; for a 10 year old it is 2.3% and for a 15 year old it is 1.3%. Such an extra weight gain is of decreasing biological significance, unless it can be sustained each year, and will require a larger sample size to detect in the older age groups. 3.8 Remedial therapy after treatment An implicit assumption of many randomised trials of anthelmintic treatment seems to be that removing worms will automatically lead to improvements in growth and nutritional status. This may happen, but only if the diet is adequate (Hall, 2007). If worms have impaired growth, the haemoglobin concentration or micronutrient status, their effects are most likely to have been due to the mechanisms outlined in Section 1.3 which are worth repeating here: by feeding on the host’s food, secretions, tissues and blood; by causing maldigestion or malabsorption; by effects on appetite and food intake; and by causing responses to infection that consume or divert resources unnecessarily. The losses or deficits caused by worms cannot be rectified or remedied simply by removing the worms alone, although halting any pathological processes is an important first step. After worms have been killed the need is for remedial treatment of the underlying nutritional deficits by providing energy, protein and micronutrients, so that catch-up growth can be achieved. This is illustrated in Figure 12 and discussed in some detail in Section 6.2. Ideally, children should be protected from moderate to heavy infections throughout their childhood by repeated treatment to keep worm burdens low, by primary measures such as sanitation to prevent faeces containing worm eggs from getting into the environment, and by secondary measures to prevent exposure to worm eggs, such personal hygiene. 40 3.9 Summary Summary There are a number of factors related to anthelmintic drugs and study design that need to be considered when either designing studies or evaluating papers that report studies. There are a number of factors related to anthelmintic drugs and study design that need to be considered when either designing studies or evaluating papers that report studies. There are a number of factors related to anthelmintic drugs and study design that need to be considered when either designing studies or evaluating papers that report studies. • Different drugs give a different cure rate and egg reduction rate for each species of intestinal nematode; • All anthelmintics are highly effective against A.lumbricoides; • Albendazole 400 mg and mebendazole 500 mg are the best treatments available for hookworms and T.trichiura; • At these dosages albendazole is more effective than mebendazole against hookworm infections, but both achieve egg reduction rates of 55 – 95%; • Neither albendazole nor mebendazole given as a single dose cures many infections with T.trichiura, but such treatment can reduce egg counts by 50% • Albendazole and mebendazole may be a less effective treatment for T.trichiura in Asia than in Africa • Three daily doses of albendazole or mebendazole may be needed to achieve high cure rates for T.trichiura. • Reinfection with worms can occur immediately after treatment, so treatment needs to be given periodically. • The interval between treatments may influence the impact on growth and nutritional status. • The subjects for study should be malnourished as well as infected. • The subjects for study should be malnourished as well as infected. • As a minority of children (ranging from 10 – 40%) have moderate to heavy infections, not all will benefit to the same degree from treatment, and the impact on the average will of any outcome measure may be diluted. • These factors should be taken into account so that the period of follow up after treatment should be sufficient to be able to detect a statistically significant effect. 41 41 • It cannot be expected that nutritional status will improve and show catch-up if the diet of subjects is inadequate to meet the extra requirements. • It cannot be expected that nutritional status will improve and show catch-up if the diet of subjects is inadequate to meet the extra requirements. 4. Aims and methods of the meta-analysis The aim of the review was to identify studies that had measured the effect of anthelmintic treatment given for infections with A.lumbricoides, T.trichiura and both species of hookworm on children’s growth and weight gain, or on haemoglobin concentration. 4.1 Search terms Table 9 shows the terms used in combination to search Medline. The search was limited to papers on humans in the following groups: infant, preschool, child and adolescent. Only papers in English were located. In addition, the reference list of each paper identified was scanned for other papers, so too was the Cochrane Review of 2000 (Dickson R, 2005, Dickson et al., 2000a, Dickson et al., 2005) and the book by Stephenson (1987) Impact of helminth infections on human nutrition (Stephenson, 1987). One unpublished paper known to the principal author, who was a co-investigator, was included (Partnership for Child Development, 2001). (Partnership for Child Development, 2001). All papers identified were summarised under the following headings: • Design. A summary of the study design, including whether placebo controlled and blinded or not. • Follow-up. The period children were studied after treatment, or the difference in time between the initial, interim and final measurements. • Location. The region and country where the study was done. • Infection prevalence at baseline. The prevalence of each species of worm recorded. • Treatments. The drugs used and the doses given. • Sample size. The number of subjects studied in each group. • Outcomes. The principal outcome measures and any derived indices. 42 • Findings. A summary of the major findings with standard deviations and the statistical significance of any differences between groups. • Notes. A summary of the prevalence of each intestinal nematode infection; a summary of the effect of treatment of worms; a summary of the degree of undernutrition estimated as z-scores, the mean haemoglobin concentration or the prevalence of anaemia; and short notes on other major or different features of the study. • In Cochrane review of 2000 (recorded as Yes or No): Dickson R, Awasthi S, Demellweek C, Williamson P. Anthelmintic drugs for treating worms in children: effects on growth and cognitive performance (Review). Cochrane Database of Systematic Reviews, 2000, Issue 2. DOI: 10.1002/14651858.CD000371. • Included in meta-analysis. Either Yes, or the reason the study was excluded is given. 4.2 Inclusion criteria 4.2 Inclusion criteria Papers were included in the analysis if the following criteria were met: • Studies of treating children and adolescents (aged 1 – 19 years) for intestinal nematode worm infections with albendazole, levamisole, mebendazole, piperazine or pyrantel (pamoate or embonate). • An experimental group given the anthelmintic alone. • An untreated control group. • Random allocation to treatment and control groups. • An initial prevalence of infection in children in the study locality with any species of intestinal nematode worm of ≥ 50%, the threshold used by the WHO to classify a “high risk” community (WHO, 2006). • A duration of follow-up of at least 12 weeks for all outcomes except the modified relative dose response test to an oral dose of retinol (Tanumihardjo et al., 1996a). • Outcomes measured included at least one primary outcome variable among weight, height, mid upper arm circumference, skinfold thickness, haemoglobin concentration, and a measure of serum retinol; 43 or secondary outcomes based on weight-for-age, height-for-age or weight-for-height such as z-scores or percentages of the median. or secondary outcomes based on weight-for-age, height-for-age or weight-for-height such as z-scores or percentages of the median. • Data given as a mean with standard deviation and sample size, or enough data to calculate the standard deviation. Data from the study of (Koroma et al., 1996) were entered separately for urban and rural children. Data from the study of (Koroma et al., 1996) were entered separately for urban and rural children. b Downloaded from http://www.cc-ims.net/RevMan 4.3 Exclusion criteria Papers were excluded from the meta-analysis for one or more of the following reasons (the references cited are examples, and is not a comprehensive list): • Data on an outcome measurement not presented in a form that could be used e.g. change in percentage weight for height (Hadidjaja et al., 1998), difference in percentage improved (Kloetzel et al., 1982), change in percentage with anthropometric indices less than -2 z-scores (Lai et al., 1995), weight gain presented as a graph (Sur et al., 2005), no data given for stated outcome measures for treated children (Olds et al., 1999); or data are unique so could not be combined with other studies (Tanumihardjo et al., 1996b). • No standard deviations of outcome measures given (Beach et al., 1999a, Fox et al., 2005, Greenberg et al., 1981, Michaelsen, 1985, Rousham and Mascie-Taylor, 1994, Willett et al., 1979) so not amenable to meta-analysis using Review Manager 4.2 software. • The initial prevalence of worms was less than the “high risk” threshold of 50% (Garg et al., 2002, Awasthi et al., 2000, Awasthi and Pande, 2001, Donnen et al., 1998, Freij et al., 1979); • Inadequate control group or method of allocation e.g. infected subjects treated and compared with subjects who were lightly infected or uninfected (Mahendra Raj and Naing, 1998, Mahendra Raj, 1998, Bhargava et al., 2003, Callender et al., 1994); a sample size of two, one village where children were treated and another control village where they were not (Fernando et al., 1983); or no untreated controls (Forrester et al., 1998). 44 • Treatment given for other conditions in addition to treatment for intestinal nematode worms e.g. metrifonate, which treats hookworm as well as Schistosoma spp (Stephenson et al., 1989a, Stephenson et al., 1989b, Stephenson et al., 1985); praziquantel to treat Schistosoma spp combined with a drug to treat intestinal nematode worms (Beasley et al., 1999, Kruger et al., 1996, Jalal et al., 1998, Jinabhai et al., 2001a); or food or micronutrient supplements given in addition to anthelmintic treatment (Cerf et al., 1981, Marinho et al., 1991, Jinabhai et al., 2001b, Mwaniki et al., 2002, Kruger et al., 1996, Palupi et al., 1997) • Short period of follow up e.g. 3 and 7 weeks (Hadju et al., 1996), 10 days (Karyadi et al., 1996). • Treatment given for other conditions in addition to treatment for intestinal nematode worms e.g. 4.3 Exclusion criteria metrifonate, which treats hookworm as well as Schistosoma spp (Stephenson et al., 1989a, Stephenson et al., 1989b, Stephenson et al., 1985); praziquantel to treat Schistosoma spp combined with a drug to treat intestinal nematode worms (Beasley et al., 1999, Kruger et al., 1996, Jalal et al., 1998, Jinabhai et al., 2001a); or food or micronutrient supplements given in addition to anthelmintic treatment (Cerf et al., 1981, Marinho et al., 1991, Jinabhai et al., 2001b, Mwaniki et al., 2002, Kruger et al., 1996, Palupi et al., 1997) • Short period of follow up e.g. 3 and 7 weeks (Hadju et al., 1996), 10 days (Karyadi et al., 1996). 5. Results of the meta-analysis A total of 58 published papers were found, summaries of which can be found in the Appendix. They were not all unique studies as some data were reported in more than one paper. A total of 40 papers were not included in the analysis either because they met exclusion criteria or did not meet inclusion criteria; this left 18 studies for analysis. One additional unpublished study, a randomised cluster trial of albendazole in Vietnamese schoolchildren, was included, (Partnership for Child Development, 2001) increasing the sample to 19 papers, as follows: A total of 58 published papers were found, summaries of which can be found in the Appendix. They were not all unique studies as some data were reported in more than one paper. A total of 40 papers were not included in the analysis either because they met exclusion criteria or did not meet inclusion criteria; this left 18 studies for analysis. One additional unpublished study, a randomised cluster trial of albendazole in Vietnamese schoolchildren, was included, (Partnership for Child Development, 2001) increasing the sample to 19 papers, as follows: Adams et al. (1994). Physical activity and growth of Kenyan school children with hookworm, Trichuris trichiura and Ascaris lumbricoides infections are improved after treatment with albendazole. Journal of Nutrition 124: 1199 – 1206. (Adams et al., 1994) Alderman et al. (2006) Effect on weight gain of routinely giving albendazole to preschool children during child health days in Uganda: cluster randomized controlled trial. British Medical Journal 333: 122 -24. (Alderman et al., 2006) Dossa et al. (2001). Impact of iron supplementation and deworming on growth performance in preschool Beninese children. European Journal of Clinical Nutrition 55: 223 – 228. (Dossa et al., 2001) Dossa et al. (2001). Impact of iron supplementation and deworming on growth performance in preschool Beninese children. European Journal of Clinical Nutrition 55: 223 – 228. (Dossa et al., 2001) Gupta et al. (1982). Effect of periodic antiAscaris and antiGiardia treatment on nutritional status of preschool children. American Journal of Clinical Nutrition 36: 79 – 86. (Gupta and Urrutia, 1982) Gupta et al. (1982). Effect of periodic antiAscaris and antiGiardia treatment on nutritional status of preschool children. American Journal of Clinical Nutrition 36: 79 – 86. (Gupta and Urrutia, 1982) Koroma et al. (1996). Effects of albendazole on growth of primary school children and the prevalence and intensity of soil-transmitted helminths in Sierra Leone. 4.4 Meta-analysis 4.4 Meta-analysis Data from suitable papers were extracted and entered manually into RevMan version 4.2.8 for Windows.b y Data from suitable papers were extracted and entered manually into RevMan version 4.2.8 for Windows.b Statistical comparisons were made between subjects treated with an anthelmintic drug and untreated controls for the outcomes listed in Table 13. For some studies the standard deviations had to be calculated from 95% confidence intervals and the sample size. For studies in which the mean and standard deviations of values before and after were given, the difference between mean values was used in the analysis with a pooled estimate of the standard deviation. The weighted mean difference between treatment and control groups was calculated with 95% confidence intervals in RevMan 4.2 software (RevMan, 2003) for each variable using a fixed effects model. Studies were disaggregated into sub-categories by treatment except that studies in which mebendazole and albendazole were given were combined, as the two drugs are chemically similar and have the same mode of action. 45 5. Results of the meta-analysis Journal of Topical Pediatrics 42: 371 – 372.(Koroma et al., 1996) Northrop-Clewes et al. (2001). Anthelmintic treatment of rural Bangladeshi children: effect on host physiology, growth, and biochemical status. American Journal of Clinical Nutrition 73: 53–60.(Northrop-Clewes et al., 2001) Partnership for Child Development (2002, unpublished). A randomised cluster trial of six-monthly deworming and its effects on the growth and 46 educational achievements of Vietnamese school children. (Partnership for Child Development, 2001) Sarkar et al. (2002). Effect of deworming on nutritional status of Ascaris infested slum children of Dhaka, Bangladesh. Indian Pediatrics 39: 1021 – 1026. (Sarker et al., 2002) Sarkar et al. (2002). Effect of deworming on nutritional status of Ascaris infested slum children of Dhaka, Bangladesh. Indian Pediatrics 39: 1021 – 1026. (Sarker et al., 2002) Simeon et al. (1995). Treatment of Trichuris trichiura infections improves growth, spelling scores and school attendance in some children. Journal of Nutrition 125: 1875 – 1883, (Simeon et al., 1995) Simeon et al. (1995). Treatment of Trichuris trichiura infections improves growth, spelling scores and school attendance in some children. Journal of Nutrition 125: 1875 – 1883, (Simeon et al., 1995) Stephenson et al. (1989c). Treatment with a single dose of albendazole improves growth of Kenyan schoolchildren with hookworm, Trichuris trichiura and Ascaris lumbricoides infections. American Journal of Tropical Medicine and Hygiene 41: 78 – 87.(Stephenson et al., 1989c) Stephenson et al. (1993a). Weight gain of Kenyan school children infected with hookworm, Trichuris trichiura and Ascaris lumbricoides is improved following once- or twice-yearly treatment with albendazole. Journal of Nutrition 123: 656 – 665. (Stephenson et al., 1993b) Stephenson et al. (1993b). Physical fitness, growth and appetite of Kenyan school boys with hookworm, Trichuris trichiura and Ascaris lumbricoides infections are improved four months after a single dose of albendazole. Journal of Nutrition 123: 1036 – 1046. (Stephenson et al., 1993a) Stoltzfus et al. (1997). School-based deworming program yields small improvement in growth of Zanzibari school children after one year. Journal of Nutrition 127: 2187 – 2193. (Stoltzfus et al., 1997) Stoltzfus et al. (1998). Effects of the Zanzibar school-based deworming program on iron status of children. American Journal of Clinical Nutrition 127: 179 -186. (Stoltzfus et al., 1998) Stoltzfus et al. (2004). Low dose daily iron supplementation improves iron status and appetite but not anemia, whereas quarterly anthelmintic treatment improves growth, appetite and anemia in Zanzibari preschool children. Journal of Nutrition 134: 348 – 356. 5. Results of the meta-analysis (Stoltzfus et al., 2004) Tanumihardjo et al. (1996) Vitamin A status of Indonesian children infected with Ascaris lumbricoides after dosing with vitamin A supplements and 47 albendazole. Journal of Nutrition 126: 451 – 457.(Tanumihardjo et al., 1996b) Tanumihardjo et al. (2004). Vitamin A status and hemoglobin concentrations are improved in Indonesian children with vitamin A and deworming treatments. European Journal of Clinical Nutrition 58: 1223 – 1230. (Tanumihardjo and Permaesih, 2004) Thein-Hlaing et al. (1991). A controlled chemotherapeutic intervention trial on the relationship between Ascaris lumbricoides infection and malnutrition in children. Transactions of the Royal Society of Tropical Medicine and Hygiene 85: 523 – 528. (Thein-Hlaing et al., 1991) Thein-Hlaing et al. (1991). A controlled chemotherapeutic intervention trial on the relationship between Ascaris lumbricoides infection and malnutrition in children. Transactions of the Royal Society of Tropical Medicine and Hygiene 85: 523 – 528. (Thein-Hlaing et al., 1991) Watkins et al. (1996). Effect of removing Ascaris on the growth of Guatemalan schoolchildren. Pediatrics 97: 871 -876.(Watkins and Pollitt, 1996) Table 10 shows a summary of the key features of the 19 studies. The studies could not be disaggregated by age into pre-school (1 – 5 y) and school age (6 – 15 y) because several had overlapping age ranges, particularly the largest study, undertaken by Alderman et al. (Alderman et al., 2006) in Uganda. 5.1 Geographic origin of studies Table 11 shows the countries represented in the papers found and used in this review. Of all 58 papers found, nearly 50% reported data from Africa. Of the 19 papers used in the analysis, a little more than 50% reported data from Africa. 5.2 Estimates of effects Table 12 presents summary data on the impact of treatment on 12 variables for all studies in which intestinal worms were treated with any effective anthelmintic. The drugs used were albendazole and mebendazole, levamisole, piperazine or pyrantel pamoate. There were statistically significant differences for most variables related to growth; neither of the outcomes for micronutrients were statistically significant. 48 5.3 The figures and how to interpret them 5.3 The figures and how to interpret them Figures 13 to 24 show the mean differences between treated and control groups for each study grouped vertically by the drugs used. The mean is shown as a box around the vertical line of zero difference, with 95% confidence intervals (95% CI) shown as bars. The diamonds represent the weighted mean difference (WMD) between the treated and control groups for each treatment group and in total. The percentage weight given to each study is shown and the numerical value of the estimated difference with 95% CI. It should be noted that the scale in all but figures except for Fig 22, 25 and 25 is from –4 (favours control) to 4 (favours treatment). Insert Table 12 here Table 13 presents summary data on the impact of treatment on 12 variables for all studies in which intestinal worms were treated with albendazole or mebendazole only, as these drugs are now the most widely used and most broadly effective anthelmintics. 5.4 Sources of error or bias The decision to exclude studies in which the initial prevalence of infection is < 50% is somewhat arbitrary, but is based on the WHO threshold which classifies them as “low risk” communities (WHO, 2006). Ideally all children taking part in studies of anthelmintic treatment would be infected with at least one species of worm, just as any drug trial would be conducted only on infected individuals. But most trials of the impact of anthelmintics on child growth tend to study groups of children rather than infected subjects only, so tend to be trials of effectiveness rather than efficacy. This type of study is driven also because of ethical problems with having infected but untreated children as controls, so studies tend to be done in communities in which the prevalence is known, and whether individual subjects are infected or usually unknown. Applying a threshold prevalence of 50% or more means that a half of all subjects or fewer will not benefit from anthelmintic treatment, and their inclusion in the study sample will to some degree dilute the impact of treatment on the infected majority. The threshold prevalence of 50% does not 49 serve to maximise the impact of treatment, a prevalence of 100% is needed to do that, but a prevalence higher than 50% serves to reduce the effect of uninfected subjects. This means that the conclusions of the meta-analysis can only be applied to circumstances in which the prevalence is more 50% and excludes what the WHO calls “low risk” communities, in which the prevalence of infection with any species of worm lies between 20 – 49% (WHO, 2006). About a half of all papers found and used in the analysis reported data from Africa. All of the papers reporting African data were from sub-Saharan Africa, reflecting concern for the effects of worms in African children. In many parts of sub-Saharan Africa children are concurrently infected with species of Schistosoma, which may cause loss of appetite, internal bleeding, and tissue damage and inflammation due to reactions to the eggs of worms when they become lodged in tissues. Studies were excluded from this analysis in which subjects were also treated with praziquantel or metrifonate, the drugs most commonly used to treat Schistosoma spp, as this would over- estimate the effect of treating intestinal nematodes. This could, however, have led to a bias against studies from sub-Saharan Africa. Insert Figures 13 to 24 here 6. Discussion This meta-analysis indicates that if the prevalence of intestinal nematodes is 50% or more then giving anthelmintic drugs leads to significant extra gains in weight, height, mid upper arm circumference and skinfold thickness in comparison with untreated controls. Table 12 shows the weighted average changes recorded in the studies found. The gains in weight and height also led to improvements in indices of anthropometric status in studies in which only these outcomes were reported. There was no evidence of a significant effect of treating intestinal nematode infections on haemoglobin concentration. Only two studies, both by the same author in Indonesia, were reported of the dehydroretinol to retinol ratio, and found no difference between treated and control groups (Tanumihardjo and Permaesih, 2004, Tanumihardjo et al., 1996b). 5.4 Sources of error or bias But this did not seem to be the case, as a larger proportion of all papers from Africa were included in the final analysis than in Africa, Asia or the Indian sub-continent. Of the papers found and the papers used, the proportion from Africa was similar in both categories (28/59 or 47% and 11/24 or 46%). Insert Figures 13 to 24 here Insert Figures 13 to 24 here 50 50 6.1 Magnitude of effects It is not possible to say anything conclusive about the absolute magnitude of any effects of giving treatment, for a number of reasons. The studies summarised were different in terms of: • the species and mixture of species of worms; • the distribution of worms between hosts; • the drug and dose used, leading to variability in the efficacy of each treatment; • the number of treatments given and intervals between them; • the initial degree of undernutrition, the age and current health of subjects; • and the period of follow-up after treatment, or the gap between the initial and final measurements. The magnitude of the extra increases shown in Figures 14 – 25 are not standardised as gains per unit time, which would have enabled them to be better compared, and would have put any improvement into some sort of perspective. A extra gain in weight of 0.2 kg in four months is much bigger and more significant in terms of growth than a extra gain of 0.2 kg over a year. Any gain per unit time will also be influenced by the frequency of treatment, 51 thereby keeping worm burdens down, perhaps. This was indicated in the study in Uganda: children who were dewormed at least every six months gained an extra 10% in weight compared with the 5% extra by children who were treated only annually (Alderman et al., 2006). However this could have been confounded by the fact that other health services were delivered at the Parish Health Fairs to children who came more often, or perhaps the regular attendance of children at Health Fairs also reflected better care at home. If the magnitude of any gain is related to the initial degree of undernutrition, then it might be better to standardise the gain per unit time as a proportion of the initial value, though this may be statistically difficult to do. This could be important for the haemoglobin concentration. For example, an increase of 10 g/L when the initial average was 90g/L (11%) is more significant than an increase of 10 g/L when the initial average was 110 g/L (9%). Nothing can be said about whether any particular drug is more effective than another at bringing about an improvement in a nutritional outcome, although egg reduction rate is likely to be a good indicator of this. 6.1 Magnitude of effects The benzimidazole drugs are currently the most efficacious treatments available for treating intestinal nematode worms. Albendazole was the most commonly used drug (Table 10), and is effective to differing degrees against the three main species of worms, as Table 7 shows. Mebendazole has a similar efficacy but was given less often, probably because it used to be given as a dose of 100 mg a day for 3 days rather than as a single dose of 500 mg, as it is now used. A single dose of an effective drug is a crucially important attribute for a helminth disease control programme. However neither albendazole nor mebendazole are highly effective against T.trichiura (Bennett, 2000), and a failure to achieve a good egg reduction rate could contribute to a smaller impact of treatment than could be achieved by giving multiple doses. 6.2 Treatment alone is not enough Whatever extra gains in nutritional or anthropometric outcomes are achieved after treatment with an anthelmintic drug, they do not occur solely as a result of that treatment. Catch-up growth, for example, requires extra nutrients and energy, and if they are not available after treatment then growth rates are 52 likely to remain unchanged. Alternatively a statistically significant difference between a treatment and control group could occur during a study because an untreated control group remain persistently diseased as a result of their infections, and thus gain less weight than the treated group. The lack of extra food to meet the needs for growth could explain the lack of impact of deworming in many studies, such as the large cluster trial in Viet Nam (Partnership for Child Development, 2001). The expulsion of worms from the gut may remove a constraint that acts through effects on appetite and food intake, digestion and absorption, or because of the diversion of nutrients in response to infection. But to achieve the maximum growth rate after treatment, energy, protein and micronutrients need to be provided to children, preferably ad libitum . Most studies did not give food supplements after treatment, but one that did showed an improved appetite measured as food intake as well as improved growth (Stephenson et al., 1993a). Catch-up growth is possible (Golden, 1994) especially if the deficits occur early in childhood and are treated adequately. Studies of children treated for Trichuris dysentery syndrome, admittedly an acute disease, have shown an average gain of nearly 11 cm in height and 4 kg in weight a year, which was more than 2 S.D. above the expected gain in height and weight of British children of the same age (Cooper et al., 1995). Some of the 5-year-old children studied showed an annual growth velocity of nearly 20 cm a year and a weight gain of 10 kg. To achieve such catch-up growth children need to be fed ad libitum, not just with energy and protein, but also with micronutrients, so that no nutrient is limiting and the maximum possible growth is achieved (Hall, 2007). If studies of deworming have failed to show an impact on growth or weight gain it could be because children were not getting enough of the food and micronutrients they needed to achieve catch-up growth. 6.2 Treatment alone is not enough And the studies that have shown a statistically significant impact of treatment may have underestimated the potential effect if children did not have an ideal and balanced diet given ad libitum. No published studies have been found of randomised controlled trials of deworming with or without any form of remedial feeding given ad libitum; most studies have only given supplementary food or supplementary micronutrients. 53 A problem with giving food supplements is that there can be substitution, in which people eat less food at home after being fed at school or work (Wolgemuth et al., 1982). Or supplementary food may benefit the better off most: a study of a school nutrition programme in Viet Nam found that better nourished children gained more weight than undernourished children (Hall et al., 2007), which is not what is hoped for in school feeding programmes. The need for adequate remedial treatment is important when the outcome being measured is the haemoglobin concentration. A study in Tanzania of giving iron supplements to anaemic children after treating their infections with hookworm and Schistosoma haematobium showed no improvement in the mean haemoglobin concentration, but the serum ferritin concentration increased, an indicator of improved iron status (Beasley et al., 2000). This suggests that iron was not the micronutrient limiting the haemoglobin concentration. This does not mean that improvements in growth and nutritional status cannot be achieved by deworming alone, especially if there are improvements in appetite and food is available to children. But to have the maximum effect on growth and nutritional status, therapeutic nutrients are needed as well. A failure to have an impact could be because of this lack, or because the worms were not an important cause of any nutritional deficit in the first place. But the costs of only giving supplementary food can be quite high: the World Food Programme estimate that it costs $34 a year to feed a child in school (World_Food_Programme, 2006). An alternative is to provide micronutrient supplements, a deficiency of which may also act to impair growth, and which have been shown to reduce the intensity of reinfection with schistosomes (Olsen et al., 2003). A multiple micronutrient supplement is a much less expensive intervention than food, and may cost only around one US cent/child/day if purchased in large quantities. 6.2 Treatment alone is not enough But the key point is that if any nutrient is not provided in sufficient amounts to meet demands, and becomes rate limiting, then growth after deworming may be constrained. The weighted average gains in parameters of growth shown in Table 12 therefore do not indicate what extra growth might be achieved after treatment or indicate the potential for growth, they only estimate what can be 54 achieved after giving treatment. If a study included in the analysis showed no statistically significant difference between the treatment and control groups, the question is: did this happen because there was no effect, or because treated children were lacking sufficient energy or nutrients to show extra growth and weight gain? A lack of effect in such studies will serve to reduce the weighted average, and so underestimate the potential impact of treatment. It is also entirely possible that anthelmintic treatment may have had no effect even if children had been adequately nourished. But, as there are plausible biological mechanisms by which moderate to heavy burdens of worms can affect nutritional status, a beneficial impact on some (but not all) children seems likely. This touches upon the aggregated distribution of worms and the uneven distribution of the impact of treatment. If disease and malnutrition are a consequence of moderate to heavy infections, then in any given sample of children a minority will benefit most from treatment, so the effect of treatment will not be evenly distributed between children. For example if 25 - 49 A.lumbricoides is arbitrarily considered to be a moderate infection in children and 50 or more worms is a heavy infection, then of the 1,069 children aged 1 – 14 years studied in an urban slum in Bangladesh, 18% were moderately heavily infected and 7% heavily infected (data from (Hall et al., 1999). If only these children benefited from treatment then the impact on this 25% would in effect be diluted in the lack of change in the remaining 75%. The average treatment effect therefore includes a large proportion of children who would not be expected to benefit, and would under-estimate the potential impact on moderately to heavily infected children. The proportion of lightly infected children will be very large when the prevalence is low, and when it is less than 50% it might be expected that very few children would benefit substantially from treatment. 6.2 Treatment alone is not enough The general consequence is that the study average will not capture the larger benefit experienced by any moderately to heavily infected children. One way to control for this would be to analyse the relationship between any outcome and an estimate of the intensity of infection, such as the concentration of eggs in faeces. This method was used by (Stephenson et al., 1993a) in their studies of the effect of anthelmintic treatment on 55 appetite. This can only be done if a prior individual diagnosis is made and, if it is, it could be considered to be unethical to leave children untreated whose infections have been diagnosed. One way to deal with this dilemma is to randomly allocate subjects to treatment and control groups but only diagnose infections in the subjects who will be treated. This was done in a study in Tanzania in which in which infections were diagnosed in the control group at the end of the study (Bhargava et al., 2003). The problem with doing this is that the intensity of infections can change in the control group as worms are lost or gained during a long study, so the treatment and control groups cannot be compared at any point during the study. Two assumptions are commonly made about the effect of the intensity of infection, First, that treatment has an impact only above a threshold worm burden, which seems reasonable, though the threshold number is likely to be dependent on factors such as the size and current health of the host. Two assumptions are commonly made about the effect of the intensity of infection, First, that treatment has an impact only above a threshold worm burden, which seems reasonable, though the threshold number is likely to be dependent on factors such as the size and current health of the host. Second, that above the threshold the impact of treatment on more heavily infected individuals is linear. This may not be the case, but is very difficult to assess without having heavily infected children as controls during a study, something that would be unethical. An alternative is to examine the nature of the relationship between the intensity of infection with a worm and an outcome that is easily measurable, such as hookworm and haemoglobin concentration. 6.2 Treatment alone is not enough Cross-sectional studies typically find a non-linear relationship so that the subjects with the heaviest infections have a markedly lower haemoglobin concentration than lightly infected individuals (Lwambo et al., 1992, Pritchard et al., 1991, Srinivasan et al., 1987, Roche and Layrisse, 1966). This meta-analysis found no statistically significant effect of treating intestinal nematode worms on the mean haemoglobin concentration of children (Tables 8 and 9). Yet the potential impact of treating worms that cause internal bleeding, dysentery and the loss of iron, is large. In Africa in particular, many children are infected with species of Schistosoma in addition to hookworms, which makes it difficult to assess the impact of treating the intestinal nematodes alone. An important study in Tanzania showed that supplements of vitamin A and iron are needed if rapid improvements in haemoglobin concentration are to be achieved after deworming (Mwanri et al., 2000). All children in the study were given albendazole and praziquantel, 56 then were divided into four groups to receive on 3 school days a week either iron and vitamin A placebo, vitamin A and an iron placebo, iron plus vitamin A, or iron and vitamin A placebos (Mwanri et al., 2000). The haemoglobin concentration of children given both placebos rose by an average of only 3.6 g/L in the 12 weeks after treatment which might have been a statistically significant increase, but there was no untreated control group to allow this to be assessed. The effects of the supplements of iron and vitamin A were partially additive and achieved an increase in haemoglobin concentration of 22 g/L over the same period compared with 13.5 g/L for vitamin A alone and 17.5 g/L for iron alone. This study illustrates the importance of giving micronutrient supplements after treatment in order to achieve a rapid increase in haemoglobin concentration and may explain why no statistically significant effect of anthelmintic treatment alone was detected in the meta-analysis. If the change of 3.6 g/L over 12 weeks in the control group reflects the effect of anthelmintic treatment alone, then it might have taken six times as long to achieve an increase of 22 g/L, or nearly a year and a half. 6.2 Treatment alone is not enough The inclusion of vitamin A in addition to iron makes the important point that not all anaemia is iron deficiency anaemia, and this could explain the results of a study in Tanzania that gave iron alone and failed to achieve an statistically significant increase in haemoglobin concentration (Beasley et al., 2000). 6.3 The Cochrane Collaboration Review 6.3 The Cochrane Collaboration Review 6.3 The Cochrane Collaboration Review The Cochrane Collaboration supported a meta-analysis in 2000 (Dickson et al., 2000a, Dickson et al., 2000b). The reviewers noted heterogeneity between the results of the trials they included, and considered that they could be due to a number of factors. These were: the prevalence and intensity of infection, so that only heavily infected children benefited from treatment; the site of the study (school, community or health facility); the age of children; the prior nutritional status of children; and the manufacturer of the drug. The reviewers concluded that “there is some limited evidence that routine treatment of children in areas where helminths are common has effects on weight gain, but this is not consistent between trials”. The heterogeneity noted by the Cochrane Review really means a lack of effect in some trials which could be due, as explained here, to the fact that anthelmintic treatment 57 alone is not sufficient to achieve improved nutritional outcomes. The review received considerable criticism concerning the methods of analysis and conclusions (Bhargava, 2000, Michael, 2000, Cooper, 2000, Savioli et al., 2000, Bundy and Peto, 2000). This review was withdrawn in 2007 and replaced by a new analysis that examined effects on growth and school performance (Taylor-Robinson et al., 2007). The review drew similar conclusions: deworming may lead to improved weight gain in some circumstances but not others, and no effect on cognition or school performance has been demonstrated (Taylor-Robinson et al., 2007). 6.4 Characteristics of an ideal study Any studies that attempt to estimate the effect of anthelmintic treatment plus any supplement should ideally have a factorial design. This is illustrated in Figure 25 in which four groups of subjects are given either deworming or not with a nutritional intervention or not, though it could be applied to any treatment. The factorial design is powerful, but requires randomisation of subjects to study groups and an untreated control group to allow the effect of secular change to be estimated and subtracted from the effect of treatment. Insert Figure 25 here Insert Figure 25 here However it is important to appreciate that it may be possible to bring about an improvement in a nutritional variable by a nutritional intervention alone, so that the effect of deworming will be masked. An analogy of a leaking bucket of water may be helpful to explain this concept. The level of water in a bucket, which is the measured outcome, can be sustained or even raised if the water flowing into the bucket is greater than the loss through any leaks. Simply plugging the leaks will not raise the water level, it is necessary to add more water to achieve this. But adding water alone could overcome or mask the effect of the leaks and lead to a rise in the water level. Once the bucket is full, the excess simply overflows but the leaks continue. This creates a paradox: once you start to fill the bucket the change in the level of water no longer accurately reflects the leakage that is occurring. This analogy may apply better to anaemia than to body weight, as the concentration of haemoglobin does not rise unchecked, although some 58 excess nutrients such as iron and vitamin A can be stored. When worms cause a loss of blood and iron and the haemoglobin concentration falls, it will do so when all stores of iron needed to make haemoglobin have been used up (Crompton and Whitehead, 1993). Treating hookworms will not lead to an increase in the haemoglobin concentration if the diet does not contain enough nutrients to supply normal turnover, which is estimated to be about 0.8% of all red blood corpuscles daily, and to provide for increased erythropoiesis to replace corpuscles lost due to worms. But the cumulative loss of iron and other nutrients could be rectified by giving supplementary food alone, provided that all other requirements are met. The rate of recovery from anaemia is likely to be very slow without nutrient supplements, and studies of haemoglobin concentration after anthelmintic treatment may need to last for a year or more to show the full effect of treatment alone. The same could be said for measuring the effect of anthelmintic treatment on growth. It would be helpful for any future meta-analysis of the effects of anthelmintic treatments if any new research studies of the impact of treatment on outcomes such as growth or haemoglobin concentration had certain key features. 1. Insert Figure 25 here The study should be done where worms and undernourished children are common. A prevalence of infections with intestinal nematode worms of more than 70% is suggested, and where > 20% of children are underweight or > 40% are anaemic. 2. A randomised, controlled design should be used. If individuals are randomised to treatment and control groups then a placebo should be used; if clusters are randomised, then a placebo is ideal, but not essential. To overcome the problem of not giving a treatment to subjects in a cluster trial without a placebo, both groups could be given some other treatment, such as vitamin A. 3. A sample size calculation should be done. As a rule of thumb there should be a minimum of 250 subjects in each group in a randomised trial. In a cluster trial there should be more than 25 clusters per group and a design effect of 2 should be applied. 3. A sample size calculation should be done. As a rule of thumb there should be a minimum of 250 subjects in each group in a randomised trial. In a cluster trial there should be more than 25 clusters per group and a design effect of 2 should be applied. 4. Anthelmintic treatment should be given at least every 6 months. Ideally initial egg counts should be reduced by >70% by the first treatment. 59 This could mean giving repeated treatment initially to achieve a high efficacy, unless an effectiveness trial is being done. 5. If more than one anthelmintic is given, such as praziquantel with albendazole, or albendazole with micronutrient supplements, a 2 x 2 factorial design should be considered. 6. The key outcomes to be measured are weight and height. It may be helpful to measure triceps skinfold thickness and mid upper arm circumference to distinguish between increased adiposity and growth in tissues. If infections with hookworms or T.trichiura are common, then the haemoglobin concentration should be measured as well with, ideally, a measure of irons status such as ferritin or transferrin receptor (WHO/CDC, 2005). 7. If growth is to be measured, the study should last a minimum of one year, and two years if possible. Specific criteria that state when a study should be stopped may need to be set. 8. Insert Figure 25 here Over the last few years the costs of albendazole and mebendazole have been reduced to less than 5 US cents a tablet when bought in large quantities and, according to the WHO, these drugs can be given safely to all children once a year in areas where the prevalence of infection is more then 20% and twice a year where the prevalence is more than 50% (WHO, 2006). Over the last few years the costs of albendazole and mebendazole have been reduced to less than 5 US cents a tablet when bought in large quantities and, according to the WHO, these drugs can be given safely to all children once a year in areas where the prevalence of infection is more then 20% and twice a year where the prevalence is more than 50% (WHO, 2006). The new recommendations are shown in Table 14. To promote mass deworming the World Health Assembly in 2001 passed resolution WHA54.19 asking countries to administer anthelmintic treatments annually to at least 75% of all school-age children at risk of morbidity by 2010 (WHO, 2002a). Insert Figure 25 here If the haemoglobin concentration is to be measured the study should last a minimum of 12 weeks, and should be continued to 52 weeks if possible if no micronutrient supplements are given after treatment. 9. Drug efficacy should be assessed 21 days after each round of treatment as a matter of good practice, to assess the development of any anthelmintic resistance. 10. The gains in any parameter related to growth should be standardised as mean gain per year. The mean and standard deviation of all outcome measures and any derived indices should be given in any report or paper for each group, with the sample sizes. If subjects are studied in clusters, then this should be taken into account during the analysis. 10. The gains in any parameter related to growth should be standardised as mean gain per year. The mean and standard deviation of all outcome measures and any derived indices should be given in any report or paper for each group, with the sample sizes. If subjects are studied in clusters, then this should be taken into account during the analysis. 6.5 Implications for programmes Deworming is an easy and inexpensive thing to do, and the drugs are very safe. Treatment can be popular with parents (Brooker et al., 2001). Most anthelmintics can be given as a single dose so no adjustment for body weight is necessary, and a single 400 mg tablet of albendazole or 500 mg of mebendazole can be given to all children older than 1 year (WHO, 2002b), 6.5 Implications for programmes Deworming is an easy and inexpensive thing to do, and the drugs are very safe. Treatment can be popular with parents (Brooker et al., 2001). Most anthelmintics can be given as a single dose so no adjustment for body weight is necessary, and a single 400 mg tablet of albendazole or 500 mg of mebendazole can be given to all children older than 1 year (WHO, 2002b), 60 though a syrup should be given to very young children. The United Nations Children’s Fund in Ethiopia have issued warnings that young children should not be forced to take tablets because of the risk of choking. Anthelmintic drugs should not be given in the first trimester of pregnancy (WHO, 1994b). Anthelmintic drugs are usually available over the counter and do not require a prescription, except in developed countries. Insert Table 14 here There are three justifications for giving mass treatment at least once a year in areas where the prevalence of infection with all species of intestinal nematode worms is greater than 50%. First, because the probability of disease increases exponentially above a prevalence of 50% (see Figure 3). Second, the cost of diagnosis is typically many times the cost of treatment. When the prevalence is greater than 50% diagnosis becomes a matter of identifying uninfected people who do not need treatment, and so the cost per case of finding them increases with the prevalence. Third, most anthelmintic drugs, and the benzimidazoles in particular, are very safe, and there is no known harm in treating people who are not infected. Benzimidazoles have been taken daily for long periods to treat hydatid disease (El-On, 2003, Horton, 2003a) and when used to treat intestinal nematodes the overall frequency of side effects is about 1% (Horton, 2000). Although one of the potential studies identified for the meta-analysis reported evidence of an adverse effect on the growth of children given 400 mg albendazole given for 3 days, there was no untreated control group (so the study was excluded from the present analysis) and the children treated with 61 albendazole were compared with children treated with another anthelmintic, pyrantel embonate (Forrester et al., 1998). The use of a 20% threshold at which to apply annual mass treatment in school-age children has cost-benefit implications. When the prevalence of infection is less than 50% the majority of children are uninfected and perhaps only a few percent of children will actually benefit from treatment. Although the cost per child treated may be small in terms of the cost of each tablet, as the prevalence drops the costs per infected child treated will increase. This is shown in Figure 26 for a drug costing 3 US cents per treatment. When the prevalence is 50% the cost per infected child treated is twice the cost per child treated, or 6 US cents per child; when the prevalence is only 20% the cost per infected child treated is five time the cost per child treated or 15 US cents per child; and if only 5% of all children actually benefit from treatment the cost per beneficiary is 20 times the cost of the treatment or 60 US cents per child. Insert Table 14 here Although these unit costs may seem small in absolute terms, the total cost to a government may be large. For example if the Ethiopian government was considering treating the 11.5 million children enrolled in primary schools in 2005 with a drug costing 3 US cents per treatment, and if the prevalence of infection according to a recent national survey is 30% (SC/US, 2007), then of the $345,000 it will cost to buy drugs, 70% or $241,500 will be spent on treating uninfected children. These costs do not take into account the additional financial or opportunity costs of delivering and administering tablets. Insert Figure 26 here The main issue for deworming programmes is how to deliver treatments at low cost to the three main groups who would benefit most from treatment: pre-school children, school-age children, and women of reproductive age. As there are many current programmes delivering treatments such as vaccinations and vitamin A to children less than 5 years, the additional cost of giving a tablet of albendazole or mebendazole is minimal. In recognition of this, the WHO and UNICEF now recommend deworming children as a part of all routine health programmes, such as when vitamin A is given (WHO/UNICEF, 2004), and anthelmintic treatment is also recommend as a 62 part of the Integrated Management of Childhood Illnesses after any acute illness has ceased. A commonly unrecognised concern arises because of the lack of over- lap between health programmes for pre-school children, which typically stop at 5 years of age, and programmes for school children, which begin when children enrol in school. As the official age of enrolment in basic education in many countries is 6 or even 7 years of age, even if all children actually enrol in school at the correct age, there is a gap of 1 – 2 years when they may fall between programmes and miss out on treatment. As late-enrolment in school is very common in sub-Saharan Africa (Partnership for Child Development, 1998b) and late enrolment is strongly associated with stunted growth and anaemia (Partnership for Child Development, 1999b, Hall et al., 2001) the gap between programmes may be as much as four or five years, and such children may be in greater need of treatment than those who are actually in school. In countries where enrolment rates are high, school health programmes offer an effective mechanism to reach a large proportion of school-age children (Hall et al., 1996), an age group that harbours a large proportion of all worms in a community. The cost of delivering anthelmintics to schoolchildren has been shown to be a few US cents per child (Partnership for Child Development, 1999a, Partnership for Child Development, 1998a). In addition to the potential nutritional benefits of treatment, periodically giving anthelmintics to school children alone has other benefits, as it can serve to bring down transmission to other, untreated groups (Butterworth et al., 1991, Bundy et al., 1992b, Asaolu et al., 1991). Insert Figure 26 here Such externalities as they are called by economists, are not captured in a meta-analysis such as this, which examines only one outcome at a time. It is also possible that, as well as affecting growth and micronutrient status, intestinal nematodes may affect children’s cognitive function and educability, although the evidence for this is mixed and often comes from cross-sectional associations, which are easily confounded (Dickson et al., 2000b, Nokes et al., 1992a, Nokes et al., 1992b, Watkins and Pollitt, 1997, Jukes et al., 2002, Sakti et al., 1999). The Cochrane Review of 2007 concluded that there was insufficient evidence that treating worms improves 63 cognitive performance, which included educational outcomes including attendance and tests of educational achievements (Taylor-Robinson et al., 2007). The controlled trial of six-monthly deworming for two years in Viet Nam found no statistically significant difference between treatment and controls in terms of tests of mathematics and Vietnamese (Partnership for Child Development, 2001). The last group that would benefit considerably from periodic anthelmintic treatment are women of reproductive age, although treatment should only be given before pregnancy or after the first trimester. Nevertheless accidental treatment does not seem to be dangerous: a study of some 400 Sri Lankan women who had taken mebendazole during their first trimester of pregnancy did not find a greater rate of congenital defects than in pregnant women who had not taken the drug (de Silva et al., 1999). But the sample size was too small to detect a greater risk of rare congenital defects, so the recommendation of the study was still to avoid treatment with anthelmintics (de Silva et al., 1999). The last group that would benefit considerably from periodic anthelmintic treatment are women of reproductive age, although treatment should only be given before pregnancy or after the first trimester. Nevertheless accidental treatment does not seem to be dangerous: a study of some 400 Sri Lankan women who had taken mebendazole during their first trimester of pregnancy did not find a greater rate of congenital defects than in pregnant women who had not taken the drug (de Silva et al., 1999). But the sample size was too small to detect a greater risk of rare congenital defects, so the recommendation of the study was still to avoid treatment with anthelmintics (de Silva et al., 1999). Insert Figure 26 here The main effect of anthelmintic treatment on pregnant women is likely to be on haemoglobin concentration as a result of treating infections with hookworm or Schistosoma spp. A randomised controlled trial in Sierra Leone of giving albendazole to pregnant women after their first trimester prevented a fall in haemoglobin concentration of 6.6 g/L (Torlesse and Hodges, 2001, Torlesse and Hodges, 2000). When albendazole was given with iron, both treatments prevented a fall in haemoglobin concentration of 13.7 g/L (Torlesse and Hodges, 2001, Torlesse and Hodges, 2000). As a general rule for all programmes in which anthelmintic treatment for worms is given, drugs should not be administered to individuals who are sick for any reason. This is not because the drug may be ineffective, though it may be if there is diarrhoea. The reason for not giving treatment is because of the possibility that the subject may die of a pre-existing illness, and it could be concluded that the anthelmintic had caused the death. In any programme in which very large numbers of children are being treated on the same day, such an event becomes increasingly statistically probable, so every effort should be made to ensure that only otherwise healthy children are treated. A possible example of this was the >30 deaths reported after the administration 64 of vitamin A to some 3 million young children in the state of Assam in India in 2001 (West and Sommer, 2002), although there was some concern that children may have been overdosed (Kapil, 2004, Kapil, 2002). Reports of deaths of schoolchildren during mass deworming have not been documented. The other concern during mass treatment campaigns is for adolescent girls who may be pregnant and either not know it, or be afraid to report it. Teratogenic effects of drugs are most likely to occur during the first few weeks of pregnancy, a period when a woman may not yet be aware that she is pregnant. It cannot be assumed that school-age girls are not sexually active. A study of 9,000 schoolchildren in grades 4 to 6 in Tanzania found that 20% of girls reported having had sex, but only 39% of 114 girls with biological markers of sexual activity such as an infection, acknowledged having had sex, indicating that such activity was greatly under-reported (Todd et al., 2004). Insert Figure 26 here In an analysis of official education statistics, also in Tanzania, pregnancy was reported to be a cause of school drop out for 6 or 7 girls per 1,000 in grades 6 and 7 respectively (Partnership for Child Development, 1998b). These data should provide a warning to programmes that give mass treatment with anthelmintic drugs to school-age children as well for those considering adding mega-dose supplements of vitamin A to treat vitamin A deficient or anaemic adolescent girls. It cannot be assumed that adolescent girls are not pregnant. 6.6 Conclusions Treating intestinal nematode worms can lead to significantly better weight gain and growth when treated children are compared with untreated controls. The magnitude of the effect of treatment is specific to local epidemiological circumstances, and not all infected children will benefit to the same degree, if at all. If no significant effect of anthelmintic treatment is detected in a study, it cannot be assumed that worms did not contribute to undernutrition; it is likely that removing worms is insufficient to lead to improved growth without extra food and nutrients. The maximum size of any effect of treating worms cannot be estimated; studies can only indicate what difference is achievable. To show a significant 65 effect it is necessary to have an untreated control group and a sufficient period of follow up. This is likely to become less tenable given the measured benefits of treating worms that have been shown. Nevertheless it would be useful to have more studies of the effectiveness of anthelmintics delivered through health programmes, especially those that focus on pre-school and school-age children, and perhaps given with multiple micronutrients to make sure that no vitamin or mineral is a factor limiting catch-up growth. 7. This review was funded by the World Bank though the International Centre for Diarrhoeal Disease Research, Bangladesh. The principal author would like to acknowledge here his gratitude to David Crompton, Lani Stephenson and Michael Latham who, in 1978 in Kenya, introduced him to the study of human nutrition and parasitic worms. 8. Tables of papers considered for review (see separate file: Worm Review Tables.doc) 9. References 9. 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References 84 ry to egetation nails ef k sect sect w fish fections of humans, their infectious stages, the oblig Infectious stage in faeces Intermediate host Stage infectio humans Egg Water snail Metacercaria o Egg Water snail Metacercaria in Egg in proglottid Cow and bovids Cysticercus in Egg in proglottid Pig and swine Cysticercus in Egg in proglottid Beetle, flea Cysticercoid in Egg in proglottid Insect Cysticercoid in Egg Fish Plerocercoid in Egg No Larva in egg sh Egg or larva No Free-living larv Egg No Larva in egg sh Egg No Larva in egg sh Egg No Free-living larv Egg No Free-living larv infe In in E E E Table 2. Estimated fecundity of fertilised females of the major species of intestinal nematode worms. Data from (Sinniah, 1982, Anderson and May, 1991, Bundy and Cooper, 1989). The numbers are based on very few data and do not take into account density dependent fecundity or geographical variation in fecundity. At best they can be considered as indicative of the relative orders of magnitude of egg production. Species Eggs/female worm Ascaris lumbricoides < 200,000 Trichuris trichiura 3,000 – 20,000 Ancylostoma duodenale 10,000 – 20,000 Necator americanus 3,000 – 6,000 Species Eggs/female worm Ascaris lumbricoides < 200,000 Trichuris trichiura 3,000 – 20,000 Ancylostoma duodenale 10,000 – 20,000 Necator americanus 3,000 – 6,000 86 Table 3. Estimates of the population and number of people infected in millions, and the prevalence of infection with the main types of intestinal nematodes by region and age group. Data extracted from (de Silva et al., 2003). Table 3. Estimates of the population and number of people infected in millions, and the prevalence of infection with the main types of intestinal nematodes by region and age group. Data extracted from (de Silva et al., 2003). 9. References Age range (years) % Ascaris lumbricoides Popul- ation 0-4 5-9 10-14 ≥ 15 Total infected Latin American & Caribbean 530 8 10 10 56 84 15.8 Sub-saharan Africa 683 28 28 25 92 173 25.3 Middle east & north Africa 313 3 3 3 14 23 7.3 South Asia 363 13 15 13 56 97 26.7 India 1027 15 18 17 89 139 13.5 East Asia & the Pacific 564 20 25 25 134 204 36.2 China 1295 35 44 51 371 501 38.7 Total 4775 122 143 144 812 1221 25.6 Age range (years) % Trichuris trichiura Popul- ation 0-4 5-9 10-14 ≥ 15 Total infected Latin American & Caribbean 530 10 12 12 66 100 18.9 Sub-saharan Africa 683 26 27 23 86 162 23.7 Middle east & north Africa 313 1 1 1 4 7 2.2 South Asia 363 10 11 10 43 74 20.4 India 1027 8 9 9 47 73 7.1 East Asia & the Pacific 564 16 19 19 105 159 28.2 China 1295 15 19 22 163 219 16.9 Total 4775 86 98 96 812 1092 22.9 Age range (years) % Hookworm Popul- ation 0-4 5-9 10-14 ≥ 15 Total infected Latin American & Caribbean 530 1 3 5 41 50 9.4 Sub-saharan Africa 683 9 18 29 142 198 29.0 Middle east & north Africa 313 0 1 1 8 10 3.2 South Asia 363 2 5 8 44 59 16.3 India 1027 2 5 8 56 71 6.9 East Asia & the Pacific 564 4 9 16 120 149 26.4 China 1295 3 9 18 173 203 15.7 Total 4775 21 50 85 584 740 15.5 87 8 ecies of soil- Silva, 2007) in the mber and percentage opulations and that Any species No x 106 % 76.6 61.7 18.3 64.3 55.5 37.7 15.8 35.2 5.8 63.3 1.1 7.1 7.3 15.5 24.4 54.7 31.7 61.1 242.8 47.3 d 5 – 9 years infected with the three mai from (de Silva et al., 2003) and (Hall and untries are missing. The estimate for the e they are endemic, they occur in the sam cies are independent. Trichuris trichiura Hookworms o x 106 % No x 106 % 27.6 22.3 12.3 9. 9.6 33.9 5.3 18. 21.2 14.4 9.1 6. 8.8 19.5 2.2 4. 3.2 35.0 0.9 9. 0.0 0.3 0.0 0. 1.4 3.0 1.2 2. 10.0 22.3 7.6 17. 9. References 16.9 32.5 10.1 19. 98.7 19.2 48.6 9. Table 5. The threshold concentrations of worm eggs in faeces used to classify infections as light, moderate and heavy proposed by a WHO Expert Committee in 2002 (WHO, 2002a). No references or data are given in support of these numbers. Intensity Ascaris lumbricoides Trichuris trichiura Hookworms Low 1- 4,999 1 – 999 1 – 1,999 Moderate 5,000 – 49,999 1,000 – 9,999 2,000 – 3,999 High ≥ 50,000 ≥ 10,000 ≥ 4,000 89 Table 6. Estimates of the life span of the major species of intestinal nematode worms of humans (data from (Anderson and May, 1991)). Table 6. Estimates of the life span of the major species of intestinal nematode worms of humans (data from (Anderson and May, 1991)). Species Life span (years) Ascaris lumbricoides 1 – 2 y Trichuris trichiura 1 – 2 y Ancylostoma duodenale 2 – 3 y Necator americanus 2 – 3 y Enterobius vermicularis < 1 y Strongyloides stercoralis > 50 ya a Resulting from auto-infection Species Life span (years) 90 Table 7. The typical range in cure rates of drugs to treat intestinal nematode infections. The doses are those recommended by the World Health Organization (WHO, 1995). The data for the drugs that paralyse worms have been derived from (Albonico et al., 2003, Gustafsson et al., 1987, Huq et al., 1982). The data for the metabolic inhibitors is summarised from (Bennett, 2000), and represents the approximate inter-quartile range in an analysis of multiple studies of drug efficacy. The data have been combined for the metabolic inhibitors albendazole and mebendazole, except for hookworm, for which there is a difference between species in efficacy. Table 6. Estimates of the life span of the major species of intestinal nematode worms of humans (data from (Anderson and May, 1991)). Drugs that paralyse Metabolic inhibitors Levamisole, 40 mg Albendazole, 400 mg Piperazine, 75 mg/kg Mebendazole, 500 mg Pyrantel pamoate, 10mg/kg Tiabendazole, 25 mg/kg, 3d Species Cure rate ERRa Cure rate ERR Ascaris lumbricoides 80 - 100 % 95 – 100% 95 – 99 % 99 – 100% Ancylostoma duodenale Necator americanus 80 – 100% 60 – 90% 50 – 70% 70 – 90%b 20 – 80%c 75 – 95%b 55 – 95%c Trichuris trichiura Poord 20 – 60% 10 – 70% 50 – 90% aERR = egg reduction rate; b Albendazole 400 mg; c Mebendazole 500 mg; dUnless pyrantel given with oxantel Drugs that paralyse Metabolic inhibitors Levamisole, 40 mg Albendazole, 400 mg Piperazine, 75 mg/kg Mebendazole, 500 mg Pyrantel pamoate, 10mg/kg Tiabendazole, 25 mg/kg, 3d Species Cure rate ERRa Cure rate ERR Ascaris lumbricoides 80 - 100 % 95 – 100% 95 – 99 % 99 – 100% Ancylostoma duodenale Necator americanus 80 – 100% 60 – 90% 50 – 70% 70 – 90%b 20 – 80%c 75 – 95%b 55 – 95%c Trichuris trichiura Poord 20 – 60% 10 – 70% 50 – 90% aERR = egg reduction rate; b Albendazole 400 mg; c Mebendazole 500 mg; dUnless pyrantel given with oxantel aERR = egg reduction rate; b Albendazole 400 mg; c Mebendazole 500 mg; dUnless pyrantel given with oxantel aERR = egg reduction rate; b Albendazole 400 mg; c Mebendazole 500 mg; dUnless pyrantel given with oxantel 91 91 Table 8. Data from a study in Zanzibar of the efficacy of treating intestinal nematode infections with either albendazole or mebendazole (Albonico et al., 1994). Even though the drugs have a very similar mode of action there were statistically significant differences in the cure rates and egg reduction rates, particularly for hookworms. Species Drug n Cure rate Egg reduction rate Ascaris lumbricoides Albendazole 1,174 98.9 99.6 Mebendazole 1,120 97.8 99.3 Hookworms Albendazole 1,174 56.8*** 97.7*** Mebendazole 1,120 22.4 82.4 Trichuris trichiura Albendazole 1,174 10.5** 73.3*** Mebendazole 1,120 14.2 81.6 ** P < 0 01; *** P < 0 001 Drug n Cure rate Egg reduction rate ** = P < 0.01; *** = P < 0.001 ** = P < 0.01; *** = P < 0.001 92 92 Table 9. The terms used to search Medline for papers. MeSH denotes a specific Medical Subject Heading used as a search term. Table 6. Estimates of the life span of the major species of intestinal nematode worms of humans (data from (Anderson and May, 1991)). specific Medical Subject Heading used as a search term. Worms Treatments Outcomes "intestinal worms" "intestinal parasites" geohelminth* helminth* Ascaris roundworm Trichuris whipworm Necator ancylostoma hookworm "Ascaris lumbricoides” [MeSH] Trichuris [MeSH] “Necator americanus” [MeSH] “Ancylostoma duodenale” [MeSH] deworm* de-worm* anthelmintic* antihelmintic* vermifuge benzimidazole albendazole mebendazole pyrantel levamisole piperazine "Ascariasis/drug therapy" [MeSH] "Ascariasis/prevention and control" [MeSH] "Ascariasis/therapy" [MeSH] "Hookworm infections/drug therapy" [MeSH] "Hookworm infections/prevention and control" [MeSH] "Hookworm infections/therapy" [MeSH] "Trichuriasis/drug therapy" [MeSH] "Trichuriasis/prevention and control" [MeSH] "Trichuriasis/therapy"[MeSH] weight height length growth skinfold "arm circumference" "nutritional status" malnutrition anthropometr* haemoglobin hemoglobin "iron status" anaemia anemia retinol 93 93 tudies included in the meta-analysis. m Anya Age range N Drugb Dose Frequency Treatme % NR 5-10 y 56 ABZ 400 mg/d x 3 d Once No % ~ 56% 1-7 y 27,995 ABZ 400 mg 2 - 5 times every 6 mo No % NR 3-5 y 177 ABZ 200 mg/d x 3 d Twice, 1 month apart I % NR 24-61 mo 159 PPZ 75 mg/kg/d x 2d Once No % NR 6-10 y 98 ABZ 400 mg Once No % NR 6-10 y 99 ABZ 400 mg Once No % NR 2-5 y 117 PP 10 mg/kg Single dose then No MBZ 500 mg bi monthly for 1 year % 93% 7-8 y 2,659 ABZ 400 mg Every 6 mo, 5 times No NR NR 2-12 y 85 PP 11 mg/kg Once No NR NR 6-12 y 407 ABZ 400 mg/d x 2 d Once No % NR 6-16 y 150 ABZ 400 mg Once No % NR 6-15 y 284 ABZ 600 mg Once or twice 3.6 m later No % NR 7-13 y 53 ABZ 600 mg Once Fo % NR Not stated 3063 MBZ 500 mg 2x or 3 x per year No % NR Not stated 2924 MBZ 500 mg 2x or 3 x per year I % NR 6-71 mo 459 MBZ 500 mg Once every 3 mo for 1 y I NR NR 0.6-6.6 y 309 ABZ 400 mg Once Reti NR NR Not stated 51 ABZ 400 mg Once Reti % NR 2-12 y 1206 LEV Not stated Once No NR NR < 12 y 228 ABZ 400 mg Once No MBZ = Mebendazole; PP = Pyrantel pamoate; PPZ = Piperazine Table 11. Table 6. Estimates of the life span of the major species of intestinal nematode worms of humans (data from (Anderson and May, 1991)). The countries from which data were published in papers on the effect of anthelmintic treatment on the growth and nutritional status of children. Also shown is the number of times each country is represented in papers found during the literature search and the papers eventually used in the meta-analysis. One paper reported data from China, the Philippines and Kenya (Olds et al., 1999) so the total number of papers is 58 rather than 60. All other papers only presented data from one country. Country Papers found Papers used Benin 1 1 Botswana 1 0 Ethiopia 2 0 Kenya 11 4 Sierra Leone 1 1 South Africa 4 0 Tanzania 6 3 Uganda 1 1 Zaire 1 0 China 1 0 Indonesia 7 2 Malaysia 3 0 Myanmar 1 1 Philippines 1 0 Viet Nam 1 1 Bangladesh 5 2 India 3 0 Sri Lanka 1 0 Brazil 2 0 Guatemala 2 2 Haiti 2 0 Jamaica 2 1 Mexico 1 0 Total 58 19 Country Papers found Papers used Benin 1 1 Botswana 1 0 Ethiopia 2 0 Kenya 11 4 Sierra Leone 1 1 South Africa 4 0 Tanzania 6 3 Uganda 1 1 Zaire 1 0 China 1 0 Indonesia 7 2 Malaysia 3 0 Myanmar 1 1 Philippines 1 0 Viet Nam 1 1 Bangladesh 5 2 India 3 0 Sri Lanka 1 0 Brazil 2 0 Guatemala 2 2 Haiti 2 0 Jamaica 2 1 Mexico 1 0 Total 58 19 Country Papers found Papers used 95 Table 12. Summary of changes in primary and secondary outcome measures for studies in which any drug to treat intestinal nematode worms was given. The drugs were: albendazole and mebendazole, levamisole, piperazine or pyrantel pamoate Table 12. Summary of changes in primary and secondary outcome measures for studies in which any drug to treat intestinal nematode worms was given. The drugs were: albendazole and mebendazole, levamisole, piperazine or pyrantel pamoate Primary outcome measure No. of studies No. of participants Estimate (95% C.I.) P Weight (kg) 11 33,860 0.21 (0.17, 0.26) <0.001 Height (cm) 9 5,801 0.11 (0.03, 0.19) 0.01 MUACa (cm) 7 3,325 0.30 (0.23, 0.37) <0.001 Triceps skinfold (mm) 5 3,021 0.11 (0.03, 0.18) 0.04 Haemoglobin (g/dL) 5 2,178 -0.93 (-2.97, 1.10) 0.37 % DR/Rb ratio 2 204 0.17 (-0.60, 0.93) 0.67 Secondary outcome measures No. of studies No. Table 6. Estimates of the life span of the major species of intestinal nematode worms of humans (data from (Anderson and May, 1991)). of participants Estimate (95% C.I.) P Weight-for-age z-score 5 557 0.06 (0.03, 0.08) <0.001 Height-for-age z-score 6 961 0.09 (0.06, 0.11) <0.001 Weight-for-height z-score 4 378 0.38 (0.30, 0.46) <0.001 Weight-for-age % median 4 401 2.52 (1.95, 3.08) <0.001 Height-for-age % median 4 401 0.27 (0.12, 0.42) <0.001 Weight-for-height % median 3 323 2.64 (1.97, 3.30) <0.001 a MUAC = mid upper-arm circumference b DR/R = dehydroretinol/retinol 96 Table 13. Summary of changes in primary and secondary outcome measures for studies in which albendazole or mebendazole were given to treat intestinal nematode worms. Outcome measure No. of studies No. of participants Estimate (95% C.I.) P Weight (kg) 8 33,275 0.18 (0.13, 0.23) <0.001 Height (cm) 6 5,227 0.08 (-0.01, 0.17) 0.07 MUACa (cm) 6 3,228 0.30 (0.23, 0.37) <0.001 Triceps skinfold (mm) 5 3,021 0.11 (0.03, 0.18) 0.004 Haemoglobin (g/dL) 5 2,178 -0.93 (-2.97, 1.10) 0.37 % DR/Rb ratio 2 204 0.17 (-0.60, 0.93) 0.67 Secondary outcome measure No. of studies No. of participants Estimate (95% C.I.) P Weight-for-age z-score 4 468 0.06 (0.03, 0.08) <0.001 Height-for-age z-score 5 855 0.09 (0.06, 0.11) <0.001 Weight-for-height z-score 4 378 0.38 (0.30, 0.45) <0.001 Weight-for-age % median 2 242 3.16 (2.51, 3.81) <0.001 Height-for-age % median 2 242 0.31 (0.14, 0.47) <0.001 Weight-for-height % median 2 242 2.73 (2.03, 3.44) <0.001 a MUAC = mid upper-arm circumference b DR/R = dehydroretinol/retinol 97 97 Table 14. The prevalence of any species of intestinal nematode that warrants mass treatment criteria for applying treatments and the target groups. Table 14. The prevalence of any species of intestinal nematode that warrants mass treatment criteria for applying treatments and the target groups. Risk category Prevalence Target groups and treatments High ≥ 50% Treat all school- age children twice a year Treat all: • pre-school children • women of child- bearing age except in first trimester • adults at risk Low ≥ 20% to < 50% Treat all school- age children once a year Treat all: • pre-school children • women of child- bearing age except in first trimester • adults at risk 98 Figure 1. The proglottid of Taenia saginata (at the end of the stick) crawling away from a human stool in a trail of mucus. The sample was collected from a Pokot man in western Kenya, an ethnic group that traditionally enjoys eating undercooked beef (Hall et al., 1981). Figure 1. Table 6. Estimates of the life span of the major species of intestinal nematode worms of humans (data from (Anderson and May, 1991)). The proglottid of Taenia saginata (at the end of the stick) crawling away from a human stool in a trail of mucus. The sample was collected from a Pokot man in western Kenya, an ethnic group that traditionally enjoys eating undercooked beef (Hall et al., 1981). Figure 1. The proglottid of Taenia saginata (at the end of the stick) crawling away from a human stool in a trail of mucus. The sample was collected from a Pokot man in western Kenya, an ethnic group that traditionally enjoys eating undercooked beef (Hall et al., 1981). 99 Figure 2. The prevalence of infection with A.lumbricoides in 11 age classes of males living in an urban slum in Dhaka, Bangladesh (data from (Hall et al., 1999)). 0 10 20 30 40 50 60 70 80 90 100 0 10 20 30 40 50 60 Mean age of age class (years) Percentage infected 0 10 20 30 40 50 60 70 80 90 100 0 10 20 30 40 50 60 Mean age of age class (years) Percentage infected 100 Figure 3 The typical relationship between the prevalence and the mean worm burden for intestinal nematode worms (see (Guyatt et al., 1990)). The shape of the curve is derived from data collected during a study of A.lumbricoides in Bangladesh (Hall et al., 1999). 0 5 10 15 20 25 0 10 20 30 40 50 60 70 80 90 100 Prevalence Mean worm burden 0 5 10 15 20 25 0 10 20 30 40 50 60 70 80 90 100 Prevalence Mean worm burden 101 Figure 4. A diagrammatic representation of the proportions of a population of 100% who are infected with one, two or three types of worms when the prevalence of infection with Ascaris lumbricoides is 60%, Trichuris trichiura is 50% and hookworm is 40%. It is assumed that the probability of each infection is independent of each other and that probability of having two or more infections is multiplicative. Uninfected 12% Ascaris only 18% Ascaris and Trichuris 12% Ascaris, Trichuris and hookworm 18% Trichuris only 12% Hookworm and Trichuris 8% Hookworm only 8% Ascaris and hookworm 12% Ascaris and Trichuris 12% Hookworm and Trichuris 8% 102 Figure 5. The average number of worms recovered from males in 11 age classes living in an urban slum in Bangladesh (Hall et al., 1999). Figure 5. Table 6. Estimates of the life span of the major species of intestinal nematode worms of humans (data from (Anderson and May, 1991)). The average number of worms recovered from males in 11 age classes living in an urban slum in Bangladesh (Hall et al., 1999). 0 5 10 15 20 25 0 10 20 30 40 50 60 Mean age of age class (years) Mean worm burden 0 5 10 15 20 25 0 10 20 30 40 50 60 Mean age of age class (years) Mean worm burden 103 Figure 6. The distribution of A.lumbricoides in 1,765 people living in an urban slum in Dhaka, Bangladesh (Hall et al., 1999). The black line shows the negative binomial distribution. Figure 6. The distribution of A.lumbricoides in 1,765 people living in an urban slum in Dhaka, Bangladesh (Hall et al., 1999). The black line shows the negative binomial distribution. 0 50 100 150 200 250 300 0 10 20 30 40 50 60 70 80 90 100 No. of worms/host No of hosts >= 80% of all worms in 40% of all hosts 50% of all worms in 16% of all hosts Mean worm burden = 16.7 worms 0 50 100 150 200 250 300 0 10 20 30 40 50 60 70 80 90 100 No. of worms/host No of hosts >= 80% of all worms in 40% of all hosts 50% of all worms in 16% of all hosts Mean worm burden = 16.7 worms 104 Figure 7. The cumulative percentage of worms recovered from 1,511 people infected with Ascaris lumbricoides in an urban slum in Bangladesh ranked according to worm burdens from zero to the maximum of 187 worms, plotted against the cumulative number of subjects from whom they were recovered (data from (Hall et al., 1999). 0 10 20 30 40 50 60 70 80 90 100 0 10 20 30 40 50 60 70 80 90 100 Cumulative percentage worms Cumulative percentage hosts 0 10 20 30 40 50 60 70 80 90 100 0 10 20 30 40 50 60 70 80 90 100 Cumulative percentage worms Cumulative percentage hosts Cumulative percentage worms Cumulative percentage worms 105 Figure 8 The prevalence of infection with A.lumbricoides at three rounds of treatment six months apart (Hall et al., 1992). Table 6. Estimates of the life span of the major species of intestinal nematode worms of humans (data from (Anderson and May, 1991)). 0 10 20 30 40 50 60 70 80 90 100 0 10 20 30 40 50 60 Mean age of age class (years) Percentage infected Round 1 Round 2 Round 3 0 10 20 30 40 50 60 70 80 90 100 0 10 20 30 40 50 60 Mean age of age class (years) Percentage infected Round 1 Round 2 Round 3 106 Figure 9. The mean worm burden with A.lumbricoides at three rounds of treatment six months apart (Hall et al., 1992). Figure 9. The mean worm burden with A.lumbricoides at three rounds of treatment six months apart (Hall et al., 1992). Figure 9. The mean worm burden with A.lumbricoides at three rounds of treatment six months apart (Hall et al., 1992). 0 5 10 15 20 25 0 10 20 30 40 50 60 Mean age of age class (years) Mean worms/person Round 1 Round 2 Round 3 0 5 10 15 20 25 0 10 20 30 40 50 60 Mean worms/person Round 1 Round 2 Round 3 Mean age of age class (years) 107 Figure 10. The prevalence of infection with A.lumbricoides among 445 school-age children in Bangladesh and the mean worm burden at baseline (0 months) and at two treatments six months apart (data from study of (Hall et al., 1999). The dotted lines represent extrapolated trend lines if treatments had been given twice more at six month intervals. 0 10 20 30 40 50 60 70 80 90 100 0 6 12 18 24 Time (months) Prevalence of infection 0 5 10 15 20 25 Mean worms/person Prevalence Mean worms/child 0 10 20 30 40 50 60 70 80 90 100 0 6 12 18 24 Time (months) Prevalence of infection 0 5 10 15 20 25 Mean worms/person Prevalence Mean worms/child Time (months) 108 Figure 11 The average and 95% confidence intervals of body weight of two hypothetical groups of 300 children, on treated regularly with an anthelmintic and an untreated control group followed up for 3 years. 10 12 14 16 18 20 22 0 6 12 18 24 30 36 Months Mean weight (kg) Experimental Control 10 12 14 16 18 20 22 0 6 12 18 24 30 36 Months Mean weight (kg) Experimental Control 109 Figure 12. Table 6. Estimates of the life span of the major species of intestinal nematode worms of humans (data from (Anderson and May, 1991)). A conceptual model of the effect of worms on an outcome measure, and the need for remedial therapy after anthelmintic treatment to bring about a recovery. 110 Figure 12. A conceptual model of the effect of worms on an outcome measure, and the need for remedial therapy after anthelmintic treatment to bring about a recovery. O U T C O M E M E A S U R E Impact of worms Anthelmintic treatment Remedial therapy Uninfected Uninfected TIME O U T C O M E M E A S U R E Impact of worms Anthelmintic treatment Remedial therapy Uninfected Uninfected TIME Anthelmintic treatment 110 Figure 13. The effects of treating intestinal worms on body weight (kg). To interpret the figure, see Section 5.3. Figure 13. The effects of treating intestinal worms on body weight (kg). To interpret the figure, see Section 5.3. 111 Figure 14 The effects of treating intestinal worms on height (cm). To interpret the figure, see Section 5.3. Figure 14 The effects of treating intestinal worms on height (cm). To interpret the figure, see Section 5.3. 112 Figure 15. The effects of treating intestinal worms on mid upper-arm circumference (mm). To interpret the figure, see Section 5.3. Figure 15. The effects of treating intestinal worms on mid upper-arm circumference (mm). To interpret the figure, see Section 5.3. 113 Figure 16. The effects of treating intestinal worms on triceps skinfold thickness (mm). To interpret the figure, see Section 5.3. Figure 16. The effects of treating intestinal worms on triceps skinfold thickness (mm). To interpret the figure, see Section 5.3. Figure 17. The effects of treating intestinal worms on z-score of weight-for- age). To interpret the figure, see Section 5.3. Figure 17. The effects of treating intestinal worms on z-score of weight-for- age). To interpret the figure, see Section 5.3. 114 115 Figure 18. The effects of treating intestinal worms on z-score of height-for- age. To interpret the figure, see Section 5.3. Figure 19. The effects of treating intestinal worms on the difference in z- score of weight-for-height. To interpret the figure, see Section 5.3. Figure 19. The effects of treating intestinal worms on the difference in z- score of weight-for-height. To interpret the figure, see Section 5.3. Figure 19. The effects of treating intestinal worms on the difference in z- score of weight-for-height. To interpret the figure, see Section 5.3. 116 Figure 20. The effects of treating intestinal worms on percentage of the median weight-for-age. To interpret the figure, see Section 5.3. Figure 20. The effects of treating intestinal worms on percentage of the median weight-for-age. To interpret the figure, see Section 5.3. 117 Figure 21. The effects of treating intestinal worms on percentage of the median height-for-age. To interpret the figure, see Section 5.3. Figure 21. The effects of treating intestinal worms on percentage of the median height-for-age. To interpret the figure, see Section 5.3. 118 Figure 22. The effects of treating intestinal worms on percentage of the median weight-for-height. To interpret the figure, see Section 5.3. Figure 22. The effects of treating intestinal worms on percentage of the median weight-for-height. To interpret the figure, see Section 5.3. Figure 23. The effects of treating intestinal worms on haemoglobin concentration (g/dL). To interpret the figure, see Section 5.3. Figure 23. The effects of treating intestinal worms on haemoglobin concentration (g/dL). To interpret the figure, see Section 5.3. Figure 23. The effects of treating intestinal worms on haemoglobin concentration (g/dL). To interpret the figure, see Section 5.3. 119 Figure 24. The effects of treating intestinal worms on the dehydroretinol/ retinol ratio expressed as a percentage. To interpret the figure, see Section 5.3. Figure 24. The effects of treating intestinal worms on the dehydroretinol/ retinol ratio expressed as a percentage. To interpret the figure, see Section 5.3. Figure 24. The effects of treating intestinal worms on the dehydroretinol/ retinol ratio expressed as a percentage. To interpret the figure, see Section 5.3. 120 Figure 25. A 2 by 2 factorial design to estimate the effect of deworming with or without a nutritional treatment or not. Anthelmintic treatment Yes No Yes A B Nutritional intervention No C D 121 121 Figure 26. The costs of anthelmintic treatment per child for a drug costing 3 US cents per child and the costs per infected child treated. 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 10 0 Prevalence of infection or percentage who will benefit from treatment Cost per infected child treated (US cents) Cost per infected child treated Cost per child treated 0 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 10 0 Prevalence of infection or percentage who will benefit from treatment 122

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A comparison study on algorithms of detecting long forms for short forms in biomedical text

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BioMed Central BioMed Central This article is available from: http://www.biomedcentral.com/1471-2105/8/S9/S5 © 2007 Torii et al; licensee BioMed Central Ltd. Open Acce Proceedings A comparison study on algorithms of detecting long forms for short forms in biomedical text Manabu Torii1, Zhang-zhi Hu2, Min Song3, Cathy H Wu2 and Hongfang Liu*1 Open Access Open Access This article is available from: http://www.biomedcentral.com/1471-2105/8/S9/S5 © 2007 Torii et al; licensee BioMed Central Ltd. This article is available from: http://www.biomedcentral.com/1471-2105/8/S9/S5 © 2007 Torii et al; licensee BioMed Central Ltd. This article is available from: http://www.biomedcentral.com/1471-2105/8/S9/S5 © 2007 Torii et al; licensee BioMed Central Ltd. g A comparison study on algorithms of detecting long forms for short forms in biomedical text Manabu Torii1, Zhang-zhi Hu2, Min Song3, Cathy H Wu2 and H f Li *1 Address: 1Department of Biostatistics, Bioinformatics, and Biomathematics, Georgetown University Medical Center, 4000 Resevoir Rd, NW, Washington, DC 20057, USA, 2Department of Biochemistry and Molecular & Cellular Biology, Georgetown University Medical Center, 3300 Whitehaven St., NW, Washington, DC 20007, USA and 3Department of Information Systems, New Jersey Institute of Technology, University Heights, Newark, NJ 07102, USA Email: Manabu Torii - [email protected]; Zhang-zhi Hu - [email protected]; Min Song - [email protected]; Cathy H Wu - [email protected]; Hongfang Liu* - [email protected] * Corresponding author from First International Workshop on Text Mining in Bioinformatics (TMBio) 2006 Arlington, VA, USA. 10 November 2006 Published: 27 November 2007 BMC Bioinformatics 2007, 8(Suppl 9):S5 doi:10.1186/1471-2105-8-S9-S5 Introduction C0014644. Terminological knowledge bases specifically listing the definitions of SFs also exist. For example, the file LRABR in the SPECIALIST lexicon provides definitions for SFs that are present in the lexicon. Much of the new knowledge relevant to biomedical research is recorded as free text in the form of journal arti- cles or annotation fields of databases. The development of reliable natural language processing (NLP) systems, which retrieve relevant documents, extract relevant infor- mation, and mine new information from free text, can help biomedical researchers to better handle the over- whelming knowledge recorded in free text [1,2]. One crit- ical component in those systems is the mapping of text strings (i.e., terms) to biomedical concepts. Because of the complexity of the biomedical domain, biomedical terms are often lengthy. They usually contain words that imply their corresponding semantic types, e.g., virus in Epstein- Barr virus or protein in latent membrane protein, or words that describe properties of referred entities such as latent in latent membrane protein. At the same time, for biomedical concepts such as genes or proteins, it may be difficult to come up with short and yet descriptive terms for them. To ease the communication, concise representations of bio- medical concepts such as acronyms, abbreviations, and symbols have been used in text for biomedical concepts that either occur frequently or are difficult to describe. However, the use of concise representations has posed great challenges to NLP systems. First, it is difficult to automatically infer their semantic categories from the rep- resentation. For example, systems can detect Epstein-Barr virus representing a kind of virus but it would be difficult to infer the semantic type virus from its acronym, EBV. Secondly, concise representations can be highly ambigu- ous. For example, besides Epstein-Barr virus, EBV can also represent estimated blood volume, among others. In the fol- lowing, we denote concise representations as short forms (SFs) and their corresponding definitions as long forms (LFs). This study was designed to answer several questions. First, how do various systems perform in detecting LFs for SFs from parenthetical expressions given a large collection of novel text? To avoid evaluating systems on their develop- ment data set, abstracts of recently published articles were used in the study. Secondly, what is the coverage for vari- ous terminological knowledge bases to record SFs as syn- onyms of their LFs? Background g In the following, we provide background information about SFs in the biomedical domain, review studies pub- lished relevant to detecting LFs for SFs in text, and summa- rize two terminological knowledge bases in biomedicine. Abstract Motivation: With more and more research dedicated to literature mining in the biomedical domain, more and more systems are available for people to choose from when building literature mining applications. In this study, we focus on one specific kind of literature mining task, i.e., detecting definitions of acronyms, abbreviations, and symbols in biomedical text. We denote acronyms, abbreviations, and symbols as short forms (SFs) and their corresponding definitions as long forms (LFs). The study was designed to answer the following questions; i) how well a system performs in detecting LFs from novel text, ii) what the coverage is for various terminological knowledge bases in including SFs as synonyms of their LFs, and iii) how to combine results from various SF knowledge bases. Method: We evaluated the following three publicly available detection systems in detecting LFs for SFs: i) a handcrafted pattern/rule based system by Ao and Takagi, ALICE, ii) a machine learning system by Chang et al., and iii) a simple alignment-based program by Schwartz and Hearst. In addition, we investigated the conceptual coverage of two terminological knowledge bases: i) the UMLS (the Unified Medical Language System), and ii) the BioThesaurus (a thesaurus of names for all UniProt protein records). We also implemented a web interface that provides a virtual integration of various SF knowledge bases. Results: We found that detection systems agree with each other on most cases, and the existing terminological knowledge bases have a good coverage of synonymous relationship for frequently defined LFs. The web interface allows people to detect SF definitions from text and to search several SF knowledge bases. Availability: The web site is http://gauss.dbb.georgetown.edu/liblab/SFThesaurus. Page 1 of 9 (page number not for citation purposes) (page number not for citation purposes) BMC Bioinformatics 2007, 8(Suppl 9):S5 http://www.biomedcentral.com/1471-2105/8/S9/S5 http://www.biomedcentral.com/1471-2105/8/S9/S5 http://www.biomedcentral.com/1471-2105/8/S9/S5 Introduction Since those terminological knowledge bases often contain rich semantic information about terms, it will be beneficial to map SFs and LFs to them. Additionally, how can we combine the results from vari- ous systems and SF knowledge bases? To answer those questions, we evaluated several LF detection systems that are publicly accessible using a corpus consisting of MEDLINE abstracts published between January 2006 and May 2006. We used two terminological knowledge bases, the UMLS and BioThesaurus, to see the coverage of LFs and the coverage of including SFs as synonyms of their LFs. In addition, we implemented a web interface that uti- lizes several SF knowledge bases for detecting or searching LFs. http://www.biomedcentral.com/1471-2105/8/S9/S5 http://www.biomedcentral.com/1471-2105/8/S9/S5 http://www.biomedcentral.com/1471-2105/8/S9/S5 BMC Bioinformatics 2007, 8(Suppl 9):S5 http://www.biomedcentral.com/1471-2105/8/S9/S5 ties such as genes or proteins are usually represented as symbols in text which can be derived by initializing their descriptive names or assigned by nomenclature commit- tees. Note that some of the symbols may never be defined in text [1,12]. the initial letters of the words in a candidate LF, and the ratio of SF letters that are aligned with the initial letters of the syllables in the words of a candidate LF, and among others. Nadeau and Turney [16] also used machine learn- ing to select the best definition phrases among the set of candidate phrases that are assembled using heuristics based on previous studies [6,17]. Collocation-based approach pp Motivated by the fact that the majority of LFs for SFs have been defined using parenthetical expressions many times in a large corpus and the parenthetical expressions can be considered as collocations, we extracted an SF knowledge base from parenthetical expressions in MEDLINE abstracts using a collocation-based approach [20]. Oka- zaki and Ananiadou [21] and Zhou et al. [22] also used a collocation-based approach to build SF knowledge bases. One advantage of collocation-based approaches is the cor- rect detection of LFs for SFs that are created through sym- bols/synonyms substitution/initialization. For example, collocation-based methods can successfully detect the def- inition for 1H-MRS is proton magnetic resonance spectroscopy where 1H is a symbol for proton. Another alignment-based algorithm is proposed by Schwartz and Hearst [4]. Given an SF candidate in paren- theses, their algorithm seeks the shortest phrase that immediately precedes the parentheses and subsumes all the letters in the SF candidate in the same order, while the leftmost letter of the phrase and that of the SF should be the same. Despite its simplicity, the performance of their algorithm is highly competitive [3,5,6]. Template/rule-based approach Existing methods for detecting LFs in text or assembling SF knowledge bases can be categorized into one or combina- tion of the following four types: (i) alignment-based approach, (ii) machine learning approach, (iii) template/ rule-based approach, and (iv) collocation-based approach. We summarize a few systems for each type in the following. Template/rule-based approach Most studies using handcrafted templates/rules are for constructing SF knowledge bases from MEDLINE abstracts such as AcroMed [6], ARGH [18], and SaRAD [19]. Another example of using templates/rules is ALICE [3] which includes templates/rules for 320 different pat- terns. ALICE assembled several sets of stop words to avoid proposing SFs and LFs containing inappropriate words. For example, one of the sets contains stop words (e.g., of) for the leftmost word of LFs. Note that some studies such as the work by Yu et al. [14] in alignment-based approach can also be considered as the template/rule-based approach. Knowledge bases As we have discussed, several knowledge bases for SFs have been constructed automatically using MEDLINE abstracts. Other resources to obtain SF knowledge are bio- medical terminology sources. Those sources contain syn- onym relationship between terms, and SFs can be considered as synonyms of corresponding LFs. Since not all SFs are defined in text, it is important to have such knowledge bases. In this study, we used two terminologi- cal knowledge bases in the biomedical domain: the Uni- fied Medical Language System (UMLS) and BioThesaurus. SFs of biomedical concepts SFs are universal phenomena, occurring in all languages and writings and they can be formed in several ways [8,9]: Usually, authors provide the corresponding LF of an SF in their writing using patterns such as parentheses or phrases such as stands for. For example, readers can know what EBV stands for in a document when introduced first as fol- lowing in the document, Epstein-Barr virus (EBV) is a mem- ber of. However, parentheses can also be used for other purposes besides defining SFs. Several systems have been developed to detect parentheses used for defining SFs and extract the corresponding LFs [3-6] with F-measures reported as over 90%. However, authors may not always define SFs especially for well-known biomedical concepts in the domain. In this situation, automated systems or readers unfamiliar with the domain would need an SF knowledge base that lists all LFs associated with a given SF, and a method to associate the SF with the correct LF in a document. Some terminological knowledge bases such as the UMLS [7] have included SFs as synonyms of their LFs. For example, EBV and Epstein-Barr virus have been assigned to the same UMLS conceptual identifier Truncating the end, e.g. adm for administration (or admin- istrator), First letter initialization, e.g. AAA for abdominal aortic aneurysm, Syllabic initialization, e.g. BZD for benzodiazepine, Syllabic initialization, e.g. BZD for benzodiazepine, Combination initialization, e.g. ad lib for ad libitum, and Symbols/synonyms substitution or initialization e.g. ASD I for Primum atrial septal defect; Fe for iron. In the clinical domain, writing favors brevity because time pressures often prevent medical specialists from describ- ing clinical findings fully. Many clinical words and phrases are long, and SFs are a way to ease the communi- cation [10,11]. In the biomedical domain, biological enti- Page 2 of 9 (page number not for citation purposes) Page 2 of 9 (page number not for citation purposes) Alignment-based approach g pp The basic assumption of the alignment-based approach is that LFs can be found in neighboring phrases that sub- sume all or almost all the letters of the corresponding SF (in the same order). For example, Taghva et al. [13] devel- oped a detection system based on the longest common subsequence (LCS) algorithm. Their system assumes that an SF consists of the initial letters of the words contained in the LF (the common letters should appear in the SF and the LF in the same order), and the system seeks candidate LFs of an SF accordingly. Yu et al. [14], in their study of abbreviations in biology and medical papers, used several patterns to detect LFs, which also reflects the alignment idea. Similarly, the method by Yoshida and colleagues [15] detects LFs based on the assumption that the first sev- eral letters of each syllable in the words of LFs constitute the corresponding SFs. Candidate SF detection To compare how well each system performs in detecting LFs, we focused on sentences containing parentheses. As we have indicated, different systems have different criteria in considering a text string as a candidate SF. For instance, CSA proposes a phrase containing a comma-space sequence as an SF, but the other two systems discard tokens after a comma or semicolon, e.g., BMI, in kg/m2 vs. BMI given body mass index (BMI, in kg/m2) in the example below. CSA also recognizes an SF with only one letter, while S&H requires SF candidates containing at least two letters. S&H does not recognize SF candidates in sentences containing nested parentheses. Machine learning approach Machine learning has also been explored for LF detection. For example, Chang et al. [5] proposed a supervised machine learning approach to extract (SF, LF) pairs from MEDLINE abstracts. The system employs the LCS algo- rithm to search for different alignments between a candi- date SF in parentheses and the text string preceding the parentheses. Alignments detected are then evaluated using a machine learning method (logistic regression), and the one yielding the highest score is considered as the LF. The features considered in their approach for machine learning include the ratio of SF letters that are aligned with The UMLS [7] contains terms from a set of large scale ter- minological knowledge sources in biomedicine. Among many components in the UMLS, we used MetaThesaurus, which associates synonyms with unique concept identifi- Page 3 of 9 (page number not for citation purposes) Page 3 of 9 (page number not for citation purposes) http://www.biomedcentral.com/1471-2105/8/S9/S5 http://www.biomedcentral.com/1471-2105/8/S9/S5 http://www.biomedcentral.com/1471-2105/8/S9/S5 BMC Bioinformatics 2007, 8(Suppl 9):S5 BMC Bioinformatics 2007, 8(Suppl 9):S5 ers, and the file LRABR in the SPECIALIST lexicon that associates SFs with the corresponding LFs. Schwartz and Hearst. Note that we did not include the col- location-based approach in the comparison study since it is not suitable for detecting LFs in text but for assembling SF knowledge bases from a large corpus. As introduced in the previous section, the CSA system proposes an LF for a given SF with a score (between 0 and 1). Higher scores indicate more confidence in detecting LFs. After reviewing the systems' outputs as well as reported performance [2,6], we chose ≥0.03 as the threshold for the system to propose SFs and LFs. BioThesaurus is a knowledge source providing mapping between gene/protein names and protein entries in Uni- ProtKB, the most comprehensive protein knowledge base [23]. Through unique accession numbers assigned to each protein entry in UniProtKB, BioThesaurus groups terms referring to the same gene/protein entities. In the general English domain, one popular SF site is Acro- nymAttic (Table 1), which consists of 2,982,000 human- edited entries and it was claimed to be the world's largest and most comprehensive dictionary of acronyms. Other online resources include Special Dictionary, http://abbre viations.com, and http://acronyma.com (see Table 1). Methods As we have discussed, there are four types of approaches for detecting LFs for SFs. However, it is not clear how well they perform given novel text. We conducted a compari- son study to evaluate their performance in detecting LFs. The method involves several steps. The first step is to iden- tify systems that are publicly accessible. The second step is to define common criteria to select candidate sentences for detecting LFs. Because different systems may use differ- ent criteria for selecting candidate sentences, it is impor- tant to include only sentences that all systems consider them as candidate sentences. The third step is to obtain a list of candidate sentences from MEDLINE abstracts pub- lished between January 2006 and May 2006. We then ran the systems on these sentences, followed by a detail assessment of the results. Example 1.The objective was to describe the association of waist circumference (WC) and body mass index (BMI; in kg/ m2) with plasma circulating oxidized LDL (ox-LDL) and C- reactive protein (CRP) [PMID:16400046]. In order to investigate how well each system associates LFs with SFs without being confused with different schemes to identify SF candidates, we only consider sentences where all three systems attempt to detect LFs. Specifically, we consider an occurrence of parentheses for detection when the text string inside parentheses consists only of alpha- betic letters, numbers or hyphen, and contains at least one upper case letters with a total length between two and ten inclusive. Table 1 provides a summary of systems and resources used in the study. We used three systems in our comparison study in detecting LFs given novel text (indicated using "*"): i) a handcrafted pattern/rule based system by Ao and Takagi, ALICE, ii) a machine learning system by Chang et al., and iii) a simple alignment-based program by Table 1: Systems and SF search engines considered in the study. The sign * indicates the system was used for the comparison study. The sign + indicates the system was included by the web interface. Results and discussion Statistics and performance comparison The MEDLINE evaluation dataset contains about 210 thousands records with a set of candidate sentences con- taining about 258 thousands candidate SF occurrences. Figure 1 shows the Venn diagram of the results where each area is labeled with Roman numerals (i.e., I, II,...,VII). For example, there are totally four areas associated with ALICE (i.e., I, II, III, V) with a total number of tuples as 226,684 (the summation of 214,886, 896, 3,978, and 6,924). For each system, we obtained a collection of tuples (PMID, SF, LF), where the pair (PMID, SF) indicates the candidate definition occurrence and LF denotes the long form proposed by the system. We derived a Venn diagram to show the overlapping information about the three col- lections. For each area in the Venn diagram, we sampled 100 instances and manually judged the detection accu- racy. Note that one candidate occurrence (PMID, SF) may correspond to multiple tuples if different systems extract different LFs. We also provided an analysis on those occurrences. From Figure 1, we can see that the three systems agreed with each other for a large portion of the tuples (over 94%). S&H detected as many tuples as the other two elab- orated systems did, though the algorithm of S&H is very simple. This significant overlap is mainly because most SFs were obtained through various kinds of initialization as discussed in the background session. For example, there are 87,057 unique pairs (SF, LF) corresponding to Area I (i.e., detected by all three systems), 61% of the SFs were formed through First Letter Initialization from their corresponding LFs, e.g., AAA for abdominal aortic aneurysm. We then predicted the recall of the systems using the UMLS and BioThesaurus as knowledge sources. For pairs (PMID, SF) where none of the systems propose any LF, we used the UMLS MetaThesaurus as a knowledge source of synonyms to find out missing LFs using the following steps: The estimation of the precision for each area is also shown in Figure 1. For example, the precision in Area I is 100% when assessed using 100 randomly sampled tuples. We found that generally the more systems detect the same LF, the more accurate the detection is. For example, the detec- tion in areas III and IV tends to be reasonably accurate Look up a given SF (e.g., APAP) candidate in the UMLS MetaThesaurus. Methods System/Resource Reference Method Website ALICE* Ao and Takagi Templates/rules http://uvdb3.hgc.jp/ALICE/program_download.html ARGH+ Wren & Garner Templates/rule http://invention.swmed.edu/argh CSA* (BAS+) Chang Machine Learning http://abbreviation.stanford.edu/ (BAS) S&H* Schwartz & Hearst Alignment http://biotext.berkeley.edu/software.html ADAM+ Zhou Collocation http://128.248.65.210/arrowsmith_uic/adam.html Acromine+ Okazaki & Ananiadou Collocation http://www.nactem.ac.uk/software/acromine/ AcronymAttic+ NA NA http://www.acronymattic.com Special Dictionary (Acronym)+ NA NA http://www.special-dictionary.com/acronyms/ Abbreviation+ NA NA http://www.abbreviations.com/ ACRONYMA+ NA NA http://www.acronyma.com SF search engines considered in the study. The sign * indicates the system was used for the comparison stud e system was included by the web interface. Table 1: Systems and SF search engines considered in the study. The sign * indicates the system was used The sign + indicates the system was included by the web interface. BMC Bioinformatics 2007, 8(Suppl 9):S5 http://www.biomedcentral.com/1471-2105/8/S9/S5 Gather all the phrase strings associated with the recorded CIDs, e.g., CID: C0000970 → {"APAP", "Acetaminophen, N-(4-Hydroxyphenyl)acetanilide",...} Evaluation data The MEDLINE records published between January 2006 and May 2006, which are not part of the development/ training corpora for any of the three systems, were used for evaluation. Look up any of the gathered strings in the text prior to the corresponding parenthetical expression. If found and it is longer than the SF candidate, propose it as the LF for the given SF. For each MEDLINE record (identified using PubMed iden- tifier PMID), we used a Perl script to extract all sentences containing parentheses with candidate SFs. Each occur- rence of parentheses with candidate SFs can be uniquely identified as (PMID, SF) where PMID is the PubMed unique identifier and SF is the text string inside parenthe- ses found in the corresponding MEDLINE record. For example, there are four parentheses associated with the sentence shown in Example 1, where three of them are considered as candidate definition occurrences (identified as (16400046, WC), (16400046, ox-LDL), and (16400046, CRP)), and one, i.e., (BMI; in kg/m2) is not considered since it contains characters other than letters, numbers, and hyphen. When looking up SFs or LFs in text or knowledge bases, we first tokenize phrases, where tokens can be words, num- bers, or special tokens such as Roman numerals, Greek let- ters, or digits. Tokens are then normalized by converted into base forms using the UMLS SPECIALIST lexicon. We also ignored case difference during the lookup. We estimated the coverage of two existing terminological knowledge bases, the UMLS and BioThesaurus, regarding to LFs and synonymous relationships between SFs and the corresponding LFs. For pairs (SF, LF) agreed by the three systems, we grouped them according to their frequencies in the result collection (i.e., the number of occurrences of the corresponding tuples (PMID, SF, LF)). For each group, we measured (i) coverage of LFs, and (ii) coverage of pairs (SF, LF) as synonyms. Assessment of LF detection After we obtained a list of candidate sentences containing at least one candidate SF occurrence, we then obtained LF detection results. For ALICE and S&H, we downloaded the programs available in their project web pages (see Table 1), and executed them locally [3,4]. For CSA, we submit- ted the sentences to the system running on their project web site (see Table 1). Venn diagr ALICE, CS Figure 1 g y , , g Venn diagram of the results obtained from three systems: ALICE, CSA, and S&H. Each area is labeled with Roman numerals (i.e., I, II,...,VII). Statistics for each area includes the number of tuples (PMID, SF, LF) and the estimation of the precision for each area. g y g Venn diagram of the results obtained from three systems: ALICE, CSA, and S&H. Each area is labeled with Roman numerals (i.e., I, II,...,VII). Statistics for each area includes the number of tuples (PMID, SF, LF) and the estimation of the precision for each area. Finally, given a precision over 100 instances for different partitions in the Venn diagram (Figure 1), we may specu- late the precision of the three systems for the entire data set, e.g., ALICE proposed 214886 (I) + 896 (II) + 3978 (III) + 6924 (V) pairs, and considering the corresponding precisions in Figure 1, 214886*1.0 + 896*0.75 + 3978*0.89 + 6924*0.91 may be correct pairs. Similarly, incorporating the recall study using the UMLS above, ALICE, CSA, and S&H would achieve recalls of 97%, 96%, and 96%. These performance measures are much higher than the precision and recall values reported before on these systems. One reason for the better performance may be our highly selective choices of sentences that were passed to the systems for evaluation. (i.e., 89% and 86%) when S&H and one other system pro- posed the same LF for a given candidate SF occurrence. Table 2 shows the statistics of SF candidate occurrences associated with multiple LFs for different combinations and the number of correct detection associated with each system when assessed using 53 pairs randomly sampled. For example, the third column of the third row indicates that there are totally 2,572 SF candidate occurrences where ALICE and CSA proposed the same LF and S&H proposed a different one. When inspecting 53 pairs ran- domly selected from 2,572 occurrences, ALICE and CSA proposed the correct LFs for 51 pairs, S&H proposed the correct LF for one pair, and none of the systems proposed the correct LF for one pair. As we have shown, most problematic cases (i.e., inconsist- ent or failed detection of LFs among the systems) are chemical/protein/gene symbols. It may be caused by the following reasons: the symbols may be assigned by nomenclature committee or be created through symbols/ synonyms initialization/substitution. http://www.biomedcentral.com/1471-2105/8/S9/S5 BMC Bioinformatics 2007, 8(Suppl 9):S5 http://www.biomedcentral.com/1471-2105/8/S9/S5 Venn diagram of the results obtained from three systems: ALICE, CSA, and S&H Figure 1 Venn diagram of the results obtained from three systems: ALICE, CSA, and S&H. Each area is labeled with Roman numerals (i.e., I, II,...,VII). Statistics for each area includes the number of tuples (PMID, SF, LF) and the estimation of the precision for each area. When predicting the recall of the systems using the UMLS and BioThesaurus, we obtained 21,657 SF candidate occurrences where none of the systems proposed LFs. After mapping, we found 1,029 pairs with a total of 396 unique pairs failed to be detected by all three systems according to the synonymous relationship in the knowl- edge bases. The most frequently observed pairs were 5-HT for serotonin (111 times), Pb for lead (44 times), CsA for cyclosporine (33 times). Additionally, several chemical names such as RDX for hexahydro-1,3,5-trinitro-1,3,5-tri- azine were observed. A few pairs were observed where the derivation of SFs from the LFs involves substitution of synonymous tokens, e.g., NIS for sodium iodide symporter (sodium → natrium), or reordering of words, e.g., PLAUfor urokinase plasminogen activator. We found that over 70% of the detected unique pairs were ones where the SFs could not be implied from LFs through initialization, e.g., 5-HT and serotonin. To identify these pairs and to properly han- dle them in various applications, it is necessary to incor- porate terminology knowledge bases such as the UMLS, or to explore the collocation-based approach [20,22]. Results and discussion Statistics and performance comparison If found, record the corresponding con- cept IDs (CIDs) (e.g., APAP → CID: C0000970.) Page 5 of 9 (page number not for citation purposes) Page 5 of 9 (page number not for citation purposes) Venn diagr ALICE, CS Figure 1 Table 2: Statistics of SF candidate occurrences when multiple LFs proposed by ALICE, CSA, and S&H. The same superscript indicates the two systems proposed the same LFs. The superscript 0 indicates the corresponding system did not propose an LF. Cases # Correct (of 53) # SF candidate occurrence ALICE1, CSA2, S&H3 17, 20, 13 53 ALICE1, CSA1, S&H3 51, 51, 1 2,572 ALICE1, CSA2, S&H1 36, 11, 36 765 ALICE1, CSA2, S&H2 33, 18, 18 901 ALICE1, CSA2, S&H0 27, 12, --- 167 ALICE1, CSA0, S&H2 37, ---, 6 160 ALICE0, CSA2, S&H3 ---, 13, 28 325 Table 2: Statistics of SF candidate occurrences when multiple LFs proposed by ALICE, CSA, and S&H. The same superscript indicates the two systems proposed the same LFs. The superscript 0 indicates the corresponding system did not propose an LF. Results of Figure 2 R l Results of the coverage study Figure 2 Results of the coverage study. X-axis is the frequency bin [2n, 2n+1) where n from 0 to 8, the first Y-axis (left side) is the coverage and the second Y-axis (right side) is the number of unique pairs. Four lines mean: Line NP – the total number of unique pairs for each bin. Line LF – the percentage of unique pairs (SF, LF) where LF can be mapped to the knowledge base. Line (SF, LF) – the percentage of pairs where the synonymous relationship between SF and LF can be inferred. Line NC – the percentage of pairs where LF cannot be mapped to knowledge bases. Results of the coverage study Figure 2 Results of the coverage study. X-axis is the frequency bin [2n, 2n+1) where n from 0 to 8, the first Y-axis (left side) is the coverage and the second Y-axis (right side) is the number of unique pairs. Four lines mean: Line NP – the total number of unique pairs for each bin. Line LF – the percentage of unique pairs (SF, LF) where LF can be mapped to the knowledge base. Line (SF, LF) – the percentage of pairs where the synonymous relationship between SF and LF can be inferred. Line NC – the percentage of pairs where LF cannot be mapped to knowledge bases. Coverage of terminological knowledge bases g f g g Figure 2 shows the coverage results where X-axis is the fre- quency bin with the [2n, 2n+1) where n from 0 to 8, the first Y-axis (left side) is the coverage and the second Y-axis (right side) is the number of unique pairs. From Figure 2, we can see that there are 66 thousands of unique pairs (SF, LF) defined only once in our data with the corresponding coverage of LFs as 31% and the corresponding coverage of pairs (SF, LF) around 11%. The coverage increases when the frequency of a pair being defined increases. For exam- ple, for pairs being defined [16, 32) times, the coverage of Page 6 of 9 (page number not for citation purposes) Page 6 of 9 (page number not for citation purposes) BMC Bioinformatics 2007, 8(Suppl 9):S5 http://www.biomedcentral.com/1471-2105/8/S9/S5 http://www.biomedcentral.com/1471-2105/8/S9/S5 BMC Bioinformatics 2007, 8(Suppl 9):S5 Results of the coverage study Figure 2 Results of the coverage study. X-axis is the frequency bin [2n, 2n+1) where n from 0 to 8, the first Y-axis (left side) is the coverage and the second Y-axis (right side) is the number of unique pairs. Four lines mean: Line NP – the total number of unique pairs for each bin. Line LF – the percentage of unique pairs (SF, LF) where LF can be mapped to the knowledge base. Line (SF, LF) – the percentage of pairs where the synonymous relationship between SF and LF can be inferred. Line NC – the percentage of pairs where LF cannot be mapped to knowledge bases. http://www.biomedcentral.com/1471-2105/8/S9/S5 http://www.biomedcentral.com/1471-2105/8/S9/S5 http://www.biomedcentral.com/1471-2105/8/S9/S5 http://www.biomedcentral.com/1471-2105/8/S9/S5 http://www.biomedcentral.com/1471-2105/8/S9/S5 BMC Bioinformatics 2007, 8(Suppl 9):S5 knowledge base, it compares retrieved LFs to the text prior to the corresponding parenthetical expression and associ- ates the one that appears in the text. When multiple over- lapping LFs found, the system returns ones with the highest precedence score in those SF knowledge bases applicable (e.g., the frequency of SF being defined as LF in MEDLINE). For example, when searching ARGH, we retrieved 229 LFs for CRP including C-reactive protein, cAMP receptor protein, cyclic AMP receptor protein. Compar- ing to the text prior to the parenthetical expression in Example 1, we consider C-reactive protein as the associated LF in the given text. according to the number of SF knowledge bases contain- ing them. Figure 3 shows the screenshots of the web inter- face. Note that the last column of the result tables indicates the existence of synonymous relationship between SF and LF in BioThesaurus (i.e., if SF and LF can be mapped to the same protein entity in BioTheasurus, we consider BioThesaurus captures the synonymous relation- ship). We do not include the UMLS in the result tables of our interface due to issues relevant to the UMLS license agreement. A web interface for LF detection and search LFs increased to 83% and the coverage of (SF, LF) pairs increased to 59%. For 19 pairs defined [256, 512) times, all LFs were mapped to the UMLS (i.e., 100% coverage) and 17 (SF, LF) pairs were mapped to the UMLS (i.e., 90% coverage). Observing that i) different LF detection methods may pro- pose different LFs, and ii) the more systems proposed the same LF, the more accurate the detection is, we have implemented a web interface so that users can search for LFs associated with a given SF from different SF knowl- edge bases. It is a virtual integration of various SF knowl- edge bases including ones assembled from MEDLINE abstracts and ones available in the general English domain (indicated using "+" in Table 1). Although the coverage of existing terminological knowl- edge bases was found to be low for less frequently defined LFs, they have an advantage of associating LFs with SFs that are not derived through simple initialization (e.g., 1H MRS for Proton Magnetic Resonance Spectroscopy). Also, the mapping of LFs to existing terminological knowledge bases provides useful additional information, such as semantic type information (e.g., UMLS Semantic Types) or links to biological sequence databases (e.g., BioThesau- rus). The web interface provides two functions. One is to use various SF knowledge bases to detect definitions in text for SFs in parenthetical expressions. Given a document, the system searches each SF candidate and retrieves corre- sponding LFs from those knowledge bases. For each Page 7 of 9 (page number not for citation purposes) Page 7 of 9 (page number not for citation purposes) Conclusion In this work, we conducted a comparison study of three LF detection systems, ALICE, CSA, and S&H, which reflect three different approaches in LF detection for SFs. We observed that the majority of (SF, LF) pairs in MEDLINE abstracts were formed in a relatively simple way (i.e., ini- Another function is to search various SF knowledge bases. For a given SF, we send queries to all search engines and provide a table summarizing the retrieved results where syntactic variants have been grouped and LFs were ranked Screen shots of the web interface for virtual integration of various SF knowledge bases Figure 3 Screen shots of the web interface for virtual integration of various SF knowledge bases. 1. The main page of the web interface which provides two functions: i) LF detection in text, and ii) LF search from various knowledge bases. 2. Results for LF detec- tion in text for Example 1. 3. Results for LF search from various knowledge bases. g g g Screen shots of the web interface for virtual integration of various SF knowledge bases. 1. The main page of the web interface which provides two functions: i) LF detection in text, and ii) LF search from various knowledge bases. 2. Results for LF detec- tion in text for Example 1. 3. Results for LF search from various knowledge bases. Page 8 of 9 (page number not for citation purposes) http://www.biomedcentral.com/1471-2105/8/S9/S5 BMC Bioinformatics 2007, 8(Suppl 9):S5 9. Liu H, Lussier YA, Friedman C: Disambiguating ambiguous bio- medical terms in biomedical narrative text: an unsupervised method. J Biomed Inform 2001, 34(4):249-261. tialization) and can be detected by almost all three sys- tems. We also investigated the coverage of existing terminology knowledge sources, namely the UMLS and BioThesaurus, and the results showed that they have bet- ter coverage for pairs that are frequently defined. We implemented a web interface to provide virtual integra- tion of SF knowledge bases derived using various detec- tion methods in the biomedical domain or those available in the general English domain. We are currently working on incorporating a semantic category classifica- tion system so that users can limit their LF search to cer- tain semantic categories. J f ( ) 10. Luxton T, Al-Qassab H: Better use of abbreviations - a lesson from a stroke. Medical Education 2000, 34(11):965. 11. Bloom DA: Acronyms, abbreviations and initialisms. BJU Inter- national 2000, 86(1):1-6. 12. Competing interests 16. Nadeau D, Turney P: A Supervised Learning Approach to Acronym Identification. 18th Conference of the Canadian Society for Computational Studies of Intelligence: 2005; Victoria, BC, Canada 2005:319-329. The authors declare that they have no competing interests. Authors' contributions 17. Park Y, Byrd RJ: Hybrid Text Mining for Finding Abbreviations and Their Definitions. Conference on Empirical Methods in Natural Language Processing: 2001; Pittsburgh, PA 2001. Manabu Torii – Overall development and implementa- tion of the software for the study and preparation of sub- stantial portions of the manuscript. Zhang-zhi Hu – Input regarding to the conceptual design and help with the manuscript preparation. Min Song – Input to the concep- tual design and assist with the manuscript preparation. Cathy Wu – Input regarding to the conceptual design and help with manuscript preparation. Hongfang Liu – Over- all conceptual design and supervision of the project, and preparation of the final manuscript. 18. Wren JD, Garner HR: Heuristics for identification of acronym- definition patterns within text: towards an automated con- struction of comprehensive acronym-definition dictionaries. Methods Inf Med 2002, 41(5):426-434. 19. Adar E: SaRAD: a Simple and Robust Abbreviation Diction- ary. Bioinformatics 2004, 20(4):527-533. 20. Liu H, Friedman C: Mining terminological knowledge in large biomedical corpora. Pac Symp Biocomput 2003:415-426. p y p p 21. Okazaki N, Ananiadou S: Building an abbreviation dictionary using a term recognition approach. Bioinformatics 2006. g g pp f 22. Zhou W, Torvik VI, Smalheiser NR: ADAM: another database of abbreviations in MEDLINE. Bioinformatics 2006, 22(22):2813-2818. 23. Bairoch A, Apweiler R, Wu CH, Barker WC, Boeckmann B, Ferro S, Gasteiger E, Huang H, Lopez R, Magrane M, et al.: The Universal Protein Resource (UniProt). Nucleic Acids Res 2005:D154-159. Acknowledgements The project was supported by IIS-0639062 from the National Science Foun- dation. We thank the authors of the three systems used in the comparison study (Hiroko Ao, Jeffrey Chang, Ariel Schwartz, and each of their col- leagues) for making their systems available. We also thank for the authors, developers, or maintainers for making their search engines available. This article has been published as part of BMC Bioinformatics Volume 8 Sup- plement 9, 2007: First International Workshop on Text Mining in Bioinfor- matics (TMBio) 2006. The full contents of the supplement are available online at http://www.biomedcentral.com/1471-2105/8?issue=S9. Conclusion Eyre TA, Ducluzeau F, Sneddon TP, Povey S, Bruford EA, Lush MJ: The HUGO Gene Nomenclature Database, 2006 updates. Nucleic Acids Res 2006:D319-321. The HUGO Gene Nomenclature Database, 2006 updates. Nucleic Acids Res 2006:D319-321. 13. Taghva K, Gilbreth J: Finding Acronyms and Their Definitions. Int Journal on Document Analysis and Recognition 1999, 1(4):191-198. J y g ( ) 14. Yu H, Hatzivassiloglou V, Rzhetsky A, Wilbur WJ: Automatically identifying gene/protein terms in MEDLINE abstracts. J Biomed Inform 2002, 35(5–6):322-330. ( ) 15. Yoshida M, Fukuda K, Takagi T: PNAD-CSS: a workbench for constructing a protein name abbreviation dictionary. Bioinfor- matics 2000, 16(2):169-175. Publish with BioMed Central and every scientist can read your work free of charge from MEDLINE. J Am Med Inform Assoc 2005, 12(5):576 586. 4. Schwartz AS, Hearst MA: A simple algorithm for identifying abbreviation definitions in biomedical text. Pac Symp Biocomput 2003:451-462. 5. Chang JT, Schutze H, Altman RB: Creating an online dictionary of abbreviations from MEDLINE. J Am Med Inform Assoc 2002, 9(6):612-620. ( ) 6. Pustejovsky J, Castaño J, Cochran B, Kotecki M, Morrell M, Rumshisky A: Extraction and Disambiguation of Acronym-Meaning Pairs in Medline. Medinfo 2001, 10:371-375. f 7. Bodenreider O: The Unified Medical Language System (UMLS): integrating biomedical terminology. Nucleic Acids Res 2004:D267-270. 8. Zahariev M: A (Acronyms). In Unpublished PhD thesis School of Com- puting Science, Simon Fraser University, USA; 2004. References 1. Hirschman L, Morgan AA, Yeh AS: Rutabaga by any other name: extracting biological names. Journal of Biomedical Informatics 2002, 35(4):247-259. ( ) 2. Hunter L, Cohen KB: Biomedical language processing: what's beyond PubMed? Mol Cell 2006, 21(5):589-594. 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 P 9 f 9 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 y ( ) 3. Ao H, Takagi T: ALICE: an algorithm to extract abbreviations from MEDLINE J Am Med Inform Assoc 2005 12(5):576 586 Publish with BioMed Central and every scientist can read your work free of charge y ( ) 3. Ao H, Takagi T: ALICE: an algorithm to extract abbreviations from MEDLINE. J Am Med Inform Assoc 2005, 12(5):576-586.

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Prioritisation of Provinces for African Swine Fever Intervention in South Africa through Decision Matrix Analysis

Pathogens

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To cite this version: Leana Janse van Rensburg, Mary-Louise Penrith, Eric M.C. Etter. Prioritisation of Provinces for African Swine Fever Intervention in South Africa through Decision Matrix Analysis. Pathogens, 2022, 11 (2), pp.135. ￿10.3390/pathogens11020135￿. ￿hal-03615744￿ Distributed under a Creative Commons Attribution 4.0 International License



 Citation: Janse van Rensburg, L.; Penrith, M.-L.; Etter, E.M.C. Prioritisation of Provinces for African Swine Fever Intervention in South Africa through Decision Matrix Analysis. Pathogens 2022, 11, 135. https://doi.org/10.3390/ pathogens11020135 Citation: Janse van Rensburg, L.; Penrith, M.-L.; Etter, E.M.C. Prioritisation of Provinces for African Swine Fever Intervention in South Africa through Decision Matrix Analysis. Pathogens 2022, 11, 135. https://doi.org/10.3390/ pathogens11020135 Keywords: African swine fever; analytic hierarchy process; multi-criteria decision analysis; pigs Article Prioritisation of Provinces for African Swine Fever Intervention in South Africa through Decision Matrix Analysis Leana Janse van Rensburg 1,2,* , Mary-Louise Penrith 3 and Eric M. C. Etter 1,4,5 Leana Janse van Rensburg 1 Department of Production Animal Studies, Faculty of Veterinary Sciences, University of Pretoria, Onderstepoort 0110, South Africa; [email protected] p 2 Directorate Animal Health, Department of Agriculture, Land Reform & Rural Development of South Africa, Pretoria 0001, South Africa p 2 Directorate Animal Health, Department of Agriculture, Land Reform & Rural Development of S Pretoria 0001, South Africa 3 Department of Veterinary Tropical Diseases, Faculty of Veterinary Sciences, University of Preto Onderstepoort 0110, South Africa; [email protected] 3 Department of Veterinary Tropical Diseases, Faculty of Veterinary Sciences, University of Pretori Onderstepoort 0110, South Africa; [email protected] 4 CIRAD, UMR AnimalS Territories Risks Ecosystems (ASTRE), 97170 Petit Bourg, France 5 ASTRE, University Montpellier, CIRAD, INRAE, 34070 Montpellier, France * Correspondence: [email protected] or [email protected] y g 5 ASTRE, University Montpellier, CIRAD, INRAE, 34070 Montpellier, France * Correspondence: [email protected] or [email protected] Abstract: South Africa has experienced an increase in the number of African swine fever (ASF) outbreaks in domestic pigs in the last ten years. Intervention will be needed in the form of control and prevention strategies to minimise the impact of this disease in the country. The aim of this study is to prioritise which provinces resources should be allocated to for ASF intervention strategies, based on the risk factors identified as pertinent in South Africa. A multi-criteria decision analysis approach was followed using an analytic hierarchy process (AHP) method to determine the perceived risk of ASF outbreaks in domestic pigs per province. Nine risk factors applicable to the South African context were identified from literature. Data on the presence of these risk factors per province were collected from records and by means of a questionnaire. The risk factors were weighted by means of an AHP. The decision matrix determined that ASF intervention and prevention resources should be focused on Mpumalanga, Free State and Gauteng provinces in South Africa. Specific intervention strategies should be focused on the confinement of pigs, swill-feeding of pigs and buying/selling of pigs at auctions through a participatory approach with stakeholders. pathogens pathogens pathogens HAL Id: hal-03615744 https://hal.inrae.fr/hal-03615744v1 Submitted on 21 Mar 2022 L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. Distributed under a Creative Commons Attribution 4.0 International License pathogens 1. Introduction Received: 22 December 2021 Accepted: 20 January 2022 Published: 22 January 2022 African swine fever (ASF) remains one of the chief limitations for pig production in Africa, causing high mortality with no available vaccine or means of treatment [1–3]. The ASF virus is the sole virus in the Asfarviridae family and infects wild suids and domestic pigs [4]. Ticks of the Ornithodoros moubata complex are the natural biological vector of the ASF virus, and all investigated species of Ornithodoros can also act as biological vectors that can maintain and transmit the virus for several years [5–7]. Primarily labelled as an African disease following its first description in 1921, it has since become one of the main transboundary diseases of concern in pigs, most recently affecting Europe [8], Asia [9] and more recently, Dominican Republic and Haiti in the Caribbean region [10]. Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affil- iations. Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affil- iations. South Africa has an ASF sylvatic cycle (between soft ticks and warthogs) present in a defined area in the north of the country, with only occasional spill-over to domestic pigs, usually due to biosecurity breaks [11–13]. South Africa has unfortunately experienced an increase in the number of ASF outbreaks in domestic pigs in the last ten years [10], with epidemics in 2012, 2016/17 and 2019/20 by means of domestic pig cycle transmission (domestic pig-to-pig transmission) [10,14]. This development of the domestic pig cycle of ASF in South Africa indicates that the country risk profile has changed [15]. As the risk of disease is not evenly distributed throughout a country, it makes sense to focus resources in Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). https://www.mdpi.com/journal/pathogens Pathogens 2022, 11, 135. https://doi.org/10.3390/pathogens11020135 Pathogens 2022, 11, 135 2 of 12 areas where they would be most effective. An example is risk-based surveillance, where fewer animals need to be tested to confirm presence of the disease [16]. areas where they would be most effective. An example is risk-based surveillance, where fewer animals need to be tested to confirm presence of the disease [16]. South Africa is divided into nine provinces. 1. Introduction In South Africa, even though animal disease control is listed as a concurrent national and provincial function in Schedule 4 of the Constitution of the Republic of South Africa, 1996, veterinary services are listed as an exclusive provincial function in Schedule 5 of the Constitution. This implies that each provincial veterinary service would need to identify which animal diseases should be prioritised in their province for prevention and management strategies. The national veterinary authority of South Africa and industry stakeholders would benefit from the knowledge of which provinces should be prioritised for engagement and assisted in ASF disease management strategies. In a situation where multiple factors must be considered to achieve objectives, a multi-criteria decision analysis (MCDA) can be used to come to a decision. All factors do not contribute equally to the ultimate decision, and thus the weighting of these criteria can be performed by using an analytic hierarchy process (AHP). In an AHP model, both physical and social criteria can be taken into account in the weighting process by measuring the criteria relatively in a hierarchical structure [17]. MCDA has been used in Southern Africa in many different spheres, such as disease risk analysis, health care and mining sectors [18–22]. The aim of this study is to identify which risk factors for ASF are of primary importance in South Africa and should be addressed by intervention strategies, as well as which provinces need to be prioritised to implement these strategies in order to manage and prevent further outbreaks of ASF in domestic pigs in the country. 2. Results The risk factors that were identified for this study and the motivation for inclusion are shown in Table 1. Table 1. Identified risk factors and motivation for inclusion. Risk Factor Reason for Inclusion References Number of ASF outbreaks reported in that province from Jan 2000 to Oct 2020 Previous outbreaks in the area may indicate that a source of ASFV could still be present in the area. [10,11,14,23] Number of pigs in the province In order for ASF outbreaks to occur, it stands to reason that domestic pigs should be present, and that the more pigs, the higher the risk of an outbreak there is. [19,24,25] Whether warthogs are present in the province Although very simplified, if wildlife reservoirs of ASF are present in the province, they could serve as a potential source/maintainer of virus. [12,24,26,27] Responsiveness to ASF questionnaire request per province This can give a crude indication of the current priority of ASF prevention (as this was combined with an ASF awareness campaign) in the particular province. [1,28] Pigs not kept confined This was found to have played a significant role in previous ASF outbreaks in both South Africa and other countries due to owners having no control over what the pigs come into contact with. [19,29–33] Feeding of uncooked swill potentially containing meat products This was found to have played a role in previous ASF outbreaks in both South Africa and other countries due to the ASF virus being able to survive well in a proteinaceous environment. [2,14,19,32,34–41] Buying and/or selling at auctions This was found to have played a role in previous ASF outbreaks in both South Africa and other countries due to the mixing of pigs of various origins, including some which may be at auction due to panic selling that occurs once pigs start dying. [1,14,34,42] Practising home slaughter When pigs are informally slaughtered, there is no meat inspection performed to detect signs of ASF. Furthermore, households slaughtering pigs often provide meat to neighbours or sell the meat in the local community, which may contribute to the spread of disease. Disposal of the remains also presents problems, especially in areas with free-roaming pigs. [1,24,33,36,37,40,42–44] Poor knowledge of ASF Where a pig keeper’s knowledge of ASF is poor to none, no measures are implemented to prevent the entry of ASF into the pig herd. [34,44]. Table 1. Identified risk factors and motivation for inclusion. 2. Results Table 1. Identified risk factors and motivation for inclusion. Pathogens 2022, 11, 135 3 of 12 2.1. Data Collected Data were collected for the presence of each of the risk factors per province (Table 2). Table 2. Results from data collection on presence of ASF risk factors per province. Province Outbreaks (2000–2020) Number of Pigs Warthogs (Present /Absent) Responsiveness (%) Pigs Not Kept Confined (%) Fed Uncooked Swill (%) Use of Auctions (%) Home Slaughter (%) Poor ASF Knowledge (%) Eastern Cape 7 536 366 Present 45.2 (57/126) 30.4 (17/56) 3.5 (2/57) - - - Free State 16 149 878 Present 228.6 (80/35) 24.1 (19/79) 48.1 (38/79) 27.8 (22/79) 53.8 (43/80) 78.9 (63/80) Gauteng 17 141 145 Present 40.0 (14/35) 28.6 (4/14) 21.4 (3/14) 50.0 7/14) 21.4 (3/14) 21.4 (3/14) KwaZulu- Natal 0 200 428 Present 88.0 (44/50) 9.1 (4/44) 38.6 (17/44) 13.6 (6/44) 50.0 (22/44) 60.0 (24/40) Limpopo 25 135 112 Present 22.6 (7/31) 14.3 (1/7) 0 (0/7) 0 (0/7) 42.9 (3/7) 28.6 (2/7) Mpumalanga 20 192 823 Present 64.4 (29/45) 20.7 (6/29) 34.5 (10/29) 93.1 (27/29) 0 (0/28) 79.3 (23/29) Northern Cape 5 13 078 Present 3000.0 (120/4) 13.2 (29/119) 62.2 (74/119) 9.6 (11/115) 63.3 (74/115) 81.4 (96/118) North West 7 127 702 Present 41.9 (13/31) 15.4 (2/13) 15.4 (2/13) 46.2 (6/13 23.1 (3/13) 7.7 (1/13) Western Cape 0 105 417 Absent 377.8 (102/27) 18.6 (19/102) 15.7 (16/102) 3.0 (3/99) 77.5 (79/102) 83.3 (85/102) 2.1. Data Collected Data were collected for the presence of each of the risk factors per province (Table 2). Table 2. Results from data collection on presence of ASF risk factors per province. There were 466 questionnaire responses received in total (Figure 1), which exceeds the determined minimum sample size, resulting in some responsiveness percentages exceeding 100%. Some of the questionnaires were not fully completed, resulting in fewer responses for some of the risk factors than the total number of questionnaires received. In the case of the Eastern Cape province, the questionnaire had been adapted for their awareness activities, which resulted in three of the risk factors being omitted from the responses (marketing, home slaughter and knowledge). For calculation purposes, a value of 0 was utilised for these three risk factors in the Eastern Cape. 2.2. Analytic Hierarchy Process Of the thirty-two experts approached to complete the pairwise comparison form, ten returned responses. Of these, seven were found to be consistent (CR < 0.2). The criteria weights of the consistent experts were then averaged to determine the final weighting of each risk factor (Table 3). 2. Results Of the 466 pig herds represented, the median herd size of the respondents was ten, with a minimum herd size of 1 pig and the largest herd representing over 4000 pigs. 2.3. Final Calculation of Priority Following the standardisation of the risk factors, a decision matrix was compiled with the standardised values, and the criteria weights and priority scores were calculated (Table 3). The province with the highest score was found to be Mpumalanga, followed by Free State and Gauteng (Figure 2). The province with the lowest score was found to be the Western Cape. Pathogens 2022, 11, 135 4 of 12 Table 3. Decision matrix for prioritisation of provinces. Risk Factors Outbreaks (2000–2020) Number of Pigs Warthogs (Present/Absent) Responsiveness (%) Pigs Not Kept Confined (%) Fed Uncooked Swill (%) Use of Auctions (%) Home Slaughter (%) Poor ASF Knowledge (%) Average criteria weights 0.07585 0.05993 0.08451 0.05373 0.22103 0.18975 0.13964 0.05823 0.11735 Province Score Eastern Cape 0.5 1 1 1 0.304 0.035 0 0 0 0.30992 Free State 1 0.5 1 0 0.241 0.481 0.278 0.538 0.788 0.49747 Gauteng 1 0.5 1 1 0.286 0.214 0.5 0.214 0.214 0.45536 KwaZulu- Natal 0 1 1 0.5 0.091 0.386 0.136 0.5 0.6 0.38317 Limpopo 1 0.25 1 1 0.143 0 0 0.429 0.286 0.31921 Mpumalanga 1 1 1 0.5 0.207 0.345 0.931 0 0.793 0.58142 Northern Cape 0.5 0.25 1 0 0.132 0.622 0.096 0.633 0.814 0.43039 North West 0.5 0.25 1 1 0.154 0.154 0.462 0.231 0.077 0.34139 Western Cape 0 0.25 0 0 0.186 0.157 0.03 0.775 0.833 0.23295 Pathogens 2021, 10, x FOR PEER REVIEW 4 of 13 Figure 1. Location of questionnaire responses received. In the case of the Eastern Cape province the questionnaire had been adapted for their Figure 1. Location of questionnaire responses received. Table 3. Decision matrix for prioritisation of provinces. 2.3. Final Calculation of Priority Risk Factors Outbreaks (2000–2020) Number of Pigs Warthogs (Present/Absent) Responsiveness (%) Pigs Not Kept Confined (%) Fed Uncooked Swill (%) Use of Auctions (%) Home Slaughter (%) Poor ASF Knowledge (%) Average criteria weights 0.07585 0.05993 0.08451 0.05373 0.22103 0.18975 0.13964 0.05823 0.11735 Province Score Eastern Cape 0.5 1 1 1 0.304 0.035 0 0 0 0.30992 Free State 1 0.5 1 0 0.241 0.481 0.278 0.538 0.788 0.49747 Gauteng 1 0.5 1 1 0.286 0.214 0.5 0.214 0.214 0.45536 KwaZulu- Natal 0 1 1 0.5 0.091 0.386 0.136 0.5 0.6 0.38317 Limpopo 1 0.25 1 1 0.143 0 0 0.429 0.286 0.31921 Mpumalanga 1 1 1 0.5 0.207 0.345 0.931 0 0.793 0.58142 Northern Cape 0.5 0.25 1 0 0.132 0.622 0.096 0.633 0.814 0.43039 North West 0.5 0.25 1 1 0.154 0.154 0.462 0.231 0.077 0.34139 Western Cape 0 0.25 0 0 0.186 0.157 0.03 0.775 0.833 0.23295 Pathogens 2021, 10, x FOR PEER REVIEW 4 of 13 Table 3. Decision matrix for prioritisation of provinces. Cape Figure 1. Location of questionnaire responses received. I h f h E h h d b d d f h Figure 1. Location of questionnaire responses received. Figure 1. Location of questionnaire responses received. I th f th E t C i th ti i h d b d t d f th i Figure 1. Location of questionnaire responses received. Figure 1. Location of questionnaire responses received. Figure 1. Location of questionnaire responses received. 5 of 12 y Pathogens 2022, 11, 135 Figure 2. Map indicating which provinces in South Africa should be prioritised for ASF interventions. Figure 2. Map indicating which provinces in South Africa should be prioritised for ASF interventions. 3. Discussion This may be because most of the pigs are located in these areas, or that provincial officials have deemed those areas to be most in need of ASF awareness, but this clustering can potentially lead to a shortcoming in the representativeness of the responses for the whole province. Other publications on pigs in the Eastern Cape rather suggest a widespread distribution of pigs in the province [45,46]. Some of the risk factors selected are crude measures, and the specificity could be greatly improved if more data were available. Instead of using pig numbers per province, it would have been even more significant if one had more information on the number of pigs kept per level of biosecurity. Similarly, instead of only the warthog presence, more specific data on the distribution of soft tick vectors (especially those harbouring the virus), would have increased the significance. Further research in this regard is recommended. For the standardisation of certain risk factors, i.e., number of outbreaks, pig population and province responsiveness, there could be many potential ways to approach the matter, but the aim for these risk factors was to try to divide the provinces into three roughly equal categories of low, medium and high risk. Warthog presence or absence was noted for this particular risk factor. This was found to be a practical approach, and can be refined in future studies. For questionnaire administration, since there was no exhaustive list of pig owners, the owners could not be randomly selected, but provincial officials selected owners they were aware of or came into contact with during the awareness campaign, which focused more on non-commercial pig farmers. The sensitivity of the data on the presence of the risk factors measured by means of the questionnaire would be considerably increased if a list of pig owners was available, and sampling could also be better stratified within the provinces. It is recommended that a census of pig owners be carried out, not only to ensure better coverage of ASF awareness activities, but also to improve the planning of surveillance activities, as well as forming the first step in developing a traceability system. g p p g y y For the AHP conducted in this study, experts with a CR < 0.2 were included in the calculations. 3. Discussion As veterinary services in South Africa are divided into provincial units, it is sensible in this context to determine in which provinces ASF should be prioritised. Deciding which province should be prioritised for ASF intervention is not straightforward, as there are many factors which need to be considered, and not all of these factors are equally important. A MCDA using an AHP method is one way to analyse the collected data and be able to come to an overall decision, even when the data are complex and include ordinal data. [17,20,22]. However, it can be modelled in different ways and this study represents only one method, with the specific aim to assist the veterinary services and industry stakeholders of South Africa to prioritise the distribution of ASF intervention and prevention resources. p p Based on the data collected on risk factor occurrence in the respective provinces, and using the weight per risk factor, it was determined that the province that needs to be prioritised the most for ASF intervention and prevention is Mpumalanga, with Free State and Gauteng following. Prior to ASF domestic pig cycles occurring in South Africa (2012), awareness of the disease was only focused in the legislated ASF controlled areas based on the endemic sylvatic cycle. This may have led to pig owners outside of the controlled areas not being aware of the risk of ASF, only viewing warthogs as potential sources of infection or thinking they were safe due to their location. Awareness outside of the controlled areas is essential. Not only has the epidemiology in South Africa changed, but the previous zoning is no longer as relevant in predicting the risk of exposure to the virus. It is therefore recommended to start with intensive awareness and ASF prevention activities, as well as increased surveillance in these three provinces. Due to the absence of responses on the marketing, home slaughter and knowledge risk factors for the Eastern Cape, it can be assumed that there is an underestimation of this Pathogens 2022, 11, 135 6 of 12 province’s risk priority. This can be demonstrated by the fact that if only the six factors for which data are available are used, the end score is much higher (0.45). The completed questionnaires from Mpumalanga and the Eastern Cape also appear to have originated from clustered locations within the provinces. 3. Discussion Saaty [47] suggested using a cut-off of 0.1; however, with the inclusion of nine risk factors to be compared pairwise (resulting in 36 comparisons), more inconsistency was to be expected. Only three experts had a CR < 0.1; if only these had been used, it would have limited representativeness, but using only the data from these three experts resulted in the same three risk factors with the highest weighting, as well as the same first-priority province as that which was found using the data from the seven experts (data not shown). A second round of expert elicitation could have been organised to reduce the irrelevance of the pairwise comparison for experts having a CR > 0.1, but this was not possible due to time constraints. Using the AHP, the experts determined that in the South African context the most important risk factors were pigs not being kept confined, pigs being fed untreated swill potentially containing meat and the buying and/or selling of pigs at auctions (Table 3). By conducting a ranking of the risk factors by means of a pairwise comparison, it is hoped that they will each be ranked on their own merit and not be influenced by the frequency of risk factor occurrence in the local context (with more frequent risk factors being seen as more important). For future studies, it may be advisable to include experts not familiar with the local context for the weighting of the risk factors, to prevent any such possible bias. Specific attention and intervention strategies should be focused on the confinement of pigs, swill-feeding of pigs and use of livestock auctions through a participatory approach with stakeholders. These three main risk factors are governed by human behaviour and are thus preventable by good biosecurity practices. Good biosecurity practices should be the main focus of the ASF prevention strategies that require implementation. This is similar to what has been proposed for the control of ASF in Europe [8]. Confining pigs to a property where the owner is in control of what the pigs are exposed to would Pathogens 2022, 11, 135 7 of 12 7 of 12 be ideal, and is essential for semi-commercial-to-commercial pig farmers. This has been found effective in the recent outbreaks in Timor-Leste [48], where fences were made from corrugated metal roof sheeting. 3. Discussion However, especially in resource-limited communities, it would not be socially acceptable to prohibit free-roaming pig systems [29]. In many instances, pigs are kept by the poor because they do not necessarily have to be fed, but can scavenge for themselves and/or convert kitchen waste into edible protein [15]. For these subsistence farming systems, it would be paramount that these communities are targeted for engagement on awareness of potential disease transmission routes and promotion of the buy-in of all pig owners in an area to prevent the spread of ASF [49]. Identification of potential ASF sources in these areas needs to be highlighted and appropriate measures on how to approach these risks should be discussed with the community using participative approaches [50]. These prevention and control measures should be perceived as positive by pig owners, and should be innovative and increase the role of the community in ASF control [29,37]. In South Africa, livestock auctions are live-animal markets where pigs are sold in lots to the highest bidder via an auctioneer. The participation in auctions as a marketing strategy is often used by small-scale pig farmers, as it is an easy way to market a small—and perhaps inconsistent—number of animals. Intervention strategies for this risk factor could look at assisting small-scale pig farmers with marketing options, such as forming cooperatives [42]. Biosecurity at auction premises, with full traceability of animals, health checks before off-loading and health declarations from the herds of origin are also recommended as intervention strategies, with the engagement and cooperation of auctioneers being paramount. As was seen in previous domestic pig cycle ASF outbreaks in South Africa, due to these anthropogenic factors involving the movement of pigs and their products, outbreaks have not spread contiguously from one area to another but have rather been seen to “jump”. This has also been seen in Russia and China [35,51,52] and emphasises that pig owners should not become complacent regarding the implementation of biosecurity measures if there have been no reports of outbreaks in their immediate vicinity. This was demonstrated very recently in South Africa, where outbreaks occurred in the Western Cape province [10], which were suspected to have spread due to anthropogenic activity from the Eastern Cape province. 4. Materials and Methods An MCDA approach was followed to determine the perceived risk of ASF outbreaks in domestic pigs per province. This entailed searching literature for risk factors associated with ASF outbreaks in domestic pigs. The risk factors were then weighted according to their relative importance by experts by means of an AHP, in order to determine which risk factors are seen as most relevant to the South African situation. Data on the presence of the selected risk factors per province were then collected, both from available records [13,23,25,27] and by means of a questionnaire. Finally, a decision matrix was compiled using the collected data and the weighting of the risk factors, in order to determine which provinces should be prioritised for ASF intervention due to the risk of ASF outbreaks in domestic pigs. 4.1. Risk Factor Identification Risk factors that lead to the introduction and spread of ASFV in domestic pig popula- tions were identified from available literature and consulted via the University of Pretoria library and their internal research engines. The risk factors were discussed with the South African veterinary services and representatives of the pig industry of South Africa to confirm applicability to the local context. 4.2. Provincial Data Collection Data on the provincial presence of these factors were collected by means of available records and a questionnaire administered to pig keepers throughout South Africa as part of an ASF awareness drive by the veterinary services of South Africa. Pathogens 2022, 11, 135 8 of 12 8 of 12 The number of previous outbreaks (2000–2020) per province was obtained from the South African Department of Agriculture, Land Reform & Rural Development (DALRRD) disease database [23] and OIE WAHIS [10]. The number of pigs present per province was obtained according to the report on the 2016 Agricultural Household of the Community Survey [25]. The presence of warthogs in the province was obtained according to the red list of mammals of South Africa, Swaziland and Lesotho [27]. The data on the other risk factors were collected by means of the questionnaire administered. Provincial veterinary officials, as well as business development managers employed by the South African Pork Producers’ Organisation, administered the questionnaires to pig keepers encountered during an ASF awareness drive. To determine the required sample size for the questionnaires in a large population (the sampling frame being primarily the smaller-scale pig keepers of South Africa), the expected frequency of the presence of risk factors was taken at 50% to maximise the sample size. A 95% confidence interval with a desired absolute precision of 5% was utilised. The formula used was adapted from Thrusfield [53]: The formula used was adapted from Thrusfield [53]: n = 1.962 Pexp 1 −Pexp
d2 (1) (1) where n = required sample size, Pexp = expected frequency of the presence of risk factors and d = desired absolute precision. This determined that a minimum of 384 questionnaire responses were required in total. This number was then stratified per province, according to the percentage of the total number of pigs in South Africa present in that province [25]. The provincial veterinary services were then requested to perform a minimum number of questionnaires with pig owners during their ASF awareness drive. Maps for this study were created using ArcGIS® software by Esri. (ArcGIS®and ArcMap™are the intellectual property of Esri and are used herein under license. Copyright © Esri. All rights reserved. www.esri.com). The questionnaire included questions on whether pigs were confined at all times; what the pigs were fed; where pigs were obtained and marketed; and whether any pigs were slaughtered at home. 4.2. Provincial Data Collection The following guidelines were provided to the administrators of the questionnaires to determine the pig owner’s level of knowledge of ASF: p g None: Does not know about ASF; None: Does not know about ASF; Poor: Has heard of ASF but does not know the clinical signs or how it is spread; Reasonable: Knows about ASF and that it causes haemorrhaging and mortality; g g y Good: Knows about ASF and the clinical signs and that it is caused by a virus and how the disease is prevented. Good: Knows about ASF and the clinical signs and that it is caused by a virus and how the disease is prevented. 4.4. Analytic Hierarchy Process 4.4. Analytic Hierarchy Process For the AHP, risk factors were compared in a pairwise manner by experts, to determine a perceived hierarchy of importance. The form for pairwise comparison of the risk factors was compiled on Google Forms, and the link sent via email to experts, in order to determine the relative importance of the risk factors in the South African context. Experts were selected based on relevant experience and knowledge on ASF and the local situation in South Africa. The experts selected were all members of the South African ASF working group (33) and included officials from DALRRD, provincial veterinary officials, researchers and academics. The method utilised was adapted from the AHP [47]. The experts were sent a questionnaire to complete on Google Forms, which compared two risk factors at a time on the Saaty scale (Figure 3). Nine is the maximum number of factors recommended to be used in an AHP, as an increase in number of factors compared leads to decreased consistency in the comparisons. EW 10 of Figure 3. Simplified Saaty scale used for comparing risk factors with the AHP method. Figure 3. Simplified Saaty scale used for comparing risk factors with the AHP method. Figure 3. Simplified Saaty scale used for comparing risk factors with the AHP metho Figure 3. Simplified Saaty scale used for comparing risk factors with the AHP method. The criteria weight of each risk factor per expert was calculated by converting th different comparisons two at a time into a pairwise comparison matrix, then normalisin the values in this matrix and finally taking the mean of the normalised values The criteria weight of each risk factor per expert was calculated by converting the different comparisons two at a time into a pairwise comparison matrix, then normalising the values in this matrix and finally taking the mean of the normalised values. the values in this matrix and finally taking the mean of the normalised values. 4.3. Standardisation of Risk Factors For each of the nine risk factors selected, rules for standardisation were determined and assigned a risk value between 0 and 1. For the data obtained from records, this was achieved by determining two or three categories which could be equated to a lower/higher risk of ASF outbreaks, thus dividing the values into roughly equal categories. The number of completed questionnaires received per province as a percentage of the minimum number requested was used for the “responsiveness” risk factor. For the data obtained from the questionnaires, the percentage of responses confirming the presence of the risk factors was utilised. The risk factors were standardised to assign a risk value between 0 and 1, as per the rules in Table 4. Pathogens 2022, 11, 135 9 of 12 Table 4. Rules for risk factor standardistion. Risk Factor Number of Outbreaks Number of Pigs Warthog Presence Responsiveness Pigs Not Kept Confined Fed Uncooked Swill Use of Auctions Home Slaughter Poor ASF Knowledge Rule 0 = 0 <10 = 0.5 >10 = 1 <140,000 = 0.25 140,000 to 190,000 = 0.5 >190,000 = 1 Present = 1 Absent = 0 >100% = 0 50–99% = 0.5 0–49% = 1 Percentage of responses received indicating the presence of this risk factor Table 4. Rules for risk factor standardistion. 5. Conclusions With the increase in ASF outbreaks in domestic pigs in South Africa, in a historically “ASF-free” area of the country, intervention will be needed in the form of control and pre- vention strategies in order to minimise the impact of this disease in the country. However, as with most animal disease control authorities worldwide, resources are limited. It is therefore sensible to establish where resources should be dedicated in order to address the highest risk areas. By means of an MCDA using the AHP method, it was found that ASF intervention and prevention resources should be focused on the Mpumalanga, Free State and Gauteng Provinces in South Africa, and more investigation is warranted for the Eastern Cape Province. Specific attention and intervention strategies should be focused on the confinement of pigs, swill-feeding of pigs and buying/selling of pigs at auctions through a participatory approach with stakeholders. Author Contributions: Conceptualisation, L.J.v.R., M.-L.P. and E.M.C.E.; methodology, L.J.v.R., M.-L.P. and E.M.C.E.; validation, M.-L.P. and E.M.C.E.; formal analysis, L.J.v.R., M.-L.P. and E.M.C.E.; investigation, L.J.v.R.; writing—original draft, L.J.v.R.; writing—review & editing, M.-L.P. and E.M.C.E.; visualisation, L.J.v.R.; supervision, M.-L.P. and E.M.C.E.; project administration, L.J.v.R.; funding acquisition, E.M.C.E. All authors have read and agreed to the published version of the manuscript. Funding: This work was supported by the project "Unraveling the Effect of Contact Networks & Socio-Economic Factors in the Emergence of Infectious Diseases at the Wild-Domestic Interface" funded by The Ecology and Evolution of Infectious Diseases Program, grant no. 2019-67015-28981 from the USDA National Institute of Food and Agriculture. Institutional Review Board Statement: Research ethics approval was obtained from the University of Pretoria, Faculty of Veterinary Science; and Section 20 approval in terms of the Animal Diseases Act, 1984 (Act 35 of 1984) was obtained from the South African Director of Animal Health. Informed Consent Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: The data presented in this study are available on request from the corresponding author. Acknowledgments: The authors wish to thank the members of the South African provincial veteri- nary services and the South African Pork Producers’ Organisation for assisting with questionnaire administration, as well as the South African ASF working group members for their expert opinion on the weighting of the risk factors and the Director of Animal Health, Mpho Maja for the use of the data as well as the support for this research project. 4.4. Analytic Hierarchy Process Each expert’s matrix was then evaluated for consistency by calculating the co sistency ratio (CR): y g Each expert’s matrix was then evaluated for consistency by calculating the consistency ratio (CR): CI 𝐶𝐼 CR = CI RI (2) stency ratio (CR): 𝐶𝐼 ( ) CR = CI RI (2) istency ratio (CR): 𝐶𝑅= 𝐶𝐼 𝑅𝐼 (2 ( ) CR = CI RI (2) where the Consistency Index (CI) (2) where the Consistency Index (CI) CI = (λmax −n) (n −1) (3) (3) 𝐶𝐼= (𝜆max − 𝑛) ( 1) with n the number of risk factors in the comparison and 𝐶𝐼= (𝜆max − 𝑛) ( 1) with n the number of risk factors in the comparison and (𝑛−1) tors in the comparison and λmax = ∑ i,j sumi ∗weightj
(4) (4) 𝜆max = ෍(𝑠𝑢𝑚௜∗𝑤𝑒𝑖𝑔ℎ𝑡௝) (4 where sum is the sum for each column i of the numerical matrix and weight is the sum for each row j of the normalised matrix. 𝜆max = ෍(𝑠𝑢𝑚௜∗𝑤𝑒𝑖𝑔ℎ𝑡௝) ( where sum is the sum for each column i of the numerical matrix and weight is the sum for each row j of the normalised matrix. ௜,௝ where sum is the sum for each column i of the numerical matrix and weight is the sum fo The Random Index (RI) is dependent on the number of risk factors and is 1.45 for nine factors [47]. where sum is the sum for each column i of the numerical matrix and weight is the sum fo each row j of the normalised matrix. The Random Index (RI) is dependent on the number of risk factors and is 1.45 fo i e fa to [47] For this study, if the CR was more than 0.20, there was an indication that the prioritisa- tion was not consistent enough to be of value, and the matrixes from these experts were excluded due to the inconsistency. nine factors [47]. For this study, if the CR was more than 0.20, there was an indication that the prior ti ti t i t t h t b f l d th t i f th The criteria weights of the consistent experts were then averaged to determine the final criteria weight for each risk factor. Pathogens 2022, 11, 135 10 of 12 10 of 12 4.5. Final Calculation of Priority A decision matrix was compiled with the standardised values per risk factors per province. These values were then multiplied with the criteria weight for that risk factor as determined through the AHP. The sum of the weighted values for all nine risk factors per province determined the final risk value for that province. 5. Conclusions Conflicts of Interest: The authors declare no conflict of interest. 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. Fasina, F.O.; Lazarus, D.D.; Spencer, B.T.; Makinde, A.A.; Bastos, A.D.S. 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Unusual markedly-dilated chorionic vessels with placentomegaly

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CASE STUDY Open Access Abstract We reported a very rare case with placentomegaly and markedly-dilated chorionic vessels. A 23-year-old Japanese pregnant woman was referred to our hospital at 32 weeks of gestation because of suspected dilatation of the umbilical vein. Ultrasound revealed fetal growth restriction with no fetal anomalies. The placenta was thick. The umbilical cord had two arteries and one vein, and marginal insertion of the umbilical cord in the placenta was suspected. A lot of remarkably tortuous tubular structures were observed on the surface of the placenta. Slow blood flow, indemonstrable with color Doppler, was observed within them. Labor started suddenly and progressed very rapidly at 33 weeks of gestation. A female infant weighing 1524 g was delivered. The infant had no malformations. However, she showed hypotension, anemia and DIC and required blood transfusion. The placenta was very large, weighing 1416 g. On the fetal surface, numerous dilated and tortuous vessels were observed, arising from a vein that was connected to the umbilical vein. These venous channels were dilated and tortuous and led into the placenta. In conclusion, prenatal diagnosis of placentomegaly and markedly-dilated chorionic vessels requires assessment of growth, anemia and DIC of the fetus and the newborn infant. Keywords: Dilation of chorionic vessels; Placentomegaly; Fetal anemia; Fetal growth restriction © 2014 Kaido 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/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. Unusual markedly-dilated chorionic vessels with placentomegaly Yoshitaka Kaido, Akihiko Kikuchi*, Tomonobu Kanasugi, Rie Oyama and Toru Sugiyama Introduction We report a very rare case with placentomegaly and markedly-dilated chorionic vessels, without cystic lesions in the placenta typical of PMD. As far as we know, only one case exactly like ours has been reported so far (Lee et al. 1991). Placentomegaly and dilation of chorionic vessels are rare anomalies of the placenta. There have been limited papers that reported on anomalies of the chorionic ves- sels, although venous anomaly of those is clinically im- portant because it may threaten the life of the fetus. Recently, placental mesenchymal dysplasia (PMD) has been reported as a benign entity of the placenta (Taga et al. 2013). PMD was described for the first time by Moscoso et al. in 1991 (Moscoso et al. 1991). It is cha- racterized by placentomegaly with hydropic villi and is often accompanied by a dilation of chorionic vessels (Moscoso et al. 1991; Sander 1993; Kinoshita et al. 2007; Koga et al. 2013). As clinical symptoms of the fetus, fetal growth restriction (FGR) (Sander 1993; Kinoshita et al. 2007; Koga et al. 2013) and hematologic disorders (Sander 1993; Koga et al. 2013) have been reported in PMD. PMD is often suspected by prenatal ultrasound showing cystic lesions in the placenta similar to those of partial mole. Kaido et al. SpringerPlus 2014, 3:146 http://www.springerplus.com/content/3/1/146 Kaido et al. SpringerPlus 2014, 3:146 http://www.springerplus.com/content/3/1/146 a SpringerOpen Journal a SpringerOpen Journal * Correspondence: [email protected] Department of Obstetrics and Gynecology, Iwate Medical University School of Medicine, 19-1 Uchimaru, Morioka, Iwate 020-8505, Japan Case report A 23-year-old gravida 0, para 0, Japanese pregnant woman was referred to our hospital from another hos- pital at 32 weeks of gestation because of suspected dila- tation of the umbilical vein. The patient had no past or familial medical history, and her pregnancy had been uneventful except for pregnancy-induced hypertension (PIH). Her blood pressure was 149/92 mmHg; urinary protein, 2+; and her lower legs were edematous. Obstetric abdominal ultrasound revealed a fetus in cephalic presentation with an estimated fetal weight of 1498 g (−1.7 standard deviation), an amniotic fluid index of 10.6 cm, resistance index of the umbilical artery of 0.65, resistance index of the middle cerebral artery of 0.87, peak systolic velocity of the middle cere- bral artery of 60.2 cm/s (<1.5 MoM). No fetal anomalies were found. The placenta was thick and located on the * Correspondence: [email protected] Department of Obstetrics and Gynecology, Iwate Medical University School of Medicine, 19-1 Uchimaru, Morioka, Iwate 020-8505, Japan Kaido et al. SpringerPlus 2014, 3:146 http://www.springerplus.com/content/3/1/146 Page 2 of 6 Figure 1 Ultrasound at 32 weeks’ gestation showing a thick placenta and tortuous tubular structures on the surface of the placenta. No cystic lesions were observed in the parenchyma of the placenta. Figure 1 Ultrasound at 32 weeks’ gestation showing a thick placenta and tortuous tubular structures on the surface of the placenta. No cystic lesions were observed in the parenchyma of the placenta. (MRI) clearly indicated numerous dilated and tortuous vessels on the surface of the placenta (Figure 3). Trans- vaginal ultrasound revealed normal cervical length. Car- diotocogram showed a reassuring fetal status. posterior uterine wall. The umbilical cord had two arteries and one vein, and marginal insertion of the um- bilical cord in the placenta was suspected. A lot of remarkably tortuous tubular structures were observed on the surface of the placenta (Figure 1). Slow blood flow, indemonstrable with color Doppler, was observed within them (Figure 2). Magnetic resonance imaging The patient was admitted to our hospital for manage- ment of PIH. Although her condition of PIH was stable, labor started suddenly and progressed very rapidly at Figure 2 Ultrasound at 32 weeks’ gestation showing a lot of remarkably tortuous tubular structures (arrows on the left pictures) on the surface of the placenta. Slow blood flow, indemonstrable with color Doppler, was observed within them (arrows on the right pictures). Case report Figure 2 Ultrasound at 32 weeks’ gestation showing a lot of remarkably tortuous tubular structures (arrows on the left pictures) on the surface of the placenta. Slow blood flow, indemonstrable with color Doppler, was observed within them (arrows on the right pictures). Kaido et al. SpringerPlus 2014, 3:146 http://www.springerplus.com/content/3/1/146 Page 3 of 6 http://www.springerplus.com/content/3/1/146 Figure 3 MRI at 32 weeks’ gestation clearly indicated numerous dilated and tortuous vessels (short arrows) on the surface of the placenta. The placenta was thickened (long arrows) with no cystic lesions in the parenchyma. Figure 3 MRI at 32 weeks’ gestation clearly indicated numerous dilated and tortuous vessels (short arrows) on the surface of the placenta. The placenta was thickened (long arrows) with no cystic lesions in the parenchyma. oxygen supply. However, her blood pressure was 39/ 21 mmHg; hemoglobin content, 10.1 mg/dl; platelet count, 71000/μl; fibrinogen, 49.2 mg/dl; FDP, 21.0 μg/ml; and D-D, 8.8 μg/ml. Because these data met the criteria of neonatal DIC, she required blood transfusion and 33 weeks of gestation. A female infant weighing 1524 g was delivered vaginally with an Apgar scores of 8 and 9 at 1 and 5 min, respectively. The infant had no malformations. She was admitted to the NICU for a preterm infant without incubation and Figure 4 Delivered placenta. The umbilical cord was inserted in a velamentous fashion (red arrow). The fetal surface of the placenta showed numerous dilated and tortuous vessels (blue arrows) on and under the chorionic membranes. These vessels branched from the umbilical vein (yellow arrows). Figure 4 Delivered placenta. The umbilical cord was inserted in a velamentous fashion (red arrow). The fetal surface of the placenta showed numerous dilated and tortuous vessels (blue arrows) on and under the chorionic membranes. These vessels branched from the umbilical vein (yellow arrows). Kaido et al. SpringerPlus 2014, 3:146 http://www.springerplus.com/content/3/1/146 Kaido et al. SpringerPlus 2014, 3:146 http://www.springerplus.com/content/3/1/146 Page 4 of 6 Figure 5 Cut surface of the delivered placenta. Thrombi (red allows) were observed in the dilated vessels on the fetal surface of the placenta. There were no cystic lesions in the parenchyma of the placenta. Figure 5 Cut surface of the delivered placenta. Thrombi (red allows) were observed in the dilated vessels on the fetal sur There were no cystic lesions in the parenchyma of the placenta. Figure 5 Cut surface of the delivered placenta. Case report Thrombi (red allows) were observed in the dilated vessels on the fetal surface of the placenta. There were no cystic lesions in the parenchyma of the placenta. administration of dopamine hydrochloride. Her general condition gradually recovered and she was transferred to a nearby hospital from her parents’ home on the 32nd day after birth. Wharton’s jelly was deficient near the insertion site. Thrombi were observed in the dilated vessels on the fetal surface of the placenta (Figure 5). Histologic examination revealed necrosis in the de- cidua. The villi were normally formed and hydropic villi were not observed (Figure 6). Part of the intervillous spaces were occluded by precipitated fibrin and the villous stroma were edematous. Infarction was widely noted in the amnion. Intraplacental vessels were ex- tremely dilated and walls of these vessels were fused The placenta was very large, weighing 1416 g. On the fetal surface, numerous dilated and tortuous vessels were observed, arising from a vein that was connected to the umbilical vein (Figure 4). These venous channels were dilated and tortuous and led into the placenta. Velamen- tous insertion of the umbilical cord was present and Figure 6 Parenchyma of the placenta. The villi were normally formed (red allows) and hydropic villi were not observed. Part of the intervillous spaces were occluded by precipitated fibrin (blue allows), compatible with PIH. Figure 6 Parenchyma of the placenta. The villi were normally formed (red allows) and hydropic villi were not observed. Part of the intervillous spaces were occluded by precipitated fibrin (blue allows), compatible with PIH. Kaido et al. SpringerPlus 2014, 3:146 http://www.springerplus.com/content/3/1/146 Page 5 of 6 Figure 7 Intraplacental vessels were extremely dilated and walls of these vessels were fused (red arrow). Figure 7 Intraplacental vessels were extremely dilated and walls of these vessels were fused (red arrow). (Figure 7). On the other hand, the umbilical arteries and vein, and parenchyma of the maternal side of the pla- centa had no anomalies. vascular obstruction. Circulatory failure due to these abnormal blood vessels and thrombosis may have caused enlargement of the placenta compensatively. But on the other hand, this explanation might not be enough because veins usually dilate not at the central site but at the peripheral site of their obstruction. Discussion We have found the only one previous report of a case with placentomegaly and dilation of the chorionic ves- sels of the placenta exactly like ours (Lee et al. 1991). Although some of the papers on PMD regarded it as a case of PMD (Kinoshita et al. 2007; Koga et al. 2013), Lee et al. described that the villi in their case were nor- mally formed microscopically. Similarities in pathologic and clinical manifestations between the case reported by Lee et al. and our case include; (1) numerous dilated and tortuous vessels on fetal the surface of the placenta, (2) very large and heavy placenta, (3) fetal growth re- striction, and (4) anemia in the infant. Sander described that the cause of FGR might be the di- version of blood away from the maternal intervillous space and that fetal anemia might be a result of a micro- angiopathic process occurring within the aberrant placen- tal vasculature (Sander 1993). In addition, thrombosis in chorionic vessels might have caused FGR associated with fetoplacental circulatory insufficiency in our case. In conclusion, we reported a very rare case with placentomegaly and markedly-dilated chorionic vessels. Prenatal diagnosis of this condition using ultrasound and MRI requires assessment of growth, anemia and DIC of the fetus and the newborn infant. The chorionic vessels are supposedly formed by the branching of the umbilical vessels (Lee et al. 1991). As the direction of flow from the fetus is established, a minimum number of necessary vessels remain, and the balance of the vascular plexus disappears (Lee et al. 1991). This regressive process may be halted or inter- rupted by indefinite factors. So it is thought that placen- tal vascular abnormality occurs very early in embryonic life (Sander 1993; Kinoshita et al. 2007; Lee et al. 1991). However, Jauniaux et al. described that the dilation of chorionic vessels is not observed until the second tri- mester and becomes characteristic during the third tri- mester (Jauniaux et al. 1997). In our case, therefore, dilation of chorionic vessels may be considered to have progressed gradually secondary to peripheral villous Consent Written informed consent was obtained from the patient for the publication of this report and any accompanying images. Competing interests The authors declare that they have no competing interests. Competing interests Authors’ contributions YK, AK and TK took care of the patient. YK and AK wrote the manuscript. All authors read and approved the final manuscript. Kaido et al. SpringerPlus 2014, 3:146 http://www.springerplus.com/content/3/1/146 Kaido et al. SpringerPlus 2014, 3:146 http://www.springerplus.com/content/3/1/146 Authors’ contributions Authors’ contributions YK, AK and TK took care of the patient. YK and AK wrote the manuscript. All authors read and approved the final manuscript. Received: 26 February 2014 Accepted: 3 March 2014 Published: 17 March 2014 Received: 26 February 2014 Accepted: 3 March 2014 Published: 17 March 2014 Page 6 of 6 Page 6 of 6 References Jauniaux E, Nicolaides KH, Hustin J (1997) Perinatal features associated with placental mesenchymal dysplasia. Placenta 18:701–706 Kinoshita T, Fukaya S, Yasuda Y, Itoh M (2007) Placental mesenchymal dysplasia. J Obstet Gynaecol Res 33:83–86 Kinoshita T, Fukaya S, Yasuda Y, Itoh M (2007) Placental mesenchymal dysplasia. J Obstet Gynaecol Res 33:83–86 Kinoshita T, Fukaya S, Yasuda Y, Itoh M (2007) Placental mesenchymal dysplasia. J Obstet Gynaecol Res 33:83–86 Koga H, Makimura M, Tanaka H, Sumioki H (2013) Placental mesenchymal dysplasia and fetal hematologic disorder. J Pediatr Hematol Oncol [Epub ahead of print] Koga H, Makimura M, Tanaka H, Sumioki H (2013) Placental mesenchymal dysplasia and fetal hematologic disorder. 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Case Rep Obstet Gynecol, doi:10 1155/2013/265159 Epub 2013 Nov 20 Taga S, Haraga J, Sawada M, Nagai A, Yamamoto D, Hayase R (2013) A case of placental mesenchymal dysplasia. Case Rep Obstet Gynecol, doi:10.1155/2013/265159. Epub 2013 Nov 20 doi:10.1186/2193-1801-3-146 Cite this article as: Kaido et al.: Unusual markedly-dilated chorionic vessels with placentomegaly. SpringerPlus 2014 3:146. 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